TLDc proteins: Stress-responsive nexus regulators of redox signaling, V-ATPase dynamics, and organellar crosstalk in neurodegeneration

Abstract

Many age-related neurodegenerative disorders are marked by progressive defects in cellular energy metabolism and protein homeostasis that converge on mitochondrial and lysosomal dysfunction. TLDc domain-containing proteins, such as OXR1, NCOA7, and related family members, have emerged as crucial modulators of organellar physiology and cellular stress responses. Growing evidence indicates that TLDc proteins physically interact with vacuolar ATPases (V-ATPases) to modulate their assembly and catalytic activity, linking TLDc function directly to the maintenance of lysosomal and Golgi lumen pH. This organellar pH homeostasis, in turn, is fundamental to intracellular iron handling and metabolic regulation, processes essential for mitochondrial bioenergetics, lysosomal functions, and cellular viability. Lysosomes maintain an acidic lumen via V-ATPase proton pumping, counterbalanced by specific ion channels, including TMEM175. This acidic environment is required for ferric iron reduction and subsequent release into the cytosol; when acidification fails, cells develop cytosolic iron deficiency, mitochondrial defects, pseudohypoxia via HIF-1α activation, and inflammation. Conversely, iron flux from lysosomes to mitochondria depends on acidic conditions and direct organelle contact, as exemplified by BDH2-driven siderophore transport, a V-ATPase-dependent but not TLDc-regulated process, which supports mitochondrial bioenergetics and sustains lysosomal acidity. Iron and pH dysregulation synergize to drive ferroptosis, lipid peroxidation, and neurotoxicity. Emerging studies link lysosomal deacidification and iron dyshomeostasis to the pathogenesis of major neurodegenerative diseases. These mechanisms collectively shape neuronal resilience, survival, and aging trajectories. This review integrates recent insights into how TLDc proteins coordinate organellar pH regulation and iron homeostasis and discusses how disruption of these interconnected pathways contributes to age-related neurodegeneration.

Graphical abstract

Highlights

• •TLDc proteins regulate V-ATPase assembly and organellar pH homeostasis.
• •TLDc proteins couple lysosomal iron handling to mitochondrial metabolism.
• •Disrupted TLDc signaling drives iron dyshomeostasis a.
• •nd neurodegeneration.
• •TLDc proteins are emerging therapeutic targets for neurodegenerative diseases.

1. Introduction

1.1. The burden of age-related neurodegeneration: multifactorial organellar failure

The progressive decline in neuronal function observed in age-related neurodegenerative disorders is increasingly attributed to failures in organellar communication and quality control. Disorders such as Alzheimer's disease (AD), Parkinson's disease (PD), amyotrophic lateral sclerosis (ALS), and Huntington's disease (HD) pose a major public health challenge and are characterized by the progressive and selective loss of vulnerable neuronal populations [1,2]. These disorders share fundamental pathogenic mechanisms, notably chronic oxidative stress, impaired proteostasis, and the progressive failure of essential cellular maintenance systems [3]. The inherent nature of neuronal cells renders them uniquely vulnerable to these accumulating stressors. The brain maintains a remarkably high metabolic rate, accounting for approximately 20% of the body's total energy consumption at rest [4]. This intense metabolic activity generates substantial by-products, specifically reactive oxygen species (ROS). When combined with the brain's relatively limited intrinsic antioxidant capacity, oxidative stress renders the central nervous system (CNS) exquisitely vulnerable [5]. Consequently, chronic oxidative stress is not merely a symptom but is implicated as a common etiological factor in neurological disease, as evidenced by consistent markers of oxidative stress in affected brain regions [5].

The maintenance of cellular quality hinges on the synchronized activity of two pivotal organelles: lysosomes and mitochondria. Lysosomes serve as the primary acidic recycling centres, degrading and processing cellular waste, while mitochondria act as central hubs for energy generation, calcium homeostasis, and apoptosis modulation [3]. The synergy between these organelles is paramount; lysosomes require mitochondrial-derived ATP for proton pumping, while mitochondria depend on lysosomal degradation for iron recycling and mitophagy. Growing evidence confirms that dysfunction in either lysosomes or mitochondria plays an essential role in cellular quality control, and defects in one often lead to impairment of the other, establishing a detrimental feedback loop that drives neurodegeneration. Age-related mitochondrial decline, for example, manifests as impaired oxidative phosphorylation, elevated ROS, and compromised mitophagy, all of which contribute directly to the pathogenesis of neurodegenerative disease [3].

This reciprocal vulnerability suggests that the pathological hallmark of the aging brain is not merely the accumulation of metabolic debris, but rather the disintegration of the functional bridges connecting these compartments. Consequently, while the accumulation of proteinopathic aggregates has traditionally defined the neurodegenerative landscape, the progressive erosion of neuronal function is increasingly recognized as a systemic failure of organellar communication and quality control [1]. This shift in perspective underscores that the critical upstream drivers of pathology reside within the endolysosomal and mitochondrial networks, where the precise orchestration of luminal pH and oxidative stress responses serves as the fundamental nexus for cellular longevity. In this context, the TLDc domain-containing proteins (OXR1, NCOA7, TBC1D24, TLDC1, and TLDC2) have emerged as critical, evolutionarily conserved guardians of neuronal health [[5], [6], [7]]. This review argues that TLDc domain–containing proteins are central molecular components of the organellar quality control network. We highlight how TLDc proteins regulate lysosomal and mitochondrial function by fine-tuning organellar pH through the V-ATPase complex, and how this regulation critically influences iron metabolism and neurodegenerative processes.

1.2. The TLDc domain containing family of proteins: guardians against oxidative stress

The TLDc domain, named after the highly conserved protein catalytic motif found across all eukaryotes (Tre2/Bub2/Cdc16 (TBC), Lysin Motif (LysM), Domain Catalytic), defines a family of proteins that are essential for cellular resilience against oxidative stress [8,9]. In mammals, this domain is shared among several proteins, including Oxidation Resistance 1 (OXR1, or TLDC3), Nuclear Receptor Coactivator 7 (NCOA7, or TLDC4), TBC1 Domain Family Member 24 (TBC1D24, or TLDC6), and TBC/LysM-associated domains containing 1 and 2 (TLDC1/mEAK7 and TLDC2) [5,10]. Although these proteins differ substantially in their domain organization, localization, and biological functions, accumulating evidence indicates that the conserved TLDc domain provides an important interface for cellular stress adaptation, particularly through regulation of V-ATPase-dependent organellar homeostasis.

Historically, TLDc proteins were investigated primarily for their robust ability to confer general neuroprotection against oxidative stress. OXR1 was first identified through a functional genomic screen for human cDNAs capable of suppressing spontaneous oxidative DNA damage in a repair-deficient Escherichia coli mutator strain [7,11]. Subsequent work in Drosophila and yeast established that loss of the OXR1 ortholog sensitizes cells to oxidative stress, with overt neurodegenerative phenotypes emerging specifically in flies and mice; in yeast loss of the oxr1 ortholog instead manifests as heightened sensitivity to oxidative stress [8,9]. In mammals, OXR1 is highly expressed in neurons within the mitochondrial and other cytosolic fractions [12]. OXR1 knockout mice display progressive ataxia and cerebellar degeneration [12]. While the deletion of OXR1, leads to neurodegeneration in mice [13] its overexpression provides protection in models of amyotrophic lateral sclerosis (ALS) [14], and a variety of other neurodegenerative diseases [8].

NCOA7 another TLDc family member interacts with endosomes/lysosomes and, when absent, cells show lysosomal dysfunction [15]. Indeed, yeast genetic studies revealed that the Drosophila mustard gene (homologous to OXR1/NCOA7) is required for lysosomal acidification: loss of mustard gene or human OXR1 yields aberrant lysosomal structures and cell death, and remarkably, expression of only the TLDc domain of human OXR1 or NCOA7 rescues the lysosomal defects [8,11]. Thus, the TLDc domain itself is sufficient for this protective role. This evolutionarily conserved requirement for TLDc-mediated lysosomal regulation extends to the nematode Caenorhabditis elegans, where LMD-3, the TLDc-domain ortholog of the OXR1/NCOA7 subfamily, was recently shown to interact directly with the V-ATPase subunit VHA-13 via its TLDc domain to control lysosomal acidification and proteostasis; loss of lmd-3 produces defective oxidation resistance, elevated cellular stress responses, and impaired proteostasis, phenocopying core features of TLDc dysfunction observed in flies, yeast, and mammals [16]. Together with NCOA7's documented role in lysosomal acidification in mammalian cells [11], these findings establish that multiple TLDc proteins across evolutionarily distant eukaryotes, from yeast and nematodes to flies and mammals, are critical for lysosomal integrity, reinforcing the premise that TLDc-mediated lysosomal regulation is a fundamentally conserved mechanism rather than one restricted to mammalian neurodegeneration.

This protective function, highly conserved across species, was initially thought to be linked to the role of TLDc proteins as scavengers of oxygen free radicals. However, the core molecular mechanisms underlying this broad protective capability remained largely undefined. Recent structural and biochemical studies have uncovered a significant paradigm shift, identifying the TLDc family not merely as generalized oxidative stress modulators, but as a class of critical V-ATPase interaction partners [10,17,18]. Unlike classical antioxidants that scavenge radicals directly, TLDc proteins function as chaperones and regulators of macromolecular complexes, ensuring their stability under stress. Their unique contribution lies in their ability to interface with the vacuolar H⁺-ATPase (V-ATPase), thereby coupling stress sensing with proton pump regulation [19].

2. The TLDc-V-ATPase regulatory axis: differential control of proton pumping

2.1. V-ATPase structure, dynamics, and TLDc interaction

The V-ATPase is a rotary motor enzyme responsible for the acidification of organelle/intracellular compartments. It is a multi-protein complex composed of a cytosolic ATP-hydrolyzing V1 subcomplex and a membrane-embedded V0 subcomplex responsible for proton transport [20]. A crucial regulatory mechanism conserved across species involves the reversible disassembly of the V1 and V0 components, which halts proton pumping to conserve cellular ATP, typically in response to metabolic stress such as glucose starvation [20]. TLDc proteins have been identified as a new class of V-ATPase interacting proteins, capable of modulating this assembly state.

The physical interaction between the V-ATPase complex and the TLDc family of proteins is now well-established. All five major mammalian TLDc family members have been shown to co-immunoprecipitate with the V-ATPase [10]. Structural studies suggest that the TLDc domain itself functions as a conserved V-ATPase interaction module, indicating that modulation of the pump is the central, ancient role of this protein family [10,17]. The structural integrity of this interaction is critical; mutations in the TLDc domain, such as those found in patients with TBC1D24-related epilepsy, disrupt this binding, leading to functional consequences [6]. However, the functional impact of this interaction varies profoundly among TLDc paralogs, demonstrating a highly specialized regulatory network [10,18].

2.2. The negative regulatory role: OXR1 and NCOA7

A functional paradox exists where certain TLDc proteins appear to inhibit V-ATPase activity to preserve cellular homeostasis. OXR1 and NCOA7 are recognized primarily as negative regulators of V-ATPase activity, functioning to moderate organellar acidification [18]. OXR1 and NCOA7 have been shown to promote the disassembly of the V-ATPase holoenzyme into its V1 and V0 subcomplexes [18]. This “tender love and disassembly” mechanism functions independently of catalytic activity, effectively silencing the pump [21]. Structural analysis, including cryo-electron microscopy (cryo-EM) of the yeast ortholog Oxidation Resistance protein 1 (Oxr1p), demonstrated that Oxr1p acts as a canonical disassembly factor for V-ATPase in live yeast [22]. Khan et al. showed in vitro that yeast Oxr1p binds directly to the V-ATPase and can disassemble the holoenzyme even in the absence of ATP, representing an “alternative disassembly” pathway distinct from canonical, glucose-starvation-induced disassembly [19]. This alternative action of Oxr1p depends on the TLDc domain of Oxr1p. Thus, Oxr1p can directly inhibit V-ATPase activity by causing dissociation of the V1 (ATPase) and V0 (proton-channel) subcomplexes [19,22]. OXR1 binding to the V1 subcomplex inhibits H⁺-ATPase activity and promotes the disassembly of the entire holoenzyme. This disassembly is mediated by OXR1 inducing a structural bend in the V1 subunit EG2, which destabilizes the interface between V1 and V0 [18]. These structural rearrangements provide a mechanistic basis for OXR1-mediated V-ATPase inhibition and holoenzyme disassembly, as illustrated in Fig. 1.

Fig. 1.

Tldc-mediated regulation of the V-ATPase complex. The V-ATPase holoenzyme consists of a cytosolic V1 sector (subunits A–H, responsible for ATP hydrolysis) and a membrane-embedded V0 sector (subunits a, c, c′, c″, d, e, responsible for proton translocation), connected by three peripheral EG stalks whose N-termini normally anchor onto subunit C-foot and the N-terminal domain of subunit a (aNT). (Left panel) TBC1D24 binds V1 via its TLDc domain at an EG peripheral stalk (subunits E and G), promoting assembly and stabilization of the V1–V0 holoenzyme. This leads to active proton (H⁺) influx into the organellar lumen, maintaining a low, acidic pH (e.g., in synaptic vesicles). (Right panel) Conversely, the TLDc domains of OXR1 and NCOA7 dock at the junction formed by the open A/B catalytic interface, subunit C, and peripheral stalk EG2. This binding forces a structural bend in EG2 that disrupts the EG2–C-foot–aNT anchor holding V1 to V0, causing V1 to release from V0 and silencing the pump. This disassembly mechanism prevents over-acidification, maintaining a neutral pH in compartments like the Golgi/TGN. This opposing action on the EG2 stalk acts as a molecular “rheostat” for pH control. Created using BioRender.com.

This inhibitory mechanism serves a critical homeostatic function, particularly within the secretory pathway. The proteins OXR1 and NCOA7 are required to maintain proper pH within the Golgi lumen and the Trans-Golgi Network (TGN). This regulation is necessary because acidification above normal levels would otherwise inactivate the function of key glycosyltransferases enzyme and severely disrupt post-translational modifications [23]. By limiting V-ATPase activity, they ensure the optimal, slightly less acidic pH (typically pH 6.0–6.5) necessary for the proper function of glycosylation enzymes. In mammals, analogous effects have been observed: the TLDc proteins OXR1 and NCOA7 localize to the Golgi and trans-Golgi network (TGN) membranes in a Rab-dependent manner, where they bind the catalytic ATPase subunit of V-ATPase via their TLDc domains [23]. Depletion of OXR1/NCOA7 leads to excessive acidification of the Golgi/TGN lumen and defective glycosylation of secretory proteins. Interestingly, OXR1/NCOA7-depleted cells also exhibit higher lysosomal pH and increased sensitivity to membrane-damaging agents, indicating broader lysosomal compromise [23]. These results indicate that the inhibitory roles of OXR1 and NCOA7 within the Golgi/TGN are indirectly essential for preventing over-acidification of secretory and endolysosomal compartments. They achieve this by modulating V-ATPase activity, which is critical for maintaining overall organelle integrity and proper pH gradients throughout the endo-lysosomal pathway [23]. Notably, this inhibitory activity of OXR1 and NCOA7, first defined using purified components in reconstituted systems [18], is broadly concordant with the loss-of-function phenotypes observed in cells and animal models: depletion of either protein produces the hyperacidification and secretory/lysosomal dysfunction predicted by loss of a molecular brake on the pump. As discussed in Section 2.3, this concordance is not observed for all TLDc family members.

2.3. The positive regulatory role: TBC1D24

In sharp contrast to the inhibitory roles of OXR1 and NCOA7, TBC1D24 acts as an apparent positive regulator or stabilizer of V-ATPase activity, particularly in specialized neuronal compartments. TBC1D24 is highly expressed in the central nervous system, localizing prominently at synaptic vesicles (SVs) and endo-lysosomal compartments in the brain.

The essential difference in TBC1D24 function becomes evident in loss-of-function models. Studies using TBC1D24 knockout mice demonstrated that the absence of this protein results in the mis-localization and cytosolic shift of V1 subunits (e.g., ATP6V1 A and ATP6V1B2) [6,10]. This detachment prevents the formation of the functional V-ATPase holoenzyme, leading to a pathological increase (alkalinization) of pH within endo-lysosomal compartments and a corresponding impairment of autophagic flux [6]. TBC1D24, particularly in neurons, interacts with the V-ATPase to maintain the acidic pH of synaptic vesicles and endosomes. Furthermore, loss of TBC1D24 severely compromises the integrity of hippocampal synapses, impairing synaptic vesicle endocytosis and, critically, SV reacidification, which is required for neurotransmitter loading and receptor recycling [6,24]. The implication is clear: TBC1D24 is essential for V-ATPase assembly or stabilization in specialized neuronal systems, ensuring the low pH gradients necessary for synaptic communication and waste clearance [18].

It is important to note, however, that this in vivo evidence for a stabilizing, positive-regulatory role stands in direct contrast to biochemical data obtained using purified, reconstituted human V-ATPase, in which TBC1D24; like OXR1, NCOA7, and TLDC2; was found to inhibit pump activity rather than stabilize it [18]. Unlike the concordance seen for OXR1 and NCOA7 (Section 2.2), this represents a genuine, currently unresolved discrepancy between reductionist biochemical assays and cellular/organismal phenotypes, and is addressed further in Section 2.4. This differential regulation, where OXR1 and NCOA7 inhibit acidification in the Golgi apparatus and lysosomes, while TBC1D24 maintains pH stability within synaptic vesicles, suggests that TLDc proteins tune pH in a compartment-specific manner. The distinct effects of TLDc proteins on pH homeostasis across secretory and endolysosomal compartments are shown in Fig. 1.

2.4. Resolving the functional paradox of TLDc-V-ATPase regulation

The functional landscape of TLDc proteins has recently become a focal point of biochemical debate, centered on a perceived “functional paradox” between their stabilizing and inhibitory effects on the V-ATPase complex. While TBC1D24 is recognized as a positive regulator essential for holoenzyme stability in neuronal compartments, based on live-cell imaging and biochemical evidence [6,24], a growing body of structurally resolved evidence demonstrates that other family members, specifically OXR1 and NCOA7, act as potent negative regulators. This inhibition occurs through a mechanism colloquially termed “tender love and disassembly,” where the TLDc domain actively promotes the dissociation of the cytosolic V1 subunit from the membrane-bound V0 subunit, effectively silencing proton translocation: a mechanism directly visualized by cryo-EM for yeast OXR1p and further resolved structurally for human OXR1, NCOA7, and mEAK7 bound to V-ATPase [18,19,21]. In a systematic biochemical comparison of purified, reconstituted human V-ATPase with five recombinant TLDc proteins (OXR1, NCOA7, TLDC2, TBC1D24, and mEAK7) summarized in Table 1, four of the five, including TBC1D24, were found to inhibit pump activity, while only mEAK7 enhanced it [18]. For OXR1, NCOA7, and TLDC2, this in vitro inhibitory activity is broadly consistent with in vivo and cellular loss-of-function phenotypes: for example, Tldc2-knockout mice display impaired urine alkalinization and lower urine pH, consistent with loss of a normal restraint on V-ATPase-driven proton secretion in kidney intercalated cells [18,25]. TBC1D24 is the clear outlier, its in vitro inhibitory activity is opposite in direction to the loss-of-assembly, loss-of-acidification phenotype observed when TBC1D24 is deleted in neurons [6,18,24], and it is this specific discrepancy that constitutes the unresolved paradox discussed below.

Table 1.

Comparative functional and pathological landscape of mammalian TLDc proteins.

Protein (Gene)	Primary localization	In vitro V-ATPase activity (purified/reconstituted system)	In vivo/cellular phenotype	Apparent paradox/concordance	Proposed mechanism	Associated pathology	References
OXR1 (TLDC3)	Cytosol, Golgi/TGN, vesicles	Inhibits V-ATPase; promotes V1/V0 holoenzyme disassembly independent of catalysis and of glucose-dependent reversible disassembly	Loss of OXR1 causes Golgi/TGN hyperacidification, defective glycosylation, and secondary lysosomal alkalinization/membrane fragility in cells; loss-of-function linked to neurodegeneration in vivo	Largely concordant: in vitro inhibition matches the “brake on acidification” role inferred from loss-of-function studies	TLDc domain binds V1, induces a conformational bend in subunit EG2 that destabilizes the V1–V0 interface	ALS-associated neurodegeneration; oxidative stress sensitivity; Golgi glycosylation defects	[[17], [18], [19],[21], [22], [23]]
NCOA7 (TLDC4)	Golgi, TGN, endosomes	Inhibits V-ATPase in the same purified system (ref. [16])	Ncoa7-knockout mice show incomplete distal renal tubular acidosis (persistently high urine pH, hypobicarbonatemia after acid loading) — consistent with reduced, not increased, net V-ATPase-driven acidification in renal intercalated cells; Golgi/TGN hyperacidification and glycosylation defects also seen in depleted cells	Largely concordant across tissues, but tissue-specific severity varies (kidney vs. Golgi/TGN), showing the same molecular brake produces different physiological outputs depending on cell type	TLDc domain-mediated V1/V0 disassembly; Rab-dependent Golgi/TGN targeting	Congenital disorders of glycosylation; incomplete distal renal tubular acidosis (dRTA)	[18,19,23,26]
TLDC2 (TLDC5)	Kidney B-type intercalated cells; broader tissue distribution	Inhibits V-ATPase in vitro (ref. [16])	Tldc2-knockout mice show lower urine pH and impaired urine alkalinization, indicating loss of a normal brake on V-ATPase-dependent proton secretion	Concordant: in vivo loss-of-function phenotype (increased net acidification capacity) matches the in vitro inhibitory activity	Presumed TLDc-domain-mediated disassembly/inhibition analogous to OXR1/NCOA7, but structural basis not yet resolved	Renal acid–base handling defects	[18,25]
TBC1D24 (TLDC6)	Synaptic vesicles, endo-lysosomes, ER–mitochondria contact sites	Inhibits V-ATPase in the same purified reconstitution assay (ref. [16])	Tbc1d24-knockout neurons show cytosolic mis-localization of V1 subunits (ATP6V1 A, ATP6V1B2), loss of holoenzyme assembly, endo-lysosomal alkalinization, and impaired synaptic vesicle reacidification	Genuine, unresolved paradox: purified-component assays classify TBC1D24 as inhibitory, yet its loss in neurons produces the opposite outcome (failure of assembly/acidification)	Unknown; may reflect a cellular co-factor, phosphorylation state, or membrane environment absent from the in vitro assay; N-/C-terminal extensions outside the TLDc domain are hypothesized to dictate binding mode	Epilepsy (DOORS syndrome), synaptic failure, DEE-spectrum disorders	[6,10,18,24,[27], [28], [29]]
mEAK7 (TLDC1)	Cytosol/vesicular, lysosomal membrane	The single exception in Ref. [16]: enhances/activates V-ATPase activity rather than inhibiting it	Stable mEAK7–V-ATPase association observed structurally (cryo-EM); functional consequences in vivo less well defined, with proposed links to mTOR signaling and cancer cell proliferation	Concordant by definition (only protein classified as activating in both structural and biochemical contexts), but downstream physiological role in vivo still emerging	Binds intact, assembled V-ATPase without promoting disassembly; stabilizes holoenzyme	Emerging role in cancer cell proliferation via mTOR; neurological role unclear	[5,10,17,18]

The controversy lies in the physiological “intent” of such inhibition. Emerging data suggest that this is not a maladaptive failure but a refined regulatory “rheostat” used to prevent hyper-acidification in specific compartments like the Golgi apparatus, where precise pH tuning is vital for enzymatic glycosylation [23]. However, in the context of neurodegeneration, critics argue that this inhibitory capacity may be pathologically hijacked or dysregulated, leading to the lysosomal alkalinization and autophagic stagnation characteristic of proteinopathies [18].

In contrast to the structurally resolved inhibitory mechanisms of OXR1, NCOA7, and mEAK7, no cryo-EM or crystallographic structure of the TBC1D24–V-ATPase complex has yet been reported. TBC1D24's proposed stabilizing role is currently supported only by live-cell imaging and biochemical/co-immunoprecipitation data in neurons, which demonstrate that TBC1D24 associates with the V-ATPase and is required for normal synaptic vesicle trafficking and holoenzyme function, but do not resolve the physical assembly state or binding interface of the complex [6,24]. The precise molecular mechanism by which TBC1D24 engages the V1/V0 interface to prevent disassembly therefore remains inferential rather than structurally established, and we explicitly identify this as an outstanding gap in the field, distinct from the structurally validated disassembly mechanisms of OXR1, NCOA7, and mEAK7. The highly conserved nature of the TLDc domain points to V-ATPase binding as the universal functional characteristic shared across family members. Therefore, the contrasting functional outcomes, stabilization versus disassembly, are hypothesized to be mediated by the unique non-TLDc sequences, such as the N- and C-terminal extensions present in TBC1D24 and TLDC1 [10]. These extensions may plausibly dictate the specific binding location on the V-ATPase complex, the affinity for specific V-ATPase isoforms (such as the B1 subunit bound by NCOA7 and OXR1), or the recruitment of accessory proteins that shift the V1/V0 assembly equilibrium [18]. However, in the absence of direct structural data for TBC1D24, this remains a hypothesis rather than a demonstrated mechanism. Structural studies, particularly cryo-EM or crystallographic resolution of the TBC1D24–V-ATPase interface, are therefore urgently required to test this model and definitively establish TBC1D24's assembly-stabilization mechanism at the molecular level, given that the purified-component assay alone appears insufficient to capture it's in vivo behavior [18].

Determining whether TLDc-mediated V-ATPase disassembly serves as a protective antioxidant response or acts as a primary trigger of organellar dysfunction remains a major unresolved question in the field, and this uncertainty is compounded by the current lack of structural data for the stabilizing arm of the pathway. This “two-faced” behavior of the TLDc–V-ATPase axis suggests that therapeutic approaches must be precisely tailored potentially enhancing V-ATPase assembly in lysosomes while allowing regulated disassembly in the Golgi to effectively restore cellular proteostasis and iron homeostasis in aging neurons [6,18]. This dual-action regulation suggests that TLDc proteins serve as a vital link between a cell's immediate biochemical state and its long-term organellar stability. To understand the physiological impact of this ‘rheostat’ mechanism, and to close the structural gap identified above, it is necessary to examine how these proteins integrate into the wider neuronal landscape, specifically how their structural nuances and post-translational modifications dictate the complex communication between lysosomes, mitochondria, and the endoplasmic reticulum [1,18].

3. TLDc regulation and organellar crosstalk in neurons

3.1. Structural complexity and post-translational modification (PTMs)

The functional diversity of the TLDc paralogs implies complex regulation extending beyond their primary sequences. While the core TLDc domain is necessary for V-ATPase interaction, the non-conserved extensions of proteins like TBC1D24 and TLDC1 are hypothesized to mediate specificity and regulate the final effect on enzyme function [6,30]. Post-translational modifications (PTMs), such as phosphorylation, are master regulators of protein activity, governing protein structure, dynamics, and interactions. PTMs are known to modulate crucial processes like liquid-liquid phase separation (LLPS), which is often implicated in the formation of protein aggregates characteristic of neurodegenerative proteinopathies [31]. It is highly probable that specific PTMs on the non-conserved regions of TLDc proteins determine whether the protein serves as a V-ATPase inhibitor (OXR1) or a stabilizer (TBC1D24). For TBC1D24, which contains a TBC domain typically associated with Rab GTPase-activating proteins, phosphorylation of specific serine or threonine residues is likely to regulate its ability to bind to the V-ATPase cytosolic V1 subunits. Identifying the regulatory kinases and phosphatases that control the PTM status of TBC1D24 and OXR1 represents a vital missing link that would reveal the upstream signaling pathways governing V-ATPase assembly and disassembly kinetics in response to metabolic or environmental cues [18].

3.2. Lysosomal consequences: autophagy and the LYPAS system

In the context of neuronal health, TBC1D24 function is tightly coupled to the effective operation of the Lysosomal Processing and Adaptation System (LYPAS). The LYPAS encompasses the entire interconnected network of processes including basal maintenance, substrate delivery, and quality control that maintain lysosomal capacity [24]. Loss of TBC1D24 severely compromises the integrity of the LYPAS. The resulting endo-lysosomal alkalization inactivates acid hydrolases, blocking the recycling process. This defect leads directly to the accumulation of aberrant lysosomes and undegraded lipid materials within neuronal tissue in TBC1D24 knockout models. Furthermore, this impairment is sufficient to cause a definitive blockade of autophagic flux, preventing the clearance of damaged organelles and aggregated proteins [32]. Since lysosomal dysfunction and subsequent autophagy impairment are central pathological features of major neurodegenerative conditions, the demonstration that TBC1D24 is a necessary component for the maintenance of LYPAS integrity positions TLDc dysfunction as a root cause for numerous proteinopathies [6].

3.3. TLDc and the endoplasmic reticulum-mitochondria contact sites (ERMCS)

The regulatory impact of TBC1D24 extends beyond the endo-lysosomal network, encompassing critical roles in mitochondrial dynamics and cellular energetics, indicating a central coordinating function for TLDc proteins [6]. TBC1D24 loss or mutation has been shown to directly impair mitochondrial function, resulting in reduced ATP synthesis and decreased mitochondrial membrane potential in affected patient cells [24]. Critically, these mitochondrial defects are associated with an alteration in the physical junctions between the endoplasmic reticulum (ER) and the mitochondria, known as the ER-Mitochondria Contact Sites (ERMCS) [33]. ERMCS are vital regulatory platforms that control processes such as mitochondrial fusion, fission, and lipid transfer, making them essential hubs for mitochondrial quality control. The dual impact of TBC1D24 loss i.e. affecting both lysosomal clearance and ERMCS dynamics suggests that TLDc proteins serve as previously unappreciated gatekeepers that coordinate the LYPAS with mitochondrial health [6]. This coordination is likely crucial for managing cellular stress, as the failure to clear damaged material (lysosome) alongside the inability to properly remodel the energetic machinery (mitochondria/ERMCS) constitutes a systemic collapse of cellular homeostasis.

4. The pathophysiological axis: pH dysregulation, iron metabolism, and oxidative stress

4.1. Maintenance of organelle pH

Acid–base homeostasis in organelles is central to their function. Lysosomal pH is maintained around 4.5–5.0 in healthy cells, primarily by the vacuolar H⁺-ATPase (V-ATPase) pumping protons into the lumen [34]. This electrogenic process generates an electrochemical proton gradient (ΔμH⁺) across the lysosomal membrane, composed of both a chemical component (ΔpH) and an electrical component (membrane potential, Δψ). Because unopposed H⁺ pumping would rapidly build up a large Δψ that stalls further V-ATPase activity, dissipation of this potential either through direct H⁺ efflux or through compensatory counter-ion flux, is required to sustain net acidification and to prevent lysosomal over-acidification [[35], [36], [37]].

TMEM175 (also referred to as LyPAP1) has been proposed to mediate this dissipative pathway, but its precise ionic mechanism remains actively debated. TMEM175 was first characterized as a voltage-independent K⁺-selective leak channel, with K⁺ efflux serving as a counter-ion current that neutralizes the charge deposited by V-ATPase and thereby indirectly sustains H⁺ pumping by limiting Δψ buildup [38]. Subsequent studies reported that under the physiologically acidic luminal pH found in lysosomes (as opposed to the near-neutral pH used in earlier recordings), TMEM175 instead behaves as a proton-activated, proton-selective channel, conducting H⁺ directly (independent of K⁺ or Na⁺ co-transport) once luminal pH drops below the normal range, thereby directly clamping ΔpH once a threshold acidity is reached [35,36,39]. Recent electrophysiological work has challenged the proton-selective model, reporting that TMEM175 loss can produce lysosomal alkalinization rather than hyperacidification in certain experimental contexts, a result more consistent with a K⁺-conductance/Δψ-dependent mechanism than with direct, independent H⁺ permeation [37]. Other recent electrophysiological analyses continue to support substantial H+ permeability and demonstrate that proton and K+ conductance are sensitive to luminal pH and can share a common permeation pathway [36]. The discrepancy may partly reflect differences in luminal pH, ionic composition, and activation state (basal vs. lipid/agonist-gated) used across studies. Taken together, current evidence indicates that TMEM175 constitutes a genuine H⁺-leak pathway that fine-tunes lysosomal pH in a feedback loop with V-ATPase, but whether this occurs via H⁺ conducted independently of other ions, or via a K⁺-coupled mechanism that acts primarily on Δψ (and thus only indirectly on ΔpH), is not yet fully resolved and likely depends on the prevailing luminal pH and gating state of the channel [35]. Consistent with TMEM175 contributing functionally to this feedback loop regardless of the precise ionic mechanism, mouse cells lacking TMEM175 develop hyper-acidic lysosomes (pH ∼4.0) and impaired degradation, phenotypes rescued by partial pH neutralization [35].

Lysosomal membrane proteins such as LAMP1/2 also influence pH [40]. Recent studies in flies and mammalian cells show that LAMP2 can restrict proton leak, so that LAMP loss may lead to either unchanged or even reduced lysosomal pH, depending on context [41]. While the exact mechanisms remain under investigation, these findings underscore that multiple factor (V-ATPase, channels, scaffolds) coordinate to maintain an optimal lysosomal pH. The cytosolic ATP supply from mitochondria is also important: experiments using oligomycin (inhibitor of mitochondrial ATP synthase) cause lysosomal alkalinization, indicating that ATP-driven V-ATPase activity depends on mitochondrial bioenergetics [42]. Conversely, mitochondrial iron uptake (via mitoferrin-1) influences lysosome acidification; blocking mitochondrial iron transport also raises lysosomal pH [42]. These results reveal interdependence: healthy mitochondria help pump protons into lysosomes (through ATP), and in turn, proper lysosomal function supports mitochondrial iron availability.

Organelle pH must be tailored to local enzymatic needs: the Golgi and early endosomes require a moderately acidic lumen (pH ∼6.0–6.7), whereas lysosomes are more acidic [23]. The TLDc proteins OXR1 and NCOA7 demonstrate that cells actively prevent excessive acidity in compartments. By inhibiting V-ATPase at the Golgi/TGN, OXR1/NCOA7 prevent Golgi lumens from becoming too acidic, which is critical for pH-sensitive processes like glycosylation [23]. Similarly, yeast OXR1p′s disassembly of V-ATPase may protect other vesicles (like endosomes or vacuoles) from hyperacidification. Thus, rather than only pumping protons in, cells use TLDc-domain “brakes” on the V-ATPase to ensure pH is neither too high nor too low.

In mitochondria, pH is also regulated: the matrix is maintained near pH 7.8 by the respiratory chain and membrane antiporters (e.g. H⁺/K⁺ exchangers). Mitochondrial pH is critical for ATP synthesis and ion homeostasis, but less is known about specific TLDc roles in mitochondrial pH. However, OXR1 is known to localize to mitochondrial membranes and its loss can destabilize mitochondrial DNA as seen in some models [43]. It is possible that OXR1 indirectly supports mitochondrial pH homeostasis by preventing excessive reactive oxygen damage to proton-coupling machinery. Overall, dynamic regulation of pH across the endolysosomal, Golgi/TGN and mitochondrial systems is essential for cellular homeostasis and metabolism. Disruption of this balance, through V-ATPase dysfunction or channel mutations can impair degradation and trafficking while perturbing iron homeostasis and stress signaling, ultimately contributing to mitochondrial dysfunction and oxidative damage (Fig. 2). Consistent with these interconnected pathological changes, defective lysosomal acidification is increasingly recognized as a hallmark and potential early marker of neurodegenerative disease [44].

Fig. 2.

The pathogenic “iron trap” and organellar domino effect. (1) TLDc loss & V-ATPase Dysfunction: Loss of functional TLDc proteins (e.g., TBC1D24) leads to V-ATPase disassembly and a failure to acidify the lysosome, resulting in a pathological rise in luminal pH. (2) The iron trap: The acidic pH required to release ferrous iron (Fe²⁺) from ferritin and degrading mitochondria (Mito) is lost. Iron becomes trapped within the lysosome, creating a “functional” iron deficiency in the cytosol. (3) Mitochondrial starvation: The lack of cytosolic iron impairs the synthesis of iron-sulfur (Fe-S) clusters and heme, which are essential for the mitochondrial respiratory chain. This leads to metabolic failure, reduced ATP production, and increased ROS generation. (4) Ferroptosis & terminal event: The combination of unchecked ROS, lipid peroxidation, and metabolic collapse sensitizes the neuron to ferroptosis, an iron-dependent form of regulated cell death, leading to neurodegeneration. Created using BioRender.com.

4.2. Lysosomal acidification failure triggers cellular iron deficiency while paradoxically loading the lysosome

Lysosomal integrity is intimately linked to the regulation of iron homeostasis, leading to the concept of the pH–iron axis. Acidic lysosomes are crucial for cellular iron homeostasis. Cellular iron is essential but highly toxic if unbound (Labile Iron Pool, or LIP) [45,46]. Iron is internalized primarily via the transferrin receptor (TfR) and released from transferrin within the acidic endosomal/lysosomal compartments [47]. Many cellular iron sources converge on lysosomes: receptor-mediated endocytosis of transferrin (Fe³⁺-bound) and autophagic degradation of ferritin both deliver ferric iron to the lysosome lumen. At low pH, ferric iron (Fe³⁺) is released from its carriers and reduced to ferrous iron (Fe²⁺) by lysosomal/endosomal ferrireductases such as STEAP3 and Cyb561a3 (LcytB), after which Fe²⁺ exits into the cytosol via pH-optimized lysosomal transporters, principally Divalent Metal Transporter 1 (DMT1)/solute carrier family 11 member 2 (SLC11A2) and Transient Receptor Potential Mucolipin 1 (TRPML1)/mucolipin TRP cation channel 1 (MCOLN1) [[48], [49], [50]].

Alkalinization does not function as a binary switch, rather, it differentially impairs iron reduction and export, with the latter becoming disproportionately compromised. Although acidic pH favors the activity of lysosomal and endosomal ferrireductases such as STEAP3 and CYB561A3, their activity is attenuated rather than completely abolished as luminal pH rises. In contrast, disruption of the proton gradient compromises Fe²⁺ efflux through DMT1 and TRPML1, with DMT1 relying directly on proton coupling and TRPML1-mediated Fe²⁺ permeation being favored by the acidic lysosomal environment. Perturbation of V-ATPase activity, TLDc function, or lysosomal acidification therefore compromises Fe²⁺ export and shifts the balance toward intraluminal iron retention. Under these conditions, Fe²⁺ generated by residual, pH-attenuated ferrireductase activity, together with Fe²⁺ produced through non-enzymatic reduction by lysosomal reactive oxygen species, can accumulate within the lumen when iron generation outpaces its export [50,51]. The elevated pH additionally blocks Fe³⁺ release from transferrin and further compromises transporter activity, ultimately reducing iron delivery to the cytosol. Thus, although cytosolic iron availability is diminished, the luminal iron pool is not a static or inert Fe³⁺ reservoir but a dynamic, chemically heterogeneous and redox-active pool. Indeed, recent work in senescent cells established that lysosomal alkalinization can cause aberrant retention of Fe²⁺ within lysosomes [52]. Therefore, lysosomal alkalinization is best viewed as a defect in iron mobilization and compartmentalization rather than as a state in which lysosomal iron is exclusively ferric.

Yambire et al. (2019) demonstrated that blocking lysosomal acidification with bafilomycin or mutating lysosomal α-glucosidase (Pompe's disease model) causes an increase in lysosomal iron while decreasing cytosolic Fe²⁺ availability, triggering HIF-1α stabilization, mitochondrial dysfunction, and cell death [53]. This deficiency is distinct from systemic iron deficiency and is reversible by supplying iron via a lysosome-independent pathway, establishing a step-by-step causal chain whereby TLDc-mediated V-ATPase failure directly precipitates localized metal dysregulation [10,53]. In vivo, a mouse model of impaired lysosomal acidification exhibited brain-wide iron deficiency and neuroinflammation [53], highlighting the importance of lysosomal pH for brain iron supply. This cytosolic starvation is not a passive endpoint; it engages a compensatory response with direct consequences for lysosomal iron loading. Falling cytosolic iron de-represses IRP/IRE-regulated gene expression, increasing transferrin receptor and ferritinophagy flux while suppressing ferritin translation, the classical cell-intrinsic response to iron scarcity [45]. Consistent with this, loss of lysosomal ferrireductase activity in macrophages increases TfR1 and DMT1 transcript levels, a signature of iron-limited cells actively trying to acquire more iron [50]. Under conditions where lysosomal export remains blocked, this compensatory response perversely increases iron trafficking into the lysosome, via TfR-mediated endocytosis and autophagic ferritin turnover, even as the cytosol remains starved and its ferritin buffering capacity is depleted. The lysosome, in other words, becomes progressively iron-loaded precisely because the cell is trying to correct a deficiency it cannot resolve through this route.

Weber et al. (2020) independently support the centrality of this axis: genetic screens showed that when lysosomal acidification is disrupted, cells become critically dependent on iron uptake and cholesterol synthesis for growth, and iron supplementation alone was sufficient to restore proliferation [54]. This finding indicates that iron acquisition, not general proteolysis, is the key limiting output of an acidic lysosome, and that lysosomal dysfunction rapidly depletes mitochondrial iron, reducing respiratory chain components and ATP output [53,55].

Organellar pH and iron homeostasis are thus tightly interwoven, and mitochondrial metabolism reciprocally supports lysosomal acidification via ATP generation. Several neurodegeneration-associated genes intersect this network: TMEM175 (a Parkinson's risk gene) encodes the lysosomal proton channel discussed above, and PD-linked variants reduce H⁺ conductance, causing lysosomal hyperacidification [35]. Brain iron dyshomeostasis is common in neurodegenerative aging (Parkinson's, Alzheimer's, and rare Neurodegeneration with Brain Iron Accumulation, NBIA, disorders) and frequently correlates with lysosomal-autophagy dysfunction [42]. In sum, acidic lysosomes and functional mitochondria form a relay for iron; disrupting this relay produces a dual lesion, a starved cytosol and an increasingly iron-loaded, redox-active lysosome, that sets the stage for the acute event described in Section 4.3.

4.3. From chronic sequestration to acute release: how a starved cytosol becomes a ferroptotic one

Section 4.2 establishes that chronic lysosomal alkalinization produces cytosolic iron starvation while, counter-intuitively, increasing the redox-active iron burden within the lysosomal lumen itself, since export is blocked but reduction and iron delivery continue at reduced but non-zero rates. The iron pool that ultimately drives ferroptosis is predominantly Fe²⁺, generated and retained inside the lysosome during the chronic phase, not a pool of inert lumenal Fe³⁺ that somehow converts en route to the cytosol.

The pathological sequestration and accumulation of non-degraded, iron-laden material within the lysosomal lumen sets the stage for the primary mechanism of neurodegenerative cell death called ferroptosis. Ferroptosis is an iron-dependent form of regulated cell death driven by lethal lipid peroxidation and is increasingly implicated in neurodegeneration [56]. Aging and senescent cells accumulate lipofuscin-rich lysosomes with diminished acidity [57]. Within such lysosomes, the trapped Fe²⁺/Fe³⁺ pool described in Section 4.2 is not chemically inert: retained Fe²⁺ continues to catalyze intraluminal Fenton chemistry with locally generated H₂O₂, driving lipid peroxidation of the lysosomal membrane itself and progressive lipofuscin formation [58,59]. This constitutes a slow, self-amplifying feedback loop, iron retention promotes membrane oxidation, which further sensitizes the lysosome to rupture, that gradually erodes the integrity of the limiting membrane [59,60].

The transition from chronic sequestration to acute crisis occurs when this cumulative oxidative damage reaches a threshold sufficient to cause Lysosomal Membrane Permeabilization (LMP) as illustrated in Fig. 3. Direct visualization of this process shows that intralysosomal lipid peroxidation precedes and triggers LMP, leading to subsequent bulk release of lysosomal iron, rather than LMP being required for the initiation of lipid peroxidation [58,60]. At the moment of rupture, the lumenal pool released is a mixture, but is functionally dominated by Fe²⁺: any co-released Fe³⁺ layers are rapidly reduced by the strongly reducing cytosolic milieu (glutathione, ascorbate, NADPH-dependent reductases), so the redox state that drives Fenton chemistry and lipid peroxidation is ferrous regardless of the exact lumenal speciation at the instant of release. Alkalinization therefore does not force iron to remain as Fe³⁺; it selectively blocks export of the Fe²⁺ that continues to be generated, allowing a redox-active ferrous pool to accumulate in situ until it is released catastrophically. The severity of the resulting insult is compounded by the chronic phase itself: because the starvation response described in Section 4.2 has already suppressed cytosolic ferritin and depleted the cell's normal iron-buffering capacity, the cytosol that receives the LMP bolus is poorly equipped to sequester it. The released Fe²⁺ therefore drives an unbuffered surge in the labile iron pool that catalyzes the Fenton reaction with membrane hydroperoxides (Fig. 3), initiating a chain reaction of lipid peroxidation [56].

Fig. 3.

Proposed mechanistic model illustrating how TLDc–V-ATPase dysfunction establishes a pH–iron axis governing two divergent lysosomal outcomes: chronic cytosolic iron deficiency versus acute iron-driven ferroptosis. TLDc domain-containing proteins (NCOA7, OXR1, TBC1D24, mEAK7/TLDC1, TLDC2) physically interact with and regulate the V-ATPase proton pump; loss of this regulation impairs V-ATPase assembly/activity and causes lysosomal deacidification (increased luminal pH). This dysfunction bifurcates into two pathways depending on lysosomal membrane integrity. Pathway 1 (intact lysosomal membrane, chronic cytosolic iron deficiency): (1) Transferrin-bound Fe³⁺ (Tf–Fe³⁺) enters the lysosome via endocytosis or ferritinophagy; (2) the alkalinized lysosomal lumen prevents Fe³⁺ release from its bound state; (3) loss of the acidic environment blocks DMT1 (SLC11A2)- and TRPML1 (MCOLN1)-mediated Fe²⁺ efflux, trapping iron within the lysosome. Downstream consequences include reduced cytosolic and mitochondrial Fe²⁺ availability (iron starvation), HIF-1α stabilization, impaired mitochondrial electron transport chain (ETC) activity and ATP production, and neuroinflammation, culminating in Outcome 1: chronic cytosolic iron deficiency. Pathway 2 (lysosomal membrane permeabilization, LMP, acute iron release and ferroptosis): prolonged lysosomal stress (excess iron/lipofuscin accumulation, oxidative stress, membrane damage) triggers (4) rupture of the lysosomal membrane, (5) release of accumulated Fe³⁺ and Fe²⁺ into the cytosol, with co-released Fe³⁺ being rapidly converted to Fe²⁺ within the highly reducing cytosolic environment through the concerted action of glutathione, ascorbate, and NADPH-dependent reductases (6) iron-catalyzed lipid peroxidation via the Fenton reaction (Fe²⁺ + H₂O₂ → ·OH + OH⁻ + Fe³⁺). Downstream consequences include a rapid expansion of the labile iron pool (LIP), peroxidation of cellular membranes, organelle damage, and cell death, culminating in Outcome 2: acute iron overload and ferroptosis. Symbols and transporters are defined in the legend panel. ATP, adenosine triphosphate; DMT1, divalent metal transporter 1; ETC, electron transport chain; HIF-1α, hypoxia-inducible factor 1-alpha; LIP, labile iron pool; LMP, lysosomal membrane permeabilization; TfR, transferrin receptor; TRPML1, transient receptor potential mucolipin 1; V-ATPase, vacuolar H⁺-ATPase. Created using BioRender.com.

Loo et al. (2025) found that senescent cells with defective lysosomal acidification can resist ferroptosis induced by cystine deprivation, because insufficient lumenal acidity impairs lipid peroxidation within the lysosome and thus delays or prevents the LMP-triggering step [52]. Conversely, recent experimental evidence substantiates a mechanistic link between lysosomal lipid peroxidation, LMP, and ferroptosis. Saimoto et al. demonstrated that intralysosomal lipid peroxidation promotes LMP and subsequent iron leakage, which amplifies cellular lipid peroxidation and ultimately precipitates ferroptotic cell death [60]. Together, these findings suggest that lysosomal lipid damage and membrane destabilization can establish a positive-feedback loop in which iron redistribution further propagates lipid peroxidation. Importantly, however, LMP should not be considered an obligatory intermediate linking lysosomal alkalinization to ferroptosis. The context dependence of lysosomal iron handling is further exemplified by studies of BDH2. In a melanoma model, BDH2-dependent lysosome-to-mitochondria iron flux influences cellular fate, with BDH2 loss promoting lysosomal iron retention and deacidification and thereby increasing susceptibility to lipid peroxidation and ferroptosis [42]. The BDH2 example is presented here as a mechanistically analogous, convergent pathway showing how V-ATPase-dependent lysosomal acidification governs inter-organellar iron flux more broadly, rather than as an established downstream effector of TLDc–V-ATPase signaling. However, a direct molecular link between TLDc proteins and BDH2 has not yet been reported and remains an open question for future study. Collectively, these studies indicate that whether chronic pH dysfunction resolves as protracted iron sequestration, with relative ferroptosis resistance, or progresses to LMP-driven ferroptosis is not a fixed outcome, but depends on the degree and duration of alkalinization, the local ROS/lipofuscin burden, and the cell's residual antioxidant reserve (GSH/GPX4 capacity). Accordingly, because direct evidence linking individual TLDc proteins to LMP-dependent ferroptosis remains limited, the proposed TLDc–V-ATPase–lysosomal iron–ferroptosis axis should be considered a mechanistic hypothesis requiring experimental validation rather than an established linear pathway. This context dependence may explain why a common upstream defect in TLDc/V-ATPase function can produce divergent outcomes across cell types and disease states. Within this framework, TLDc dysfunction may initially impair lysosomal iron mobilization and contribute to chronic cytosolic iron deficiency, while, in cells that cross the LMP threshold, the same iron pool may become a source of redox-active iron that drives acute lipid peroxidation and ferroptotic cell death.

4.4. Iron deficiency drives mitochondrial pathology and inflammation

The functional consequences of the compartmentalized iron starvation are severe and directly explain many symptoms associated with TLDc deficiencies and aging. Cytosolic iron deficiency rapidly impairs mitochondrial function, as the production of critical iron-sulfur clusters and heme components necessary for the respiratory chain is disrupted [48]. In mouse models of impaired lysosomal acidification, mitochondrial malfunction manifests as instability of mitochondrial DNA (mtDNA) and general energetic deficit [61].

Furthermore, the state of cellular iron deficiency is sufficient to trigger a pervasive and chronic inflammatory signaling signature in cultured primary neurons and in mouse brain tissue [62]. This chronic neuroinflammation, coupled with mitochondrial failure, links TLDc-V-ATPase dysfunction directly to the core mechanisms of sporadic neurodegenerative diseases, where inflammation and oxidative stress are common features [5,[63], [64], [65]] The conclusion is that the general neuroprotective function initially ascribed to TLDc proteins fundamentally derives from their role in regulating organellar homeostasis necessary for iron partitioning, thereby preventing the H⁺-iron-mitochondrial pathogenic cascade [10,66].

4.5. Resolving the iron paradox in neurodegeneration

A central paradox in neurodegeneration research lies in the contrasting spatial profiles of iron dysregulation. While major brain pathologies including AD, PD, and frontotemporal dementia (FTD) are characterized by regional lysosomal-autophagic impairment and pathological iron accumulation [[67], [68], [69], [70], [71]], defects in TLDc domain-containing proteins paradoxically drive cytosolic iron deficiency. The TLDc-V-ATPase axis provides a mechanistic resolution to this apparent contradiction by reconciling altered organellar trafficking with systemic intracellular iron allocation [48,72,73]. The localized iron accumulation observed in neurodegenerative tissues is likely sequestrated, undegraded iron trapped within the dysfunctional, alkalized lysosomes [56,72]. This is a failure of compartmentalization, not simple overload. The actively metabolizing compartments of the cell, the cytosol and mitochondria are simultaneously experiencing critical iron starvation required for respiratory chain activity. This reinterpretation fundamentally alters the therapeutic strategy, suggesting that indiscriminate iron chelation, which reduces total iron, may exacerbate the deficiency, whereas targeted strategies to restore lysosomal iron efflux or increase cytosolic iron availability are necessary [74].

5. TLDc dysfunction in age-related neurodegeneration and inherited disorders

5.1. TBC1D24 related neurodevelopmental epilepsies

Mutations in TBC1D24 are responsible for a spectrum of severe, overlapping neurological conditions, including developmental epileptic encephalopathies, movement disorders, and DOORS syndrome (Deafness, Onychodystrophy, Osteodystrophy, Renal dysfunction, and Seizures) [[27], [28], [29]]. These disorders are characterized by a loss-of-function in TBC1D24, leading to the functional collapse of the V-ATPase assembly, as confirmed by the detection of V1 subunit mis-localization and increased intra-organelle pH [6].

The pathogenesis of these TBC1D24 disorders is directly linked to the consequences of pH dysregulation, particularly the failure of synaptic vesicle (SV) reacidification. Since SV reacidification is essential for neurotransmitter packaging, its impairment leads to the synaptic defects, decreased SV density, and failed neurotransmission that underpin the severe, early-onset seizures observed in patients [6]. The fact that mutations in V-ATPase subunits (such as ATP6V0C) result in strikingly similar clinical syndromes reinforces the conclusion that the loss of pH homeostasis is the common pivotal mechanism driving disease pathogenesis in this class of disorders [6,75].

5.2. OXR1 in ALS and age-related cerebellar ataxia

OXR1 deficiency also leads to neurodegeneration, causing pediatric seizures and cerebellar neurodegeneration in patients and mouse models. This outcome highlights a critical distinction in functional vulnerability based on TLDc paralog localization [13]. While TBC1D24 function is critical for high-turnover processes like SV recycling [28], OXR1's broader role is maintaining systemic organelle equilibrium and providing generalized protection against oxidative stress [6,8,13,76]. OXR1 mutations demonstrate the consequences of disrupted pH/oxidative regulation. Wang et al. (2019) reported that human recessive loss of OXR1 causes a novel cerebellar atrophy syndrome with lysosomal storage phenotypes [11]. This parallels the Drosophila phenotype: mustard null flies (lacking the OXR1 homologue) accumulate aberrant lysosomes and die, but can be rescued by expressing only the TLDc domain of human OXR1 or NCOA7 [11]. Similarly, Wilson et al. (2024) found that OXR1 expression declines with age and that OXR1 is required to maintain the retromer complex, which supports endolysosomal trafficking [43]. Loss of OXR1 in fly or patient cells destabilized retromer, caused endolysosomal defects, and led to neurodegeneration (visual decline, tau pathology), all of which could be rescued by bolstering retromer or restoring OXR1 [43]. Thus, OXR1 deficiency impairs lysosomal/autophagic pathways, linking it to aging and neurodegeneration. Other TLDc proteins also play roles: NCOA7 is upregulated by interferon and has been implicated in innate immunity and endosome function [5,77].

The observed neuroprotective effect of OXR1 overexpression in models of ALS must be interpreted within the V-ATPase regulatory framework [23,78]. Rather than acting as a traditional antioxidant, OXR1's protection may stem from its ability to regulate the dynamic assembly/disassembly of V-ATPase under metabolic stress, thereby preventing wholesale organelle dysfunction that would otherwise activate the H⁺-iron-mitochondrial cascade [19,22,72,73]. The functional outcome of TLDc dysfunction appears to be differentiated: TBC1D24 causes acute, developmental catastrophic failure due to synaptic defects [24], while OXR1 regulates long-term resistance to aging and acquired cellular stresses [43], and NCOA7 has been implicated in innate immunity and endosome function [77].

5.3. Implications for neurodegenerative diseases such as AD and PD

Lysosomal dysfunction, manifesting as generalized lysosomal alkalization and impaired autophagic clearance, is a consistent feature of cellular senescence and age-related neurodegenerative diseases, including PD and AD [[79], [80], [81]].

The TLDc-V-ATPase axis provides a strong mechanistic link that unifies lysosomal pathology across these sporadic disorders. Dysfunctional TLDc activity, potentially reduced by aging, oxidative damage, or PTM alterations, could lead to generalized lysosomal alkalization [82,83]. This failure to maintain acidic pH [44] then impairs the degradation of pathological proteins such as α-synuclein, β-amyloid, and Tau in both AD and PD, while simultaneously triggering the devastating downstream consequences of mitochondrial iron starvation and chronic inflammatory signaling [81,84,85]. Consequently, TLDc dysfunction emerges as a plausible early trigger that destabilizes neuronal homeostasis, initiating a cascade of events such as autophagy failure, iron dysregulation, and mitochondrial impairment-that define age-related neurodegeneration [5,64,[86], [87], [88], [89]].

5.4. The kinetic imperative of organellar pH and the crisis of neurodegeneration

Cellular survival hinges on the stringent regulation of proton concentration [H⁺] across various organelle membranes. Within both the lysosome and the mitochondrion, the H⁺ gradient dictates functional activity, electrochemical energy storage, and macromolecular conformation [35]. The lysosome, functioning as the cell's primary recycling center, requires an intensely acidic environment (typically pH 4.5–5.0) for the optimal activity of hydrolytic enzymes, such as Cathepsins. This acidification is predominantly driven by the V-type H⁺-translocating ATPase (V-ATPase), which actively pumps protons into the lumen [35]. Conversely, mitochondria rely on maintaining a steep electrochemical gradient across the inner membrane, the proton-motive force, to drive ATP synthesis. In essence, H⁺ homeostasis represents the foundational energetic and degradative capacity of the eukaryotic cell.

A central feature of age-related pathology is the disruption of this tightly regulated H⁺ homeostasis. Perturbation of lysosomal pH, whether through excessive alkalinization (hypo-acidification) or excessive acidification (hyper-acidification), compromises lysosomal proteolysis, cargo degradation, and efficient cellular waste recycling. In parallel, mitochondrial dysfunction, characterized by impaired membrane potential and increased oxidative stress, compromises bioenergetic capacity and cellular energy production [44]. The convergence of defective lysosomal processing and mitochondrial bioenergetics represents a fundamental feature of cellular aging and neurodegenerative diseases [90] as illustrated in Fig. 4.

Fig. 4.

The pH-iron axis: a proposed sequential lysosome-to-mitochondria relay linking TLDc domain dysfunction to neurodegeneration. Schematic representation of the proposed mechanistic cascade linking loss-of-function mutations in TLDc domain-containing proteins (TBC1D24, OXR1, NCOA7) to progressive neurodegeneration through sequential organellar dysfunction. In the lysosomal compartment, TLDc domain dysfunction impairs V-ATPase holoenzyme assembly, resulting in failure of lysosomal acidification (increased luminal pH). This, in turn, stalls ferritinophagy and promotes lysosomal iron accumulation. Disrupted iron hand-off at lysosome-mitochondria membrane contact sites (pH-iron axis relay) propagates dysfunction to the mitochondrial compartment, where mitochondrial iron starvation impairs Fe-S cluster biogenesis and electron transport chain (ETC) function. Concurrent OXR1 deficiency reduces mitochondrial antioxidant defense, promoting reactive oxygen species (ROS) accumulation, oxidative stress, and bioenergetic failure (impaired oxidative phosphorylation, reduced ATP output). A reinforcing feedback loop is proposed whereby ROS-induced lysosomal membrane permeabilization (LMP) further impairs V-ATPase function, sustaining and amplifying the cycle. This cascade culminates in progressive neurodegeneration, with clinical correlates including Parkinson's disease, Alzheimer's disease, dementia, amyotrophic lateral sclerosis (ALS), and DOORS syndrome. ETC, electron transport chain; Fe-S, iron-sulfur; LMP, lysosomal membrane permeabilization; ROS, reactive oxygen species. Created using BioRender.com.

6. Conclusion and therapeutic perspective

The TLDc family of proteins has emerged as an indispensable, evolutionarily conserved regulatory hub governing V-ATPase assembly dynamics and organellar pH homeostasis. Recent studies reveal that TLDc-domain proteins integrate organelle acidification with iron metabolism to sustain cellular health. Specifically, OXR1 and NCOA7 directly bind to and modulate V-ATPase activity to maintain optimal pH in lysosomes, the Golgi, and related compartments, whereas TBC1D24 stabilizes V-ATPase activity in neuronal lysosomes and synapses, thereby preserving organellar integrity [13,21,23]. This pH regulation is essential for nutrient recycling and for the release of iron from lysosomes, which is required to support mitochondrial bioenergetics [42,54]. When lysosomal acidification fails, iron becomes trapped within lysosomes, leading to mitochondrial dysfunction, pseudohypoxia, and inflammatory signaling [53,55]. In neurons, age-related decline in TLDc function or lysosomal acidity promotes waste accumulation, redox imbalance, and activation of ferroptotic pathways, contributing to neurodegenerative disorders such as AD and PD [42,44,91].

Notably, accumulating evidence suggests that lysosomal pH dysregulation is an early pathogenic event that precedes classical protein aggregation (including β-amyloid, Tau, and α-synuclein) in neurodegenerative disease. Lo et al. (2023) highlight genetic risk factors, including V-ATPase subunits and endosomal Na⁺/H⁺ exchangers that converge on lysosomal de-acidification in AD and PD, underscoring lysosomal pH as a potential prognostic marker and therapeutic target [44]. Given these insights, strategies that restore organellar pH and iron handling represent promising therapeutic avenues, and translational progress is now moving in several distinct but complementary directions.

Among small-molecule and biophysical approaches, lysosome-targeted acidic nanoparticles, including Poly d,l-lactide-co-glycolide (PLGA)-based formulations, have been shown to directly lower luminal pH and restore autophagic-lysosomal flux in presenilin-1-deficient and α-synuclein-overexpressing neurodegeneration models, offering a physicochemical route to re-acidification that bypasses defective V-ATPase assembly itself [44,82]. In Parkinson's disease models, acidic nanoparticles improved lysosomal function and protected dopaminergic neurons from neurodegenerative insults [92]. More recently, lysosome-acidifying nanoparticles based on a biodegradable poly (ethylene tetrafluorosuccinate-co-succinate) copolymer restored lysosomal acidification, enhanced autophagic clearance of α-synuclein, improved mitochondrial function, and reduced neuronal toxicity in cellular and Drosophila models of A30P α-synucleinopathy [93]. These findings provide proof-of-concept that direct correction of the lysosomal pH defect can influence both lysosomal proteostasis and downstream mitochondrial homeostasis. Nevertheless, the translational development of such nanotherapeutics will require optimization of brain delivery, cellular targeting, pharmacokinetics, long-term safety, and avoidance of excessive lysosomal acidification.

A complementary strategy is to enhance endogenous lysosomal signaling and trafficking rather than directly supplying acidity. In this regard, transient receptor potential mucolipin 1 (TRPML1; MCOLN1) represents a promising pharmacological target. Pharmacological activation of the endolysosomal calcium channel TRPML1 has emerged as a genetically and mechanistically well-supported strategy. The synthetic TRPML1 agonist ML-SA1 reverses APOE ε4-and PIKfyve inhibition-associated endosomal-autophagic-lysosomal defects in models of late-onset AD [94], and this approach has now advanced to brain-penetrant clinical candidates (e.g., CSM-101, LW-1017) targeting Parkinson's disease and related lysosomal storage-linked neurodegeneration. Thus, TRPML1 agonism should be viewed as a lysosomal functional-restoration strategy that can improve the efficiency of the autophagy–lysosome pathway and the generation of functionally competent acidic autolysosomes. Because TLDc proteins regulate V-ATPase-dependent organellar homeostasis, it will be important to determine experimentally whether TRPML1 activation can compensate for, or synergize with, impaired TLDc–V-ATPase signaling in TLDc-deficient neurons.

Beyond direct pharmacological acidification, metabolic and nutritional interventions are increasingly recognized as complementary levers on the lysosome-mitochondria axis. Caloric restriction, which modulates OXR1 levels, delays retromer decline and brain aging in dietary-restriction models [43]. More specifically to TLDc biology, recent work by Zhai et al. in Caenorhabditis elegans demonstrates that loss of the TLDc-domain protein LMD-3 (the invertebrate ortholog of OXR1) disrupts V-ATPase-dependent lysosomal acidification and elevates oxidative stress; strikingly, supplementation with methylcobalamin (meCbl), the active form of vitamin B12, fully rescues lysosomal acidity and mitochondrial membrane potential in this model, acting in part by reprogramming propionate-shunt metabolism (via acdh-1/hphd-1) [16]. The mechanistic significance of this observation is particularly relevant to the lysosome–mitochondria axis. Vitamin B12 is required for key metabolic reactions involved in propionate utilization, and disruption of B12-dependent metabolism can promote accumulation of potentially deleterious metabolic intermediates and compromise mitochondrial function. While this mechanistic link has so far been established in an invertebrate TLDc model rather than mammalian neurons, it identifies vitamin B12 status and propionate/one-carbon metabolism as a plausible, readily testable nutritional axis for rescuing TLDc-associated lysosomal dysfunction, complementing iron chelation and other metabolic interventions already implicated in this pathway [43].

In summary, the TLDc domain serves as a molecular nexus linking organellar pH regulation with oxidative and iron stress management. This conceptual redefinition of TLDc biology argues for a shift from nonspecific antioxidant or chelation strategies to precision approaches that target organellar pH control and V-ATPase regulation, spanning direct pharmacological re-acidification (acidic nanoparticles, TRPML1 agonists), genetic/structural stabilization of V-ATPase assembly, and metabolic reprogramming (caloric restriction, vitamin B12/propionate metabolism). Future therapeutic efforts should focus on restoring compartment-specific pH gradients, correcting lysosomal iron efflux defects, and testing whether these convergent strategies act synergistically when TLDc function itself is compromised. Viewing lysosomal pH, iron homeostasis, and TLDc function as an integrated network will be essential for developing biomarkers and interventions to preserve neuronal health and combat age-related neurodegenerative disease.

CRediT authorship contribution statement

Mohammad Sajid Ghufran: Writing – review & editing, Writing – original draft, Visualization, Validation, Methodology, Investigation, Formal analysis, Data curation, Conceptualization. Priyanka Soni: Writing – review & editing, Writing – original draft, Visualization, Validation, Methodology, Investigation, Formal analysis, Data curation, Conceptualization. Bobby Thomas: Writing – review & editing, Writing – original draft, Visualization, Validation, Supervision, Resources, Project administration, Methodology, Investigation, Funding acquisition, Formal analysis, Data curation, Conceptualization.

Declaration of competing interests

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Abbreviations

AD:

Alzheimer's disease

ALS:

Amyotrophic lateral sclerosis

CNS:

Central Nervous System

DMT1:

Divalent Metal Transporter 1

DOORS:

Deafness, Onychodystrophy, Osteodystrophy, Renal dysfunction, and Seizures

ERMCS:

Endoplasmic reticulum-mitochondria contact sites

FTD:

Frontotemporal Dementia

LIP:

Labile Iron Pool

LLPS:

Liquid-liquid phase separation

LMP:

Lysosomal Membrane Permeabilization

LYPAS:

Lysosomal Processing and Adaptation System

LysM:

Lysine motif

meCbl:

methylcobalamin

mtDNA:

mitochondrial DNA

MCOLN1:

Mucolipin TRP cation channel 1

NCOA7:

Nuclear Receptor Coactivator 7

OXR1:

Oxidation Resistance 1

PD:

Parkinson's disease

PLGA:

Poly(d,l-lactide-co-glycolide

PTMs:

Post-translational modifications

ROS:

Reactive Oxygen Species

SLC11A2:

Solute carrier family 11 member 2

TBC1D24:

TBC1 Domain Family Member 24

TGN:

Trans-Golgi Network

TLDc:

Tre2/Bub2/Cdc16 (TBC), Lysin Motif (LysM), and Domain catalytic

TRPML1:

Transient Receptor Potential Mucolipin 1

V-ATPases:

Vacuolar ATPases

Data availability

No data was used for the research described in the article.