Epigenetic Modulation Is a Key Mechanism in Cognitive Deficit: Unveiling Garcinol as a Potent HAT Inhibitor in Improving Alzheimer’s Disease

Abstract

Alzheimer’s disease (AD) is a global concern. Conventional FDA-approved drugs mostly provide symptomatic relief but largely fail to address the underlying pathophysiology of AD. Further, chronic use of prescribed anti-AD drugs causes unpredictable, countless side effects, which thus evokes exploration for novel, safer, nontoxic, affordable, yet potent alternative modalities. Among various predicted pathophysiological hall marks in the progression of AD, aberrant histone acetylation has been identified as one of the major culprits, which leads to dysregulated gene expression and gradual neuronal degeneration. Thus, histone acetyl transferase (HAT) inhibitors are considered lucrative anti-AD agents. Active phyto constituents in recent times have occupied the center stage in addressing challenging neurodegenerative disorders in place of routine drug therapy. Garcinol (GL), a phytoactive constituent from Garcinia indica, possesses potent HAT inhibitory effect. Reports show that GL exhibits profound antioxidant, anti-inflammatory, anticancer, and neuroprotection effects. Neuroprotection of GL could be attributed to its ability to reduce oxidative stress and modulate important signaling pathways involved in neuronal degeneration. Present review unveils potentiality of HAT inhibitors in improving memory functions and neuroprotection; with a special focus on GL. Mechanical insights into activity of GL in ameliorating neurodegeneration have been discussed with rationalized evidence of various preclinical reports. Side by side, a portion has been devoted to update the trending drug delivery approaches investigated in recent times for GL toward improved pharmacological performance.

Introduction

Alzheimer’s disease (AD), a chronic neurological disorder is the most common and serious cause of dementia in older adults. Senile plaques, neurofibrillary tangles, reduced cholinergic transmission, gradual cognitive decline, psycho behavioral problems, with gradual decline of memory have been identified as major hallmarks of AD. According to the World Health Organization, there are around 57 million instances of AD worldwide, with nearly 10 million new cases occurring annually. Though the exact cause of the AD is yet to be unveiled, key factors that have been linked to the development of AD include elevated levels of beta (β)-amyloid (Aβ), metal ion deregulation, hyperphosphorylated tau protein, oxidative stress, inflammatory processes, failure of the cell cycle regulatory mechanism, and mitochondrial dysfunction. , Further, alteration in acetylcholine leads to dysregulation in histone modulations and subsequent cholinergic neuron degeneration in the brain, which has been one of the recognized neuropathological hallmark of AD. , Thus, therapeutic moieties targeting histone acetyl transferase (HAT) can be potential anti-AD agents.

In view of poor pathophysiology of AD and multitude biological factors, design of any specific medication has been a challenging task. At present, there is no established treatment regimen for AD to address its uncontrolled global incidence, necessitating exploration of novel therapeutic agents and unique delivery strategies. It is a well-known fact that discovery/development of new drug is extremely laborious, time-consuming, and expensive. Hence, recent scientific focus has been shifted toward repurposing of approved drugs or application of innovative techniques to ensure improved clinical outcomes. , As of now, the USFDA has only approved a handful number of drugs for treatment of AD, including donepezil, galantamine, lecanemab, aducanumab, etc. , Majority of these drugs in fact target antagonists of an N-methyl-d-aspartic acid (NMDA) receptor or Aβ or acetylcholinesterase inhibitors, which mostly bring symptomatic relief rather than targeting the root mechanism of the disease. , Additionally, the majority of marketed drugs often result in adverse effects such as hepatotoxicity, diarrhea, or insomnia on chronic use. Since AD is a chronic disorder, any patient once diagnosed with AD has used the prescribed medication lifelong, which in turn leads to moderate-to-severe bizarre and unpredictable symptoms. To address these issues associated with routine drug therapy and to improve the quality of life in AD patients, potent, compatible, affordable therapeutic agents with limited side effects are the need of the hour.

Over the recent years, phytoactive components derived from various medicinal plants have been a trending buzz among formulation scientists and in medical fraternity, thanks to their numerous therapeutic activities along with biocompatible, biodegradable, and nontoxic properties. Array of phytocomponents (essential oils/plant tissue extracts) have evidence of potent antimicrobial, anti-inflammatory, and antioxidant properties as documented in several in vitro/in vivo studies. − Isolated active phytoconstituents like curcumin, ginkgolides, epigallocatechin-3-gallate, silymarin, baicalein, lycopene, and many more have been extensively researched over the years for various neurological diseases, including AD. , Unlike modern medicines, which are usually expensive with a limited safety window, active phytoconstituents offer potentially advantageous alternatives to address chronic brain disorders. At present, active phytoconstituents of the Clusiaceae family have also been gaining interest as potential therapeutic options owing to their diverse medicinal benefits and nontoxic nature. Many phytoactive constituents derived from this family possess efficient anti-inflammatory, antiamyloidogenic, anticholinesterase, and antioxidant activities , and thus are being largely investigated for several brain disorders.

Garcinol (GL) is one such trending active constituent from various Garcinia species; mostly Garcinia indica (Family: Clusiaceae), which has been instigating interest among researchers in recent times in view of its countless therapeutic properties. Chemically, GL is a polyisoprenylated benzophenone (molecular weight 602.812 g/mol), possessing strong anti-inflammatory and antioxidant properties. Several studies have demonstrated its capacity to scavenge free radicals and to stop the inflammatory cascade. − Investigations on the pharmacological impact of GL on neurological diseases are a trending buzz in the drug development arena. GL can rescue brain precursor cells, encourage their fast development, and successfully restore the equilibrium among the neurotransmitters glutamate and γ-aminobutyric acid (GABA). Moreover, it suppresses HAT effectively. Both histone acetylase and deacetylase inhibitors have been used to target accelerated neurodegeneration in AD brain caused by the disruption in histone dysregulation status. Through chromatin tightness, histone hypoacetylation in AD decreases the production of neuroprotective genes. Memory loss occurs when synaptic plasticity genes like the brain-derived neurotrophic factor are suppressed by decreased HAT activity and histone deacetylase (HDAC) activity. Through boosting oxidative stress, encouraging tau hyperphosphorylation, and hindering memory formation, neurodegeneration progresses. GL, as a HAT inhibitor in this context, plays a crucial role in suppressing the tau protein. Additionally, it has been found to inhibit cyclin-dependent kinase-5 (CDK5) and glycogen synthase kinase-3-β (GSK3β), the main mediators in between tau hyperphosphorylation and amyloid precursor protein (APP) processing. Additionally, GL suppresses the signaling pathway of nuclear factor kappa B (NF-κB), another crucial modulator of neuroinflammation. At the present time, within the array of naturally derived HAT inhibitors, GL has garnered significant interest owing to its robust epigenetic modulatory capacity and extensive pharmacological profile. Distinct from other natural HAT inhibitors, GL displays a unique blend of potent HAT inhibition and the capacity to influence multiple pathological hallmarks of AD, such as oxidative stress, neuroinflammation, and Aβ aggregation. Its well-defined chemical architecture, coupled with evidence of modulation across diverse signaling pathways, further underscores its potential as a therapeutic agent. Application of GL rebalances neurotransmitters, rescues brain precursor cells, and accelerates their regeneration. Thus, in light of these attributes and the growing body of preclinical data substantiating its neuroprotective effects, GL has been chosen as the model therapeutic entity to deliver a comprehensive mechanistic exploration of its role in epigenetic regulation and its prospective utility in AD management.

Although several reviews have addressed epigenetic regulation in AD, the present Perspective provides a distinct perspective by specifically focusing on GL as a potential epigenetic modulator and its emerging relevance in neurodegenerative disorders. Unlike previous reviews that broadly discuss epigenetic mechanisms, this article integrates evidence from in silico, in vitro, and in vivo studies to summarize the multitarget therapeutic actions of GL, including histone acetyltransferase inhibition, antioxidant and anti-inflammatory effects, and neuroprotective mechanisms. No such review on the HAT inhibition potential of GL and its mechanistic insights modulating AD pathology is available so far. In addition, this review highlights recent advances in nanocarrier-based delivery systems that may improve the bioavailability and brain-targeting potential of GL. By combining mechanistic insights with translational perspectives, the present review aims to provide a comprehensive update on preclinical research evidence including both in vivo and in vitro studies, as well as translational perspectives on potential application of GL in AD.

HAT and Its Family: Role in Memory Function

HATs are considered the epigenome’s workhorses, participating in a variety of activities such as gene silencing, transcription activation, repair of DNA, and the progression of the cell cycle. They catalyze the transfer of acetyl groups from acetyl-CoA to ε-amino groups of particular lysine residues. According to estimates, there are 15,000 acetylation sites in mammalian tissues and 2000–4000 acetylated proteins on both nonhistone and histone substrates. About 30 HATs have been identified in humans, which have been further divided into two major groups based on their subcellular locations. Dysregulations of histones in chromatin are caused by type A HATs, which are found in the nucleus. In contrast to that, type B HATs are found in the cytoplasm, which helps to repair DNA double-strand breaks and to modify freshly translated histones. Further, based on domain structures and sequence similarity, HATs can be subdivided into different groups, viz., transcription factor-related HATs, nuclear receptor coactivators (NCOAs), p300/cAMP-response element-binding proteins (CBPs), etc. Camello proteins are a new set of HATs that have been discovered recently in a zebrafish model, which are essential for development of perinuclear localization, and acetylate H4. An in-depth description of HAT families and their functions is depicted in Table .

1. Various Histone Acetyl Transferase Families Along with Their Role in Targeting Histone/Non-Histone Proteins towards the Development of AD .

family	representative protein	amino acid length	histone targets	non-histone targets	function	references
MYST family	MOZ/KAT6A	2004	H3K14	p53, NF-κB	acetylates histones and plays a key role in hematopoiesis and normal development
	TIP60/KAT5	513	H4K5, K8, K12, K16	p53	regulation of diverse cellular activities, including chromatin remodeling, gene transcription, DNA damage responses, and tumorigenesis
	MORF/KAT6B	2073	H3K14	--	acetylating H3K9, having a vital function in controlling gene expression, hematopoiesis, and brain development
	MOF/KAT8	457	H4K16, H4	p53, Nrf2, TIP5, MSL3	plays a crucial role in DNA damage repair, embryonic stem cell development, and gene expression regulation
	ESA1	445	H4, H3, H2A, H2B	--	the catalytic subunit of the NuA4 HAT complex is responsible for acetylating histone H4
	HBO1/KAT7	611	H4k5, K12, K8, H4, H3	ORC2, MCM2, CDC6, and geminin	acetylating histone proteins H3 and H4, thereby regulating gene transcription by altering chromatin structure
	SAS2	338	H4K16, H3, H2A	--	catalytic subunit of a HAT complex responsible for acetylating H4K16, regulates gene silencing by preventing silencing proteins from at euchromatin–heterochromatin boundaries
	Ybf2/SAS3	831	H3, K9, K14	--	regulate gene expression
p300/CBP family	p300	2413	H3, H3K27, H3K18	MyoD, Rb, p53,NF-κB, E2F1, HMG proteins, adenovirusE1A	key transcriptional coactivator of HAT, regulate a plethora of fundamental biological processes including cell growth, development, oncogenesis, and apoptosis
	CBP	2441	H3, H4, H3K27	MyoD, p53, HMG proteins, NF-κB	coactivators in transcription, acetylate histones, and transcription factors
NCOA family	NCOA1	1440	H3	--	assists nuclear receptors in the upregulation of DNA expression as a coactivator
	NCOA2	1463	H3, H4	--	NCOA2 encodes a nuclear receptor coactivator, which aids in the function of nuclear hormone receptors
	NCOA3	1423	--	--	NCOA3 interacts with nuclear receptors to enhance gene transcription
transcription-related HATs	TAF1	1872	H2B, H3, H4	--	mediate triggered transcription by giving different activators places to interact
	GTF3C1/TFIIIC220	2109	--	--	involved in RNA polymerase III transcription complex assembly
	GTF3C4/TFIIIC90	822	H3, H4	DDX31, MED22, TTF1	RNA polymerase III transcription and gene expression regulation
	GTF3C2/TFIIIC110	911	--	--	involved in RNA polymerase III transcription complex assembly
GNAT family	PCAF	832	H3K9, H3K14, H4K8, H4K16	P53, E2F1, EV11, DTCF	regulates gene expression, acetylates histones, coactivates transcription, and influences cellular differentiation
	ELP3	547	H3, H4	--	it is crucial for tRNA modification, transcriptional elongation, histone acetylation, and neuronal development
	HPA-2	551	H3K4, H3K14, H4K5, H4K12	--	it regulates platelet function, affecting blood clotting, immune response, and disease susceptibility, including thrombocytopenia
	HPA-3	160	H3, H4	--	it is a human platelet antigen involved in platelet adhesion, immune response, and transfusion compatibility
	GCN5	836	H3K9, H3K14, H3K18, H3K27, H4K5, H4K8, H4K12	P53, HY5, c-MYC	act as the catalytic subunit, directly responsible for the enzymatic acetylation of histone, which leads to chromatin relaxation and increased transcription of associated genes
	ATF-2	505	H2B, H4	cyclin A, cyclin D1, GADD45	directly modify histone proteins by adding acetyl groups, altering chromatin structure, and promoting gene transcription

^aAbbreviations: MYSTMoz, Ybf2/Sas3, Sas2, Tip60; MOZMonocytic leukemia zinc finger protein; KATLysine acetyltransferase; TIP60Tat-interacting protein, 60 kDa; MORFMOZ-related factor; MOFMales absent on the first; SASSomething about silencing; ESA1Essential SAS-related acetyltransferase 1; HBO1Histone acetyltransferase binding to origin recognition complex 1; PCAFp300-/CREB-binding protein-associated factor; TAF1TATA-box-binding protein-associated factor 1; GTF3C1General transcription factor IIIC Subunit; ELP3Elongator Protein 3; HPAHistone acetyltransferase p300-/CBP-associated factor; GCN5General control nondepressible 5; GNATGcn5-related N-acetyltransferase; ATF-2Activating transcription Factor 2.

Histone Dysregulation in the Progression of AD

Epigenetic processes, such as long noncoding RNAs (lncRNAs), DNA methylation, and histone modifications, dynamically control gene expression without modifying the DNA sequence. While long noncoding RNAs and DNA methylation contribute to AD, histone modifications offer distinct benefits, including rapid adaptability to environmental changes, potential for pharmacological intervention, and synergistic regulation of transcription. − Histone modification has a substantial impact on gene expression because it alters the chromatin structure and influences the accessibility of transcription factors to gene promoters. These are pertinent to neurogenesis and synaptogenesis, which play an important role in learning and memory functions. The predominant histone modifications encompass acetylation, methylation, ubiquitination, and phosphorylation. Histone methylation modifies chromatin structure, enabling its involvement in DNA-related activities like transcription, replication, and DNA repair. Histone acetylation is the process by which HATs attach acetyl groups to the N-terminal lysine residues of histones, leading to a more accessible chromatin configuration that generally facilitates gene expression. Histone phosphorylation often transpires at threonine or serine residues of histones, and its recognized purpose is to contribute to the physiological response to DNA damage. Histone ubiquitination frequently causes histones to be destroyed by proteasomes or alters their location or activities in a variety of cellular processes. Two recently discovered post-translational modifications of histones are crotonylation and lactylation, which both involve the enzymatic transfer of a crotonyl or lactyl group to lysine residues. The disruption in gene expression and memory is caused by the dysregulation of histone modifications, which contributes to AD and cognitive loss. As histone modifications control gene expression through changes in chromatin structure, the variation in acetylation, methylation, etc., can either activate or suppress genes. A close association with memory loss and cognitive decline in neurodegenerative diseases such as AD has been associated with these epigenetic changes, with the imbalance of HATs and HDACs as the driving force of the pathologies.

Histone Acetylation

Histone acetylation in the hippocampus increases transiently during learning in normal mice, and disturbed equilibrium is related with memory deficits. − Histone acetylation, particularly on histones H3 and H4, is the major controller of gene expression. This process is regulated by HATs and HDACs. Lysine acetylation and deacetylation of histones, respectively, attenuate and activate gene expression. HDACs usually inhibit transcription by removing acetyl groups from histone tails and compressing the chromatin. HDAC2, which is extensively expressed in the central nervous system, adversely modifies memory and synaptic plasticity in this way. To inhibit the expression of a number of synaptic plasticity genes, including glutamate ionotropic receptor AMPA-type subunit 1, brain-derived neurotrophic factor (BDNF), early growth response 1, and C-Fos, HDAC2 was mechanistically enriched in their promoters. HDAC3 causes changes in APP processing, while HDAC4 and HDAC6 have an impact on synaptic plasticity and neuronal survival. As a result, there is less H3 and H4 acetylation around these genes’ transcription start sites, which suppresses gene expression.

Proteomic study demonstrated that increased levels of H3K27Ac and H3K9Ac were seen throughout AD-associated single-nucleotide polymorphisms, and these higher levels of H3K27Ac and H3K9Ac enabled Aβ42-induced neurotoxicity in vivo. In a different investigation, H3K27Ac was examined in the entorhinal cortex of AD patients and low-pathology controls. H3K27Ac was found in areas linked to late-onset AD-associated variations and genes linked to tau and Aβ pathology. Histone acetylation is caused by the lysine acetyltransferase CBP/P300 complex, which has a neuroprotective role in the development of AD, whereas its dysregulated expression results in neuronal death and neurodegenerative disorders. In the AD cell model, P300 directly interacted with the promoter regions of two genes linked to the metabolism of APP: progerin 1 and Aβ precursor protein lyase 1 (BACE1). This resulted in increased histone H3 acetylation in these regions and increased expression of both progerin-1 and BACE1. According to a study, memory impairments and synaptic dysfunction are linked to acetyltransferase CBP/p300 deficiency. Furthermore, P300 mediates Tau acetylation in a concentration-dependent way that inhibits Tau assembly. Additionally, increased HDAC2 levels in AD patients and mouse models were linked to cognitive dysfunction through suppression of H4K12Ac. Another study showed that APP-mediated reduction of H4K5Ac and H4K12Ac on the promoters of immediate early genes downregulated these genes’ transcription in a way reliant on CREB and HDAC2, respectively, which consequently impacted memory function and synapse formation.

Histone Methylation

One of the highly notable post-translational modifications is histone methylation, which is involved in the physiological processes, including chromatin compaction, transcription control, DNA damage responses, alternative splicing, DNA replication, and genome stability in AD. One of the most studied histone methylation indicators is H3K4 methylation, which is associated with gene activation. H3K4me3 is found in the nucleosomes of the promoter regions of genes that are actively transcribed, whereas H3K4me2 is found in the gene bodies and enhancers of active genes. , A class of methyltransferases mediates H3K9 methylation, which inhibits gene expression. Notably, transcriptional suppression results from G9A’s catalysis of H3K9me2 in euchromatin areas. Further, H3K27 trimethylation and monomethylation mostly result in the activation and inactivation of genes, respectively.

In a study, abnormal histone methylation patterns have been identified in the hippocampus/cortex regions in AD, which elevated H3K9me2 and the activity of enzymes like G9a, leading to memory loss, distortion of chromatin structure, and cellular signaling. , In another study, an elevation of H3K9me2 and H3K9me3 on the promoters of immediate early genes in AD is associated with impaired memory and learning functions. Inhibition of histone methyltransferases, including SUV39H1, reduced histone methylation and generated BDNF, which subsequently increased memory and learning performance. Cao et al. reported that both AD patients and a mouse model of tauopathy have higher levels of the gene activation-related histone H3K4me3 in the prefrontal cortex. These alterations were associated with phosphorylated tau, which was reversed in the mouse model with targeted H3K4me3 reduction, as well as impairment of memory-related behaviors and synaptic functioning. Furthermore, H3K4Me3 alteration was seen in the cytoplasm of AD patients’ hippocampi, and the degree of this modification was strongly associated with a rising Braak stage. This cytoplasmic H3K4Me3 interacted with phosphorylated Tau, suggesting that it may play a part in the phosphorylation process of Tau. In another work, Gjoneska et al. evaluated H3K4me3 levels in the CK-p25 AD mouse model and found a correlation with learning and the immunological response. The hippocampal regions of AD patients also showed comparable levels of H3K4me3. Interestingly, APP/Preseniline-1 (PS1) mouse’s neurofilament-labeled and calretinin-positive interneurons showed an age-related increase in H3K27me3 when compared to wild-type mice.

Histone Phosphorylation

Histone phosphorylation refers to a significant alteration associated with the regulation of genes, repairing their DNA, and apoptosis, as well as the remodeling of the chromatin. Histone phosphorylation is often linked to active transcription and, like acetylation, neutralizes the positive charge on histones to encourage chromatin structural relaxation. The abnormal histone phosphorylation has been reported in neurons as well as glial cells, which indicates its role in the development of AD. Interestingly, the enhancement of phosphorylation of H4S47p in amyloid-treated neurons and in the AD brain samples and the augmentation of phosphorylation of H2AXS139p in astrocytes causes DNA damage. Similarly, H3S10p phosphorylation has been observed in hippocampal neurons, resulting in the disruption of mitosis and the death of neurons. Histone phosphorylation through CREB signaling is also associated with the kinase and kinase mitogen and stress-activated protein kinase-1.

Neurons treated with Aβ and cells expressing an APP isoform showed an increased level of phosphorylation of H4S47p. The results showed that histone phosphorylation in AD was dysregulated by APP and/or Aβ, with H4S47p somewhat elevated in mild cognitive impairment and significantly elevated in AD brain samples. S47 phosphorylation levels of H4 have been shown to be increased in Aβ-overexpressing neuroblastoma. In brain samples from AD patients, phosphorylation of histone variant H2AX has also been observed. This variant histone is phosphorylated on Ser-139 in response to DNA damage. The observation of higher levels of H2AX in the hippocampus and cortical astrocytes of AD patients emphasized the role of astrocytes and DNA damage responses in AD. Additionally, Ogawa et al. investigated whether the hippocampus tissue of AD patients had higher levels of histone H3 phosphorylation, particularly at H3S10p. Susceptible neurons in AD have phosphorylated H3 in their cytoplasm, rather than their nuclei. These results were linked to abnormal cell cycle activation and mitotic machinery, suggesting a potential mechanism causing AD neurodegeneration. Also, the kinase mitogen and stress-activated protein kinase-1, which phosphorylates histone H3 residues, mediates the phosphorylation and subsequent activation of CREB. These results taken together indicate that histone phosphorylation dysregulation is linked to neuronal and glial dysfunction, impaired gene expression, and neurodegeneration in AD.

Histone Ubiquitination

In the brains of AD, histone ubiquitination is markedly changed, particularly of H2A and H2B. In terms of ubiquitylation, histones H2A and H2B can be either mono- or polyubiquitylated. , Conversely, polyubiquitylation has been connected to interactions between histones inside the nucleosome, whereas monoubiquitylation has been connected to the identification of DNA damage sites. In an investigation, the levels of H2B ubiquitylation, namely, H2BK120, were elevated in AD patients’ frontal cortex. The ubiquitin–proteasome system facilitates the clearance of proteins, appears to be compromised in AD, and correlates with the formation of Aβ plaque and hyperphosphorylated tau. The ubiquitination of H2Aubi is facilitated by polycomb repressive complex 1’s B-cell specific Moloney murine leukemia virus integration site 1. This protein expression and H2Aubi are both decreased in AD brains and iPSC-derived AD neurons, targeting the INK4A/ARF gene, which encodes essential proteins for cellular senescence. This results in deposition of Aβ, accumulation of Tau, and neurodegeneration. , Furthermore, H2B monoubiquitylation is essential for an increase in H3K4me3 on the hippocampus learning-related gene c-Fos, and deletion of H2Bubi inhibits learning-induced synaptic plasticity and memory formation.

Other Histone Modifications

Histone alterations, including crotonylation and lactylation, are becoming increasingly important in regulating the pathology of AD, yet are far less studied in AD. One kind of histone modification was identified as histone crotonylation. An increasing amount of research suggests that endocytosis-related gene expression is impacted by histone crotonylation, which, in turn, affects the microglia-mediated removal of Aβ in AD. A unique histone modification called histone lactylation was first reported. It is dynamically controlled by lactate created by cellular metabolism and can directly alter transcription, translation, and gene replication processes, which, in turn, affects the biological consequences of cells. A previous study found that downregulating lncRNA nuclear enriched abundant transcript-1 in AD reduced neuroglial cell-mediated Aβ uptake and clearance by lowering expression levels of transforming growth factor beta 2, caveolin 2 and transforming growth factor beta receptor 1, etc. Further mechanistic studies demonstrated that nuclear-enriched abundant transcript-1 is connected to P300, and that its suppression influences P300-mediated H3K27 crotonylation near the transcriptional activity of these genes. , The brains of AD patients and AD mice had higher levels of histone H4K12 lactylation, according to a study. H4K12la caused aberrant microglial activation and neuroinflammation in AD microglia by activating the transcription of glycolytic genes such as lactate dehydrogenase A, pyruvate kinase M2, and hypoxia-inducible factor 1-alpha. Further, it was shown that the hippocampus area and senescent microglia in the AD mouse model had increased H3K18 lactylation. It triggered the NF-κB signaling pathway by binding to the promoters of Rela and NF-κB1 genes, leading to increased neuroinflammation and senescence-associated secretory phenotypes. A recent study found that lncRNA EPB41L4A-AS1 plays a crucial role in autophagy-mediated Aβ clearance. EPB41L4A-AS1 influences the transcription of three autophagy-related genes by altering pan histone lysine lactylation levels near their transcription start sites. Aβ plaques, APP, PS1, BACE-1, and tau tangles are overexpressed in AD due to dysregulated histone modification, which also promotes oxidative stress and altered gene regulation, followed by proteasomal dysfunction (Figure ).

1.

A schematic illustration of a representative histone post-translational modification involved in the progression of AD. Histone dysregulations include acetylation (Ac), crotonylation (Cr), methylation (Me), ubiquitination (UB), phosphorylation (P), and lactylation (La). These alterations in turn lead to dysregulated transcriptional processes facilitated by a series of histone-modifying enzymes (CBP/p300, Tip60, PCAF/GCN5, MOZ, MOF, and SAS2). Proteasomal dysfunction associated with histone dysregulation results in increased DNA damage, oxidative stress, and proteostasis, which collectively contribute toward the progression of AD. GL, being a HAT inhibitor, can modulate these crucial pathogenic processes, like histone hyperacetylation, proteasomal dysfunction, oxidative stress, neuroinflammation, acetylcholinesterase activity, and AD. Created with www.biorender.com.

Therapeutic Implications of Histone Modification in AD

In recent years, research has gathered data indicating that epigenetic regulatory systems are vital in the development and prognosis of several ailments, including AD. A number of histone-modifying enzyme inhibitors have been used to treat AD in murine models. For instance, UNC0642 and BIX01294, two small-molecule inhibitors of the G9A/GLP complex, reduce H3K9me2 levels, leading to memory restoration. Moreover, they dramatically improve the expression of nerve growth factor-inducible genes, nerve growth factor, synaptophysin, and BDNF while concurrently lowering Aβ plaques, increasing synaptic plasticity, neuroinflammation, and oxidative stress in the 5xFAD mouse model. , Synaptic function and memory-related behaviors were restored in the prefrontal cortex of 5xFAD animal models when the compound WDR5–0103 inhibited the H3K4-specific methyltransferases SETD1a/b and MLL1–4. Further, in APP/PS1 and Tg2576 mouse models, HDAC inhibitors such as valproic acid, vorinostat, sodium butyrate, and trichostatin A enhance synaptic functioning, restore hippocampal Tau phosphorylation, and promote cognitive formation. −

Histone chemical changes can be utilized as both therapeutic targets and indicators through the application of epigenetic modulators of HAT and HDAC. It has been demonstrated that inhibitors of both mechanisms effectively lower total neurotoxicity. Unlocking the function of HAT inhibition in AD has been made possible, nonetheless, by mounting evidence in favor of HAT inhibition. Here, we present a handful of crucial reports that support the use of HAT inhibitors as a potential modality for AD. CBP, p300, and PCAF are three HATs that have been extensively implicated in memory function, which may represent more precise targets than HDACs. The role of CBP has been found crucial for memory function, as mice lacking CBP showed poor long-term memory development. Mutations in the CBP gene cause mental retardation. When CBP was expressed in the brains of 3 × Tg-AD triple transgenic AD mice, the animals’ memory impairments were restored. However, acetylated tau and phosphorylated tau at Ser202 decreased in vitro when the HAT p300 was inhibited with p300 inhibitor C646. Phase II clinical trial conducted using curcumin (a natural p300/CBP HAT inhibitor) depicted enhancement of cognitive performance and lowering Aβ-deposition in AD patients. Curcumin also reduced damage from oxidative stress and Aβ pathology in transgenic animal models via regulating anti-inflammatory pathways.

Since AD is also connected with altered acetylation of nuclear/cytoplasmic nonhistone proteins, such as NF-κB, p53, alpha tubulin, and tau, thus HAT inhibitors could be implicated as effective control mechanism in AD. Recent research has shown that aging-related oxidative stress increased the activity of the CBP. Eventually, these molecules trigger transcription factor NF-κB, which is a key activator of the inflammatory response. Additionally, oxidative stress increased the p65 component of NF-κB’s binding ability to the HAT, CBP. Further, the tip60 (an important protein of the MYST family) plays a major role in the synthesis of Aβ-fibers following its interaction with APP.

Some of the novel epigenetic drugs employed for the effective management of AD are summarized in Table .

2. Therapeutic Strategies to Treat AD through Novel Epigenetic Candidates.

drug	epigenetic target	experimental model	therapeutic activity	references
trichostatin A	HDAC1, 2	Slc9a6 knockout mice	participate in Aβ degradation via the endosomal route and Nhe6 restoration through HDAC1, 2 inhibitions
sodium butyrate	HDAC	APP-PS1–21 mice	improved associative memory even in more severe stages of AD
curcumin	p300	APPSwe-transfected murine brain-derived neuroblastoma	through p300 inhibition, suppressed the BACE1 and progerin-1 hyperacetylation
entinostat	HDAC	drosophila AD model	Tip60 is activated and downstream genes are hyperacetylated by HDAC inhibition
vorinostat	HDAC	APP/PS1 miceTg2576 AD mouse model	through HDAC inhibition, alleviated cognitive deficits in AD mice
C-30–27	PCAF	BV-2 murine microglial cells and Neuro-2A neuroblastoma cellsMale Sprague–Dawley rats	specific PCAF inhibition evades the NF-kB activation and suppressed neuronal apoptosis and cytokine release
garcinol	p300, PCAF	Sf21 and Hella cell line model	effectively inhibited p300 and PCAF and downregulated gene expression
C646	p300/CBP	primary cortical neurons; TauP301L-expressing neurons; HEK293T cells	restored ubiquitination, accelerated the hyperphosphorylation of tau clearance, and increased the rate of tau degradation through proteasomes
gallic acid	p300/CBP and PCAF	ICR mice with intracerebroventricular Aβ1–42 injection	the inhibition of Aβ-induced microglial activation, pro-inflammatory cytokine reduction, and an improvement in cognitive impairment
anacardic acid	p300, PCAF	in vitro HAT assays using purified human p300/PCAF and HeLa core histones	inhibition of p300- and PCAF-mediated HAT suppressed HAT-dependent transcriptional activity of chromatin templates without disrupting DNA transcription

Garcinol, a Potent HAT Inhibitor: Role in Neuroprotection

GL, has been used in a variety of industrial, medicinal, and culinary applications. Their potential efficacy in preventing and treating chronic diseases that are not transmissible has garnered significant interest in recent decades. Additionally, GL possesses a variety of biological activities, including antibacterial, anti-inflammatory, antioxidant, anticancer, and antiobesity properties. It has also showed neuroprotective qualities primarily through HAT inhibition and antioxidant benefits through high free radical and superoxide anion scavenging activity. Moreover, recent investigations depicted potential anticancer properties of GL by causing cell cycle arrest and apoptosis, preventing angiogenesis, and controlling the expression of certain genes in cancerous cells. Some of the important therapeutic effects of GL pertain to its neuroprotective and cognitive amelioration property.

Neuroprotective Effect

GL has been investigated in a number of neurological diseases due to its potential impact on oxidative stress and inflammation. The hyperactivation of NMDA glutamate receptors brought on by changes in extracellular glutamate and GABA levels sets off a series of signaling cascades that promote neuronal death through excitotoxicity. GL inhibited the hyperactivation of NMDA receptors and increased the expression of GABA_A and glutamic acid decarboxylase 65. In C57BL/6 mice, it has also been shown to control neuronal degeneration and significantly lower epileptic seizure scores while improving memory and cognition.

The neuroprotective effect of GL on mice with MPTP-induced Parkinson’s disease was examined by Chetia Phukan et al. Results depicted that GL (25 mg/kg) produced considerably more rears in the behavior test and significantly inhibited akinesia and catalepsy in comparison to parkinsonian mice. In the striatal brain areas, GL markedly increased the depleted dopamine levels and its metabolite 3,4-dihydroxyphenylacetic acid by 1.28 and 1.36 times, respectively, in contrast to the parkinsonian group of mice. Additionally, GL significantly decreased the degradation of glial fibrillary acidic protein, dopaminergic cell bodies, and neuroinflammation in the substantia nigra region. In another research, GL at a dose of 5 mg/kg effectively reduced the axial, limb, and orofacial scores for dyskinesia by cotreatment with l-DOPA in hemiparkinsonian mice with unilateral 6-hydroxydopamine lesioning. Additionally, GL reduced the expression of c-Fos, FRA-2, and ARC genes, which are often overactivated in l-DOPA-induced dyskinesia.

The neuroprotective activities of GL were confirmed in another rat model involving middle cerebral artery occlusion/reperfusion. The results showed that GL (20 mg/kg) substantially decreased the infarct size, volume, and neurological impairment score compared to ischemia-reperfusion treated rats. Furthermore, GL lowered oxidative stress and cerebral ischemia-reperfusion-induced inflammatory cytokines. It also decreased superoxide dismutase, malonaldehyde, and nitric oxide (NO) levels as well as the production of IL-1β, IL-6, and TNF-α. Further, it was found to reduce NF-κB, p65, phosphorylated inhibitor of κB-alpha (p-IκB-α), and toll-like receptor 4 (TLR4) expression in vivo shown by the immunohistochemistry assay (Figure ). In silico research on the molecular interactions between GL and the active sites of catechol-O-methyltransferase and monoamine oxidase type B depicted successful suppression of the selected enzyme activity. Since the inhibition of monoamine oxidase type B and catechol-O-methyltransferase has been linked to increased dopamine availability and the prevention of the production of harmful dopamine metabolites such as homocysteine, 3-o-methyldopa, 3-methoxytyramine, 3,4-dihydroxyphenylacetaldehyde, etc., the results thus indicated potent neuroprotective action of GL toward successful management of Parkinson’s disease.

2.

(I) GL enhanced neurological function and reduced brain damage in ischemia-reperfusion rats. Rats in the sham group, the ischemia-reperfusion group (caused by blockage and reperfusion of the middle cerebral artery, followed by reperfusion), and the GL groups (administered with 5, 10, and 20 mg/kg/d of GL for 3 days). (A) Neurologic deficiency was assessed. (B) The infarct tissues and (C) the infarct size is displayed in a histogram (n = 5 per group). *P <0.05 in comparison to the sham group, and #P <0.05 in comparison to the ischemia-reperfusion group. (II) GL inhibited the activation of TLR4/NF-κB signaling caused by cerebral ischemia reperfusion. (A) Western blot measurements of TLR4, p-IκB-α, and nuclear p65 are shown. The histogram displayed the relative protein expression of TLR4 (B), p-IκB-α (C), and nuclear p65 (D). Immunohistochemistry test (×200) was used to assess the expression and localization of TLR4 (E) and p65 (F). *P <0.05 in comparison to the sham group, and #P <0.05 in comparison to the I/R group. Reprinted with permission from [Kang, Y.; Sun, Y.; Li, T.; Ren, Z. Garcinol Protects Against Cerebral Ischemia-Reperfusion Injury In Vivo and In Vitro by Inhibiting Inflammation and Oxidative Stress. Molecular and Cellular Probes 2020, 54, 101672]. Copyright [2020 ELSEVIER B.V.].

Preclinical Evidence of GL in Ameliorating Memory Functions

Among all HAT inhibitors, GL has been recognized as the first cell-permeable HAT inhibitor with nontoxic and tissue-compatible nature. In animals, even at high doses of GL of up to 2000 mg/kg, there were no aberrant reactions in terms of reproductive, hematological, or developmental abnormalities. Basically, the higher lipophilicity and reasonable molecular weight of GL work in favor of its excellent BBB permeability. , The HATs, p300 (IC₅₀ >7 μm) and PCAF (IC₅₀ >5 μm), have recently been shown to be strongly inhibited by GL, both in vitro and in vivo. GL successfully decreased the transcription of genes that were triggered by elevated HAT activity. GL has been reported to block the histone acetylase GCN5 in the environmental fungus Cryptococcus neoformans. This enzyme otherwise controls the expression of genes related to different conditions in the environment. Due to its impact on DNA damage responses, this clinically relevant characteristic of GL is further expanded to include its potential application in radio sensitization. Therefore, it is proposed that GL can effectively regulate and inhibit the hyperacetylation of histones, a crucial mechanism in AD pathology (Figure ).

The capacity of GL to prevent oxidative stress and free radicals provides a roadmap for its possible use in neurological diseases since the high levels of cellular oxidative load in AD remain the main cause of neuronal death. As oxidative stress is one of the main factors in AD progression, GL having potent antioxidant activity is explored widely ameliorate AD. At an inhibitory dose of 0.32 μM, GL scavenges superoxide anions, hydroxyl, and methyl radicals, preventing their disruption of normal cellular biomolecule functioning and causing a favorable downshift in reactive oxygen species. GL’s β-diketone moiety and phenolic hydroxyl groups contribute toward its potential antioxidant ability. Experimental research showed that GL’s superoxide radical scavenging ability was more effective than tea catechins and as powerful as gallic acid. Compared to α-tocopherol (a lipid-soluble antioxidant), the emulsified GL reduced the levels of the free-radical DPPH three times more effectively. GL at 25 μg/mL strongly inhibited the reactive thiobarbituric acid created by peroxynitrite (100 μM).

Zou and their colleagues have demonstrated potent antioxidant activity of GL toward hydrogen peroxide-induced excessive oxidative stress and bone marrow mesenchymal stem cell dysfunction. Results showed that 10 μM GL greatly reduced the elevated ROS level in bone marrow mesenchymal stem cells. The potential mechanisms of GL and its derivatives’ antioxidant activity on arachidonic acid metabolism and NO radical production have also been examined by Hong et al. At doses (>1 μM), the peak plasma and urine concentration levels of GL in CD-1 female mice were 12 and 2.7 μM, respectively. GL (1 μM) significantly inhibited the creation of ROS in cells by activating the antioxidant pathway controlled by DJ-1/SIRT1 and PGC-1α in human neuroblastoma (SH-SY5Y) cells as reported elsewhere. In another work, potential neuroprotective action of GL was reported. Even at 5 μM concentration, GL greatly lowered the production of lipopolysaccharide (LPS)-induced inflammatory mediators, including inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2), and decreased the buildup of NO radicals in LPS-treated astrocytes. These findings implied that GL exhibits potential neuroprotective effect via multiple pathways including blocking iNOS in astrocytes.

Neuroinflammation is a fundamental process in AD that results in cellular impairment and death. It has been found that glial cell activation, which results in inflammatory responses, is influenced by impaired NO radical clearance and arachidonic acid metabolism. It has been demonstrated that inflammation is influenced by abnormal arachidonic acid metabolism and the production of NO radicals. The enzyme COX, lipoxygenase (LOX), and cytochrome P450 pathways further break down arachidonic acid when it is liberated from membrane phospholipids by phospholipase A2. Inhibiting the COX and LOX enzymes has been thought to be an effective way to modify the metabolism of arachidonic acid in order to cure inflammation. Through the regulation of iNOS production by modification of the JAK/STAT-1 signaling pathway and the inhibition of extracellular ERK1/2 kinase activation, GL modulates the metabolism of arachidonic acid by delaying the phosphorylation of cytosolic phospholipase A2. GL inhibited NF-κB activation and COX-2 expression by blocking LPS’s ability to bind to toll-like receptors when it was administered before LPS. The NO radical moiety is implicated in a number of physiological activities, such as host defense, synaptic neurotransmission, platelet function inhibition, and vasodilation. Endothelial nitric oxide synthase (NOS), neuronal NOS, and iNOS are the three kinds of NOS enzymes that catalyze the biological system’s production of NO radicals from arginine. Inflammatory cytokines trigger the iNOS enzyme, which in turn triggers the generation of NO by macrophages and several other cell types. GL has been shown to suppress the expression of iNOS and COX-2 in LPS-activated macrophages. It was shown that GL effectively inhibited the activation of the eukaryotic transcription factor NF-κB caused by LPS. Inhibition of NF-κB leads to phosphorylation of IκB-α and p38 mitogen-activated protein kinase which thus improves memory functions.

A recent study found that GL had anti-inflammatory activities on Raw 264.7 and LPS-activated THP-1 macrophages, reducing the release of TNF-α, IL-6, IL-1β, prostaglandin E2, and NO. Furthermore, it inhibited the NF-κB signaling pathway and reduced the generation of pro-inflammatory substances (Figure ). Jacob et al. found that pH-sensitive biodegradable GL-loaded polymeric nanoparticles significantly lowered lactate dehydrogenase and myeloperoxidase activity and also inhibited TNF-α, (IL)-8, and NF-κB activity. GL’s DPPH, H₂O₂, and NO scavenging activity has already been documented elsewhere. GL at 8 μM significantly suppressed the production of pro-inflammatory cytokines such IL-6 and TNF-α and reduced the expression of iNOS and COX-2. In addition, GL inhibited the expression of thrombospondin motifs 5 and metalloproteinases, both of which control extracellular matrix breakdown. According to a different study, GL efficiently inhibited NF-κB activation in rat spinal nerve ligation-induced neuropathic pain and LPS-stimulated primary cultured microglia. GL at 10 μM inhibited the production of COX-2/prostaglandin E2, IL-6, iNOS/NO, and IL-1β in the spinal cord of rats as well as microglial activation. Additionally, it decreased the expression of the acetyl-p65 protein, which lowers the nuclear translocation of NF-κB. These findings suggest that GL protects against AD by lowering neuroinflammation and neuropathic pain.

3.

GL (10, 20, and 30 μM) effects on associated molecule expression in the NF-κB signaling pathway in LPS-treated THP-1 cells (A–C) and RAW 264.7 cells (D–F) were identified by using Western blot analysis. For normalization, β-actin was used. The information is displayed as the mean ± SD of three separate experiments’ triplicate analysis (n = 9). Dunnett’s test and one-way ANOVA were employed for statistical analysis. *p <0.05, **p <0.01, ***p <0.001, and ****p <0.0001. Reprinted from [Chantree, P.; Martviset, P.; Thongsepee, N.; Sangpairoj, K.; Sornchuer, P. Anti-Inflammatory Effect of Garcinol Extracted from Garcinia Dulcis VIA Modulating NF-ΚB Signaling Pathway. Nutrients 2023, 15 (3), 575.], distributed under Creative Commons Attribution 4.0 International License, which permits unrestricted use, distribution, and reproduction in any medium. https://creativecommons.org/licenses/by/4.0/ (accessed on 29 March 2025).

GL prevented acrylamide-induced cognitive deficits in zebrafish larva’s brains by blocking amyloidogenic APP processing and subsequent tau hyperphosphorylation. A balance between phosphatases and tau kinases, such as GSK3β, CDK5, or c-Jun N-terminal protein kinase, controls its phosphorylation. Increased amyloidogenesis and the accompanying neuroinflammation are correlated with the activation of CDK5. GL inhibited GSK3β and CDK5, which is the connection between tau hyperphosphorylation and APP processing. Additionally, GL treatment considerably improved the uncoordinated swimming patterns of zebra fish larvae caused by acrylamide as compared to the disease control group. Further, larvae exposed to acrylamide had cartilage abnormalities such as the lack of posterior pharyngeal arches and impaired curvature of the first pharyngeal arches, whereas the GL-treated group almost showed reversal in the above behavioral pattern (Figure ). Furthermore, GL demonstrated favorable anticholinesterase characteristics by inhibiting the acetylcholinesterase enzyme with an IC₅₀ value of 0.66 μM, which is comparable to the IC₅₀ value of galantamine, a well-known cholinesterase inhibitor, which is 0.50 μM. Table displays research pertinent to the neuroprotection provided by GL or garcinia extracts.

4.

Evaluation of acrylamide-induced brain damage and recuperation with GL therapy. (A) Each experimental group’s swim route as captured by Toxtrac software. (B) The average swimming speed and total distance covered in 1 min inside the designated arena zone. (C) Presynaptophysin (SYP) and postsynaptic density protein 95 (PSD-95) synaptic neuronal protein expression. (D) Densitometry analysis of the matching SYP and PSD-95 protein levels (E) Heart output (heartbeat/min) following each treatment. (F) Morphological disruption following ACR therapy. (G) The craniofacial configuration is shown in the ventral view of an 8dpf zebrafish larva dyed with alcian blue. Meckel’s cartilage (m), palatoquadrate (pq), ceratohyal (ch), hyosymplectic (hs), and ceratobranchials 3–7 (cb) are among the craniofacial cartilage elements that are normally arranged. Scale bar: 200 μm, magnification: ×10. Reprinted with permission from [Sharma, C.; Kang, S. C. Garcinol Pacifies Acrylamide Induced Cognitive Impairments, Neuroinflammation and Neuronal Apoptosis by Modulating Gsk Signaling and Activation of PCREB by Regulating Cathepsin B in the Brain of Zebrafish Larvae. Food and Chemical Toxicology 2020, 138, 111246.]. Copyright [2020 ELSEVIER B.V.].

3. Important In Vitro, In Vivo, and In Silico Outcomes of GL in Various Neurodegenerative Disorders.

sl. no	experimental model	level of evidence	therapeutic outcomes	strengths	limitations	translational relevance	references
1	in vivo (Sprague–Dawley rats) post-traumatic stress disorder model	in vivo	GL prevented histone H3 from being acetylated when it was injected into the brain’s lateral amygdale (10 mg/kg), which hindered the consolidation of earlier fear memories	multilevel strategy that incorporates electrophysiological, molecular, and behavioral data	restricted to rodent model; lack of human validation	emphasizes the use of epigenetic modifications as a treatment approach
				mechanistic understanding of how memory functions is regulated by epigenetics	short-term examination; no long-term safety/toxicity study	dosage, safety, and long-term effectiveness in people require more studies
2	in vitro cell line (Hella cells)	in vitro	GL at a dosage of 10 μM was shown to be a highly effective inhibitor of PCAF and p300 (HATs)	specificity toward HATs; quantitative IC50 values and mechanistic understanding of enzyme inhibition	exclusive to isolated enzyme systems, it does not account for the complexity of cells	shows promise for treatment of cancer and potentially other epigenetic diseases
				demonstrates epigenetic modification in vivo	absence of long-term toxicity evidence; inadequate model description	demonstrates a wide range of regulatory potential that is pertinent to complicated diseases like cancer and neurodegeneration
3	in vitro cell line LPS induced (RAW 264.7 cells)	in vitro	in LPS-treated cell lines, GL at a dose of 1 μM inhibited the phosphorylation of STAT1, iNOS, COX-2, PLA2, and activated NF-κB	cross-validation across several cell lines demonstrates the reproducibility of the anti-inflammatory effects	restricted to in vitro models; lacks in vivo validation	reveals GL as a potential drug candidate for further phases of development
				comprehensive molecular understanding of anti-inflammatory pathways; assessment of many signaling targets, evidence of dose-specific effects	absence of pharmacokinetic and long-term toxicity studies	validates effective anti-inflammatory potency against several systemic diseases
4	in vitro cell line LPS induced (RAW 264.7 cells) and in silico analysis	in vitro and in silico	GL effectively decreased COX-2 and iNOS’s relative mRNA expressions. Furthermore, GL’s molecular docking against COX-2 and iNOS targets demonstrated strong protein–ligand binding affinities	reliability is enhanced by the use of a well-established anti-inflammatory model	limited to in vitro studies, absence of in vivo and pharmacokinetic/bioavailability assessments	exhibits significant anti-inflammatory efficacy against iNOS/COX-2 pathways; however, it requires clinical validations to confirm safety and efficacy
				multilevel assessment, like gene expression, biochemical markers, and dose-dependent analysis, which shows the potential of GL
5	in vitro	in vitro	in a system that produces free radicals, GL has been demonstrated to scavenge a superoxide anion, hydroxyl radical, and methyl radical, among other hydrophilic and hydrophobic free radicals	strong antioxidant potencies in comparison with frequently used assays	limited to in vitro conditions, lacks in vivo validations	reveals the antioxidant potential in relation to lipid peroxidation in neurodegenerative diseases
				direct link to the production of advanced glycation end-products, a key component of AD pathogenesis		shows promise for preventing advanced glycation end-product-mediated neurodegeneration
6	in vitro	in vitro	GL (10–20 μM) reduced iNOS expression, MMP activity, and NO and H2O2 generation in HL-60 cell lines	biological evaluation of several targets, such as oxidative stress and inflammation, by using both parent and oxidative derivatives of GL	restricted to in vitro models, in the absence of in vivo, long-term toxicity, and pharmacokinetic studies	helps in understanding molecular antioxidant mechanisms but requires validation in in vivo studies
7	in vitro cell lines (MDA-MB-231 and MCF-7)	in vitro	GL (25 μmol/L) demonstrated to effectively inhibit NF-κB and inflammation	generalizability is enhanced by using both estrogen-positive and estrogen-negative cell lines	limited to in vitro models, lacks in vivo studies	GL’s cytotoxicity and NF-κB targeting, it is a promising option for treating neuroinflammation; however, in vivo studies are needed to validate its safety, efficacy, and chronic toxicity
8	in vivo (Wistar rats)	in vivo	in primary cortical cell cultures, including glial cells, GL (5 μM) increased neurite outgrowth and neuronal survival. Furthermore, it decreased growth factor-deficient cell death and markedly increased glial fibrillary acidic protein and microtubule-associated protein 2, which controls the ERK pathway	mechanistic insights into ERK and NMDA receptor involvement	specific to the in vitro model only, the absence of long-term toxicity, and in vivo and pharmacokinetic data	suggests an effective result in neurodegeneration and requires validation in in vivo and clinical studies
				use of multiple neuronal and glial markers
9	in vivo (C57BL/6J mice) l-DOPA-induced dyskinesia of Parkinson’s disease model	in vivo	in mice with l-DOPA-induced dyskinesia and 6-hydroxydopamine-induced parkinsonism, GL (5 mg/kg) reduced the dyskinetic behavior. Additionally, it prevented the ERK cascade caused by prolonged l-DOPA treatment and the overexpression of c-Fos, Fra-2, and Arc	both behavioral and molecular end points were evaluated by using a well-established PD dyskinesia model	lack of chronic toxicity and pharmacokinetic data	revealed the potential of GL to reduce l-DOPA-induced dyskinesia and supports epigenetic modulation as a therapeutic strategy
10	in silico model	in silico	GL inhibited monoamine oxidase-B, which is anticipated to reduce dopamine depletion through the production of its harmful metabolites and catalysis mediated by MAO-B	enables comparison with established MAO-B inhibitors, which support the multitarget potential of GL	lacks both in vitro and in vivo validations	exhibits a preliminary mechanistic basis for GL as an MAO-B inhibitor; further in vitro and in vivo validation are needed before clinical translation
11	in silico model/in vitro	in silico/in vitro	through epigenetic modulation, antioxidant and anti-inflammatory actions, Aβ inhibition, and neuroprotection, GL has multitarget therapeutic effectiveness against AD	identifies key molecular targets and interaction patterns along with dynamic stability and interaction	lacks a long-term toxicity study	supports antioxidant and preliminary safety, which is relevant to AD pathogenesis but requires in vivo validation
				direct assessment of cytocompatibility, which ensures the safety of GL	does not mimic the in vivo oxidative stress environment

Safety and Toxicological Reports on GL for AD

A few preclinical studies have indicated the safety and tolerability of GL. In an acute toxicity study, using Wistar rats, a 40% GL formulation, administered at a single high dose of 2000 mg/kg, did not result in any observed adverse effect. Moreover, repeated-dose studies spanning 28 and 90 days, together with dedicated reproductive and developmental toxicity assessments, revealed no treatment-related abnormalities in the treated animal groups even at a daily exposure of 100 mg/kg dose. Additionally, another 90 day clinical trial of GL combination therapy in patients suffering from nonalcoholic steatohepatitis reported no significant adverse effects nor any clinically relevant alterations in hematological or biochemical parameters in the GL-treated group. Collectively, these data underscore notable nontoxic and tolerability profile of GL for in vivo applications.

Despite these encouraging preclinical/clinical safety profiles, further extensive studies on dose optimization, toxicity profile, drug–drug interaction, and bioequivalent studies are warranted. Specifically, future research endeavors should incorporate long-term toxicity studies in suitable animal models to align its feasible clinical application in AD therapy. Additionally, well-designed phase-I clinical trials in human populations to delineate GL’s pharmacokinetics, biodistribution, and elimination half-life are also needed. Such varied investigations are required to provide a reliable and therapeutically relevant long-term safety profile for GL to validate its suitability as an anti-AD drug.

Novel Trends in Efficient Delivery of GL

Recent works have sufficiently documented potential application of GL including antimicrobial, antioxidants, anticancer, anti-inflammatory, hepatoprotective, antiobesity, and neuroprotective. However, low aqueous solubility, limited oral absorbance, limited plasma half-life, higher protein binding, and faster metabolic clearance leading to poor bioavailability of GL stand as main drawbacks to attain desirable therapeutic performance. The bioavailability studies of GL have also been scarcely reported. To address these inherent issues associated with GL, nanocarriers have progressively gained popularity over conventional macro drug delivery systems to improve delivery of GL to the brain. Various nanotechnology-based approaches like nanoliposomes, nanoparticles, solid-lipid nanostructures, niosomes, nanobilosomes, micelles, etc., have already shown promising avenues in enhancing brain delivery of active molecules. − Nanocarriers can modulate pharmacokinetic/pharmacodynamic properties of loaded cargo(s) and thus ensure improved plasma half-life and bioavailability than conventional dosage forms. − Delivery of GL through several nanocarrier-based formulations has been reported by some researchers. Further, to make an efficient nanodelivery system for GL, selection of suitable polymer/lipid/excipients and optimization of critical manufacturing steps and in-process parameters are crucial as they regulate crucial characteristics such as solubility, drug loading, stability, drug release profile, etc.

In a recent work, the delivery of GL was attempted through pH-sensitive biodegradable PLGA nanoparticles. Experimental GL-loaded nanoparticles were developed using the solvent evaporation approach. The results depict that the nanoparticles showed a sustained release of GL in vitro. A homogeneous size distribution with an average particle size of 295 nm was achieved for the GL-loaded nanoparticle. Additionally, experimental nanoparticles effectively reduced the activity of lactate dehydrogenase, TNF-α, IL-8, and NF-κB, which depicted potential anti-inflammatory action. Paul et al. encapsulated GL in PLGA nanoparticles coupled with the iRGD peptide on the particles’ surface to investigate its impact on colorectal cancer cell lines HCT116 and HT-29 both in vitro and in vivo. Experimental nanoparticles were spherical, closely associated, and between 90 to 210 nm in size. iRGD-modified GL nanoparticles depicted greater cytotoxic potency than GL nanoparticles on HCT116 and HT-29 cell lines in the MTT assay. When iRGD-GL-nanoparticles were applied to HCT116 cells, the IC₅₀ value was 2.3 times lower than that of GL nanoparticles indicating improvement in cytotoxic potential of GL following nanoencapsulation. Moreover, after 2 and 5 h after injection, the radioactivity of iRGD-GL-nanoparticles in the tumor site was considerably greater than that of GL nanoparticles, as shown by scintigraphic imaging. Further, 2.5 times more apoptosis in HCT116 cells was observed for iRGD–GL nanoparticles as compared to GL nanoparticles. FACS analysis depicted preferential cell cycle arrest in the G0/G1 phase by the nanoparticles (Figure ). In another study, GL-mediated silver nanoparticles were synthesized and assessed using a time kinetic analysis, broth microdilution assay, and well diffusion test. The morphology and size range of silver nanoparticles affected their antibacterial activity. The stability and biocompatibility were also dependent on the size. A preferentially higher antibacterial activity was detected for GL silver nanoparticles than GL alone. Further, nanoparticulate encapsulation increased GL’s solubility and depicted synergistic therapeutic efficacy. In another work, GL was encapsulated into PLGA nanoparticles; prepared using the nanoprecipitation technique with d-α-tocopheryl polyethylene glycol 1000 succinate and vitamin E. Data exhibited a negative zeta potential of −28.1 ± 2.1 and PDI value of 0.170 ± 0.05 with a sustained release pattern of GL in vitro. Higher anticancer effectiveness of GL-encapsulated nanoparticles was validated by in vitro cytotoxic activity. Data showed comparatively lower IC₅₀ values of 3, 5, and 3 μg/mL of the GL nanoparticle against HepG2, HCT-116, and KB cell lines, as compared to plain GL (IC₅₀: 9.6, 20, and 11 μg/mL) in the 48 h study period. Furthermore, GL nanoparticles significantly increased B16F10 cell apoptosis (63%) and the necrotic impact (33%) in comparison to GL.

5.

Synthesis and morphological analysis of GL nanoparticles and iRGD–GL nanoparticles. (A) AFM images of GL nanoparticles and iRGD–GL nanoparticles showing their topography, amplitude, and three-dimensional view (3D). (B) Transmission electron microscopy images of GL nanoparticles and iRGD–GL nanoparticles at two different magnifications (2,50,000× and 6,25,000×). (C) Bar diagram representing the biodistribution of ^99mTc-GL nanoparticles and ^99mTc-iRGD–GL nanoparticles in colorectal cancer-bearing Sprague Dawley rats at 1, 2, and 5 h of postinjection. (D) Scintigraphic images of colorectal cancer-bearing SD rats at 2 and 5 h postinjection of ^99mTc-GL nanoparticles and ^99mTc-iRGD–GL nanoparticles. Reprinted with permission from [Paul, B.; Gaonkar, R. H.; Dutta, D.; Dasi, R.; Mukherjee, B.; Ganguly, S.; Das, S. K. Inhibitory Potential of IRGD Peptide-Conjugated Garcinol-Loaded Biodegradable Nanoparticles in Rat Colorectal Carcinoma. Biomaterials Advances 2022, 134, 112714. ]. Copyright [2022 ELSEVIER B.V.].

Conclusion and Future Perspectives

Stagnant state of AD therapies encourages researchers in shifting focus from existing medications to novel phytoactive constituents as alternative, cost-effective, biocompatible therapeutic options with limited side effects. While Alzheimer’s therapy is on the verge of a paradigm change in view of technological advancement coupled with novel formulation design, a voluminous issue is yet to be addressed. HAT modulators in recent times have become a cutting-edge avenue in targeting the molecular pathway of AD. Hence, GL, a potent HAT inhibitor, has become a favorable therapeutic candidate toward the management of AD. Owing to its natural origin, biocompatible, and nontoxic profile, GL could be a lucrative entity for translational research and futuristic clinical trials. Though, various cell lines and animal-based models have evidenced the remarkable potential of GL in ameliorating neurodegeneration; however, novel technology-based targeted approaches of GL toward AD are very limited. Though various nanocarriers are being designed for GL to achieve desirable therapeutic effectiveness in challenging diseases like cancer, similar reports on GL-loaded nanocarriers in suitable AD models are scarce. Though GL possesses potent anti-inflammatory and antioxidant effects, its mechanistic insights in modulating AD pathophysiology through HAT inhibition have not been explored so far. Additionally, BBB permeation, in vivo half-life, chronic toxicity, dose optimization, bioavailability, pharmacokinetic profile, degradation kinetics, etc., of GL are yet to be studied in detail. Thus, preclinical research to comprehend long-term safety and efficacy of GL in various neurodegenerative disease models seems to be the need of the hour.

To address the inherent limitations of GL, nanocarrier-based approaches in view of their modulated physicochemical properties and pharmacokinetic characteristics possess the required potential to improve delivery of GL into the brain. Also, while designing suitable nanocarriers for GL, important formulation and process variables, including composition, particle size, surface charge, loading efficiency, colloidal stability in biorelevant media, and potential neurotoxic effects, necessitate meticulous optimization. Thus, future research should be directed to gather sufficient preclinical data on long-term safety and efficacy to facilitate clinical trials. Further, the effect of GL/GL-loaded nanocarriers across different neurodegenerative disease microenvironments needs to be tested. Advanced approaches, such as ligand-functionalized nanoparticles, receptor-mediated transcytosis, and intranasal delivery strategies utilizing olfactory and trigeminal pathways, are some of the evolving strategies to surmount these limitations. Rigorous validation of GL-loaded nanocarriers in pertinent AD animal models and in vitro–in vivo correlation analysis are also warranted to initiate a phase-1 clinical trial. Hence, a combined multifaceted approach encompassing nanotechnology, pharmacology, toxicology, and in silico simulation tools is essential to establish GL as a futuristic potent anti-AD drug.

Glossary

Abbreviations

AD

Alzheimer’s disease

Aβ

beta amyloid

NMDA

N-methyl-d-aspartic acid

HAT

histone acetyl transferase

GL

garcinol

GABA

γ-aminobutyric acid

HDAC

histone deacetylase

CDK5

cyclin-dependent kinase-5

GSK3β

glycogen synthase kinase-3-β

APP

amyloid precursor protein

NF-κB

nuclear factor kappa B

NCOA

nuclear receptor coactivator

CBP

p300/cAMP-response element-binding protein

MYST

Moz, Ybf2/Sas3, Sas2, Tip60

MOZ

monocytic leukemia zinc finger protein

KAT

lysine acetyltransferase

TIP60

tat-interacting protein, 60 kDa

MORF

MOZ-related factor

MOF

males absent on the first

SAS

something about silencing

ESA1

essential SAS-related acetyltransferase 1

HBO1

histone acetyltransferase binding to origin recognition complex 1

PCAF

p300-/CREB-binding protein-associated factor

TAF1

TATA-box-binding protein-associated factor 1

GTF3C1

general transcription factor IIIC Subunit

ELP3

elongator protein 3

HPA

histone acetyltransferase p300-/CBP-associated factor

GCN5

general control nonderepressible 5

PS1

preseniline-1

BDNF

brain-derived neurotrophic factor

GNAT

Gcn5-related N-acetyltransferase

lncRNAs

long noncoding RNAs

ATF-2

activating transcription factor 2

p-IκB-α

phosphorylated inhibitor of κB-alpha

TLR4

toll-like receptor 4

D-Gal

d-galactosamine

LPS

lipopolysaccharide

IL

interleukin

NO

nitric oxide

iNOS

inducible nitric oxide synthase

NOS

nitric oxide synthase-

COX-2

cyclooxygenase-2

LOX

lipoxygenase.

The authors so declare that there are no existing ethical concerns, all of the data in the work can be passed on for scientific purposes. No legitimate privacy, ethical, or safety concerns are violated by these data, nor do they compromise the confidentiality of human subjects. The data can be made available from corresponding author’s orcid id 0000–0002–8805–8682.

§.

Bhabani Sankar Satapathy has equal authorship rights with the author of the correspondence. Abhishek Mishra: Writingoriginal draft, Writingreview and editing, Visualization, Methodology. Bhabani Sankar Satapathy: Writingreview and editing, Writingoriginal draft, Validation, Investigation, Supervision, Data curation. Pratap Kumar Sahu: Writingreview and editing, Visualization, Validation, Supervision, Investigation, Conceptualization. Aruna Ghose: Writingoriginal draft, Validation, Methodology, Data curation. Sudhir Kumar Paidesetty: Writingreview and editing, Supervision, Investigation, Visualization.

The authors did not receive support from any organization for the submitted work.

All the authors of the manuscript have extended their consent for the publication.

The authors declare no competing financial interest.