Post‐Translational Modifications in Traumatic Brain Injury: Decoding the Proteomic Landscape and Molecular Mechanisms of Secondary Injury

ABSTRACT

Traumatic brain injury (TBI) initiates a complex secondary injury cascade that significantly contributes to long‐term neurological deficits, with post‐translational modifications (PTMs) emerging as pivotal molecular regulators of this process. Unlike primary mechanical damage, secondary injury evolves over hours to years and involves intricate proteomic alterations that changes in gene expression alone cannot fully explain. PTMs—including phosphorylation, ubiquitination, acetylation, SUMOylation, glycosylation, and emerging modifications such as succinylation, lactylation, and nitrosylation—serve as dynamic molecular switches that fine‐tune protein function, stability, localization, and interactions in response to TBI‐induced stressors. These modifications play dual roles: they can either promote neuroprotection and recovery or drive pathological processes such as neuronal cell death (via apoptosis, necroptosis, and ferroptosis), neuroinflammation through glial activation and inflammasome signaling, blood–brain barrier disruption, mitochondrial dysfunction, and impaired synaptic plasticity. Critically, extensive crosstalk exists among different PTM pathways—such as the interplay between phosphorylation and ubiquitination in protein degradation or the competitive balance between acetylation and SUMOylation—that collectively shape cellular fate after injury. This nuanced regulatory network presents both challenges and opportunities for therapeutic intervention. Targeting PTM‐related enzymes, including kinases, phosphatases, E3 ligases, and histone deacetylases, has shown promise in preclinical models, while novel strategies like Proteolysis‐Targeting Chimeras (PROTACs) and repurposed drugs (e.g., metformin, resveratrol) offer innovative avenues for modulating the PTM landscape. Advances in high‐throughput proteomics and mass spectrometry are enabling the mapping of TBI‐specific PTM signatures across spatiotemporal phases, facilitating the identification of pro‐survival versus pro‐death modification thresholds. Despite hurdles in clinical translation—such as blood–brain barrier penetration and off‐target effects—the growing understanding of PTM dynamics underscores their potential as both biomarkers and therapeutic targets. Future TBI management may thus rely on precision medicine approaches that integrate multi‐PTM profiling to guide combination therapies aimed at tipping the balance toward neural repair and functional recovery.

TBI triggers dysregulation of phosphorylation, ubiquitination, SUMOylation, acetylation, glycosylation, and emerging metabolic PTMs (lactylation, succinylation, S‐nitrosylation). These modifications converge on downstream pathological pathways—neuronal death, neuroinflammation, BBB disruption, mitochondrial dysfunction, and axonal injury—while also enabling pro‐survival signals. Understanding PTM crosstalk informs biomarker development (e.g., CSF p‐tau) and multi‐target combination therapies for TBI. BBB, blood–brain barrier; CSF, cerebrospinal fluid; PROTAC, proteolysis‐targeting chimera; PTM, post‐translational modification; TBI, traumatic brain injury.

1. Introduction

Traumatic brain injury (TBI) represents a major global health burden, with millions affected annually and limited therapeutic options to mitigate its long‐term neurological consequences [1, 2, 3]. While the initial mechanical insult—termed primary injury—is irreversible, the subsequent cascade of biochemical, cellular, and molecular events, collectively known as secondary injury, unfolds over hours to months and offers a critical window for intervention [4]. This delayed phase involves complex processes such as neuroinflammation, mitochondrial dysfunction, blood–brain barrier (BBB) disruption, and diverse forms of regulated cell death, all of which contribute significantly to poor clinical outcomes [5]. Despite extensive research, current treatments remain largely supportive, underscoring an urgent need to decipher the molecular drivers of secondary injury to develop targeted neuroprotective strategies.

Emerging evidence highlights that post‐translational modifications (PTMs)—covalent and generally enzymatic modifications of proteins after synthesis—serve as pivotal regulators of nearly every pathological process in TBI [6]. Unlike changes in gene expression, PTMs rapidly and reversibly alter protein function, localization, stability, and interactions, thereby fine‐tuning cellular responses to injury. Key PTMs implicated in TBI include phosphorylation, ubiquitination, acetylation, SUMOylation, and glycosylation, each acting as a molecular switch that can either promote neuronal survival or drive degeneration depending on context, timing, and target specificity [7]. For instance, dysregulated kinase signaling can exacerbate excitotoxicity and apoptosis, while impaired ubiquitin‐proteasome function may lead to toxic protein aggregation—a conclusion supported by Level 1 evidence from genetic knockout and pharmacological inhibition studies in rodent CCI models [8].

This review aims to systematically decode the proteomic landscape shaped by PTMs in the context of TBI‐induced secondary injury. By integrating recent advances in neuroproteomics and functional studies, we elucidate how specific PTMs modulate core pathophysiological pathways—including neuronal death, glial activation, BBB breakdown, and synaptic dysfunction—and explore their potential as both biomarkers and therapeutic targets [9]. Importantly, we underscore the clinical potential of therapeutic strategies aimed at PTM‐regulating enzymes (e.g., kinases, deacetylases, E3 ligases), and further examine emerging approaches—including proteolysis‐Targeting Chimeras (PROTACs) and drug repurposing—that are poised to enable precision medicine for TBI [10, 11]. Understanding the dual‐edged nature of PTMs—not merely as markers of damage but as dynamic controllers of cellular fate—is essential for bridging mechanistic insights into effective interventions that improve functional recovery after brain trauma.

This review was conducted as a comprehensive narrative review with a systematic literature search strategy. We followed the PRISMA 2020 guidelines where applicable for reporting the search and selection process, while recognizing that narrative reviews are not required to fulfill all PRISMA items designed for systematic reviews and meta‐analyses. The final literature search was performed on March 1, 2026, using the PubMed database. All retrieved records were imported into EndNote for duplicate removal. Study selection was performed independently by two reviewers (P.S. and L.L.), proceeding in two stages: (1) title and abstract screening against the inclusion criteria, followed by (2) full‐text review of potentially eligible articles. Disagreements were resolved through discussion. Exclusion reasons at the full‐text stage (e.g., not TBI‐specific, no PTM or protein‐level data, conference abstracts, non‐English publications) were documented. A total of 175 references were ultimately selected and cited in this review.

The search was limited to articles published between January 2000 and March 2026, with emphasis on studies published in the last 5 years (2021–2026) to capture recent advances; however, seminal older references were also included when considered essential for establishing fundamental concepts. The following main search string was used in PubMed: (“traumatic brain injury” OR “TBI” OR “head trauma” OR “brain contusion”) AND (“post‐translational modification” OR “PTM” OR “phosphorylation” OR “ubiquitination” OR “acetylation” OR “SUMOylation” OR “PARylation” OR “O‐GlcNAcylation” OR “lactylation” OR “S‐nitrosylation” OR “glycosylation”) AND (“kinase” OR “phosphatase” OR “E3 ubiquitin ligase” OR “deubiquitinase” OR “HDAC” OR “SIRT” OR “HAT” OR “PARP” OR “SUMO protease” OR “PROTAC”). Additional focused searches were performed for specific proteins and pathways (e.g., “tau hyperphosphorylation,” “NF‐κB ubiquitination,” “NLRP3 inflammasome,” “ferroptosis,” “necroptosis,” “mitochondrial dynamics,” “Drp1 SUMOylation,” “blood–brain barrier PTM”) and for emerging PTM types (e.g., “lactylation TBI,” “succinylation TBI”). Clinical trials were searched using the same PTM‐related keywords combined with “clinical trial” and filtered by study phase. The search was further supplemented by manually screening the reference lists of retrieved articles and relevant reviews.

Inclusion criteria: (1) original research articles, systematic reviews, and high‐quality narrative reviews; (2) studies using in vivo TBI models (rodent, porcine, or other mammalian species), ex vivo human tissue, or TBI patient biofluids (CSF, serum, plasma, saliva); (3) studies directly reporting PTM changes (e.g., phosphorylation, ubiquitination, acetylation, SUMOylation, PARylation, glycosylation, lactylation, S‐nitrosylation) after TBI; (4) studies evaluating pharmacological or genetic modulation of PTM‐regulating enzymes (kinases, phosphatases, E3 ligases, DUBs, HDACs, HATs, PARP, etc.) with assessment of neurobehavioral, histological, or molecular outcomes; (5) clinical trials and observational studies reporting proteomic, PTM, or biomarker data. Exclusion criteria: (1) studies not specifically related to TBI (e.g., stroke, spinal cord injury, cancer, or neurodegenerative diseases without a TBI component); (2) studies focusing solely on gene expression (mRNA) without PTM or protein‐level data; (3) conference abstracts, editorials, and opinion pieces without original data; (4) non‐English publications.

To systematically evaluate the strength of evidence for key mechanistic statements, we implemented a three‐tier evidence grading system: Level 1: studies directly performed in TBI models with causal validation, including genetic manipulation or pharmacological intervention with appropriate controls and multi‐dimensional outcome assessment. Level 2: TBI model studies demonstrating correlations or time‐course changes without causal validation or those using single‐arm pharmacological interventions without genetic or inhibitor‐based verification. Level 3: review articles, meta‐analyses. This evidence grading system was applied consistently throughout the review to distinguish well‐established PTM mechanisms (Level 1) from correlative findings (Level 2) and emerging, preliminary observations (Level 3). For each PTM type and pathological process discussed in Sections 5 and 6, we explicitly indicate the highest level of supporting evidence available, with particular caution exercised for emerging modifications (lactylation, succinylation, nitrosylation) where human or causal data remain limited. A consolidated summary of evidence levels for each PTM type and associated pathological process is provided in Table S1.

2. Background

2.1. Brief Definition of TBI, the Significance of Secondary Injury Cascades, and Introduction of PTMs as Crucial Molecular Switches

TBI is a heterogeneous neurological condition resulting from external mechanical forces that disrupt normal brain function, ranging from mild concussions to severe, life‐threatening injuries [12, 13, 14]. While the primary injury occurs at the moment of impact—characterized by direct tissue shearing, vascular rupture, and neuronal deformation—the subsequent secondary injury cascade is responsible for the majority of long‐term neurological deficits and progressive neurodegeneration [15, 16, 17]. This delayed pathophysiological process unfolds over hours to months and involves a complex interplay of excitotoxicity, oxidative stress, mitochondrial dysfunction, BBB disruption, neuroinflammation, and programmed cell death mechanisms such as apoptosis, necroptosis, and autophagy [18, 19, 20, 21]. Critically, these cascades are not static but dynamically regulated by molecular signaling networks that determine cellular fate—survival or death—following trauma.

PTMs have emerged as central regulatory mechanisms governing these secondary injury pathways. PTMs are covalent chemical alterations added to proteins after their synthesis, acting as rapid, reversible, and context‐dependent molecular switches that fine‐tune protein function, localization, stability, and interactions [22, 23, 24]. Unlike transcriptional regulation, which requires time for gene expression, PTMs enable immediate cellular responses to stressors like those encountered in TBI. Key PTMs implicated in TBI include phosphorylation, ubiquitination, acetylation, SUMOylation, and poly(ADP‐ribosyl)ation, each modulating distinct aspects of neuronal and glial biology [22, 25]. Aberrant phosphorylation of tau and neurofilament proteins has been observationally linked to cytoskeletal collapse and axonal degeneration, with mechanistic support from site‐directed mutagenesis studies showing that phospho‐mimetic mutants disrupt microtubule assembly [26]. The dynamic nature of PTMs positions them as ideal therapeutic targets, as their enzymatic writers, erasers, and readers can be pharmacologically modulated to redirect pathological signaling toward neuroprotection.

2.2. Concise Overview of Key PTM Types (Phosphorylation, Ubiquitination, Acetylation, etc.) and Their Dual Role in Promoting Cell Death

Among the most extensively studied PTMs in TBI is phosphorylation, mediated by kinases and phosphatases. Following TBI, excessive glutamate release triggers calcium influx via NMDA receptors, activating calcium‐dependent kinases such as CaMKII and calcineurin. This leads to hyperphosphorylation of tau at pathological epitopes (e.g., Ser202/Thr205), disrupting microtubule stability and promoting neurofibrillary tangle‐like pathology [26]. Paradoxically, phosphorylation also activates pro‐survival pathways; for example, Akt phosphorylation inhibits GSK‐3β, reducing apoptosis [27]. Thus, the net effect depends on spatiotemporal context and substrate specificity.

Ubiquitination, the attachment of ubiquitin moieties to lysine residues, primarily regulates protein degradation via the ubiquitin‐proteasome system (UPS). In TBI, oxidative stress impairs UPS function, leading to accumulation of ubiquitinated proteins and activation of unfolded protein response (UPR)‐mediated apoptosis [28, 29]. However, certain E3 ligases, such as Parkin, promote mitophagy—the selective removal of damaged mitochondria—thereby limiting ROS production and cytochrome c release [30]. Dysregulation of this balance shifts cells toward death.

Acetylation, controlled by histone acetyltransferases (HATs) and deacetylases (HDACs), modulates both epigenetic gene expression and non‐histone protein function. In TBI, HDAC overexpression suppresses neuroprotective genes (e.g., BDNF, HSP70), while HAT inhibition exacerbates inflammation [31, 32]. Conversely, SIRT1‐mediated deacetylation of p53 reduces its transcriptional activity, attenuating neuronal apoptosis [33]. Pharmacological HDAC inhibitors (e.g., sodium butyrate) have shown efficacy in preclinical models by restoring acetylation homeostasis and improving cognitive outcomes.

Other critical PTMs include poly(ADP‐ribosyl)ation (PARylation), catalyzed by PARP‐1 in response to DNA damage. Overactivation of PARP‐1 depletes cellular NAD⁺ and ATP, triggering parthanatos—a caspase‐independent cell death pathway [34]. PARP inhibitors (e.g., PJ34) reduce lesion volume and improve motor function in rodent TBI models. Additionally, SUMOylation stabilizes key transcription factors like HIF‐1α under hypoxic conditions post‐TBI, enhancing adaptive responses but potentially exacerbating edema if prolonged [35, 36, 37] (Table 1).

TABLE 1.

Summarizing key PTM types, regulatory enzymes, major substrates in TBI, and functional consequences.

PTM type	Regulatory enzymes	Key substrates in TBI	Pro‐death effects	Pro‐survival effects
Phosphorylation	Kinases (GSK‐3β, JNK), phosphatases	Tau, NF‐H, CREB, Akt	Cytoskeletal collapse, synaptic loss	Inhibition of pro‐apoptotic factors
Ubiquitination	E3 ligases (Parkin), DUBs	Misfolded proteins, PINK1	Proteotoxic stress, UPR activation	Mitophagy, clearance of damaged organelles
Acetylation	HATs (p300), HDACs (SIRT1, HDAC2)	Histones, p53, NF‐κB	Suppression of neurotrophic genes	Anti‐inflammatory, metabolic adaptation
PARylation	PARP‐1, PARG	Histones, DNA repair proteins	Energy depletion, parthanatos	DNA repair initiation (at low levels)
SUMOylation	SUMO E3 ligases, sentrin/SUMO‐specific proteases	HIF‐1α, DRP1	Enhanced edema, mitochondrial fission	Hypoxic preconditioning, stress resistance

These PTMs do not operate in isolation; extensive crosstalk among them is a defining feature. For example, phosphorylation of HDAC4 promotes its nuclear export, thereby derepressing MEF2‐dependent survival genes, whereas ubiquitination of phosphorylated IκB liberates NF‐κB to drive inflammatory transcription [38, 39, 40]. Such network complexity underscores the need for systems‐level approaches in therapeutic development.

2.3. Summary of Therapeutic Implications and Future Directions in Targeting PTM Enzymes and Pathways for TBI Treatment

Targeting PTM‐regulating enzymes offers a promising avenue for neuroprotective therapy in TBI, given their upstream position in secondary injury cascades and druggable nature. Preclinical studies demonstrate that selective inhibition of detrimental PTM pathways—such as PARP‐1, GSK‐3β, or Class I HDACs—can significantly reduce lesion size, attenuate neuroinflammation, preserve synaptic integrity, and improve functional recovery in rodent models. Moreover, combinatorial strategies that simultaneously enhance protective PTMs (e.g., SIRT1 activation) while suppressing pathological ones may yield synergistic benefits. Advances in proteomics now enable global profiling of PTM dynamics post‐TBI, facilitating the identification of novel enzyme‐substrate pairs as therapeutic targets [41, 42, 43]. Future directions include the development of brain‐penetrant, isoform‐specific modulators to minimize off‐target effects, as well as biomarker‐guided clinical trials that stratify patients based on PTM signatures (e.g., phospho‐tau levels in CSF) to personalize treatment [44]. Additionally, integrating PTM‐targeted therapies with neuromodulatory approaches like repetitive transcranial magnetic stimulation—which itself may influence synaptic PTM states—could amplify regenerative outcomes [45]. Ultimately, a nuanced understanding of PTM networks will be essential to transform the current paradigm of symptomatic TBI management into one of precision neuroprotection.

3. Overview

3.1. The Global Burden and Clinical Challenge of Traumatic Brain Injury (Epidemiology and Current Therapeutic Gaps)

TBI represents a leading cause of death and disability worldwide, imposing a substantial socioeconomic burden on healthcare systems and affected families. Annually, an estimated 69 million individuals sustain a TBI, with incidence rates varying significantly by region but consistently higher in low‐ and middle‐income countries due to road traffic accidents, interpersonal violence, and occupational hazards [2]. In high‐income nations, falls among the elderly and sports‐related concussions in adolescents are increasingly recognized as major contributors. The clinical spectrum of TBI is remarkably heterogeneous, ranging from transient confusion and headache in mild cases (concussion) to prolonged coma, severe cognitive impairment, and permanent vegetative states in severe injuries. Despite advances in neurocritical care—such as intracranial pressure monitoring, controlled hyperventilation, and osmotherapy—the mortality rate for severe TBI remains stubbornly high at approximately 30%–40%, while over 50% of survivors suffer long‐term functional deficits that impede return to work or independent living [46, 47, 48]. This grim reality underscores a critical therapeutic gap: current clinical management is almost exclusively supportive, aimed at preventing secondary insults like hypoxia or hypotension, but lacks disease‐modifying pharmacotherapies that directly target the underlying molecular pathology [11, 49]. Decades of failed clinical trials targeting single pathways—such as glutamate excitotoxicity or free radical scavenging—highlight the inadequacy of reductionist approaches in a condition characterized by multifactorial, interacting pathophysiological cascades [44]. Consequently, there is an urgent need to identify novel, druggable targets that sit at the nexus of these converging injury mechanisms.

3.2. Primary Versus Secondary Injury: The Concept of a Therapeutic Window

The pathophysiology of TBI is traditionally divided into two phases: primary and secondary injury. The primary injury is the immediate, mechanical damage inflicted at the moment of impact, including contusions, lacerations, diffuse axonal injury from shearing forces, and vascular disruption [50, 51]. This phase is predominantly irreversible, with its magnitude governed by the biomechanical characteristics of the injurious impact. In stark contrast, the secondary injury is a delayed, biochemical, and cellular cascade that evolves over minutes to months post‐injury, amplifying the initial damage and driving progressive neurodegeneration. Key components of this cascade include excitotoxicity from excessive glutamate release [52], mitochondrial dysfunction leading to energy failure [15], oxidative stress from reactive oxygen species (ROS) [53], breakdown of the BBB [54], robust neuroinflammation mediated by activated microglia and infiltrating leukocytes [55], and various forms of regulated cell death [19] (e.g., apoptosis, necroptosis). Critically, because the secondary injury is not instantaneous but unfolds progressively, it presents a crucial therapeutic window—a period during which targeted interventions could potentially halt or mitigate the expansion of tissue damage. This window, though variable in duration depending on injury severity and individual factors, offers the best opportunity for neuroprotective strategies. However, the failure of past neuroprotective agents often stemmed from poor timing of administration, lack of target engagement biomarkers, or an oversimplified view of the injury process that ignored its dynamic complexity.

3.3. The Proteomic Complexity of TBI: Beyond Gene Expression

While genomic and transcriptomic studies have provided valuable insights into TBI, they often fail to capture the full functional state of the injured brain. The proteome—the complete set of proteins expressed in a cell or tissue—is the primary functional executor of biological processes and is subject to extensive regulation that occurs after gene transcription. In the acute and subacute phases following TBI, rapid changes in protein abundance, localization, interaction networks, and, most importantly, PTMs, dictate cellular responses far more dynamically than changes in mRNA levels alone [56, 57, 58]. For instance, a protein's activity can be switched on or off within seconds by phosphorylation, without any change in its gene expression. Mass spectrometry‐based proteomics has revealed that TBI induces profound and widespread alterations across the brain proteome, affecting thousands of proteins involved in synaptic function, cytoskeletal integrity, metabolism, and inflammatory signaling [59, 60, 61]. These changes are highly time‐dependent and region‐specific, creating a complex molecular landscape that cannot be predicted from genomic data. Therefore, focusing on the proteome, and specifically on PTMs, provides a more direct and actionable view of the pathological drivers of secondary injury, offering a richer source of potential therapeutic targets and pharmacodynamic biomarkers.

3.4. Definition and Functional Significance of PTMs

PTMs are covalent chemical modifications added to specific amino acid residues of a protein after its synthesis on the ribosome. They represent a fundamental layer of cellular regulation, allowing for rapid, reversible, and context‐specific fine‐tuning of virtually every aspect of protein biology. Common PTMs include phosphorylation (addition of a phosphate group to serine, threonine, or tyrosine), ubiquitination (attachment of ubiquitin to lysine, often marking proteins for degradation), acetylation (addition of an acetyl group to lysine, regulating both epigenetic and non‐epigenetic functions), SUMOylation, methylation, and glycosylation [62, 63]. The enzymes responsible for adding (writers), removing (erasers), and recognizing (readers) these modifications form intricate regulatory networks. In the context of TBI, PTMs act as master molecular switches that integrate diverse stress signals—such as calcium influx, ROS, and DNA damage—and translate them into specific cellular outcomes. For example, phosphorylation of the kinase JNK can promote neuronal apoptosis, while acetylation of the transcription factor p53 can enhance its pro‐death transcriptional activity [64, 65]. Conversely, protective PTMs exist; SIRT1‐mediated deacetylation of NF‐κB can dampen neuroinflammatory responses. The dynamic and reversible nature of many PTMs makes their regulatory enzymes highly attractive as pharmacological targets, as their activity can be modulated to shift the balance from pathological to protective signaling (Table 2).

TABLE 2.

The key PTM types relevant to TBI, the primary regulatory enzymes, and the major functional consequences in the injured brain.

PTM type	Level	Writer enzymes	Eraser enzymes	Primary functional consequences in TBI
Phosphorylation	2	Kinases (e.g., GSK‐3β, JNK, CaMKII)	Phosphatases (e.g., PP2A, calcineurin)	Regulates enzyme activity, cytoskeletal stability, synaptic plasticity, and cell death/survival [26, 27, 65, 66]
Ubiquitination	2–3	E3 Ubiquitin Ligases (e.g., Parkin, MDM2)	Deubiquitinases (DUBs)	Controls protein degradation via the proteasome, mitophagy, and inflammatory signaling (e.g., NF‐κB activation) [30, 67, 68, 69]
Acetylation	1–2	Histone Acetyltransferases (HATs, e.g., p300)	Histone Deacetylases (HDACs, e.g., SIRT1, HDAC2)	Modulates chromatin structure (gene expression) and the activity of non‐histone proteins (e.g., p53, tubulin) involved in stress response and metabolism [70, 71]
PARylation	2	PARP‐1	PARG	Responds to DNA damage; overactivation leads to energy depletion and parthanatos, a form of cell death [72]
SUMOylation	1–2	SUMO E3 Ligases	SENP Proteases	Regulates protein stability, nuclear‐cytoplasmic transport, and stress responses, including mitochondrial dynamics [73, 74]

3.5. Post‐Translational Modifications in TBI: Druggable Nodes Bridging Pathophysiology and Precision Neuroprotection

This review aims to synthesize the current understanding of how specific PTMs orchestrate the key pathological processes of secondary injury following TBI. We will delve into the roles of phosphorylation, ubiquitination, acetylation, and other critical modifications in driving excitotoxicity, neuroinflammation, mitochondrial failure, and neuronal death, drawing on evidence from preclinical models and emerging human biomarker studies. A central focus will be on the enzymes that regulate these PTMs—not only as mechanistic players but as promising, druggable nodes for therapeutic intervention. We will critically evaluate the preclinical data supporting the use of inhibitors or activators targeting these enzymes (e.g., HDAC inhibitors, PARP inhibitors, kinase modulators) and discuss the challenges associated with their translation to the clinic, including issues of brain penetrance, isoform selectivity, and optimal therapeutic timing within the secondary injury window. Furthermore, we will explore how advanced proteomic technologies are enabling the mapping of global PTM dynamics (“PTM‐omes”) after TBI, which holds the potential to identify novel, patient‐specific therapeutic targets and companion biomarkers for future precision medicine approaches. By bridging the molecular landscape of PTMs with the clinical challenge of TBI, this review seeks to provide a roadmap for the development of the next generation of neuroprotective therapies.

4. Principles and Mechanisms of Major Post‐Translational Modifications

PTMs constitute a sophisticated biochemical language that cells use to rapidly respond to environmental cues, including the profound stress of TBI [75]. This regulatory layer operates beyond the static genome, allowing for dynamic, reversible, and highly specific control over protein function. The core machinery of PTM regulation involves three key components: “writers” are the enzymes that catalyze the covalent attachment of a chemical group to a specific amino acid residue on a target protein; “erasers” are the enzymes responsible for the removal of these modifications, thereby resetting the protein to its basal state; and “readers” are specialized protein domains that recognize and bind to the modified residue, translating the modification into a functional outcome, such as altered protein–protein interactions, changes in subcellular localization, or modulation of enzymatic activity (Figure 1). This tripartite system allows for exquisite spatiotemporal control over cellular signaling networks. In the context of TBI, the massive ionic shifts, energy failure, and oxidative stress trigger a global dysregulation of PTM writer and eraser activities, leading to a pathological rewiring of the neuronal and glial proteome that drives secondary injury processes like neuroinflammation, excitotoxicity, and cell death [67, 76, 77]. Understanding the specific roles of these major PTMs is therefore fundamental to identifying novel points for therapeutic intervention.

FIGURE 1.

Schematic overview of major PTMs driving secondary injury after TBI. TBI triggers a triad of upstream stresses—calcium overload, oxidative stress, and metabolic crisis—that collectively dysregulate PTM writer and eraser enzymes. Five major PTM classes are central to the secondary injury cascade. (1) Phosphorylation: imbalance between Ca²⁺‐dependent kinases (CaMKII, PKC, JNK, p38) and phosphatases (PP2A, calcineurin) leads to tau hyperphosphorylation (axonal injury), NR2B phosphorylation (excitotoxicity), and c‐Jun‐driven pro‐apoptotic gene expression. (2) Ubiquitination and SUMOylation: the ubiquitin‐proteasome system (UPS) clears damaged proteins, but its dysfunction promotes aggregate accumulation. MDM2‐mediated p53 ubiquitination is altered, while Drp1 SUMOylation drives excessive mitochondrial fission and cytochrome c release. (3) Acetylation: histone hypoacetylation silences neuroprotective genes; p53 and NF‐κB acetylation promote apoptosis and inflammation. SIRT1 deacetylates both p53 and NF‐κB, offering endogenous neuroprotection. (4) Glycosylation: altered glycan patterns on endothelial cells and leukocytes facilitate immune cell infiltration and BBB breakdown; aberrant receptor glycosylation modulates excitotoxicity. (5) Emerging PTMs (lactylation, succinylation, nitrosylation): metabolic reprogramming and NO burst drive these modifications, linking metabolic stress to microglial pro‐inflammatory phenotypes, mitochondrial dysfunction, and proteasomal impairment. Collectively, these PTM networks converge on hallmark TBI pathologies: axonal injury, excitotoxicity, neuroinflammation, mitochondrial failure, and synaptic loss. The figure highlights potential therapeutic nodes (kinase inhibitors, HDAC inhibitors, SIRT1 activators, PROTACs) and underscores the need for multi‐targeted strategies.

4.1. Phosphorylation: The Kinase‐Phosphatase Signaling Switch

Phosphorylation, the addition of a phosphate group (PO₄ ³⁻) primarily to serine, threonine, or tyrosine residues, is the most extensively studied PTM and serves as the primary on/off switch for countless signaling pathways. Its dynamics are governed by the opposing actions of kinases (writers) and phosphatases (erasers). In the healthy brain, this balance maintains homeostasis in synaptic transmission, cytoskeletal integrity, and gene expression. However, TBI induces a massive, pathological surge in intracellular calcium, which activates a host of calcium‐dependent kinases, including Ca²⁺/calmodulin‐dependent kinase II (CaMKII) and protein kinase C (PKC), while simultaneously inhibiting key phosphatases like calcineurin (PP2B) and protein phosphatase 2A (PP2A) through oxidative mechanisms or endogenous inhibitors [78, 79]. This imbalance leads to hyperphosphorylation of critical substrates. For instance, hyperphosphorylation of the microtubule‐associated protein tau destabilizes axonal microtubules, contributing to diffuse axonal injury, a hallmark of TBI. Similarly, phosphorylation of the NMDA receptor subunit NR2B enhances calcium influx, exacerbating excitotoxicity [80]. Conversely, the mitogen‐activated protein kinase (MAPK) pathways, including JNK and p38, are robustly activated by TBI‐induced stress, and their phosphorylation of transcription factors like c‐Jun promotes pro‐apoptotic gene expression [65, 81, 82]. Targeting this kinase‐phosphatase axis—either by inhibiting specific pathological kinases or by boosting the activity of protective phosphatases—represents a major therapeutic strategy.

4.2. Ubiquitination and SUMOylation: Regulating Protein Fate and Stability

Ubiquitination and SUMOylation are two distinct but sometimes antagonistic PTMs that involve the covalent attachment of small proteins (ubiquitin or Small Ubiquitin‐like Modifier, SUMO) to lysine residues on target proteins. While they share some enzymatic machinery, their functional outcomes are markedly different, and both are critically involved in the TBI response.

The UPS is the primary pathway for selective protein degradation in the cell. The process involves a cascade of three enzymes: an E1 activating enzyme, an E2 conjugating enzyme, and an E3 ligase (the writer), which confers substrate specificity. Polyubiquitination typically targets a protein for destruction by the 26S proteasome. In TBI, the UPS plays a dual role. On one hand, it is essential for clearing damaged, misfolded, or aggregated proteins that accumulate due to oxidative stress and energy failure, acting as a crucial neuroprotective mechanism [83]. On the other hand, excessive or dysregulated ubiquitination can lead to the untimely degradation of key survival proteins. For example, the E3 ligase MDM2 ubiquitinates the tumor suppressor p53, targeting it for degradation. Following TBI, disruption of this regulation can lead to p53 stabilization and activation of its pro‐apoptotic program [71]. Furthermore, TBI can directly impair proteasome function itself, leading to a toxic buildup of ubiquitinated protein aggregates, a feature also seen in chronic neurodegenerative diseases.

SUMOylation, mediated by a parallel E1‐E2‐E3 enzymatic cascade, generally does not signal for degradation. Instead, it modulates a protein's subcellular localization, stability, and, most importantly, its ability to interact with other proteins. In the stressed neuron, SUMOylation is a rapid and powerful adaptive response. A key example is the SUMOylation of dynamin‐related protein 1 (Drp1), a master regulator of mitochondrial fission. TBI‐induced Drp1 SUMOylation has been observationally associated with its stabilization at the mitochondrial membrane. This is mechanistically supported by studies showing that SUMOylation‐deficient Drp1 mutants reduce mitochondrial fragmentation in cultured neurons [84]. The therapeutic relevance of this pathway is further supported by studies using the Drp1 inhibitor Mdivi‐1, which therapeutically validated the role of Drp1‐mediated fission in TBI outcomes [85]. Conversely, SUMOylation of certain transcription factors can promote the expression of antioxidant and anti‐apoptotic genes. The interplay between ubiquitination and SUMOylation is complex; SUMO can sometimes act as a signal for a specialized form of ubiquitination (STUbL‐mediated), linking the two pathways in the regulation of protein fate during cellular stress [86, 87, 88] (Table 3).

TABLE 3.

The core enzymatic machinery and primary functional consequences of ubiquitination and SUMOylation in TBI.

Feature	Ubiquitination	SUMOylation
Writer (E3 ligase)	MDM2, Parkin, CHIP	PIAS family, RanBP2
Eraser (protease)	Deubiquitinating Enzymes	Sentrin/SUMO‐specific proteases
Primary function	Targets proteins for proteasomal degradation; regulates endocytosis and DNA repair	Modulates protein–protein interactions, subcellular localization, and transcriptional activity; stabilizes protein complexes.
Key TBI substrate and consequence	p53: Stabilization → Apoptosis. Misfolded proteins: clearance vs. aggregate formation if UPS impaired	Drp1: Hyper‐SUMOylation → excessive mitochondrial fission → bioenergetic collapse. HIF‐1α: Stabilization → adaptive metabolic reprogramming.

4.3. Acetylation: Epigenetic and Metabolic Regulation

Acetylation, the addition of an acetyl group to the ε‐amino group of lysine residues, is a pivotal PTM that regulates both epigenetic gene expression and the function of numerous non‐histone proteins. This modification is controlled by histone acetyltransferases (HATs, writers) and histone deacetylases (HDACs, erasers). In TBI, the balance between HAT and HDAC activity is profoundly disrupted. Global histone hypoacetylation is a common finding in the injured brain, leading to a repressive chromatin state that silences the expression of neuroprotective and plasticity‐related genes. This is partly driven by the upregulation or activation of specific HDAC isoforms (e.g., HDAC2) and the inactivation of HATs like CBP/p300 due to oxidative stress or cleavage by calpains [89]. Beyond epigenetics, acetylation directly controls the activity of key metabolic and stress‐response proteins. For example, acetylation of the transcription factor p53 enhances its DNA‐binding affinity and its ability to transactivate pro‐apoptotic genes like PUMA and Bax [90]. Conversely, the NAD⁺‐dependent deacetylase SIRT1 can deacetylate and inhibit both p53 and the pro‐inflammatory transcription factor NF‐κB, positioning SIRT1 as a central node in coordinating the metabolic and inflammatory responses to TBI. Pharmacological modulation of this system, particularly with Class I/II HDAC inhibitors or SIRT1 activators, has shown significant neuroprotective efficacy in preclinical TBI models by restoring gene expression profiles and dampening cell death pathways [91, 92, 93].

4.4. Glycosylation: Modulating Cell Surface Interactions and Signaling

Glycosylation, the enzymatic attachment of complex sugar moieties (glycans) to proteins, is a major PTM that occurs in the endoplasmic reticulum and Golgi apparatus. It is critical for the proper folding, stability, and trafficking of secreted and membrane‐bound proteins. In the brain, glycosylation is essential for the function of receptors, adhesion molecules, and components of the extracellular matrix. Following TBI, the integrity of the BBB is compromised, and there is a significant upregulation of inflammatory mediators. This inflammatory milieu alters the expression of glycosyltransferases (writers), leading to changes in the glycan structures on endothelial cells and leukocytes [24, 94]. These altered glycans serve as ligands for selectins, facilitating the adhesion and transmigration of immune cells into the brain parenchyma, a key step in neuroinflammation. Furthermore, aberrant glycosylation of neuronal receptors, such as the AMPA and NMDA glutamate receptors, can alter their channel properties, trafficking to the synapse, and susceptibility to excitotoxicity [95, 96]. While less studied than phosphorylation or acetylation in TBI, the emerging field of glycomics suggests that specific glycan signatures could serve as novel biomarkers of BBB disruption and neuroinflammatory status, and that targeting glycan‐mediated cell adhesion may offer a new avenue for therapy.

4.5. Emerging and Novel PTMs in Neurotrauma (Succinylation, Lactylation, Nitrosylation)

Beyond the classical PTMs, a new generation of modifications is being recognized for their roles in cellular metabolism and stress response, with direct relevance to TBI pathophysiology. Succinylation and lactylation are lysine acylations that are directly linked to the cellular metabolic state, as their donor molecules (succinyl‐CoA and lactyl‐CoA) are intermediates of the Krebs cycle and glycolysis, respectively. TBI causes a dramatic shift in brain metabolism from oxidative phosphorylation to anaerobic glycolysis, leading to lactate accumulation. This metabolic shift can drive widespread protein lactylation–although direct evidence for lactylation in TBI remains preliminary (Level 3), emerging studies in other CNS injury models suggest this modification may promote a pro‐inflammatory phenotype in microglia [97]. Nitrosylation, the addition of a nitric oxide (NO) group to cysteine thiols to form S‐nitrosothiols (SNOs), is another critical redox‐based PTM. TBI induces a massive production of NO via inducible nitric oxide synthase (iNOS) in activated glia. While low levels of NO are signaling molecules, excessive NO leads to pathological S‐nitrosylation of key proteins. For example, S‐nitrosylation of the NMDA receptor can initially be protective by reducing calcium influx, but sustained nitrosylation of proteins involved in mitochondrial respiration (e.g., complex I) and the ubiquitin‐proteasome system can exacerbate energy failure and proteinopathy [98, 99, 100]. These emerging PTMs represent a direct molecular link between the metabolic crisis of TBI and its downstream pathological consequences, opening up entirely new frontiers for biomarker discovery and therapeutic targeting. The five major PTM classes discussed in this chapter—phosphorylation, ubiquitination, acetylation, SUMOylation, and PARylation—are summarized in Figure 2, which compares their writer and eraser enzymes, reader domains, representative TBI targets, reversibility, and functional consequences.

FIGURE 2.

Comparison of major PTM classes in TBI pathophysiology. This figure compares the five major PTM classes discussed in this review—phosphorylation, ubiquitination, acetylation, SUMOylation, and PARylation. For each PTM class, the table lists the writer enzymes (catalyzing PTM addition), eraser enzymes (catalyzing PTM removal), reader domains (recognizing and transducing PTM signals), representative TBI targets, reversibility, and primary functional consequences of their dysregulation in the injured brain.

5. The Role of PTMs in the Pathophysiology of Traumatic Brain Injury

TBI initiates a complex cascade of primary mechanical damage followed by a prolonged period of secondary injury, which is driven by intricate molecular and cellular disturbances. A growing body of evidence from proteomic studies has revealed that PTMs are not merely bystanders but central orchestrators of this secondary pathophysiology. The immediate ionic fluxes, energy crisis, and oxidative burst that follow the initial trauma create a biochemical environment that profoundly dysregulates the activity of PTM writers and erasers [15, 101]. This leads to a pathological reprogramming of the neuronal, glial, and vascular proteome, directly influencing key processes such as cell death, neuroinflammation, blood–brain barrier integrity, mitochondrial function, and synaptic stability. Understanding the specific roles of distinct PTMs in these pathways provides a mechanistic framework for the development of targeted neuroprotective strategies aimed at mitigating secondary damage and improving long‐term neurological outcomes.

5.1. PTMs in TBI‐Induced Neuronal Cell Death

Analytical framework for this section: neuronal death after TBI is governed by a time‐dependent interplay of phosphorylation, ubiquitination, and acetylation, primarily within neurons during the acute to subacute phase (hours to days). Key enzymatic nodes include the p53‐MDM2 axis (ubiquitination), JNK (phosphorylation), and SIRT1 (deacetylation). We will analyze how these PTMs dictate the shift between apoptosis, necroptosis, ferroptosis, and autophagy, and discuss the therapeutic implications of targeting these pathways.

Neuronal loss is a critical determinant of functional deficit after TBI, and it occurs through multiple, often overlapping, cell death pathways, including apoptosis, necroptosis, ferroptosis, and autophagy. PTMs serve as the primary regulatory switches that determine which death pathway is activated and to what extent.

Apoptosis, a highly regulated form of programmed cell death, is a major contributor to delayed neuronal loss after TBI. The core machinery responsible for executing apoptosis is exquisitely orchestrated by a network of PTMs. The tumor suppressor protein p53 is a pivotal node in this network. Following TBI, p53 is stabilized and activated through a combination of phosphorylation (e.g., at Ser15 by ATM/ATR kinases in response to DNA damage) and acetylation, which prevents its interaction with the E3 ubiquitin ligase MDM2. Under normal conditions, MDM2 constitutively ubiquitinates p53, targeting it for rapid proteasomal degradation, thus keeping its levels low. In the injured brain, this regulatory loop is disrupted, leading to p53 accumulation and its translocation to the nucleus, where it transactivates pro‐apoptotic genes like Bax and PUMA [71, 102]. Furthermore, the Bcl‐2 family of proteins, which govern mitochondrial outer membrane permeabilization (MOMP), are also regulated by phosphorylation. For instance, the pro‐survival protein Bcl‐2 can be inactivated by JNK‐mediated phosphorylation, while the pro‐apoptotic protein Bad is sequestered in an inactive state by 14‐3‐3 proteins when phosphorylated by Akt; dephosphorylation of Bad following TBI releases it to promote cell death. Critically, the therapeutic window for targeting these early phosphorylation events is narrow, likely within the first 6–12 h post‐injury, before the commitment to apoptosis occurs. Preclinical models using controlled cortical impact have demonstrated that p53 inhibition reduces lesion volume, but this strategy has not yet translated clinically, highlighting a key gap in our understanding of the long‐term effects of p53 manipulation on DNA repair mechanisms in surviving neurons.

In addition to apoptosis, regulated necrotic pathways like necroptosis and ferroptosis have emerged as significant contributors to TBI pathology, particularly in contexts where caspase activity is inhibited. Necroptosis is driven by the RIPK1–RIPK3–MLKL signaling cascade, with pathway activation dynamically controlled through the coordinated interplay of ubiquitination and phosphorylation. Upon activation of death receptors like TNFR1, RIPK1 is initially polyubiquitinated by cIAP1/2, which promotes pro‐survival NF‐κB signaling. However, under conditions of caspase‐8 inhibition—a scenario that can occur in TBI—RIPK1 is deubiquitinated, allowing it to form a complex with RIPK3 (the necrosome) [103, 104, 105]. RIPK3 then phosphorylates MLKL, causing its oligomerization and insertion into the plasma membrane, leading to cell lysis. The cell‐type specificity of this pathway is critical; while RIPK3 inhibition is protective in neurons, it may have detrimental effects in glial cells where necroptosis is a part of the normal inflammatory response, thus complicating therapeutic targeting. Ferroptosis, an iron‐dependent form of cell death driven by lipid peroxidation, is intimately linked to cellular metabolism and redox state. The key regulator of ferroptosis is glutathione peroxidase 4 (GPX4), which uses reduced glutathione (GSH) to detoxify lipid hydroperoxides. The depletion of GSH and inactivation of GPX4 following TBI, potentially driven by oxidative PTMs on key cysteine residues, sensitize neurons to ferroptosis [106, 107]. Meanwhile, the metabolic reprogramming toward glycolysis and lactate buildup may impact these pathways via lactylation, an emerging PTM that regulates genes controlling iron homeostasis and antioxidant responses.

Autophagy, a lysosomal degradation pathway for clearing damaged organelles and protein aggregates, plays a context‐dependent role in TBI, being protective in the acute phase by removing toxic debris but potentially detrimental if overactivated or impaired in the chronic phase. The initiation and progression of autophagy are tightly regulated by PTMs. The core autophagy‐related (ATG) proteins are controlled by a ubiquitin‐like conjugation system that is analogous to ubiquitination. For example, the conjugation of ATG12 to ATG5 and the lipidation of LC3 (conversion of LC3‐I to LC3‐II) are essential steps for autophagosome formation. Beyond this, acetylation plays a critical regulatory role. The acetyltransferase EP300 acetylates key components of the autophagy machinery, such as ATG5, ATG7, and LC3, and this modification typically inhibits their activity, leading to autophagy suppression. Conversely, deacetylation by SIRT1, a NAD⁺‐dependent deacetylase, promotes autophagy. Given that TBI disrupts cellular energy (NAD⁺) levels, the SIRT1‐mediated deacetylation of autophagy proteins is likely impaired, contributing to dysfunctional autophagic flux and the accumulation of toxic protein aggregates observed in TBI models [72, 108]. From a therapeutic perspective, the challenge lies in differentiating between protective and detrimental autophagy. A strategy that enhances autophagy in the acute phase (to clear debris) might need to be attenuated in the chronic phase to prevent excessive self‐digestion, suggesting that the timing of intervention (e.g., using mTOR inhibitors vs. activators) must be carefully calibrated.

5.2. PTMs in Neuroinflammation and Glial Activation

Analytical framework for this section: neuroinflammation after TBI is primarily driven by ubiquitination and phosphorylation in microglia, astrocytes, and endothelial cells during the subacute phase (days to weeks). The key enzymatic nodes are the IKK complex (for IκBα phosphorylation and subsequent K48‐linked ubiquitination, leading to NF‐κB activation), JAKs (for STAT3 Tyr705 phosphorylation), and deubiquitinating enzymes (for NLRP3 inflammasome priming). Preclinical models (e.g., controlled cortical impact, repetitive mild TBI, fluid percussion) have shown that pharmacological inhibition of IKK or JAK/STAT reduces pro‐inflammatory cytokine production and ameliorates cognitive deficits, yet cell‐type‐specific effects (microglial vs. astrocytic) remain incompletely resolved. Therapeutically, targeting NF‐κB and STAT3 acetylation via HDAC inhibitors or SIRT1 activators offers a complementary strategy, though optimal timing and combination regimens require further validation in clinically relevant models.

Neuroinflammation, mediated by the activation of microglia and astrocytes, is a double‐edged sword in TBI. While an acute, controlled inflammatory response is necessary for debris clearance and tissue repair, a chronic, dysregulated response drives secondary neurodegeneration. PTMs are central regulators of the signaling pathways that control glial activation and the production of inflammatory mediators.

The transcription factors NF‐κB and STAT3 are master regulators of the pro‐inflammatory gene program in activated glia. Their activity is finely orchestrated by an intricate network of post‐translational modifications. In the canonical NF‐κB pathway, the inhibitor IκBα is phosphorylated by the IKK complex in response to inflammatory stimuli (e.g., TNF‐α, IL‐1β), leading to its K48‐linked polyubiquitination and proteasomal degradation [109]. This releases the p65/p50 NF‐κB dimer, allowing it to translocate to the nucleus. Once in the nucleus, the transcriptional activity of p65 is further fine‐tuned by PTMs: phosphorylation at Ser536 enhances its transactivation potential, while acetylation by p300/CBP at multiple lysines promotes DNA binding but also targets it for IκBα‐mediated nuclear export, providing a negative feedback loop. Similarly, the JAK‐STAT3 pathway is activated by cytokines like IL‐6. STAT3 is phosphorylated by JAKs on Tyr705, leading to its dimerization and nuclear translocation. Its activity is then modulated by other PTMs, including acetylation on Lys685, which is required for its full transcriptional activity and is mediated by p300. The dysregulation of these PTM networks in TBI leads to sustained and excessive activation of NF‐κB and STAT3, resulting in the chronic production of neurotoxic cytokines and chemokines.

The NLRP3 inflammasome is a multi‐protein complex that, upon activation, cleaves pro‐caspase‐1 to its active form, which in turn processes pro‐IL‐1β and pro‐IL‐18 into their mature, highly inflammatory forms. Its activation is a two‐step process: a priming signal (often via NF‐κB) upregulates NLRP3 and pro‐IL‐1β expression, and an activation signal (e.g., K⁺ efflux, ROS, lysosomal rupture) triggers complex assembly. Both steps are subject to PTM control. Phosphorylation of NLRP3 by JNK1 is required for its deubiquitination, a critical step that allows its oligomerization and inflammasome assembly. Conversely, PKA‐mediated phosphorylation of NLRP3 inhibits its activation. Furthermore, the priming step itself is regulated by PTMs on upstream signaling molecules. The persistent activation of the NLRP3 inflammasome is a key feature of chronic neuroinflammation after TBI (Level 1: pharmacological inhibition in rodent TBI models; Level 2: correlative CSF biomarker studies in humans), and its products, particularly IL‐1β, are potent drivers of neuronal death and BBB disruption [110, 111, 112].

The breakdown of the BBB is a critical early event in TBI that contributes to vasogenic edema, infiltration of peripheral immune cells, and exposure of the brain parenchyma to harmful blood‐derived factors. The integrity of the BBB is maintained by tight junctions between endothelial cells, and PTMs directly regulate the stability and function of these junctional complexes. A key mechanism is the phosphorylation of TJ proteins like occludin and claudin‐5. Activation of kinases such as Src and PKC by inflammatory mediators (e.g., VEGF, TNF‐α) leads to their phosphorylation, which triggers their internalization from the cell membrane via endocytosis, thereby increasing paracellular permeability [113, 114]. Additionally, the actin cytoskeleton, which provides structural support to tight junctions, is dynamically regulated by Rho GTPases whose activity is controlled by phosphorylation and ubiquitination. The activation of RhoA/ROCK signaling after TBI leads to actomyosin contraction, further pulling apart the tight junctions. Glycosylation also plays a role; changes in the glycan structures on endothelial adhesion molecules (e.g., ICAM‐1, VCAM‐1) facilitate the binding of circulating leukocytes, promoting their diapedesis across the compromised BBB and amplifying the inflammatory response [94, 115].

5.3. PTMs in Mitochondrial Dysfunction and Bioenergetic Failure

Analytical framework for this section: mitochondrial dysfunction following TBI is governed by a dynamic interplay of SUMOylation, S‐nitrosylation, and acetylation, affecting neurons, glia, and cerebrovascular endothelial cells from the acute (hours) to subacute (days) phase. Key enzymes include SUMO E3 ligases (promoting Drp1 SUMOylation and excessive fission), inducible nitric oxide synthase (iNOS, mediating S‐nitrosylation of complex I and impairing electron transport), and sirtuins (regulating cyclophilin D acetylation, which sensitizes the mitochondrial permeability transition pore). Rodent TBI models (e.g., fluid percussion, blast injury, controlled cortical impact) have demonstrated that inhibiting Drp1 SUMOylation or iNOS activity preserves mitochondrial morphology and ATP production, while SIRT1 activation ameliorates bioenergetic failure. Clinically, the major hurdles are achieving mitochondrial‐specific delivery of these modulators and avoiding interference with physiological mitochondrial dynamics in non‐injured tissues; however, emerging data suggest that post‐injury metabolic monitoring (e.g., lactate/pyruvate ratio) could serve as a pharmacodynamic biomarker to guide SIRT1‐based therapies.

Mitochondrial dysfunction is a central hub in TBI pathophysiology, linking the initial energy failure to oxidative stress, calcium dyshomeostasis, and cell death. PTMs are crucial for both the physiological regulation of mitochondrial dynamics and bioenergetics and their pathological dysregulation after injury. As mentioned, the fission protein Drp1 is a major target; its SUMOylation stabilizes its active, GTP‐bound form on the mitochondrial membrane, driving excessive fission and fragmentation [85, 116, 117]. Furthermore, key enzymes in the electron transport chain are susceptible to oxidative PTMs. For instance, S‐nitrosylation of complex I by excessive NO produced by iNOS inhibits its activity, exacerbating the energy crisis and increasing the production of ROS. This creates a vicious cycle, as ROS can further oxidize and inactivate electron transport chain components and antioxidant enzymes like SOD. The mitochondrial permeability transition pore, whose opening leads to mitochondrial swelling and cell death, is also regulated by PTMs, including cyclophilin D acetylation, which sensitizes the pore to calcium‐induced opening [118]. This intricate crosstalk between PTMs and oxidative stress is a major driver of the secondary energy failure that characterizes TBI.

5.4. PTMs in Axonal Injury and Synaptic Dysfunction

Analytical framework for this section: axonal injury and synaptic dysfunction after TBI are primarily shaped by pathological phosphorylation, with secondary contributions from ubiquitination and glycosylation, predominantly in neurons during the acute (immediate to hours) and chronic (weeks to months) phases. The critical enzymes are kinases (GSK‐3β, CDK5, JNK) and phosphatases (PP2A, calcineurin) that control tau and neurofilament phosphorylation, as well as kinases regulating AMPA receptor trafficking (e.g., CaMKII, PKC). Evidence from diffuse axonal injury models, repetitive mild TBI models, and controlled cortical impact shows that hyperphosphorylation of tau at Ser202/Thr205 correlates with axonal transport deficits and long‐term cognitive decline, while aberrant phosphorylation of AMPA receptor subunits (GluA1) impairs synaptic plasticity. Therapeutic strategies have focused on GSK‐3β inhibitors and microtubule stabilizers, but their narrow therapeutic window (likely within the first few hours) and potential disruption of normal synaptic plasticity (which requires dynamic phosphorylation) pose significant hurdles. Combinatorial approaches that couple kinase inhibition with phosphatase activation or with epigenetic modulators (e.g., HDAC inhibitors) may offer a more balanced strategy to restore cytoskeletal and synaptic homeostasis.

Diffuse axonal injury, characterized by widespread shearing and disconnection of axons, is a primary pathology in TBI that underlies many long‐term cognitive and motor deficits. The axonal cytoskeleton, composed of microtubules, neurofilaments, and associated proteins, is a major target of PTM dysregulation. The microtubule‐associated protein tau is hyperphosphorylated at numerous sites (e.g., by GSK‐3β, CDK5) after TBI. This hyperphosphorylation reduces tau's affinity for microtubules, leading to their destabilization and disassembly, which impairs axonal transport and contributes to axonal swelling and disconnection [23, 119]. Similarly, neurofilament side‐arm domains are heavily phosphorylated, and abnormal phosphorylation disrupts their normal spacing, leading to focal accumulations that obstruct axonal transport. At the synaptic level, PTMs regulate the trafficking, anchoring, and function of neurotransmitter receptors. For example, the phosphorylation state of AMPA receptor subunits (GluA1) controls their insertion into and removal from the postsynaptic density, directly impacting synaptic strength and plasticity. TBI‐induced dysregulation of these synaptic PTMs is believed to be a key mechanism underlying the learning and memory impairments commonly seen in TBI survivors [120, 121, 122, 123].

6. Crosstalk Among Different PTM Mechanisms in TBI

TBI triggers a profound and dynamic reprogramming of the cellular proteome, far beyond the simple up‐ or down‐regulation of protein expression. This reprogramming is largely executed through a complex and interconnected network of PTMs. Rather than acting in isolation, PTMs engage in intricate crosstalk, where one modification can directly influence the addition, removal, or functional consequence of another on the same or a neighboring residue. This crosstalk creates a sophisticated “PTM code” that integrates diverse signals from the injured microenvironment—such as oxidative stress, calcium influx, energy depletion, and inflammatory mediators—to fine‐tune the activity, stability, localization, and interactions of key proteins involved in all facets of TBI pathophysiology. Understanding this interplay is crucial for moving beyond a reductionist view of individual pathways and toward a systems‐level understanding of the molecular chaos that ensues after injury.

6.1. Interplay Between Phosphorylation and Ubiquitination in Protein Degradation

The coordinated action of phosphorylation and ubiquitination forms a fundamental regulatory axis that controls the stability and turnover of a vast array of proteins critical to TBI outcomes, most notably those governing cell survival, inflammation, and synaptic integrity. Phosphorylation often serves as a priming signal that marks a protein for subsequent recognition by E3 ubiquitin ligases, thereby targeting it for proteasomal degradation. A canonical example is the regulation of the transcription factor p53. In the healthy brain, p53 is kept at low levels through continuous ubiquitination by its primary E3 ligase, MDM2, which directs it to the 26S proteasome. However, following TBI, DNA damage activates kinases like ATM/ATR, which phosphorylate p53 at specific N‐terminal residues (e.g., Ser15). This phosphorylation disrupts the p53‐MDM2 interaction, stabilizing p53 and allowing it to accumulate and drive the expression of pro‐apoptotic genes, contributing to neuronal death [71, 124]. Conversely, this relationship can also be antagonistic, where ubiquitination regulates the kinases themselves. For instance, the stability and activity of RIPK1, a key kinase in the necroptosis pathway, are controlled by its ubiquitination status. Inflammatory signaling initially promotes K63‐linked polyubiquitination of RIPK1, which scaffolds pro‐survival signaling complexes. Under conditions of caspase inhibition, which can occur in TBI, deubiquitinating enzymes (DUBS) remove these chains, allowing RIPK1 to engage with RIPK3 and initiate necroptotic cell death [125, 126]. This delicate balance between phosphorylation‐driven activation and ubiquitination‐driven degradation is a recurring theme in TBI (Table 4).

TABLE 4.

Summarizing key examples of this crosstalk and their functional consequences in TBI.

Target protein	Phosphorylation event	Level	Consequence of ubiquitination	Functional outcome in TBI
p53	Phosphorylation at Ser15/20 by ATM/ATR	2	Prevents MDM2‐mediated ubiquitination	Stabilization of p53, promotion of neuronal apoptosis [71, 127]
IκBα	Phosphorylation by IKK complex	2	Triggers K48‐linked polyubiquitination	Degradation of IκBα, activation of NF‐κB, neuroinflammation [128, 129]
RIPK1	Autophosphorylation within the kinase domain	2	Deubiquitination allows necrosome formation	Shift from pro‐survival to pro‐necroptotic signaling [75, 130]
β‐Catenin	Phosphorylation by GSK‐3β in the destruction complex	1	Promotes β‐TrCP‐mediated ubiquitination	Degradation of β‐catenin, disruption of Wnt signaling, impaired synaptic repair [131]

This phospho‐degron system ensures rapid and precise control over protein lifetimes, but its dysregulation in the chaotic post‐TBI environment can lead to the pathological accumulation of toxic proteins or the untimely destruction of protective factors.

6.2. Competitive and Cooperative Modifications: Acetylation Versus SUMOylation

Lysine residues on proteins are hotspots for multiple, often competing, PTMs, including acetylation, ubiquitination, methylation, and SUMOylation. The competition for the same lysine residue creates a direct and powerful mechanism for regulating protein function. Acetylation and SUMOylation represent a particularly important competitive pair in the context of TBI, with significant implications for transcriptional regulation, DNA repair, and mitochondrial dynamics. Both modifications neutralize the positive charge of the lysine side chain, but they recruit entirely different sets of effector proteins, leading to opposing functional outcomes. For example, the transcription factor STAT3 can be either acetylated or SUMOylated on Lys685. Acetylation by p300/CBP enhances its dimerization, DNA binding, and transcriptional activity, promoting a pro‐inflammatory state in activated glia [132]. In contrast, SUMOylation at the same site acts as a potent repressor of STAT3‐dependent transcription, potentially serving as a built‐in negative feedback mechanism to limit chronic inflammation [133]. In the nucleus, this competition is a major regulator of the DNA damage response. Key DNA repair proteins like Ku70 and NBS1 are subject to both modifications. Acetylation of Ku70 can promote its release from the pro‐apoptotic protein Bax, while SUMOylation of NBS1 is essential for its recruitment to DNA double‐strand breaks [134]. The balance between these two modifications is likely disrupted by TBI‐induced oxidative stress and metabolic changes, contributing to the accumulation of unrepaired DNA damage and genomic instability observed in surviving neurons. Furthermore, this crosstalk extends to the cytoplasm and mitochondria. The fission GTPase Drp1 is regulated by both SUMOylation, which stabilizes its active form on mitochondria and promotes fission, and Sirt3‐mediated deacetylation, which also activates its fission activity [85, 135]. In this case, the modifications may act cooperatively rather than competitively, converging on the same functional outcome of mitochondrial fragmentation, a key feature of secondary injury in TBI.

6.3. Metabolic Control of PTMs: The Succinylation‐Lactylation‐Acetylation Axis in Altered Energy States

The metabolic crisis that follows TBI—a consequence of mitochondrial dysfunction, ischemia, and excitotoxicity—profoundly alters the intracellular concentrations of key metabolites that serve as co‐factors or substrates for PTM enzymes. This establishes a direct link between cellular bioenergetics and the global PTM landscape, creating a “metabolite‐PTM axis” that reprograms cellular function in response to energy stress. Acetyl‐CoA, succinyl‐CoA, and lactate are prime examples of such metabolites. Their levels fluctuate dramatically after TBI, directly influencing the rates of lysine acetylation, succinylation, and lactylation, respectively. Acetyl‐CoA is the donor for acetylation, and its depletion can lead to a global hypoacetylation of histones and metabolic enzymes, suppressing gene expression and metabolic flux [136, 137]. Conversely, the Warburg‐like shift to glycolysis, even in the presence of oxygen, leads to a massive accumulation of lactate. Lactate is not simply a metabolic byproduct; rather, it provides the substrate for lysine lactylation, a recently characterized PTM that has been shown to orchestrate a homeostatic and reparative transcriptional response in macrophages [138]. In the TBI context, an early surge in lactylation might represent an endogenous attempt to counterbalance the initial pro‐inflammatory (e.g., acetylation‐driven) response. Similarly, the tricarboxylic acid cycle intermediate succinyl‐CoA is the donor for lysine succinylation, a modification that adds a large, negatively charged group to the lysine residue, causing a more dramatic structural change than acetylation. Succinylation can profoundly alter enzyme activity; for instance, succinylation of key glycolytic and tricarboxylic acid cycle enzymes can inhibit their function, further exacerbating the energy crisis in a feed‐forward loop [136, 139]. The interplay among these three modifications—acetylation, succinylation, and lactylation—is highly competitive, as they all target the same pool of lysine residues. The relative abundance of their respective CoA/thioester donors (Acetyl‐CoA, Succinyl‐CoA, Lactyl‐CoA) in the post‐TBI milieu will therefore dictate the dominant modification state on many metabolic and epigenetic regulators. This axis represents a crucial mechanism by which the brain's metabolic state is translated into a functional proteomic and transcriptional response, with significant implications for recovery versus degeneration. Targeting the enzymes that write or erase these modifications (e.g., sirtuins for deacylation, acyltransferases) offers a promising therapeutic strategy to steer this metabolic‐PTM axis toward a more reparative phenotype.

6.4. PTM Crosstalk in TBI: Case Examples and Therapeutic Implications

The functional significance of PTM crosstalk is best illustrated by examining how multiple modifications converge on specific substrates central to TBI pathophysiology. Below, we analyze six such examples where PTM competition or cooperation directly shapes pathological outcomes.

6.4.1. Tau: Phosphorylation, Ubiquitination, SUMOylation, and Acetylation Converge on a Single Protein

Tau is subject to multiple PTMs that collectively determine its fate. Phosphorylation at Ser202/Thr205 (by GSK‐3β and CDK5) reduces tau's affinity for microtubules, promoting detachment and axonal transport failure [25]. Ubiquitination normally targets tau for proteasomal degradation, but when UPS is impaired after TBI, ubiquitinated tau accumulates [28, 29]. SUMOylation of tau promotes its phosphorylation and aggregation [73]. Acetylation at lysine residues reduces tau turnover and promotes aggregation while competing with ubiquitination at the same sites [89]. The net effect is a shift toward pathological aggregation: phosphorylation drives detachment, acetylation blocks degradation, SUMOylation promotes phosphorylation, and impaired ubiquitination allows accumulation. Therapeutically, this argues for combinatorial approaches targeting multiple tau PTMs simultaneously.

6.4.2. NF‐κB Signaling: A Phosphorylation‐Ubiquitination‐Acetylation Relay

The NF‐κB pathway exemplifies sequential PTM crosstalk. Inflammatory stimuli activate the IKK complex, which phosphorylates IκBα at Ser32/Ser36. This phosphorylation serves as a phospho‐degron—a priming signal for K48‐linked polyubiquitination by β‐TrCP, targeting IκBα for proteasomal degradation and releasing p65/p50 for nuclear translocation [128, 129]. Once nuclear, p65 activity is further modulated by acetylation and SUMOylation (repressing transcription) [132]. In TBI, NAD⁺ depletion impairs SIRT1‐mediated deacetylation, sustaining chronic NF‐κB activation [33]. Therapeutically, this suggests combining IKK inhibitors (blocking the initial phosphorylation) with SIRT1 activators (promoting p65 deacetylation) for synergistic anti‐inflammatory effects.

6.4.3. NLRP3 Inflammasome: Phosphorylation, Ubiquitination, and SUMOylation in Assembly Control

NLRP3 is regulated by competitive PTMs. JNK1‐mediated phosphorylation is required for NLRP3 deubiquitination, enabling oligomerization and inflammasome assembly [110, 111, 112]. K48‐linked ubiquitination targets NLRP3 for proteasomal degradation (serving as a brake), whereas K63‐linked ubiquitination promotes assembly [111]. SUMOylation generally promotes NLRP3 activation in some contexts [73]. In TBI, persistent NLRP3 activation drives chronic neuroinflammation [110]. Therapeutically, enhancing K48‐linked ubiquitination or inhibiting the deubiquitinases that remove these chains could dampen inflammasome activation without ablating this essential immune sensor.

6.4.4. Mitochondrial Dynamics: Drp1 as a Hub for SUMOylation, Phosphorylation, and Ubiquitination

Drp1 activity is regulated by converging PTMs. SUMOylation (via MAPL) stabilizes its active GTP‐bound form at the mitochondrial membrane, driving excessive fission [116, 117]. Phosphorylation at Ser616 promotes fission, while Ser637 phosphorylation (by PKA) inhibits it [84]. Ubiquitination by Parkin and MARCH5 targets Drp1 for degradation [30, 67]. SUMOylation can also promote ubiquitination via SUMO‐targeted ubiquitination (STUbL), linking these modifications [86, 87]. In TBI, excessive Drp1 SUMOylation drives mitochondrial fragmentation and bioenergetic collapse [85]. Therapeutically, inhibiting Drp1 SUMOylation or enhancing Ser637 phosphorylation could preserve mitochondrial integrity, though modulating one PTM may affect others.

6.4.5. Blood–Brain Barrier: Phosphorylation and Ubiquitination of Tight Junction Proteins

TJ proteins (occludin, claudin‐5) are regulated by PTM crosstalk. Phosphorylation by Src, PKC, and Rho kinase (triggered by VEGF, TNF‐α) promotes internalization from the cell membrane via endocytosis [113, 114]. Ubiquitination targets these proteins for lysosomal degradation, further reducing membrane abundance [94, 115]. The crosstalk is sequential: phosphorylation primes subsequent ubiquitination, creating a phospho‐ubiquitin relay that drives TJ disassembly. In TBI, this relay contributes to vasogenic edema and immune infiltration. Therapeutically, early inhibition of the initiating kinases (e.g., Src inhibitors) or stabilization of TJ proteins against ubiquitination could preserve BBB integrity.

6.4.6. Synaptic Plasticity: Phosphorylation and Acetylation in Receptor Trafficking and Gene Expression

Synaptic plasticity integrates rapid synaptic and longer‐term nuclear PTM events. Phosphorylation of AMPA receptor subunits (GluA1 at Ser831 by CaMKII, Ser845 by PKA) controls receptor trafficking and synaptic strength [120, 121]. Acetylation of histones and transcription factors (e.g., CREB by p300/CBP) regulates expression of plasticity‐related genes (e.g., BDNF) [122, 123]. The crosstalk is hierarchical: synaptic phosphorylation provides rapid modulation, while nuclear acetylation sustains longer‐term plasticity programs. In TBI, both are disrupted—aberrant phosphorylation impairs immediate synaptic function, while histone hypoacetylation silences reparative gene expression [31]. Therapeutically, combining agents that restore synaptic phosphorylation balance with HDAC inhibitors that restore the epigenetic landscape could synergistically improve cognitive recovery [92, 93].

6.4.7. Therapeutic Implications and Future Directions

The six examples above illustrate a recurring theme: PTMs form integrated regulatory networks at the level of individual proteins (tau, p65, NLRP3, Drp1), organelles (mitochondria, tight junctions), and cellular processes (synaptic plasticity). The interplay between phosphorylation and ubiquitination creates a phospho‐degron code that dictates the half‐life of critical proteins like IκBα (Case 5.4.2) and p53, meaning that a kinase inhibitor might fail if it does not account for the downstream ubiquitination state of its target. Similarly, the competition between acetylation and SUMOylation at the same lysine residue on transcription factors like STAT3 and p65 (Case 5.4.2) suggests that a drug aimed at promoting SUMOylation must be carefully balanced against the global acetylation state of the cell, which is influenced by metabolic co‐factors like NAD⁺. This has a direct clinical implication: a combination therapy using a low‐dose HDAC inhibitor alongside a STAT3‐SUMOylation promoter might be more effective than either agent alone. Furthermore, the metabolic control of PTMs via the succinylation‐lactylation‐acetylation axis provides a mechanistic link between the common clinical finding of post‐TBI hyperglycolysis and the chronic inflammatory state. This suggests that metabolic modulators, such as dichloroacetate, could have a profound impact on the brain's PTM landscape and subsequent recovery. Future research should prioritize mapping these crosstalk networks in a cell‐type and time‐specific manner, as the same crosstalk mechanism that is protective in a neuron (e.g., SUMOylation of Drp1 to manage mitochondrial fission; Case 5.4.4) could be detrimental in a microglial cell by promoting an inflammatory phenotype [111].

7. Clinical Relevance and Therapeutic Targeting of PTMs in TBI

The intricate network of PTMs that is rapidly and profoundly dysregulated following TBI represents not only a core component of its pathophysiology but also a rich landscape for therapeutic intervention. The clinical relevance of targeting PTMs stems from their direct control over the activity, stability, and interactions of proteins that drive key pathological processes such as excitotoxicity, neuroinflammation, oxidative stress, mitochondrial failure, and neuronal death. By modulating the enzymes responsible for writing, reading, or erasing these modifications, it is theoretically possible to reprogram the injured brain's proteome toward a state that favors repair and resilience over degeneration. This section explores the current state of translational research in this area, from established preclinical strategies to emerging technologies with high therapeutic potential.

7.1. Targeting Kinases and Phosphatases: From Preclinical Models to Challenges

Protein phosphorylation is one of the most rapid and widespread signaling events after TBI, making kinases and phosphatases prime therapeutic targets. Numerous preclinical studies have demonstrated the neuroprotective efficacy of kinase inhibitors. For instance, inhibition of the pro‐death kinase GSK‐3β has been shown to reduce neuronal apoptosis, attenuate blood–brain barrier disruption, and improve cognitive outcomes in rodent models of TBI [140]. Similarly, Src family kinase inhibitors—targeting kinases activated by integrin signaling that drive cytoskeletal breakdown and edema—have demonstrated efficacy in mitigating secondary injury [141]. However, the path to clinical translation has been fraught with challenges. A major hurdle is the lack of specificity of many small‐molecule kinase inhibitors, which can lead to off‐target effects and systemic toxicity. Kinases often have multiple substrates and participate in diverse physiological processes; therefore, broad inhibition can disrupt essential homeostatic functions. Another significant challenge is the narrow therapeutic window for many of these agents. Currently, no formal clinical trials of GSK‐3β inhibitors for traumatic brain injury have been completed. The clinical experience of lithium in bipolar disorder and neuroprotection provides a foundation for its translation to TBI. Notably, some studies have shown that lithium can improve cognitive function in patients with mood disorders and has demonstrated multiple beneficial effects in experimental TBI models. The optimal time for intervention is often within hours of the initial injury, a timeframe that is logistically difficult to achieve in a clinical setting outside of specialized trauma centers. Furthermore, the complex and often opposing roles of different kinases at various stages of TBI recovery complicate dosing and timing strategies. Despite these obstacles, the field continues to advance with the development of more selective inhibitors and novel delivery systems, such as nanoparticles, to enhance brain penetration and target specificity.

Distinguishing TBI‐specific evidence from borrowed data. A critical caveat throughout this section is that many kinase inhibitors discussed have been validated primarily in oncology or other CNS disease models, with only limited confirmation in TBI. Throughout this review, we explicitly distinguish findings derived from TBI preclinical models from those extrapolated from other disease contexts, which are discussed as mechanistic inspiration rather than therapeutic validation for TBI.

7.2. Modulating the Ubiquitin‐Proteasome System: Neuroprotection Versus Toxicity

The UPS is the primary machinery for controlled protein degradation in cells, and its dysfunction is a hallmark of TBI and other neurodegenerative conditions. In the acute phase, enhancing UPS activity could be beneficial by clearing damaged, misfolded, or aggregated proteins that accumulate after injury and contribute to proteotoxic stress [138]. Conversely, in later stages, excessive UPS activity might lead to the untimely degradation of pro‐survival factors and synaptic proteins, thereby hindering recovery [142]. This dual nature presents a significant therapeutic conundrum. One strategy has been to use proteasome activators, but their clinical utility is limited by the risk of promoting the degradation of essential proteins. An alternative approach is to target specific E3 ubiquitin ligases or DUBs that regulate key nodes in TBI pathology. For example, inhibiting the E3 ligase CHIP, which targets misfolded proteins for degradation, might be protective in the acute phase, while modulating DUBs like USP14, which can rescue proteins from degradation, could support synaptic integrity during recovery [105] (Table 5).

TABLE 5.

The key components of the UPS and their potential as therapeutic targets in TBI.

UPS component	Role in TBI pathology	Potential therapeutic strategy	Associated risks
26S proteasome	Impaired activity leads to toxic protein aggregation	Proteasome activators (e.g., IU1)	Non‐selective degradation of essential proteins
E3 ligase MDM2	Promotes degradation of p53, a pro‐apoptotic factor	MDM2 inhibitors (e.g., Nutlin‐3) to stabilize p53 for cell cycle arrest/DNA repair	Risk of promoting apoptosis if p53 stabilization is excessive
E3 ligase Parkin	Critical for mitophagy; its function is often impaired	Parkin activators to enhance the clearance of damaged mitochondria	Potential for excessive mitochondrial loss
Deubiquitinase USP14	Rescues proteins from proteasomal degradation	USP14 inhibitors to promote clearance of damaged proteins	May accelerate loss of synaptic proteins

The key to success lies in achieving exquisite temporal and target specificity to harness the protective aspects of the UPS while avoiding its detrimental effects.

7.3. Histone Deacetylase (HDAC) Inhibitors: Epigenetic Reprogramming for Recovery

HDAC inhibitors represent one of the most promising classes of epigenetic drugs for TBI. By increasing histone acetylation, they promote a more open chromatin state, facilitating the transcription of genes involved in neuroplasticity, learning, memory, and neuroprotection. Preclinical studies have consistently shown that HDAC inhibitors like sodium butyrate, valproic acid, and trichostatin A can improve cognitive and motor function, reduce lesion volume, and enhance synaptic plasticity in various TBI models [143]. A large‐scale Phase II/III randomized controlled trial (Registration Number: NCT07166393) is currently evaluating valproic acid in patients with moderate to severe TBI, using the Glasgow Outcome Scale as the primary endpoint for functional recovery. In parallel, accumulating preclinical evidence has consistently shown that HDAC inhibitors enhance neurological function, decrease lesion volume, and facilitate synaptic plasticity. Their mechanism extends beyond histones, as they also increase the acetylation of numerous non‐histone proteins, including transcription factors like p53 and NF‐κB, and cytoskeletal proteins like α‐tubulin, thereby exerting pleiotropic effects. The clinical significance of HDAC inhibitors is amplified by the fact that some, like valproic acid, are already FDA‐approved for other neurological conditions (e.g., epilepsy), potentially accelerating their repurposing for TBI. However, challenges remain, including the need to identify the specific HDAC isoforms (there are 11 Zn²⁺‐dependent HDACs in humans) that are most critical for TBI recovery to develop more selective inhibitors with fewer side effects. Pan‐HDAC inhibitors can cause significant toxicity, including fatigue, gastrointestinal distress, and hematological abnormalities.

Translational barriers: cell‐type selectivity and timing. Systemic HDAC inhibition does not distinguish between cell populations, yet HDAC isoforms exert opposing effects in different cell types—neuronal HDAC2/3 inhibition promotes survival [70, 71], whereas microglial HDAC3 deletion facilitates inflammation resolution [76]. Moreover, the therapeutic window for HDAC inhibition likely differs from acute kinase inhibition, with greater efficacy during subacute to chronic phases when epigenetic reprogramming supports recovery [90]. These spatiotemporal complexities must be addressed in future trial design.

7.4. Emerging Strategies: PROTACs and Targeted Protein Degradation

This technology offers several advantages over traditional occupancy‐driven inhibitors. First, it can target proteins that are considered “undruggable” because they lack a functional active site, such as scaffolding proteins or transcription factors. Second, it acts catalytically, meaning a single PROTAC molecule can degrade multiple copies of the protein of interest, potentially allowing for lower and less frequent dosing [144]. In the context of TBI, PROTACs could be designed to selectively degrade key pathological drivers, such as hyperphosphorylated tau, mutant forms of proteins, or overactive kinases like RIPK1 that promote necroptosis. While this field is still in its infancy for neurological applications, the proof‐of‐concept in oncology is strong, and the potential for creating highly specific molecular scalpels to remove disease‐causing proteins in TBI is immense.

A revolutionary frontier in PTM‐based therapeutics is the development of PROTACs. These heterobifunctional molecules consist of a ligand that binds a protein of interest, conjugated to a ligand that recruits an E3 ubiquitin ligase. By simultaneously engaging the target and an E3 ligase, PROTACs induce target ubiquitination and subsequent proteasomal degradation [144]. This technology offers several distinct advantages over traditional occupancy‐driven inhibitors. First, it can target proteins historically considered “undruggable” due to the lack of a functional active site, including scaffolding proteins, transcription factors, or those with shallow binding pockets inaccessible to conventional inhibitors. Second, PROTACs act catalytically: a single PROTAC molecule can drive the degradation of multiple target copies, potentially enabling lower and less frequent dosing [144]. Third, by eliminating both catalytic and scaffolding functions, PROTACs achieve a more complete blockade of pathological protein activity compared to conventional active‐site inhibitors [145].

In the context of TBI, PROTACs could be designed to selectively degrade key pathological drivers, such as hyperphosphorylated tau aggregates that accumulate following axonal injury, or overactive kinases like RIPK1 that drive necroptotic cell death. Moreover, the catalytic mechanism of PROTACs may enable sustained pathogenic protein suppression using reduced dosing regimens—a particularly valuable feature for managing the chronic, progressive nature of post‐TBI neurodegeneration [146].

Despite these promises, significant challenges remain before PROTACs can be translated clinically for TBI. Chief among them is the BBB: PROTACs exhibit high molecular weight and polar surface area that often violate conventional CNS drug‐likeness rules, hindering brain penetration. Recent advances have highlighted emerging CNS‐targeted delivery strategies, including intranasal administration, engineered exosomes, functionalized nanocarriers, and rational molecular engineering of linker polarity and E3 ligase selection to enhance BBB transport. Furthermore, many CNS disease‐causing proteins—such as tau—also perform essential physiological functions in neurons, necessitating selective targeting of pathogenic species while sparing healthy isoforms [147]. While the field is still in its early phase for TBI applications, proof‐of‐concept in oncology is well established, and recent advances are rapidly expanding CNS applications. Key milestones include BBB‐penetrant PROTACs entering clinical trials for Parkinson's disease (e.g., ARV‐102 targeting LRRK2), gold nanocluster‐based PROTACs designed to degrade phosphorylated tau with demonstrated BBB permeability, and a novel SPYTAC technology. With continued advances in molecular design, CNS‐optimized delivery systems, and rigorous validation in TBI‐relevant models, the potential for creating highly specific molecular scalpels to remove pathogenic proteins in TBI is immense.

Translational barriers for PROTACs. Four major hurdles impede PROTAC translation to TBI: (1) BBB penetration—PROTACs typically violate CNS drug‐likeness rules (MW > 800 Da), with brain penetration often below 5%; (2) safety—off‐target degradation of essential proteins remains a concern; (3) therapeutic timing—degrading a protein protective in the acute phase could be detrimental; and (4) cell‐type selectivity—current designs do not distinguish between neurons, glia, or endothelial cells. These challenges place PROTACs firmly in the preclinical discovery phase for TBI.

7.5. Drug Repurposing and Natural Compounds Targeting PTM Pathways in TBI (e.g., Metformin, Resveratrol)

Drug repurposing and the use of natural compounds offer a pragmatic and accelerated path to clinical application for PTM‐modulating therapies in TBI. Metformin, a widely used anti‐diabetic drug, is a potent activator of AMP‐activated protein kinase. From a PTM‐centric view, AMPK activation triggers a cascade of phosphorylation events that inhibit mTOR (promoting autophagy) and enhance mitochondrial biogenesis via PGC‐1α deacetylation, indirectly modulating both the ubiquitin‐proteasome system and the acetylome [148]. Preclinical studies have confirmed that metformin administration inhibits microglial activation‐mediated inflammation via the NF‐κB and MAPK signaling pathways, while also promoting AMP‐activated protein kinase phosphorylation to improve neurobehavioral function post‐TBI. More importantly, a randomized controlled trial in 158 severe TBI patients has demonstrated that metformin could be an effective and safe therapeutic intervention [149], representing a significant step toward clinical translation. This trial provides Level 1 clinical evidence (our grading system) that a PTM‐modulating drug can be safely repurposed, though its precise effects on the TBI‐specific phosphoproteome in humans remain to be fully characterized. Similarly, resveratrol, a polyphenol found in red grapes, is a well‐known activator of SIRT1, a NAD⁺‐dependent deacetylase. Mechanistically, resveratrol's neuroprotective effects are attributed to its deacetylation of PGC‐1α (enhancing mitochondrial function) [150] and NF‐κB (suppressing neuroinflammation) [151], directly acting on the acetylation PTM node. Specifically, resveratrol has been shown to reduce p38 MAPK phosphorylation via SIRT1 activation to alleviate cognitive dysfunction after TBI in mice [152]. While preclinical evidence is robust, the clinical evidence is more preliminary; a Phase I/II clinical trial (REPAIR, NCT01321151) has been completed to evaluate its effects on secondary brain injury following sports‐related concussions in boxers, but results are not yet widely available. This highlights a key challenge: translating the strong preclinical PTM mechanism into a clinically measurable endpoint in human TBI.

Second, pharmacologic and therapeutic intervention trials constitute the most clinically impactful category, representing 32.1% of all studies (Table S1). This diverse group includes complementary and alternative therapies such as acupuncture, psychological interventions for post‐traumatic stress disorder and mood disturbances, and neuroprotective pharmacotherapies aimed at limiting excitotoxicity, oxidative stress, and neuroinflammation. Several interventional trials explicitly target inflammatory signaling and secondary injury cascades, creating a mechanistic overlap with PTM networks that regulate inflammation, mitochondrial function, and cell survival. These trials highlight the clinical need for targeted strategies that disrupt pathological signaling while promoting reparative pathways.

Other natural compounds, such as curcumin and epigallocatechin gallate, also exhibit multi‐target effects on PTM pathways, including the inhibition of kinases and modulation of acetylation. For curcumin, a double‐blind RCT of severe TBI patients has shown that oral nanocurcumin supplementation significantly improves brain edema and level of consciousness [153]. A 2024 systematic review and meta‐analysis further revealed that curcumin significantly reduces IL‐1β, IL‐6, TNF‐α, and MDA, while increasing the activity of SOD, GPx, Nrf2, BDNF, and Beclin‐1, thereby alleviating oxidative stress, inflammation, autophagy, and apoptosis in TBI animal models [154]. The major advantages of these agents lie in their established safety profiles, but their pleiotropic mechanisms also make it difficult to attribute clinical effects solely to a single PTM pathway, necessitating more targeted biomarker studies in future trials.

Quality of evidence for repurposed drugs. The evidence base for repurposed candidates is heterogeneous. Metformin and resveratrol have demonstrated efficacy in TBI rodent models [148], but many other candidates are supported primarily by evidence from other disease contexts. We have distinguished TBI‐specific findings from borrowed evidence throughout this section. While established safety profiles facilitate accelerated testing, efficacy in TBI requires confirmation in adequately powered, biomarker‐stratified trials. The complete translational pipeline from PTM discovery to clinical implementation is summarized in Figure 3.

FIGURE 3.

Translational pipeline of PTM‐targeted therapies in TBI. The figure illustrates the four‐stage pathway from PTM discovery to clinical implementation: (1) PTM discovery and proteomic profiling—identification of TBI‐specific PTM signatures via high‐throughput proteomics; (2) Identification of disease mechanisms—causal validation of PTM‐driven pathology in preclinical TBI models; (3) Pharmacological intervention—therapeutic strategies including small molecule inhibitors, PROTACs, drug repurposing, and combination therapies; and (4) Clinical validation and translation—biomarker‐guided trials, patient stratification, and precision medicine implementation. The bottom timeline (Discovery → Preclinical → Clinical → Implementation) indicates the progression from basic research to clinical use. This figure provides a roadmap for translating PTM‐based mechanistic insights into clinically actionable strategies for TBI.

7.6. PTMs as TBI Biomarkers: Current Evidence and Barriers

Several PTM‐modified proteins have been detected in TBI biofluids. In CSF, phosphorylated tau (p‐tau) is elevated in severe TBI, with p‐tau/threonine‐217 showing AUC of 0.78–0.83 for discriminating TBI from controls. Ubiquitination markers include CSF ubiquitin (increased > 4‐fold) [7] and UCH‐L1, which is elevated in both CSF and serum after severe TBI [9] and is FDA‐cleared for mild TBI detection [14]. Acetylation: acetylated tau (Ac‐tau) has been identified as a potential blood biomarker of TBI [3]; mass spectrometry has also detected trauma‐specific acetylated and citrullinated GFAP fragments in TBI CSF [10]. S‐Nitrosylation: CSF S‐nitrosothiols and S‐nitrosoalbumin are elevated ~1.7‐fold after pediatric severe TBI [11]. For SUMOylation and lactylation, biofluid validation in humans remains lacking [4, 6, 15, 17].

Assay feasibility varies: p‐tau, UCH‐L1, and GFAP utilize established immunoassay platforms (ELISA, automated chemiluminescence) with FDA clearance for certain indications [3, 7, 8, 10], while emerging PTM biomarkers lack validated high‐throughput assays. Specificity remains a challenge—many markers are also elevated in stroke and subarachnoid hemorrhage [7, 9]—though site‐specific PTM signatures (e.g., trauma‐specific GFAP citrullination patterns) may improve discrimination [10, 13]. Correlations with severity/outcome have been reported: pTau‐217 inversely correlates with 6‐month GOSE [8, 12], UCH‐L1 predicts mortality [9], and GFAP‐BDP trajectories distinguish good from poor outcomes [10].

Clinical implementation faces six barriers: (1) BBB penetration limits peripheral detection of brain‐derived PTM proteins [3]; (2) lack of standardized assays for emerging PTMs [7, 8]; (3) limited specificity for TBI versus other neurological injuries [7, 9, 10, 13]; (4) temporal dynamics complicating interpretation [8, 12]; (5) hemolysis confounds measurements [7]; and (6) undefined regulatory/reimbursement pathways [3, 14].

8. Technological Advances and Future Perspectives in PTM Research for TBI

The landscape of post‐translational modification research in TBI is being fundamentally reshaped by a confluence of technological breakthroughs. These advances are moving the field beyond descriptive cataloging toward a predictive, mechanistic, and ultimately therapeutic understanding of how PTMs orchestrate the complex pathophysiology from the moment of impact through to chronic neurodegeneration. The ability to map, quantify, and functionally validate PTM signatures with unprecedented resolution is now providing a powerful toolkit to identify novel biomarkers and druggable targets. This section will explore how these cutting‐edge technologies are illuminating the spatiotemporal dynamics of PTMs, defining critical thresholds between pro‐survival and pro‐death signaling, and outlining the formidable but not insurmountable challenges that lie ahead in translating these discoveries into effective clinical therapies for TBI patients.

8.1. Methodological Considerations in PTM Proteomics

While MS‐based PTM profiling has provided key insights into TBI pathophysiology, the technical complexity of these workflows introduces substantial methodological challenges that must be critically evaluated [41, 42].

8.1.1. Enrichment Methods and Biases

Most PTMs occur at low stoichiometry, necessitating enrichment prior to MS analysis [56]. For phosphorylation, IMAC and TiO₂ are widely used, though they preferentially recover multiply phosphorylated peptides while missing singly phosphorylated species [57]. For ubiquitination, anti‐diGlycine antibodies are standard, but this method cannot distinguish between different ubiquitin linkage types [58]. For acetylation, anti‐acetyl‐lysine antibodies are employed [59]. Each enrichment strategy has inherent biases; absence of a PTM in a dataset does not equate to absence in the biological sample [60].

8.1.2. Quantitative Strategies

Label‐free quantification (LFQ) and tandem mass tag (TMT)‐based multiplexing are the dominant approaches [41]. LFQ offers a broad dynamic range but suffers from higher technical variability; TMT enables multiplexed analysis but introduces ratio compression due to co‐isolation of interfering ions [61]. The choice affects which PTM changes are detectable [41].

8.1.3. Site Localization and FDR Control

Identifying the exacts modified residue is critical for biological interpretation [56]. Tools such as Ascore, PhosphoRS, and PTMProphet provide localization scores [57]. However, false localization rates (FLR) are often higher than reported; many studies apply a 1% PSM FDR threshold without adequate FLR control, meaning a significant fraction of reported PTM sites may be mislocalized [58].

8.1.4. Validation Approaches

Orthogonal validation is essential: (1) site‐directed mutagenesis; (2) modification‐specific immunoblotting; (3) synthetic peptide standards; and (4) metabolic labeling [59]. However, validation is often incomplete—antibodies may cross‐react, and mutagenesis can disrupt protein structure beyond the PTM site itself [60].

8.1.5. Limitations for Low‐Abundance and Labile PTMs

PTMs are inherently challenging due to their substoichiometric and labile nature [22]. Phosphorylation is labile under CID, leading to neutral loss of the phosphate group; ETD preserves labile modifications but is less efficient for doubly charged peptides [24]. Glycosylation and SUMOylation are even more challenging due to heterogeneity and poor ionization [24]. Low‐abundance PTMs suffer from ion suppression by more abundant unmodified peptides [25]. The practical implication is that many biologically relevant PTMs—particularly those with low stoichiometry or rapid turnover—are systematically under‐reported in TBI proteomic studies [61], cytoskeletal integrity, and cell death pathways [41, 57]. Similarly, global acetylome analyses have uncovered widespread changes in mitochondrial and metabolic enzymes, linking TBI to bioenergetic failure [155]. The clinical significance of this approach lies in its potential to discover unique PTM “fingerprints” that correlate with specific injury severities, predict long‐term outcomes, or serve as pharmacodynamic markers to monitor a patient's response to a targeted therapy.

8.2. Spatiotemporal Dynamics of PTMs: From Acute Injury to Chronic Neurodegeneration

A critical insight from recent research is that PTMs are not static markers but dynamic regulators whose roles evolve dramatically over the course of TBI pathology, with distinct temporal trajectories and cell‐type‐ and region‐specific patterns. Understanding this precise spatiotemporal choreography is essential for designing phase‐ and target‐specific therapies.

Temporal dynamics: acute phase (minutes to hours). In the immediate aftermath of TBI, PTMs act as rapid emergency signals. The acute phase is characterized by massive calcium influx, excitotoxicity, and oxidative burst, leading to rapid phosphorylation events. For example, tau is hyperphosphorylated at Ser202/Thr205 within minutes to hours after injury, driven by GSK‐3β and CDK5 activation, contributing to early axonal transport failure [25, 156, 157]. This hyperphosphorylation occurs across multiple brain regions, including the ipsilateral parietal cortex, contralateral hippocampus, and prefrontal cortex, and can persist for at least 4 weeks post‐injury. Ubiquitination of junctional proteins such as occludin and claudin‐5 occurs within hours, promoting early BBB permeability. S‐Nitrosylation of NMDA receptors and mitochondrial complex I is also an acute event, driven by the immediate surge of nitric oxide from neuronal and endothelial NOS [98, 99, 100]. PARP‐1 activation is triggered within hours of injury in response to DNA damage, initiating poly(ADP‐ribosyl)ation and, if sustained, leading to NAD⁺/ATP depletion and parthanatos. Glycome analysis has revealed significant alterations in N‐glycosylation patterns as early as day 1 post‐injury in both serum and CSF, including increased sialylation and altered fucosylation.

Temporal dynamics: subacute phase (days to weeks). As the injury progresses, the focus shifts to inflammatory and repair processes. During this phase, ubiquitination plays a central role in regulating the activation of NF‐κB in microglia and astrocytes, driving sustained neuroinflammation [69]. Ubiquitin‐conjugated protein levels are markedly increased in the ipsilateral cerebral cortex by day 7 and in the hippocampus by days 3–7 post‐TBI, while free ubiquitin protein levels remain significantly reduced. Microglial activation peaks during this subacute window, with HDAC3‐mediated epigenetic changes examined at 3–5 days after controlled cortical impact. HDAC inhibitors have been shown to increase histone H3 acetylation and reduce microglial inflammatory responses as early as 24 h after lateral fluid percussion TBI. Acetylation changes become prominent: global histone hypoacetylation leads to the silencing of neuroprotective and plasticity‐related genes, while specific non‐histone proteins (e.g., p53, NF‐κB) undergo acetylation that modulates their pro‐apoptotic or pro‐inflammatory activities [89, 91]. SUMOylation of Drp1 peaks during this subacute window, driving mitochondrial fragmentation and bioenergetic collapse [73, 116]. PARP‐1 overactivation continues in this phase, with blast‐induced modulation of the PARP‐SIRT‐NRF2 axis observed in astrocytes and microglia‐. Glycosylation changes are also prominent: N‐glycan alterations have been documented at days 1, 3, and 5 post‐injury, including increased sialylation and decreased bisection, galactosylation, and fucosylation.

Temporal dynamics: chronic phase (weeks to months to years). In the chronic phase, persistent PTM alterations lay the groundwork for progressive neurodegeneration. Chronic tau hyperphosphorylation at multiple epitopes (e.g., AT8, PHF‐1) may persist and spread transsynaptically. In human tau transgenic mice, hyperphosphorylated tau was detected in the ipsilateral hippocampus at 6 weeks after CCI, though not at 7 months, suggesting that tau pathology may be transient in some models. However, repetitive mTBI models have shown increased tau phosphorylation and microglial activation within the cortex at 12 weeks post‐injury, indicating long‐term changes in axonal integrity. Sustained histone acetylation/methylation changes maintain an epigenetically repressed state, limiting the expression of genes essential for synaptic plasticity and repair [158]. Chronic ubiquitin‐proteasome system impairment leads to the accumulation of ubiquitinated protein aggregates, with ubiquitin and Ref2P changes detectable at 4 weeks post‐TBI. Microglia‐specific HDAC3 knockout has been shown to facilitate functional recovery for up to 6 weeks after TBI, highlighting the prolonged therapeutic window for epigenetic modulation. Emerging evidence also suggests that histone lactylation‐driven astrocyte metabolic reprogramming may occur in chronic TBI, with increased glycolytic flux observed 1 month post‐injury.

Regional specificity. PTM patterns are not uniform across brain regions. The hippocampus, a key structure for memory function, appears particularly vulnerable to tau hyperphosphorylation and histone acetylation changes. Following TBI, tau hyperphosphorylation has been detected in the CA1/CA3 subregion of the hippocampus, as well as in the caudal hippocampus on day 1 post‐injury. Ubiquitin transcript levels are significantly reduced in both the ipsilateral cerebral cortex and hippocampus for up to 7 days after TBI, with ubiquitin‐conjugated proteins accumulating in the hippocampus on days 3–7. The cortex, especially the perilesional penumbra, exhibits robust inflammatory PTM signatures (e.g., NF‐κB ubiquitination, STAT3 phosphorylation) driven by activated microglia and infiltrating immune cells. Phosphorylated tau increases have been observed in the cerebral cortex beneath the impact site following single or repetitive mTBI, with repetitive mTBI animals showing increased tau phosphorylation and microglial activation within the cortex at 12 weeks. White matter tracts are particularly affected by neurofilament phosphorylation and tau hyperphosphorylation, which disrupt axonal transport and contribute to diffuse axonal injury. Microglial activation in white matter structures follows distinct temporal patterns after experimental mTBI. Some regionally enriched signaling pathways (e.g., hippocampal Akt/GSK‐3β, cortical JNK/c‐Jun, white matter CDK5) have been identified in TBI models, indicating distinct regional PTM regulatory networks.

Cell‐type specificity. PTMs also exhibit distinct cell‐type profiles. Neurons are the primary targets of tau phosphorylation (axonal injury), p53 phosphorylation/acetylation (apoptosis), and CREB phosphorylation (survival/plasticity) [25, 71]. Microglia are the main sites of NF‐κB ubiquitination, STAT3 phosphorylation, and NLRP3 inflammasome‐associated PTMs during neuroinflammation [110, 112]. HDAC3 has been identified as a key epigenetic regulator in microglia, with microglia‐specific deletion promoting inflammation resolution and functional recovery. HDAC inhibitors significantly inhibit microglial transformation to phagocytes in the CA2/3 hippocampal region at 24 h after injury. Astrocytes exhibit histone acetylation changes that affect their reactivity and neurotrophic support [69], as well as SUMOylation of transcription factors involved in stress responses. PARP‐SIRT‐NRF2 axis modulation has been observed in both astrocytes and microglia following blast overpressure. Endothelial cells are characterized by occludin/claudin‐5 phosphorylation (tight junction disruption) and glycosylation changes (leukocyte adhesion) [113, 114]. SOCS2 has been shown to alleviate TBI‐induced mitochondrial damage and parthanatos in endothelial cells by inhibiting the JAK2/STAT3 signaling pathway. Oligodendrocytes, though less studied, are susceptible to protein aggregation‐related ubiquitination and ferroptosis‐associated oxidative modifications that may contribute to white matter loss [159, 160, 161] (Table 6). A summary of these spatiotemporal and cell‐type‐specific PTM patterns is provided in Table S2.

TABLE 6.

The necessity for phase‐specific therapeutic strategies that account for the shifting roles of PTMs throughout the TBI continuum.

TBI phase	Dominant pathological process	Level	Key PTM examples	Functional consequence
Acute (0‐24 h)	Excitotoxicity, ionic dysregulation, primary axotomy	2	Hyperphosphorylation of Tau, CaMKIIα; S‐Nitrosylation of NMDA receptors	Synaptic dysfunction, Cytoskeletal collapse, neuronal hyperexcitability [162]
Subacute (1–7 days)	Neuroinflammation, blood–brain barrier breakdown, edema	2	Ubiquitination of IκB (activating NF‐κB); phosphorylation of tight junction proteins (e.g., occludin)	Microglial/astrocyte activation, increased vascular permeability, vasogenic edema [128, 163]
Chronic (> 1 week)	Neurodegeneration, synaptic loss, circuit reorganization	1	Persistent Tau hyperphosphorylation and aggregation; altered histone acetylation/methylation; lipid peroxidation (a PTM‐like event in ferroptosis)	Cognitive/mood deficits, epigenetic silencing of plasticity genes, progressive neuronal loss [158, 164]

8.3. Identifying Pro‐Survival Versus Pro‐Death PTM Thresholds in TBI

One of the most profound challenges in targeting PTMs is their inherent duality. The same modification on the same protein can have opposite effects depending on the context, site, and extent of modification. For example, phosphorylation of the transcription factor CREB at Ser133 is widely recognized as a pro‐survival signal that promotes the expression of neurotrophic factors such as BDNF; however, emerging evidence suggests that excessive or prolonged CREB activation under severe stress conditions may paradoxically contribute to neuronal dysfunction or death. Similarly, the tumor suppressor p53 is regulated by a complex network of PTMs. Its acetylation promotes cell cycle arrest and DNA repair (pro‐survival functions), whereas its phosphorylation at distinct sites can drive potent pro‐apoptotic programs in response to severe stress [165].

The concept of a “PTM threshold” is therefore critical. There likely exists a quantitative and qualitative tipping point where the balance of modifications on a key signaling node flips its output from a reparative to a degenerative state. This concept is observationally supported by studies showing divergent outcomes at different PTM levels (e.g., low vs. high Drp1 SUMOylation) and mechanistically validated by site‐directed mutagenesis studies that define the functional consequences of specific PTM sites [165]. However, therapeutic validation of PTM threshold modulation in TBI models remains limited. Defining these thresholds requires experimental approaches that extend beyond detection to functional validation. Site‐directed mutagenesis—mutating a specific PTM site to a non‐modifiable residue (e.g., alanine for phosphorylation) or a phosphomimetic residue (e.g., aspartate)—enables researchers to directly test the causal role of individual PTMs in cellular or animal models of TBI. By systematically mapping these critical nodes and their thresholds, the field can advance toward developing “smart” therapeutics that fine‐tune PTM pathway activity, keeping it within a pro‐survival window rather than fully activating or inhibiting it.

8.4. Challenges in Clinical Translation: BBB Penetration and Off‐Target Effects

Despite the immense promise of PTM‐targeted therapies, their journey from bench to bedside is obstructed by significant translational hurdles, foremost among which is the blood–brain barrier (BBB). This highly selective interface protects the brain from circulating toxins but concurrently blocks the entry of approximately 98% of small‐molecule drugs [163]. Many promising kinase inhibitors and HDAC inhibitors developed for oncology exhibit poor BBB penetration, rendering them ineffective for CNS disorders such as TBI. Overcoming this barrier requires innovative delivery strategies, including conjugating therapeutic agents to molecular Trojan horses that undergo receptor‐mediated transcytosis (e.g., via transferrin or insulin receptors), or using nanocarriers such as liposomes, polymeric nanoparticles, and dendrimers that facilitate BBB transport.

Clinical research in TBI (Table S1) has evolved into a multifaceted field spanning diagnostic imaging, therapeutic intervention, prognostic biomarker identification, rehabilitation, and systems biology. Analysis of recent clinical trials conducted between 2020 and 2025 reveals several dominant directions, reflecting both established clinical priorities and emerging mechanistic insights.

1. Imaging‐based diagnostics and characterization (~25% of trials). These studies focus on structural, functional, and metabolic neuroimaging to characterize brain–skull biomechanical coupling, monitor axonal injury, quantify cerebral hemodynamics, and evaluate neural connectivity patterns. Imaging trials provide critical translational endpoints for assessing secondary injury progression—including edema, intracranial hypertension, and long‐term neurodegeneration—and serve as essential correlates for molecular and proteomic alterations.

2. Pharmacologic and therapeutic interventions (32.1% of trials). This diverse and clinically impactful category includes complementary and alternative therapies (e.g., acupuncture), psychological interventions for post‐traumatic stress disorder and mood disturbances, and neuroprotective pharmacotherapies aimed at limiting excitotoxicity, oxidative stress, and neuroinflammation. Several interventional trials explicitly target inflammatory signaling and secondary injury cascades, creating a mechanistic overlap with PTM networks that regulate inflammation, mitochondrial function, and cell survival. These trials highlight the clinical need for targeted strategies that disrupt pathological signaling while promoting reparative pathways.

3. Proteomic, metabolomic, and biomarker‐oriented studies (7.1% of trials). Although comprising a small fraction, these trials hold unique relevance to the molecular landscape of secondary injury. Representative examples include proteomic profiling of patients with TBI‐associated intracranial hypertension and salivary biomarker analyses during concussion recovery. These studies validate body‐fluid proteomics as a feasible approach to capture dynamic protein changes after TBI and support the concept of a TBI‐specific proteomic landscape. Their relative scarcity underscores the novelty and translational importance of global proteomic and PTM‐focused investigations in TBI.

4. Rehabilitation trials. These focus on restoring physical activity, cognitive function, and quality of life, emphasizing the role of exercise, behavioral training, and lifestyle modification in long‐term recovery. Notably, exercise‐based rehabilitation directly intersects with mitochondrial bioenergetics and redox regulation—pathways heavily governed by PTMs, including acetylation and phosphorylation.

5. Microbiome–gut–brain axis studies. A small but innovative group of trials explores systemic metabolic and immunological shifts after severe TBI, revealing that TBI pathophysiology extends beyond the central nervous system. These findings expand the potential reach of PTM and metabolic regulatory networks into peripheral organ systems.

Collectively, the current clinical trial landscape demonstrates a strong emphasis on diagnosis, symptomatic management, and rehabilitation, with relatively few trials centered on the upstream molecular mechanisms that govern secondary injury. This gap underscores the value of proteomic and PTM‐directed studies in illuminating reversible regulatory events that may serve as novel biomarkers and therapeutic targets for improving long‐term neurological outcomes after TBI.

A second major challenge is achieving target specificity. As noted throughout this review, PTM‐regulating enzymes (kinases, HDACs, etc.) often belong to large families with highly conserved catalytic domains. A drug designed to inhibit one kinase may inadvertently affect dozens of others, triggering off‐target effects and systemic toxicity. The development of next‐generation isoform‐selective inhibitors, or alternative modalities such as PROTACs that degrade specific disease‐driving proteins without broadly inhibiting entire enzyme families, represents a promising frontier.

Finally, the heterogeneity of human TBI—encompassing varied mechanisms, severities, and anatomical locations—means that a single “magic bullet” is unlikely to be universally effective. Future clinical trials must incorporate rigorous patient stratification based on molecular biomarkers, including PTM signatures derived from biofluids, to identify subpopulations most likely to benefit from a specific targeted therapy. Advances in neuroimaging and blood‐based biomarkers are already enabling more precise TBI phenotyping and are poised to facilitate the next generation of mechanism‐based clinical trials. By integrating PTM biomarker panels into patient selection strategies, we can move toward a precision medicine framework for TBI management.

Beyond BBB penetration and target specificity, therapeutic timing and cell‐type selectivity represent equally critical barriers. Kinase inhibition may be most beneficial within hours post‐injury, whereas HDAC inhibition may exert greater efficacy during subacute to chronic phases [91, 92]. Moreover, the same PTM‐modulating enzyme can exert opposing effects in different cell populations. These complexities must be addressed in future therapeutic design and trial protocols.

8.5. Technical Limitations in PTM Profiling and Interpretation

First, sample preparation and enrichment efficiency are major bottlenecks. PTM‐modified peptides are typically low in abundance, and enrichment strategies (e.g., IMAC, TiO₂) exhibit differential selectivity that may bias the detected repertoire. Antibody‐based enrichments are further limited by specificity and batch variability.

Second, site localization remains challenging. Precisely determining which residue carries a modification requires sufficient fragment ion coverage; in complex TBI samples, localization confidence is often suboptimal, and a substantial proportion of reported sites may be falsely localized despite algorithmic improvements.

Third, antibody specificity for PTM detection remains a concern, with cross‐reactivity and epitope masking complicating orthogonal validation. A critical distinction exists between global PTM abundance and site‐specific functional regulation: a high‐occupancy site may lack functional consequence, while a low‐occupancy site on a key regulatory protein could be biologically decisive. Functional validation via mutagenesis is therefore essential.

Fourth, orthogonal validation is frequently lacking; most TBI PTM studies rely on single‐platform discovery proteomics without independent confirmation. Finally, longitudinal human samples are scarce, with most studies confined to acute time points in animal models or single patient time points, limiting our understanding of PTM trajectories from injury to recovery or chronic neurodegeneration.

These technical constraints underscore that many PTM findings reviewed here remain observational until subjected to rigorous orthogonal validation and site‐specific functional characterization.

9. The Dual‐Edged Nature of PTMs in TBI: Implications for Multi‐Targeted Therapeutics

The intricate world of post‐translational modifications in traumatic brain injury presents a compelling paradox: these molecular events are simultaneously the architects of neuronal demise and the potential saviors of neurological recovery. This section synthesizes the central theme of their dual‐edged nature, argues for the strategic necessity of multi‐targeted therapeutic approaches, and envisions a future where precision medicine—guided by dynamic PTM biomarkers—transforms the clinical management of TBI from a reactive discipline into a proactive, personalized one.

9.1. Summary of the Dual‐Edged Sword Nature of PTMs in TBI

The fundamental challenge and opportunity in targeting PTMs for TBI therapy lie in their profound context‐dependency. A single modification can be a harbinger of death or a beacon of survival, its ultimate effect dictated by a complex interplay of factors including the specific amino acid residue modified, the temporal phase of the injury, the cellular compartment involved, and the overall signaling milieu. For example, phosphorylation is a rapid and reversible switch that governs virtually every aspect of neuronal function. In the immediate aftermath of TBI, the pathological hyperphosphorylation of tau protein at sites like Ser202/Thr205 leads to its dissociation from microtubules, causing cytoskeletal collapse and contributing to the hallmark axonal injury seen in diffuse axonal injury [25, 26, 162]. This is a clear pro‐death signal. Conversely, the phosphorylation of the transcription factor CREB at Ser133 in surviving neurons within the penumbra can activate a pro‐survival gene program, upregulating neurotrophic factors like BDNF that support synaptic repair and plasticity during the subacute recovery phase [166, 167, 168]. Similarly, the process of ubiquitination, which typically tags proteins for degradation by the proteasome, can be protective by clearing damaged or misfolded proteins that accumulate after injury. However, excessive or dysregulated ubiquitination can lead to the untimely destruction of critical survival proteins, tipping the balance toward apoptosis. This duality extends to other PTMs, such as acetylation, which can regulate both metabolic enzymes in mitochondria (impacting energy production) and histones in the nucleus (influencing epigenetic programs for long‐term plasticity or degeneration) [158]. Recognizing this “double‐edged sword” nature is not a reason for therapeutic nihilism but rather a call for a more sophisticated and nuanced approach—one that seeks not to bluntly inhibit or activate a pathway, but to precisely modulate it to restore homeostasis.

9.2. The Necessity of Combination Therapies Targeting Multiple PTM Pathways

Given the interconnected and redundant nature of the signaling cascades that drive secondary injury in TBI, a monotherapeutic strategy targeting a single PTM node is unlikely to yield significant clinical benefit. The pathophysiology of TBI is a multifaceted storm involving excitotoxicity, neuroinflammation, oxidative stress, mitochondrial dysfunction, and various forms of cell death, all of which are regulated by distinct yet overlapping PTM networks [24, 128, 169, 170]. An effective therapeutic intervention must therefore be a coordinated counter‐offensive that addresses multiple fronts simultaneously. For instance, a rational combination therapy might pair an inhibitor of a pro‐death kinase (e.g., one that drives pathological tau phosphorylation) with an activator of a pro‐survival phosphatase or a compound that enhances the activity of the ubiquitin‐proteasome system to clear toxic protein aggregates. Another promising avenue is to combine a therapy that targets a key PTM in the acute inflammatory response (e.g., an inhibitor of IκB kinase to dampen NF‐κB activation in microglia) with a later‐intervention agent that promotes a reparative PTM signature, such as an HDAC inhibitor to open chromatin and facilitate the expression of genes involved in synaptic remodeling [92, 171, 172] (Table 7).

TABLE 7.

A conceptual framework for such a multi‐pronged approach, linking specific pathological processes to their key regulatory PTMs and potential therapeutic agents.

Pathological process	Key regulatory PTMs	Potential therapeutic agent class	Therapeutic goal
Acute excitotoxicity and axonal injury	Pathological Tau phosphorylation; CaMKIIα dysregulation	Selective kinase inhibitors (e.g., GSK3β inhibitors)	Stabilize microtubules, prevent axonal disconnection
Subacute neuroinflammation	Ubiquitination of IκB; phosphorylation of STAT3	IKK inhibitors; JAK/STAT pathway modulators	Shift microglia from pro‐inflammatory (M1) to anti‐inflammatory (M2) phenotype
Oxidative stress and ferroptosis	Lipid peroxidation; inactivation of GPX4 via oxidation	Ferroptosis inhibitors (e.g., Ferrostatin‐1, Liproxstatin‐1); NRF2 activators	Protect lipid membranes, restore redox balance
Chronic synaptic dysfunction and cognitive decline	Histone hypoacetylation; altered synaptic protein phosphorylation	HDAC inhibitors; positive allosteric modulators of synaptic receptors	Enhance synaptic plasticity, promote cognitive recovery

This combinatorial strategy acknowledges the complexity of TBI and aims to create a synergistic effect that is greater than the sum of its parts, thereby increasing the likelihood of a meaningful clinical outcome.

9.3. Outlook on Precision Medicine Approaches Guided by PTM Biomarkers in TBI

The ultimate realization of effective PTM‐targeted therapies for TBI hinges on the development and integration of robust, dynamic biomarkers that can guide precision medicine. TBI is not a single disease but a heterogeneous syndrome, with vast differences across etiology, severity, and individual patient biology. A “one‐size‐fits‐all” treatment is destined to fail in such a diverse population [47, 173]. The future, therefore, lies in using PTM signatures—detectable in accessible biofluids like cerebrospinal fluid or, more ideally, blood—as real‐time readouts of an individual's specific pathophysiological state. Advances in ultrasensitive immunoassays and mass spectrometry are making it increasingly feasible to detect and quantify specific, brain‐derived PTM‐modified proteins (e.g., phosphorylated tau species, acetylated histones) in plasma.

By serially monitoring these PTM biomarkers, clinicians could stratify patients into molecular endophenotypes, predict their risk for developing chronic neurodegenerative sequelae, and, most importantly, select the most appropriate combination therapy for their unique molecular profile. For example, a patient whose blood biomarker panel shows a dominant signature of ferroptosis‐related PTMs would be a prime candidate for a ferroptosis inhibitor [174], while another with a strong inflammatory PTM signature might benefit more from an immunomodulatory regimen [175]. Furthermore, these same biomarkers could serve as pharmacodynamic markers in clinical trials, providing early evidence of target engagement and biological effect, thereby accelerating drug development. This vision of a precision medicine pipeline—from molecular diagnosis via PTM biomarkers to the selection of a tailored, multi‐targeted therapeutic cocktail—represents the most promising path forward to finally deliver on the long‐standing promise of neuroprotection and functional restoration for individuals suffering from TBI.

10. Conclusion

The intricate and dynamic landscape of PTMs in TBI represents a fundamental layer of molecular regulation that dictates the trajectory from initial trauma to long‐term neurological outcome. PTMs such as phosphorylation, ubiquitination, and acetylation are increasingly recognized not merely as passive markers of damage but as active regulators of the secondary injury cascade. While much of the current evidence is observational or mechanistically supported by in vitro and in vivo functional studies, therapeutic validation in clinically relevant TBI models remains an ongoing effort. Their dual‐edged nature—capable of driving both neurodegeneration and neuroprotection—underscores the complexity of TBI pathophysiology and the inadequacy of simplistic, single‐target therapeutic approaches. The extensive crosstalk among PTM pathways creates a highly interconnected signaling network where dysregulation at one node can have cascading effects across multiple cellular processes, including neuronal death, neuroinflammation, mitochondrial failure, and synaptic dysfunction.

Despite this progress, several critical gaps remain. First, most studies examine individual PTM types in isolation, yet TBI simultaneously rewires phosphorylation, ubiquitination, acetylation, SUMOylation, and PARylation. Systematic mapping of synergistic or antagonistic PTM interactions is urgently needed. Second, the quantitative thresholds that separate pro‐survival from pro‐death PTM signaling remain largely undefined, hampering rational dose and timing decisions. Third, the blood–brain barrier continues to exclude the vast majority of PTM‐modulating agents; innovative delivery strategies—nanocarriers, prodrugs, and PROTACs—must be prioritized. Fourth, while PTM signatures in biofluids hold promise as dynamic biomarkers for patient stratification, prospective clinical validation is lacking. Finally, preclinical models rarely capture the full heterogeneity of human TBI—age, comorbidities, repetitive injury, and polypharmacy—contributing to translational failures.

While the evidence base for classical PTMs (phosphorylation, ubiquitination, acetylation) in TBI is substantial, emerging modifications such as lactylation, succinylation, and nitrosylation remain at an exploratory stage. For these nascent fields, the majority of evidence is currently Level 2 or Level 3, derived from correlative or non‐TBI models, highlighting critical knowledge gaps that warrant future investigation. We have systematically indicated the evidence level for each major claim throughout this review, providing readers with a transparent framework to assess the robustness of the underlying data.

Addressing these gaps requires a paradigm shift toward rationally designed combination therapies that simultaneously modulate several key PTM nodes to restore proteomic homeostasis and tip the balance toward repair. Critically, the future of TBI management lies in precision medicine, where advanced proteomic technologies enable the identification of patient‐specific PTM signatures in accessible biofluids. These dynamic biomarkers can guide the selection of tailored therapeutic regimens, ensuring that interventions are not only mechanistically sound but also precisely timed and targeted to an individual's unique molecular pathology. While formidable challenges in blood–brain barrier penetration, target specificity, and clinical trial design remain, the growing understanding of the PTM code in TBI offers a powerful roadmap for developing the first truly disease‐modifying therapies that move beyond symptomatic support to actively promote neural recovery and functional restoration.

Author Contributions

PeiPei Shen: writing – original draft. Liping Li: preparing tables and figures. Lin Zong: writing – review and editing. Zhonghua Zhang: conceptualization, funding acquisition, visualization, project administration, resources, and writing – review and editing.

Funding

This work was supported by the Postdoctoral Science Foundation of China (2024M754280).

Conflicts of Interest

The authors declare no conflicts of interest.

Supporting information

Table S1: Evidence levels for major PTMs and associated pathological processes in TBI.

Table S2: Spatiotemporal and cell‑type‑specific PTM patterns in TBI.

Data Availability Statement

No new data. Data sharing not applicable to this article as no datasets were generated or analyzed during the current study.