Cellular pathways for experimental discovery provide a comprehensive overview of sirtuin biology and its critical involvement in HIV-associated neurocognitive disorders (HAND) and related neurodegenerative diseases, highlighting the translational potential of sirtuin-targeted therapeutic strategies. As NAD+-dependent deacetylases and ADP-ribosyl transferases, sirtuins regulate diverse cellular processes, including stem cell maintenance, cellular proliferation, metabolic homeostasis, apoptosis, autophagy, oxidative stress responses, and genomic stability, all of which contribute to neuronal dysfunction and disease progression. This chapter focuses on key mammalian sirtuins, including SIRT1 and SIRT2, which are primarily localized within the nucleus and cytosol; mitochondrial sirtuins SIRT3, SIRT4, and SIRT5; and nuclear/nucleolar sirtuins SIRT6 and SIRT7. Here, a method with a detailed protocol to isolate compartment-specific sirtuin expression and activity was used: subcellular fractionation was performed using a subcellular fractionation kit to obtain cytosolic and nuclear fractions, while mitochondrial isolation was carried out using Tom20 antibody-conjugated magnetic microbeads. These approaches were applied to brain tissues from HIV-positive individuals, as well as to HIV-Tat-treated human microglial (HMC3) cells and astrocytes. This experimental framework enables accurate assessment of compartment-resolved sirtuin regulation in disease-relevant models. Collectively, the chapter highlights the protective roles of sirtuins in mitigating key pathogenic mechanisms underlying HAND and related neurodegenerative diseases. These findings support the emerging concept that sirtuins represent promising pharmacological targets for the development of novel therapeutic interventions in neurodegeneration and HIV-associated brain disorders.
Perinatal depression (PND) is a prevalent and multifactorial mood disorder affecting approximately 10-20 % of women globally, with higher burdens reported in low- and middle-income countries. Despite the availability of screening tools such as the Edinburgh Postnatal Depression Scale, these approaches primarily identify risk without elucidating underlying biological mechanisms. Emerging evidence highlights the role of epigenetic regulation particularly DNA methylation as a key mediator linking genetic susceptibility and environmental exposures during the perinatal period. This review synthesizes current knowledge on DNA methylation dynamics in maternal depression, emphasizing both candidate gene and epigenome-wide association study (EWAS) approaches. Candidate gene studies have identified differential methylation in stress-related pathways, including HPA axis genes (NR3C1, FKBP5), serotonergic signalling (SLC6A4), and oxytocin pathways (OXTR), though findings remain limited by poor reproducibility and small sample sizes. In contrast, EWAS provides a hypothesis-free framework, identifying novel differentially methylated positions and regions associated with PND, including predictive CpG panels with potential diagnostic utility. The review also highlights the importance of tissue specificity, temporal epigenetic remodeling across pregnancy, and the interplay between maternal and fetal epigenomes. Furthermore, methodological challenges such as heterogeneity in study design, lack of replication, and analytical inconsistencies remain barriers to clinical translation. Integrating genetic, epigenetic, and environmental data through multi-omics approaches may enhance predictive accuracy and improve early intervention strategies. Overall, DNA methylation represents a promising avenue for understanding the biological underpinnings of PND and developing robust biomarkers for risk prediction and personalized care.
Mitochondrial dysfunction is one of the significant aspects of Parkinson's disease (PD) pathophysiology, marked by a gradual decline in oxidative phosphorylation, an abnormal increase in free radical species, dysfunctional mitochondrial quality control, and faulty mitochondrial biogenesis. Sirtuin 1 (SIRT1), a NAD+-dependent class-III deacetylase, acts as a crucial metabolic sensor that orchestrates transcriptional programs related to mitochondrial biogenesis, respiratory chain assembly, and stress resilience, mainly by way of deacetylation and activation of peroxisome proliferator-activated receptor gamma coactivator-1 alpha (PGC-1α). While screening for the small molecule activators, we have identified that 2,4-dihydroxy-Azaflavanone allosterically activates SIRT1. This chapter outlines a detailed, multi-layered methodological framework for assessing the allosteric activation of SIRT1 by 2,4-dihydroxy-azaflavanone and its downstream effects in a cellular models of PD. The validation process involves synthesis of small molecules, molecular docking studies utilizing crystallographic SIRT1 coordinates (PDB: 5BTR), in vitro fluorometric deacetylase assays with recombinant enzyme, and cellular thermal shift assays (CETSA) to confirm direct, isoform-selective target engagement. The activation of downstream pathways is evaluated by immunoblotting and quantitative PCR for PGC-1α, TFAM, and quantification of mitochondrial DNA (mtDNA) copy number. Functional restoration of mitochondria in cells is analyzed by assessing the overall mitochondrial bioenergetics parameters using Seahorse extracellular flux analyzer. Overall, this integrated approach offers robust, reproducible results for exploring SIRT1-activators in the mechanisms mediating neurodegenerative disease models.
HDAC4, a class II epigenetic regulator, plays a critical role in adult hippocampal neurogenesis and determines the fate of newly proliferating and/or differentiating neural stem cells (NSCs). However, repetitive mild traumatic brain injury (rmTBI) impairs neurogenesis, contributing to the pathophysiology of cognitive deficits observed in neurodegenerative and neuropsychiatric disorders. Here, we present a protocol: (1) to induce rmTBI in mice using a closed head injury (CHI) device and to validate the model by assessing cognitive/behavioral performances and (2) to perform intracranial injection of FLAG-HDAC4 into the dentate gyrus of adult mice using a stereotaxic apparatus, in order to investigate the role of HDAC4 in impaired adult neurogenesis. This is achieved by analyzing BrdU⁺ and Nestin⁺ cells (markers of NSC proliferation), DCX⁺ and ND1⁺ cells (markers of neuronal differentiation), along with immunohistochemistry, fluorescence microscopy, and image analysis.
Lipid metabolism plays a crucial role in cellular health and physiology by acting as an energy storehouse, cell membrane component, brain development and signaling molecules. Crucial steps to metabolize dietary fat take place within the hepatic tissue. Any abnormalities in the hepatic fatty metabolic pathways cause abnormal accumulation of lipid inside the liver, causing MAFLD, ranging from simple steatosis to more complex steatohepatitis and fibrosis. During high-fat-diet-induced hepatic inflammation, systemic proinflammatory cytokines disrupt the blood-brain barrier, resulting in neuroinflammation, cognitive impairment, brain damage and even neurodegeneration. Further, during this altered metabolic scenario, circulating metabolites pass through the impaired BBR and deregulate the epigenetic landscape of the central nervous system. Thus, it becomes crucial to understand the epi-metabolic crosstalk between two crucial organs of our body: the liver and the brain. Here in this chapter, we demonstrate the approach that we are using in our laboratory to study the epigenetic reprogramming in the context of metabolic gene expression in the liver, which is the causal for life style disorders like MAFLD. Remarkably, we intend to understand how liver dysfunction can have an implication in the brain function. Here, we discuss the concept of developing a diet-induced steatosis and steatohepatitis mouse model to understand the disease progression and its interconnection with brain physiology. Further, we also demonstrate 2D and 3D cell culture models to study the liver-brain cross-talk in greater molecular detail. Collectively, these approaches can provide a template for studying the role of epi-metabolic cross-talk in liver-guided brain dysfunction upon MAFLD.
Epigenetic dysregulation is recognized as a primary contributor to the pathogenesis of neurodegenerative disorders, especially Alzheimer`s disease (AD). Epigenetic mechanisms, including DNA methylation, histone modifications, and non-coding RNA regulation, govern gene expression without altering the DNA sequence, which plays a crucial role in neuronal development, synaptic plasticity, and memory formation. In AD, abnormal epigenetic alterations disrupt neuronal homeostasis, promote Aβ aggregation, tau hyperphosphorylation, and neuroinflammation, which leads to cognitive impairment. This chapter explores the assays performed and their processes using epigenetic modulators, along with their therapeutic potential in neurodegenerative diseases, especially AD. HDAC inhibitors, DNMT inhibitors, and emerging approaches, such as PROTACs for selective degradation of epigenetic enzymes, were discussed in the context of neurocognitive disorders. Preclinical and clinical evidence suggest that targeting specific HDAC isoforms (HDAC3, HDAC6, etc) can restore synaptic plasticity and improve cognitive function. This chapter further discusses recent advances, challenges in drug specificity, BBB permeability, and off-target epigenetic effects, which remain barriers to clinical translation. Case studies highlighting successful epigenetic interventions in AD models were presented to demonstrate therapeutic feasibility. Overall, epigenetic modulators present a promising therapeutic approach for neurodegeneration, and continued research integrating various assays like DNA methylation analysis, histone modification analysis, non-coding RNA analysis, neuroinflammation analysis, and functional and behavioral assays in AD models is significant in harnessing the full potential for AD treatment.
p300/CBP is a lysine acetyltransferase enzyme that modifies the lysine residue of histones by adding acetyl groups from acetyl-CoA. Dysregulation of p300/CBP KAT activity has been implicated in several neurological disorders. Pharmacological activation of p300/CBP via small-molecule provides a promising strategy to restore acetylation balance and promote neuronal repair. Our laboratory has reported that the p300/CBP-specific small molecule KAT activator TTK21 (N-[4-chloro-3-(trifluoromethyl)phenyl]-2-propoxy-benzamide), when conjugated to a glucose-derived carbon nanosphere (CSP), efficiently crossed the plasma membrane and induced acetylation. Surprisingly, its systemic administration in mice resulted in increased acetylation levels in the frontal cortex and hippocampus. Furthermore, it induced axon regeneration and improved functional recovery after spinal cord injury. Interestingly, oral administration of CSP-TTK21 also induced acetylation and promoted motor recovery in a rat model of spinal cord injury. In this chapter, we illustrate a method for the chemical synthesis of TTK21, glucose-derived carbon nanosphere (CSP), and their conjugate (CSP-TTK21). In addition, we systematically describe the methodologies for the chemical and biological characterization of the KAT activator, including the evaluation of histone acetylation in vitro and in vivo. Lastly, we present detailed experimental procedures to assess the therapeutic efficacy of CSP-TTK21 in models of spinal cord injury. Collectively, this chapter provides comprehensive protocols and practical guidance for the characterization and biological evaluation of p300/CBP activators for spinal cord injury repair.
Aging is accompanied by progressive alterations in chromatin organization that contribute to transcriptional dysregulation, genomic instability, and loss of cellular identity. Among the epigenetic mechanisms implicated, histone modifications play a central role in regulating chromatin accessibility and gene expression during aging. Changes in key histone marks, including H3K9me3, H3K27me3, H4K20me3, and histone acetylation signatures, are associated with heterochromatin loss, increased transcriptional noise, and altered cellular function across diverse tissues and organisms. These modifications are dynamically regulated by histone-modifying enzymes and are responsive to metabolic and stress signaling pathways, linking environmental cues to chromatin state and longevity. Genome-wide profiling of histone modifications is essential to understand these age-associated chromatin changes. ChIPmentation, a streamlined approach that integrates chromatin immunoprecipitation with transposase-mediated tagmentation, enables rapid and sensitive mapping of histone marks with reduced input requirements and simplified library preparation. This method is particularly advantageous for aging studies, where sample availability is often limiting. In this chapter, we outline the principles and experimental workflow of ChIPmentation, including crosslinking, chromatin fragmentation, immunoprecipitation, tagmentation, library preparation, sequencing, and data analysis. Additionally, we discuss computational strategies for processing and analyzing ChIPmentation data, including alignment, normalization, peak calling, and reproducibility assessment. Together, this chapter provides a comprehensive methodological framework for studying histone modification landscapes in aging systems, enabling robust investigation of epigenetic mechanisms underlying age-associated phenotypes.
Ischemic stroke induced cerebral edema is a devastating complication which leads to excessive swelling, increased intracranial pressure (ICP) and rapid neurological deterioration. The mortality rate of post-stroke cerebral edema is approximately 80 %. However, therapeutic options for post-stroke cerebral edema are sparce, emphasizing an urgent need to explore new therapeutic strategies for post-stroke edema. In past few decades stem cell therapy has been a promising therapeutic strategy in decreasing infarct volume and promoting functional recovery by various mechanisms following stroke. Previous studies from our lab demonstrated that IA-MSCs administration were beneficial in mitigating post-stroke edema by regulating PKCδ/ MMP9/AQP4 axis and promoting blood-brain-barrier (BBB) integrity. It has also been reported that IA-MSCs administration mitigate post-stroke edema by maintaining BBB integrity, mitochondria function and regulating AQP4 expression by modulation of the SIRT-1/PKCδ/NF-κB axis. In this chapter, we illustrated a multi-level methodological approach to assess therapeutic outcomes of IA-MSCs in alleviating post-stroke cerebral edema by utilizing behavioural analyses, histological staining, biochemical assays, mitochondrial functional assays and molecular investigations by RT-PCR, western blotting and immunofluorescence staining.
Development of Alzheimer's is promoted through the accumulation of Tau and Amyloid Beta (Aβ) proteins at various neuronal as well as glial junctions. Tau, a microtubule-associated protein, is localized at the axonal region of neurons under physiological conditions. The main function of Tau protein is to stabilize microtubules, mediated by the electrostatic interaction of their surface with the repeat domains of Tau. The post-translational modifications (PTMs) are necessary for the physiological functioning of protein, but upon abnormal phosphorylation of Tau, Neuro-fibrillary Tangles of protein are generated disrupting normal brain functionality. Cell migration is a physiological functioning of the cell necessary for various signalling from development, cellular communication, immune function etc. cellular microenvironment affect the behaviour of the cell, which can be detected by its migration propensity. The in vitro assays assist in understanding the adhesion, migration and invasion strategies of migratory cells in response to extracellular stimuli. The migration property can be linked to phenotypic changes of microglia as anti-inflammatory phenotype of microglia display increase migration and invasion, hence understanding migration propensity over Tau and HDAC6 exposure is necessary.
Histone tails undergo various post-translational modifications that aid in recruiting effector proteins involved in diverse transcriptional responses. Our previous work explored the role of histone H3 N-terminal residues and their modifications on the transcriptional regulation of the CUP1 metallothionein gene during cellular copper stress. To pinpoint critical histone residues, we used complementary methods, including growth assays and gene expression kinetics. We screened a library of synthetic histone H3 and H4 mutants for copper response defects by assessing their sensitivity to copper stress and quantifying CUP1 transcript levels via real-time PCR. We further investigated the mechanisms of impaired CUP1 transcription by assessing the recruitment of Ace1, a copper-sensing transcription factor that activates CUP1 in response to high intracellular copper, and of TATA-binding protein (TBP), using chromatin immunoprecipitation (ChIP) assays. Our results showed that mutations in the H3 N-terminal tail impair the recruitment of Ace1 and TBP to the CUP1 promoter, causing defects in its induction. Collectively, these findings highlight the significance of histone residues and their modifications in regulating metal homeostasis. This chapter outlines the methods used to study histone-mediated regulation of copper homeostasis, with a focus on the chromatin immunoprecipitation assay.
Sirtuins are NAD+ dependent class of histone deacetylase enzymes responsible for post-translational modifications regulating transcription, cell cycle, metabolism, DNA repair and apoptosis. Substrates of sirtuins are histones, α-tubulin, p65, FOXO1 etc. all of which have multiple NƐ-acetyl lysine residues taking part in catalytic reactions of sirtuins with the help of NAD+. In this chapter, we present methods for Solid Phase Peptide Synthesis and solution phase cyclization of a series of substrate-based peptide inhibitors of sirtuins and their cyclic and CPP conjugated derivatives KP 1, Cyc KP 1, Tat KP 1, KP 2, Cyc KP 2, Tat KP 2, KP 3, Cyc KP 3 and Tat KP 3. Further, describe methods that could be used to study sirtuin inhibition activity and cytotoxicity properties of peptides and small molecules. We present data of three peptides from this family Tat KP 1, Tat KP 2 and Tat KP 3 that show promising yeast sirtuin and mammalian SIRT1 inhibition potential (IC50 6-12 µM) comparable to known sirtuin inhibitors suramin and splitomicin and also showed cytotoxicity against HeLa and BE(2)-C cells. Finally, the morphological studies using SEM and TEM showed membrane disruption potential and evidence of apoptosis of Tat conjugated derivatives. This study documents the one of the initial reports of Tat conjugation modification to enhance the sirtuin inhibition potential. Collectively, this chapter provides comprehensive protocols and practical guidance for the characterization and biological evaluation of sirtuin inhibitors which could be used to identify and characterize novel peptides inhibitors of sirtuins with cell penetrating properties.
HDACs (histone deacetylases) are components of multiprotein complexes that remove the acetyl group from lysine residues (and other acyl groups) to regulate protein function. This important post-translational modification can affect the local hydrophobic environment of a specific protein surface. When the protein substrates are histones, the regulation of lysine acetylation influences chromatin accessibility and gene expression. Defining the different genomic regions under the influence of specific HDACs is fundamental to understanding how these enzymes control the epigenetic status of cells. Furthermore, it is important to clarify the mechanisms of action of HDAC inhibitors from a therapeutic perspective. ChIP-seq (Chromatin Immunoprecipitation followed by sequencing) is a powerful technique to identify where histone acetylations regulated by HDACs or influenced by HDAC inhibitors occur in the genome. Here, we describe a protocol to perform a ChIP-seq experiment to detect the genome-wide distribution of histone acetylation.
SYNGAP1 encodes a Ras GTPase-activating protein essential for neurodevelopment and synaptic plasticity, and heterozygous loss-of-function mutations are strongly associated with intellectual disability (ID) and autism spectrum disorder (ASD). While synaptic and circuit-level impairments resulting from SYNGAP1 haploinsufficiency have been well characterized, the contribution of epigenetic dysregulation remains poorly understood. In this chapter, we describe experimental approaches to investigate histone acetylation-dependent mechanisms underlying SYNGAP1-associated neurodevelopmental phenotypes. Using Syngap1+/- mice, we demonstrate a robust reduction in p300/CBP-specific histone acetylation marks in the adolescent hippocampus, accompanied by impaired dendritic arborization of adult-born doublecortin-positive (DCX+) neurons, indicative of disrupted adult hippocampal neurogenesis. To establish causality between altered chromatin state and behavioural and circuit dysfunction, we employed a glucose-derived carbon nanosphere-conjugated small-molecule activator of p300/CBP (CSP-TTK21) in young adult Syngap1+/- mice (2-4 months). Pharmacological enhancement of p300/CBP activity restored histone acetylation, rescued synaptic and structural plasticity, normalized experience-dependent cortical circuit reorganization, and significantly improved behavioural performance to levels comparable with wild-type littermates. Transcriptomic profiling by hippocampal RNA sequencing revealed reversal of dysregulated gene expression programs, including key regulators of synaptic plasticity and neurogenesis. Collectively, the methods described in this chapter provide a framework for dissecting epigenetic contributions to SYNGAP1-associated neurodevelopmental disorders and establish chromatin-targeted modulation of p300/CBP as a reversible and therapeutically actionable mechanism for ID/ASD-related circuit dysfunction.
Histone deacetylase 6 is a unique cytoplasmic deacetylase implicated in cellular functions such as microtubule dynamics, protein quality control, ubiquitin-mediated degradation and neurodegenerative disorders. The ZnF UBP (zinc finger ubiquitin binding protein) domain of HDAC6 is known to be directly modulate several cellular processes linked to neurodegeneration such as sequestering polyubiquitinated aggregates and regulating protein aggregate clearance mechanisms in neurons. Microtubule associated protein Tau (MAP Tau) undergoes aggregation in neurodegenerative conditions like Alzheimer's disease (AD) and several other tauopathies. Tau is a natively disordered protein which is functionally regulated by wide array of post-translational modifications (PTMs) as well as by interacting with several proteins. This methodological study aims to understand the molecular interaction between HDAC6 ZnF UBP domain and Tau protein in order to elucidate the role of HDAC6 ZnF UBP domain in Tau aggregation and stability. We employed an integrated biochemical, biophysical and computational workflow to characterize the interaction between HDAC6 ZnF UBP and Tau. NMR spectroscopy, isothermal titration calorimetry and pull-down assay with purified HDAC6 ZnF UBP and Tau proteins demonstrated direct interaction between the two, with interaction associated structural perturbations and favourable binding kinetics as observed in NMR and ITC respectively. Computational analyses further suggest the formation of Tau-HDAC6 ZnF UBP complex and provided underlying molecular interactions involved in the binding of these two proteins. The findings in this study helps to advance the current understanding of regulatory role of HDAC6 specifically in Tau biology and further provides a useful framework for investigating the modulation of aggregation prone proteins via protein-protein interaction in neurodegenerative diseases.
