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An enduring deficit in neurogenesis largely contributes to the development of severe post-traumatic psychiatric disorders such as anxiety, depression, and memory impairment following traumatic brain injury (TBI); however, the mechanism remains obscure. Here we have shown that an imbalance in the generation of γ-aminobutyric acid (GABA)ergic and glutamatergic neurons due to aberrant induction of vesicular glutamate transporter 1 (vGlut1)-positive glutamatergic cells is responsible for impaired neuronal differentiation in the hippocampus following TBI. To elucidate the molecular mechanism, we found that TBI activates a transcription factor, Pax3, by increasing its acetylation status, and subsequently induces Ngn2 transcription. This event, in turn, augments the vGlut1-expressing glutamatergic neurons and accumulation of excess glutamate in the hippocampus that can affect neuronal differentiation. In our study the acetylation of Pax3 was increased due to loss of its interaction with a deacetylase, histone deacetylase 4 (HDAC4), which was downregulated after TBI. TBI-induced activation of GSK3β was responsible for the degradation of HDAC4. We also showed that overexpression of HDAC4 before TBI reduces Pax3 acetylation by restoring an interaction between HDAC4 and Pax3 in the hippocampus. This event prevents the aberrant induction of vGlut1-positive glutamatergic neurons by decreasing the Ngn2 level and subsequently reinforces the balance between GABAergic and glutamatergic neurons following TBI. Further, we found that overexpression of HDAC4 in the hippocampus improves anxiety, depressive-like behavior, and memory functions following TBI.

Tau, a microtubule-associated protein promotes assembly and stability of microtubules which is related to axoplasmic flow and critical neuronal activities upon physiological conditions. Under neurodegenerative condition such as in Alzheimer’s Disease (AD), tau-microtubule binding dynamics and equilibrium are severely affected due to its aberrant post-translational modifications including acetylation and hyperphosphorylation. This event results in its conformational changes to form neurofibrillary tangles (NFT) after aggregation in the cytosol. The formation of NFT is more strongly correlated with cognitive decline than the distribution of senile plaque, which is formed by polymorphous beta-amyloid (Ax) protein deposits, another pathological hallmark of AD. In neurodegenerative conditions, other than AD, the disease manifestation is correlated with mutations of the MAPT gene. In Primary age-related tauopathy (PART), which is commonly observed in the brains of aged individuals, tau deposition is directly correlated with cognitive deficits even in the absence of Aβ deposition. Thus, tauopathy has been considered as an essential hallmark in neurodegeneration and normal brain aging. In this review, we highlighted the recent progress about the tauopathies in the light of its posttranslational modifications and its implication in AD and the aged brain.

In Alzheimer’s disease (AD), human Tau is phosphorylated at S199 (hTau-S199-P) by the protein kinase glycogen synthase kinase 3β (GSK3β). HTau-S199-P mislocalizes to dendritic spines, which induces synaptic dysfunction at the early stage of AD. The AKT kinase, once phosphorylated, inhibits GSK3β by phosphorylating it at S9. In AD patients, the abundance of phosphorylated AKT with active GSK3β implies that phosphorylated AKT was unable to inactivate GSK3β. However, the underlying mechanism of the inability of phosphorylated AKT to phosphorylate GSK3β remains unknown. Here, we show that total AKT and phosphorylated AKT was sulfhydrated at C77 due to the induction of intracellular hydrogen sulfide (H(2)S). The increase in intracellular H(2)S levels resulted from the induction of the proinflammatory cytokine, IL-1β, which is a pathological hallmark of AD. Sulfhydrated AKT does not interact with GSK3β, and therefore does not phosphorylate GSK3β. Thus, active GSK3β phosphorylates Tau aberrantly. In a transgenic knockin mouse (AKT-KI(+/+)) that lacked sulfhydrated AKT, the interaction between AKT or phospho-AKT with GSK3β was restored, and GSK3β became phosphorylated. In AKT-KI(+/+) mice, expressing the pathogenic human Tau mutant (hTau-P301L), the hTau S199 phosphorylation was ameliorated as GSK3β phosphorylation was regained. This event leads to a decrease in dendritic spine loss by reducing dendritic localization of hTau-S199-P, which improves cognitive dysfunctions. Sulfhydration of AKT was detected in the postmortem brains from AD patients; thus, it represents a posttranslational modification of AKT, which primarily contributes to synaptic dysfunction in AD.

The brain undergoes several changes at structural, molecular, and cellular levels leading to alteration in its functions and these processes are primarily maintained by proteostasis in cells. However, an imbalance in proteostasis due to the abnormal accumulation of protein aggregates induces endoplasmic reticulum (ER) stress. This event, in turn, activate the unfolded protein response; however, in most neurodegenerative conditions and brain injury, an uncontrolled unfolded protein response elicits memory dysfunction. Although the underlying signaling mechanism for impairment of memory function following induction of ER stress remains elusive, recent studies have highlighted that inactivation of a transcription factor, CREB, which is essential for synaptic function and memory formation, plays an essential role for ER stress–induced synaptic and memory dysfunction. In this review, current studies and most updated view on how ER stress affects memory function in both physiological and pathological conditions will be highlighted.

Persistent endoplasmic reticulum (ER) stress in neurons is associated with activation of inflammatory cells and subsequent neuroinflammation following traumatic brain injury (TBI); however, the underlying mechanism remains elusive. We found that induction of neuronal-ER stress, which was mostly characterized by an increase in phosphorylation of a protein kinase R-like ER kinase (PERK) leads to release of excess interferon (IFN)β due to atypical activation of the neuronal-STING signaling pathway. IFNβ enforced activation and polarization of the primary microglial cells to inflammatory M1 phenotype with the secretion of a proinflammatory chemokine CXCL10 due to activation of STAT1 signaling. The secreted CXCL10, in turn, stimulated the T-cell infiltration by serving as the ligand and chemoattractant for CXCR3+T-helper 1 (Th1) cells. The activation of microglial cells and infiltration of Th1 cells resulted in white matter injury, characterized by impaired myelin basic protein and neurofilament NF200, the reduced thickness of corpus callosum and external capsule, and decline of mature oligodendrocytes and oligodendrocyte precursor cells. Intranasal delivery of CXCL10 siRNA blocked Th1 infiltration but did not fully rescue microglial activation and white matter injury after TBI. However, impeding PERK-phosphorylation through the administration of GSK2656157 abrogated neuronal induction of IFNβ, switched microglial polarization to M2 phenotype, prevented Th1 infiltration, and increased Th2 and Treg levels. These events ultimately attenuated the white matter injury and improved anxiety and depressive-like behavior following TBI.SIGNIFICANCE STATEMENTA recent clinical study showed that human brain trauma patients had enhanced expression of type-1 IFN; suggests that type-1 IFN signaling may potentially influence clinical outcome in TBI patients. However, it was not understood how TBI leads to an increase in IFNβ and whether induction of IFNβ has any influence on neuroinflammation, which is the primary reason for morbidity and mortality in TBI. Our study suggests that induction of PERK phosphorylation, a characteristic feature of ER stress is responsible for an increase in neuronal IFNβ, which, in turn, activates microglial cells and subsequently manifests the infiltration of T cells to induce neuroinflammation and subsequently white matter injury. Blocking PERK phosphorylation using GSK2656157 (or PERK knockdown) the whole cascade of neuroinflammation was attenuated and improved cognitive function after TBI.

Traumatic Brain Injury (TBI) affects more than 1.7 million Americans each year and about 30% of TBI-patients having visual impairments. The loss of retinal ganglion cells (RGC) in the retina and axonal degeneration in the optic nerve have been attributed to vision impairment following TBI; however, the molecular mechanism has not been elucidated. Here we have shown that an increase in histone di-methylation at lysine 9 residue (H3K9Me2), synthesized by the catalytic activity of a histone methyltransferase, G9a is responsible for RGC loss and axonal degeneration in the optic nerve following TBI. To elucidate the molecular mechanism, we found that an increase in H3K9Me2 results in the induction of oxidative stress both in the RGC and optic nerve by decreasing the mRNA level of antioxidants such as Superoxide dismutase (sod) and catalase through impairing the transcriptional activity of Nuclear factor E2-related factor 2 (Nrf2) via direct interaction. The induction of oxidative stress is associated with death in RGC and oligodendrocyte precursor cells (OPCs). The death in OPCs is correlated with a reduction in myelination, and the expression of myelin binding protein (MBP) in association with degeneration of neurofilaments in the optic nerve. This event allied to an impairment of the retrograde transport of axons and loss of nerve fiber layer in the optic nerve following TBI. An administration of G9a inhibitor, UNC0638 attenuates the induction of H3K9Me2 both in RGC and optic nerve and subsequently activates Nrf2 to reduce oxidative stress. This event was concomitant with the rescue in the loss of retinal thickness, attenuation in optic nerve degeneration and improvement in the retrograde transport of axons following TBI.

Acetylation of the microtubule-associated protein tau promotes its polymerization into neurofibrillary tangles that are implicated in the pathology of Alzheimer's disease (AD). The gaseous neurotransmitter nitric oxide (NO) regulates cell signaling through the nitrosylation of proteins. We found that NO production and tau acetylation at Lys280 occurred in the brain tissue in mice and in cultured mouse cortical neurons in response to exposure to amyloid-β1-42 (Aβ1-42), a peptide that is also implicated in AD. An increased abundance of NO facilitated the S-nitrosylation (SNO) of glyceraldehyde-3-phosphate dehydrogenase (GAPDH). S-nitrosylated GAPDH (GAPDH-SNO) promoted the acetylation and activation of the acetyltransferase p300 and facilitated the nitrosylation and inactivation of the deacetylase sirtuin 1 (SIRT1). The abundance of GAPDH-SNO was increased in postmortem brain samples from AD patients. Preventing the increase in GAPDH-SNO abundance in both cultured neurons and mice, either by overexpression of the nitrosylation mutant of GAPDH (GAPDH C150S) or by treatment with the GAPDH nitrosylation inhibitor CGP3466B (also known as omigapil), abrogated Aβ1-42-induced tau acetylation, memory impairment, and locomotor dysfunction in mice, suggesting that this drug might be repurposed to treat patients with AD.

Traumatic brain injury (TBI) is one of the major cause of morbidity and mortality and it affects more than 1.7 million Americans each year. Depending on its location and severity, TBI leads to structural and functional damage in several parts of the brain such as cranial nerves, optic nerve tract or other circuitry involved in vision, and occipital lobe. As a result, the function associated with vision processing and perception are significantly affected and cause blurred vision, double vision, decreased peripheral vision and blindness. In this mini-review, we will focus the recent progress made to understand the pathology and underlying cellular/molecular mechanisms involved in the impairment of the integrity of visual systems following TBI.

Induction of a proinflammatory cytokine, interleukin-1β (IL-1β) plays a role in memory impairment associated with various neurological disorders and brain injury. Here we show that IL-1β-induced memory impairment in brain is mediated by hydrogen sulfide (H2S) synthesized by cystathionine beta-synthase (CBS). H2S modifies GAPDH essentially via sulfhydration in dendrites, which promotes its binding to the E3 ligase protein, Siah. Then Siah binds to a critical synaptic scaffolding molecule, PSD95, and leads it to degradation via ubiquitination. In CBS heterozygous mice (cbs(+/-)) and primary neurons depleted with either CBS or IL-1R, IL-1β-induced loss of PSD95 was rescued along with a decrease in the level of GAPDH sulfhydration. Moreover, decrease in the loss of PSD95 in cbs(+/-) mice results in improvement of IL-1β-induced cognitive deficits and neurobehavioral outcomes. Thus, our findings reveal a mechanism where GAPDH sulfhydration appears to be a physiologic determinant of cytokine-induced memory impairment in brain.

Synaptic plasticity is one of the most fundamental properties of neurons that underlie the formation of the memory in brain. In recent years, epigenetic modification of both DNA and histones such as DNA methylation and histone acetylation and methylation emerges as a potential regulatory mechanism that governs the transcription of several genes responsible for memory formation and behavior. Furthermore, the recent identification of nitrosylation of proteins has shown to either activate or repress gene transcription by modulating histone methylation or acetylation status in mature neuron. Recent studies suggest that the use of major substrates of abuse, e.g., cocaine, induces alterations in molecular and cellular mechanisms of epigenetics that underlie long-term memories in the striatum and prefrontal cortex. Moreover, downregulation of genes due to alterations in epigenetics leads to cognitive deficiencies associated with neurological disorders such as Alzheimer's disease, Huntington's disease, psychiatric disorder such as Rett's syndrome and aging. In this review, I will discuss the evidence for several epigenetic mechanisms in the coordination of complex memory formation and storage. In addition, I will address the current literature highlighting the role of acetylation and methylation of chromatin in memory impairment associated with several neurological disorders, aging, and addiction.

Increased cranial pressure due to development of edema contributes significantly to the pathology of traumatic brain injury (TBI). Induction of an astrocytic water channel protein, Aquaporin 4 (AQP4), is known to predominantly contribute to cytotoxic edema following TBI. However, the mechanism for the increase in AQP4 following 24 h of TBI is poorly understood. Here we show that transcriptional activation of a ubiquitously expressed mammalian forkhead transcription factor, Foxo3a, induces cerebral edema by increasing the AQP4 level in the controlled cortical impact model of TBI in mice. TBI stimulates nuclear translocation of Foxo3a in astrocytes and subsequently augments its binding to AQP4 promoter in pericontusional cortex. Nuclear accumulation of Foxo3a is augmented by a decrease in phosphorylation at its Ser256 residue due to inactivation of Akt after TBI. Depletion of Foxo3a in mice rescues cytotoxic edema by preventing induction of AQP4 as well as attenuates memory impairment after TBI in mice.

Profound induction of immediate early genes (IEGs) by neural activation is a critical determinant for plasticity in the brain, but intervening molecular signals are not well characterized. We demonstrate that inositol polyphosphate multikinase (IPMK) acts noncatalytically as a transcriptional coactivator to mediate induction of numerous IEGs. IEG induction by electroconvulsive stimulation is virtually abolished in the brains of IPMK-deleted mice, which also display deficits in spatial memory. Neural activity stimulates binding of IPMK to the histone acetyltransferase CBP and enhances its recruitment to IEG promoters. Interestingly, IPMK regulation of CBP recruitment and IEG induction does not require its catalytic activities. Dominant-negative constructs, which prevent IPMK-CBP binding, substantially decrease IEG induction. As IPMK is ubiquitously expressed, its epigenetic regulation of IEGs may influence diverse nonneural and neural biologic processes.

Purpose: Cystathionine β-synthase (CBS), a key enzyme in the transsulfuration metabolic pathway, converts homocysteine to cystathionine, which is converted to cysteine required for the synthesis of major retinal antioxidant glutathione (GSH). Enzyme activity assays suggest that CBS is present in human and pig retina, however recent studies reported that CBS is not expressed in mouse retina. We found this species difference puzzling. Given the plethora of studies using mouse retina as a model system, coupled with the importance of GSH in retina, we investigated CBS expression in mouse retina at the molecular and cell biological level.Wildtype (WT) mice or mice lacking the gene encoding CBS (cbs(-)(/)(-)) were used in these studies. RNA and protein were isolated from retinas and liver (positive control) for the analysis of cbs gene expression by RT-PCR and CBS protein expression by Western blotting, respectively. CBS was analyzed by immunofluorescence in retinal cryosections and primary retinal cells (ganglion, Müller, retinal pigment epithelial). CBS enzyme activity was measured in primary Müller cells.RT-PCR revealed robust cbs expression in WT liver, brain and retina. Western blotting detected CBS in retina, brain and liver of WT mice, but not in cbs(-)(/)(-) mice liver. In immunohistochemical studies, CBS was present abundantly in the ganglion cell layer of retina; it was detected also in primary isolations of Müller, RPE and ganglion cells. CBS activity was detected in Müller cells by fluorescent detection of H2S.We have compelling molecular evidence that CBS is expressed in mouse retina at the gene and protein level. Our immunofluorescence data suggest that it is present in several retinal cell types and the data from the enzyme activity assay suggest activity in Müller cells. These findings set the stage to investigate the role of CBS and the transsulfuration pathway in the generation of GSH in mouse retina.

Hydrogen sulfide (H2S) a novel gasotransmitter is endogenously synthesized by multiple enzymes that are differentially expressed in the peripheral tissues and central nervous systems. H2S regulates a wide range of physiological processes ranging from cardiovascular, neuronal, immune, respiratory, gastrointestinal, liver, and endocrine systems by influencing cellular signaling pathways and sulfhydration of target proteins. This review focuses on the recent progress made in H2S signaling that affects mechanistic and functional aspects of several biological processes such as autophagy, inflammation, proliferation and differentiation of stem cell, cell survival/death, and cellular metabolism under both physiological and pathological conditions. Moreover we highlighted the crosstalk between nitric oxide (NO) and H2S in several bilogical contexts.

Anesthetics including isoflurane are known to induce neuronal dysfunction in the developing brain, however, the underlying mechanism is mostly unknown. The transcriptional activation of CREB (cyclic AMP response element binding protein) and the alterations in acetylation of histones modulated by several histone deacetylases such as HDAC4 (histone deacetylase 4) are known to contribute to synaptic plasticity in the brain. Here we have shown that administration of isoflurane (1.4%) for 2h leads to transcriptional inactivation of CREB which results in loss of dendritic outgrowth and decreased expression level of proteins essential for memory and cognitive functions, such as bdnf, and c-fos in the developing brain of mice at postnatal day 7 (PND7). To elucidate the molecular mechanism, we found that exposure to isoflurane leads to an increase in nuclear translocation of HDAC4, which interacts with CREB in the nucleus. This event, in turn, results in a decrease in interaction between an acetyltransferase, CBP, and CREB that ultimately leads to transcriptional inactivation of CREB. As a result, the expression level of bdnf, and cfos were significantly down-regulated after administration of isoflurane in PND7 brain. Depletion of HDAC4 in PND7 brain rescues the transcriptional activation of CREB along with augmentation in the level of the expression level of bdnf and c-fos. Moreover, administration of lentiviral particles of HDAC4 RNAi in primary neurons rescues neurite outgrowth following isoflurane treatment. Taken together, our study suggests that HDAC4- induced transcriptional inactivation of CREB is responsible for isoflurane-induced cognitive dysfunction in the brain.

The PKR-like ER kinase (PERK), a transmembrane protein, resides in the endoplasmic reticulum (ER). Its activation serves as a key sensor of ER stress, which has been implicated in traumatic brain injury (TBI). The loss of memory is one of the most common symptoms after TBI, but the precise role of PERK activation in memory impairment after TBI has not been well elucidated. Here, we have shown that blocking the activation of PERK using GSK2656157 prevents the loss of dendritic spines and rescues memory deficits after TBI. To elucidate the molecular mechanism, we found that activated PERK phosphorylates CAMP response element binding protein (CREB) and PSD95 directly at the S129 and T19 residues, respectively. Phosphorylation of CREB protein prevents its interaction with a coactivator, CREB-binding protein, and subsequently reduces the BDNF level after TBI. Conversely, phosphorylation of PSD95 leads to its downregulation in pericontusional cortex after TBI in male mice. Treatment with either GSK2656157 or overexpression of a kinase-dead mutant of PERK (PERK-K618A) rescues BDNF and PSD95 levels in the pericontusional cortex by reducing phosphorylation of CREB and PSD95 proteins after TBI. Similarly, administration of either GSK2656157 or overexpression of PERK-K618A in primary neurons rescues the loss of dendritic outgrowth and number of synapses after treatment with a PERK activator, tunicamycin. Therefore, our study suggests that inhibition of PERK phosphorylation could be a potential therapeutic target to restore memory deficits after TBI.SIGNIFICANCE STATEMENT Traumatic brain injury (TBI) is the leading cause of death and disability around the world and affects 1.7 million Americans each year. Here, we have shown that TBI-activated PKR-like ER kinase (PERK) is responsible for memory deficiency, which is the most common problem in TBI patients. A majority of PERK's biological activities have been attributed to its function as an eIF2α kinase. However, our study suggests that activated PERK mediates its function via increasing phosphorylation of CAMP response element binding protein (CREB) and PSD95 after TBI. Blocking PERK phosphorylation rescues spine loss and memory deficits independently of phosphorylation of eIF2α. Therefore, our study suggests that CREB and PSD95 are novel substrates of PERK, so inhibition of PERK phosphorylation using GSK2656157 would be beneficial against memory impairment after TBI.

Anesthetics including isoflurane are known to induce neuronal dysfunction in the developing brain, however, the underlying mechanism is mostly unknown. The transcriptional activation of CREB (cyclic AMP response element binding protein) and the alterations in acetylation of histones modulated by several histone deacetylases such as HDAC4 (histone deacetylase 4) are known to contribute to synaptic plasticity in the brain. Here we have shown that administration of isoflurane (1.4%) for 2 h leads to transcriptional inactivation of CREB which results in loss of dendritic outgrowth and decreased expression level of proteins essential for memory and cognitive functions, such as BDNF, and c-fos in the developing brain of mice at postnatal day 7 (PND7). To elucidate the molecular mechanism, we found that exposure to isoflurane leads to an increase in nuclear translocation of HDAC4, which interacts with CREB in the nucleus. This event, in turn, results in a decrease in interaction between an acetyltransferase, CBP, and CREB that ultimately leads to transcriptional inactivation of CREB. As a result, the expression level of BDNF, and c-fos were significantly down-regulated after administration of isoflurane in PND7 brain. Depletion of HDAC4 in PND7 brain rescues the transcriptional activation of CREB along with augmentation in the level of the expression level of BDNF and c-fos. Moreover, administration of lentiviral particles of HDAC4 RNAi in primary neurons rescues neurite outgrowth following isoflurane treatment. Taken together, our study suggests that HDAC4-induced transcriptional inactivation of CREB is responsible for isoflurane-induced cognitive dysfunction in the brain.

Nuclear factor κB (NF-κB) is an anti-apoptotic transcription factor. We show that the anti-apoptotic actions of NF-κB are mediated by hydrogen sulfide (H2S) synthesized by cystathionine gamma-lyase (CSE). TNFα treatment triples H2S generation by stimulating binding of SP1 to the CSE promoter. H2S generated by CSE stimulates DNA binding and gene activation of NF-κB, processes that are abolished in CSE deleted mice. As CSE deletion leads to decreased glutathione levels, resultant oxidative stress may contribute to alterations in CSE mutant mice. H2S acts by sulfhydrating the p65 subunit of NF-κB at cysteine-38, which promotes its binding to the co-activator ribosomal protein S3 (RPS3). Sulfhydration of p65 predominates early following TNFα treatment, then declines and is succeeded by a reciprocal enhancement of p65 nitrosylation. Anti-apoptotic influences of NF-κB, which are markedly diminished in CSE mutant mice. Thus, sulfhydration of NF-κB appears to be a physiologic determinant of its anti-apoptotic transcriptional activity.

Tumor protein (TP)-p53 family members often play proapoptotic roles, whereas nuclear factor κB (NF-κB) functions as a proapoptotic and antiapoptotic regulator depending on the cellular environment. We previously showed that the NF-κB activation leads to the reduction of the TP63 isoform, ΔNp63α, thereby rendering the cells susceptible to cell death upon DNA damage. However, the functional relationship between TP63 isotypes and NF-κB is poorly understood. Here, we report that the TAp63 regulates NF-κB transcription and protein stability subsequently leading to the cell death phenotype. We found that TAp63α induced the expression of the p65 subunit of NF-κB (RELA) and target genes involved in cell cycle arrest or apoptosis, thereby triggering cell death pathways in MCF10A cells. RELA was shown to concomitantly modulate specific cell survival pathways, making it indispensable for the TAp63α-dependent regulation of cell death. We showed that TAp63α and RELA formed protein complexes resulted in their mutual stabilization and inhibition of the RELA ubiquitination. Finally, we showed that TAp63α directly induced RelA transcription by binding to and activating of its promoter and, in turn, leading to activation of the NF-κB-dependent cell death genes. Overall, our data defined the regulatory feedback loop between TAp63α and NF-κB involved in the activation of cell death process of cancer cells.

Epigenetic regulation of histones mediates neurotrophin actions with histone acetylation enhancing cAMP response element-binding (CREB)-associated transcription elicited by brain-derived neurotrophic factor (BDNF) and nerve-growth factor (NGF). Roles for histone methylation in CREB's transcriptional activity have not been well characterized. We show that depletion of the histone methyltransferase suppressor of variegation 3–9 homolog 1 (SUV39H1) selectively augments BDNF- and NGF-mediated neurite outgrowth. SUV39H1 is the principal enzyme responsible for trimethylation of histone H3 at lysine 9, a molecular mark associated with transcriptional silencing. BDNF and NGF act via a signaling cascade wherein degradation of SUV39H1 down-regulates trimethylation of H3K9 in a nitric oxide-dependent pathway. BDNF activates neuronal NOS with the nitrosylated GAPDH/seven in absentia (Siah) homolog complex translocating to the nucleus. Degradation of SUV39H1 by Siah facilitates histone H3 on lysine 9 acetylation, CREB binding to DNA, enhanced expression of CREB-regulated genes and neurite outgrowth.

DNA topoisomerases of kinetoplastids represent a family of DNA processing enzymes that essentially solve the topological problems not only in nuclear DNA but also in kinetoplast DNA. We have, for the first time, identified aLeishmania donovanihomologue of bacterial and eukaryotic IA type of topoisomerase III protein and termed asLdTopIIIβ. Complementation study of wild-type and mutantLdTopIIIβwith slow-growing topoisomerase III mutant yeastS. cerevisiaerevealed the functional conservation of the leishmanial counterpart of topoisomerase IIIβprotein, the 327 tyrosine being the active site amino acid. A C-terminal deletion construct ofLdTopIIIβcould not suppress the slow-growth phenotype of mutant yeast, indicating the requirement of C-terminal region for the enzyme functionin vivo.LdTopIIIβlocalized inside the nucleus and kinetoplast of the parasite. Taken together, our study indicates functional conservation and possible role ofLdTopIIIβin parasite DNA processing.

Strategies to address resistance to platin drugs are greatly needed in human epithelial cancers (e.g., ovarian, head/neck, and lung) where platins are used widely and resistance occurs commonly. We found that upon ΔNp63α overexpression, AKT1 and phospho-AKT1 levels are upregulated in cancer cells. Investigations using gel-shift, chromatin immunoprecipitation and functional reporter assays implicated ΔNp63α in positive regulation of AKT1 transcription. Importantly, we found that ΔNp63α, AKT1, and phospho-AKT levels are greater in 2008CI3 CDDP-resistant ovarian cancer cells than in 2008 CDDP-sensitive cells. siRNA-mediated knockdown of ΔNp63α expression dramatically decreased AKT1 expression, whereas knockdown of either ΔNp63α or AKT1 decreased cell proliferation and increased death of ovarian and head/neck cancer cells. Conversely, enforced expression of ΔNp63α increased cancer cell proliferation and reduced apoptosis. Together, our findings define a novel ΔNp63α-dependent regulatory mechanism for AKT1 expression and its role in chemotherapeutic resistance of ovarian and head/neck cancer cells. Cancer Res; 71(3); 1167–76. ©2011 AACR.

Covalent modifications of intracellular proteins, such as phosphorylation, are generally thought to occur as secondary or tertiary responses to neurotransmitters, following the intermediation of membrane receptors and second messengers such as cyclic AMP. By contrast, the gasotransmitter nitric oxide directly S-nitrosylates cysteine residues in diverse intracellular proteins. Recently, hydrogen sulfide has been acknowledged as a gaso-transmitter, which analogously sulfhydrates cysteine residues in proteins. Cysteine residues are also modified by palmitoylation in response to neurotransmitter signaling, possibly in reciprocity with S-nitrosylation. Neurotransmission also elicits sumoylation and acetylation of lysine residues within diverse proteins. This review addresses how these recently appreciated protein modifications impact our thinking about ways in which neurotransmission regulates intracellular protein disposition.

S-nitrosylation of proteins by nitric oxide is a major mode of signalling in cells. S-nitrosylation can mediate the regulation of a range of proteins, including prominent nuclear proteins, such as HDAC2 (ref. 2) and PARP1 (ref. 3). The high reactivity of the nitric oxide group with protein thiols, but the selective nature of nitrosylation within the cell, implies the existence of targeting mechanisms. Specificity of nitric oxide signalling is often achieved by the binding of nitric oxide synthase (NOS) to target proteins, either directly or through scaffolding proteins such as PSD-95 (ref. 5) and CAPON. As the three principal isoforms of NOS--neuronal NOS (nNOS), endothelial NOS (eNOS) and inducible NOS (iNOS)--are primarily non-nuclear, the mechanisms by which nuclear proteins are selectively nitrosylated have been elusive. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is physiologically nitrosylated at its Cys 150 residue. Nitrosylated GAPDH (SNO-GAPDH) binds to Siah1, which possesses a nuclear localization signal, and is transported to the nucleus. Here, we show that SNO-GAPDH physiologically transnitrosylates nuclear proteins, including the deacetylating enzyme sirtuin-1 (SIRT1), histone deacetylase-2 (HDAC2) and DNA-activated protein kinase (DNA-PK). Our findings reveal a novel mechanism for targeted nitrosylation of nuclear proteins and suggest that protein-protein transfer of nitric oxide groups may be a general mechanism in cellular signal transduction.

Chemotherapy is the primary means of treating protozoan parasitic infections. A problem for chemotherapy is to find a novel and potential molecule in protozoa, which could be exploited as drug target. To reach this goal, mitochondrion of protozoa can be considered as the most valuable and potential organelle because of its unique structure and function compared to their natural host habitat. In fact, the respiratory systems of parasitic protozoa typically show greater diversity in electron pathways than do their host animals. These unique aspects of electron transport chain (ETC) complexes and their related enzymes represent promising targets for chemotherapy. A cytochrome independent Alternative Oxidase (AOX) in parasites is a leading drug target. Topoisomerases play key functions in replication and organization of kDNA, which is present in a specialized region of unique mitochondria known as kinetoplast. They are considered as potential targets for anti-parasitic drugs. Moreover, a novel pathway of type II Fatty acid synthesis in mitochondria of trypanosomatids provides a new array of inhibitors that could be effective against these parasites. Recent studies on the emergence of drug resistance severely limit the arsenal of available drugs against protozoan parasites. Particularly, mutations of cytochrome b gene of ETC or changes in iron homeostasis by mitochondrial enzyme aconitase alter sensitivity of MDR1 and regulate resistance level to anti-parasitic drugs. This review summarizes recent state of our knowledge and understanding of the action of various therapeutically applied substances on mitochondria and their potential application in the future.

The ubiquitious enzyme topoisomerase I can be targeted by drugs which turn these enzymes into cellular poisons and subsequently induce cell death. Drugs like staurosporine, which do not target topoisomerase I directly, can also lead to stabilization of topoisomerase I–DNA cleavable complexes by an indirect process of reactive oxygen species (ROS) generation and subsequent oxidative DNA damage. In this study, we show that betulinic acid, a catalytic inhibitor of topoisomerases, inhibits the formation of apoptotic topoisomerase I–DNA cleavable complexes in prostate cancer cells induced by drugs like camptothecin, staurosporine, and etoposide. Although events like ROS generation, oxidative DNA damage, and DNA fragmentation were observed after betulinic acid treatment, there is no topoisomerase I–DNA cleavable complex formation, which is a key step in ROS-induced apoptotic processes. We have shown that betulinic acid interacts with cellular topoisomerase I and prohibits its interaction with the oxidatively damaged DNA. Using oligonucleotide containing 8-oxoguanosine modification, we have shown that betulinic acid inhibits its cleavage by topoisomerase I in vitro. Whereas silencing of topoisomerase I gene by small interfering RNA reduces cell death in the case of staurosporine and camptothecin, it cannot substantially reduce betulinic acid–induced cell death. Thus, our study provides evidence that betulinic acid inhibits formation of apoptotic topoisomerase I–DNA complexes and prevents the cellular topoisomerase I from directly participating in the apoptotic process. [Cancer Res 2007;67(24):11848–58]

We have shown that treatment with luteolin in leishmanial cells causes loss of mt-DNA and induces apoptosis through mitochondria dependent pathway [Sen, N., Das, B.B., Ganguly, A., Banerjee, B., Sen, T., Majumder, H.K., 2006. Leishmania donovani: intracellular ATP level regulates apoptosis-like death in luteolin induced dyskinetoplastid cells. Experimental Parasitology, in press]. Here, we report that mitochondrial DNA depleted leishmanial cells require exogenous sources of pyruvate and uridine to survive and proliferate. The presence of pyruvate and uridine in a growing media help them to produce sufficient amount of glycolytic ATP to maintain the mitochondrial membrane potential in the absence of their functional ETC. Treatment of wild type cells with CPT causes generation of ROS that leads to apoptosis. But unlike the normal cells ROS was not generated in these mt-DNA depleted cells after treatment with CPT. Taken together we have shown for the first time that dyskinetoplastid cells are auxotrophic for pyruvate and uridine and apoptosis cannot be induced in these cells in the presence of CPT. Therefore, the presence of mitochondrial DNA is absolutely necessary for the cytotoxicity of CPT in kinetoplastid parasites.

Traumatic brain injury (TBI), a major global health and socioeconomic problem, is now established as a chronic disease process with a broad spectrum of pathophysiological symptoms followed by long-term disabilities. It triggers multiple and multidirectional biochemical events that lead to neurodegeneration and cognitive impairment. Recent studies have presented strong evidence that patients with TBI history have a tendency to develop proteinopathy, which is the pathophysiological feature of neurodegenerative disorders such as Alzheimer disease (AD), chronic traumatic encephalopathy (CTE), and amyotrophic lateral sclerosis (ALS). This review mainly focuses on mechanisms related to AD, CTE, and ALS that are induced after TBI and their relevance to the advancement of these neurodegenerative diseases. This review encompasses acute effects and chronic neurodegenerative consequences after TBI for a better understanding of TBI-induced neuronal death and to design therapies that will effectively treat patients in the primary or secondary progressive stages.

Leishmaniasis presents a spectrum of diseases ranging from benign cutaneous lesions to the often-fatal visceralizing form. Luteolin, a dietary flavone induces apoptosis-like death in both promastigote and amastigote forms of Leishmania, the causative agent of the diseases. Here, we have elucidated the mechanism of action of luteolin by analyzing the mitochondrial and cytosolic changes associated with apoptosis-like death of leishmanial cells. In Leishmania donovani, treatment with luteolin induces the loss of both maxicircles and minicircles which resulted in the formation of dyskinetoplastid cells. The loss of mitochondrial DNA causes reduction in the activities of complex I, II, III, and IV of electron transport chain. However, the mitochondrial ATPase activity of complex V remains almost unaltered during treatment with luteolin but the sensitivity to oligomycin is lost. The inactivation of ETC complex is associated with decrease in mitochondrial as well as glycolytic ATP production, which is responsible for depolarization of Deltapsi(m) and alteration in mitochondrial structure. This event is followed by the release of cytochrome c from mitochondria in mt-DNA depleted leishmanial cells and causes an activation of caspase like proteases. Collectively our results provide the first insight into the mechanistic pathway of apoptosis-like death where inhibition of glycolytic ATP production is an essential event responsible for depolarization of Deltapsi(m) in mt-DNA depleted cells to propagate apoptosis-like death in leishmanial cells.

Protein kinase C (PKC) is an important constituent of the signaling pathways involved in apoptosis. We report here that like staurosporine, withaferin A is a potent inhibitor of PKC. In Leishmania donovani, the inhibition of PKC by withaferin A causes depolarization of DeltaPsim and generates ROS inside cells. Loss of DeltaPsim leads to the release of cytochrome c into the cytosol and subsequently activates caspase-like proteases and oligonucleosomal DNA cleavage. Moreover, in treated cells, oxidative DNA lesions facilitate the stabilization of topoisomerase I-mediated cleavable complexes, which also contribute to DNA fragmentation. However, withaferin A and staurosporine cannot induce cleavable complex formation in vitro with recombinant topoisomerase I nor with nuclear extracts from control cells. Taken together, our results indicate that inhibition of PKC by withaferin A is a central event for the induction of apoptosis and that the stabilization of topoisomerase I-DNA complex is necessary to amplify apoptotic process.

Leishmania donovani topoisomerase I is an unusual bi-subunit enzyme. The activity of the enzyme has been detected when the genes of the individual subunits were co-expressed in yeast [J. Biol. Chem. 278 (2003) 3521]. Here, we report for the first time, the in vitro reconstitution of the two recombinant proteins, LdTOP1L and LdTOP1S, corresponding to the large and small subunits and localization of the active enzyme in both the nucleus and kinetoplast. The proteins were purified from bacterial extract and the activity was measured by plasmid DNA relaxation assay. LdTOP1L and LdTOP1S form a direct 1:1 heterodimer complex through protein–protein interaction. Under standard relaxation assay condition (50 mM KCl and 10 mM Mg 2+ ), reconstituted enzyme (LdTOP1LS) showed reduced processivity as well as 2-fold reduced affinity for DNA compared to eukaryotic monomeric rat liver topoisomerase I (RLTOP1). Cleavage assay at various salt concentrations reveals that Camptothecin (CPT) enhanced the formation of “cleavable complex” at low salt. Interaction between the two subunits leading to the formation of an active complex could be explored as an insight for development of new therapeutic agents with specific selectivity.

SummaryCocaine’s behavioral-stimulant effects derive from potentiation of synaptic signaling by dopamine and serotonin leading to transcriptional alterations in postsynaptic cells. We report that a signaling cascade involving nitric oxide (NO) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mediates cocaine’s transcriptional and behavioral actions. Lower, behavioral-stimulant doses enhance the cAMP response element-binding (CREB) signaling system, while higher, neurotoxic doses stimulate the p53 cytotoxic system. The drug CGP3466B, which potently and selectively blocks GAPDH nitrosylation and GAPDH-Siah binding, prevents these actions as well as behavioral effects of cocaine providing a strategy for anticocaine therapy.

An error in immunoblotting data in Fig. 5C is corrected.

The tumor suppressor protein p53 is a critical stress response transcription factor that induces the expression of genes leading to cell cycle arrest, apoptosis, and tumor suppression. We found that mammalian inositol polyphosphate multikinase (IPMK) stimulated p53-mediated transcription by binding to p53 and enhancing its acetylation by the acetyltransferase p300 independently of its inositol phosphate and lipid kinase activities. Genetic or RNA interference (RNAi)-mediated knockdown of IPMK resulted in decreased activation of p53, decreased recruitment of p53 and p300 to target gene promoters, abrogated transcription of p53 target genes, and enhanced cell viability. Additionally, blocking the IPMK-p53 interaction decreased the extent of p53-mediated transcription. These results suggest that IPMK acts as a transcriptional coactivator for p53 and that it is an integral part of the p53 transcriptional complex facilitating cell death.

Tyrosyl DNA phosphodiesterase 1 (Tdp1) is a member of phospholipase D superfamily, which cleaves a broad range of 3'-DNA adducts, the best characterized of which is the phosphodiester bond formed between DNA and topoisomerase IB. This study describes cloning and functional characterization of the enzyme, termed as LdTdp1 in the kinetoplastid parasite Leishmania donovani. Sequence analysis confirmed conservation of the active site motifs typical for all Tdp1 proteins. LdTdp1 activity was detected in the parasite nucleus as well as in the kinetoplast. The enzyme harbours a nuclear localization signal at its C-terminus. Overexpression of the active enzyme protected the parasites against topoisomerase IB inhibitor camptothecin (CPT) and oxidative agent H(2)O(2)-mediated cytotoxicity and its downregulation rendered the parasites hypersensitive to CPT. Trapping of mutant LdTdp1 on DNA takes place following CPT treatment in L. donovani cells. The expression level and associated activity of LdTdp1 were found to be higher in CPT-resistant L. donovani parasites. Altogether, this is the first report of Tdp1 from the kinetoplastid parasite L. donovani, which actively participates in topoisomerase I-mediated DNA damage repair process and thereby counteracts the cytotoxic effect of topoisomerase I inhibitors.

Hydrogen sulfide (H2S), a messenger molecule generated by cystathionine gamma-lyase, acts as a physiologic vasorelaxant. Mechanisms whereby H2S signals have been elusive. We now show that H2S physiologically modifies cysteines in a large number of proteins by S-sulfhydration. About 10 to 25% of many liver proteins, including actin, tubulin, and glyceraldehyde-3-phosphate dehydrogenase (GAPDH), are sulfhydrated under physiological conditions. Sulfhydration augments GAPDH activity and enhances actin polymerization. Sulfhydration thus appears to be a physiologic posttranslational modification for proteins.

DNA topoisomerases of kinetoplastid parasites represent a family of DNA processing enzymes that essentially solve the topological problems not only in nuclear DNA but also in kinetoplastid DNA. Due to their indispensable function in cell biology they are valuable potential targets for many antileishmanial drugs to induce programmed cell death (PCD). PCD is thought to have evolved to regulate growth and development of multicellular organisms. Kinetoplastid organisms have developed an altruistic mechanism to promote and maintain the clonality within their population. Characterization of PCD by topoisomerase inhibitors could provide information regarding their pathogenesis which could be exploited to develop new targets to limit their growth and treat the disease they cause.

Besides its role in glycolysis, glyceraldehyde-3-phosphate dehydrogenase (GAPDH) initiates a cell death cascade. Diverse apoptotic stimuli activate inducible nitric oxide synthase (iNOS) or neuronal NOS (nNOS), with the generated nitric oxide (NO) S-nitrosylating GAPDH, abolishing its catalytic activity and conferring on it the ability to bind to Siah1, an E3-ubiquitin-ligase with a nuclear localization signal (NLS). The GAPDH-Siah1 protein complex, in turn, translocates to the nucleus and mediates cell death; these processes are blocked by procedures that interfere with GAPDH-Siah1 binding. Nuclear events induced by GAPDH to kill cells have been obscure. Here we show that nuclear GAPDH is acetylated at Lys 160 by the acetyltransferase p300/CREB binding protein (CBP) through direct protein interaction, which in turn stimulates the acetylation and catalytic activity of p300/CBP. Consequently, downstream targets of p300/CBP, such as p53 (Refs 10,11,12,13,14,15), are activated and cause cell death. A dominant-negative mutant GAPDH with the substitution of Lys 160 to Arg (GAPDH-K160R) prevents activation of p300/CBP, blocks induction of apoptotic genes and decreases cell death. Our findings reveal a pathway in which NO-induced nuclear GAPDH mediates cell death through p300/CBP.

A noticeable loss of cardiolipin, a significant accumulation of fluorescent products of lipid peroxidation and an increased ability to produce reactive oxygen species in vitro are characteristics of aged rat brain mitochondria, as has been demonstrated in this study. In contrast mitochondrial electron transport chain activity is not significantly compromised except a marginal decline in complex IV activity in aged rat brain. On the other hand, a striking loss of mitochondrial membrane potential occurs in brain mitochondria during aging, which may be attributed to peroxidative membrane damage in this condition. Such mitochondrial dysfunctions as reported here may lead to uncoupling of oxidative phosphorylation, ATP depletion and activation of apoptotic cascade in aged rat brain.

Protozoan parasites of the order Kinetoplastida cause severe diseases primarily in the tropical and subtropical areas. The enormous development of molecular and cellular biology in recent times have provided opportunities for discovering newer molecular targets for drug designing, which now form a rational basis for the development of improved anti-parasitic therapy. DNA topoisomerases play a key role in cellular processes affecting the topology and organization of intracellular DNA. Recently, emergence of the bi-subunit topoisomerase I in the kinetoplastid family has brought a new twist in topoisomerase research related to evolution, functional conservation and as a potential target that can be exploited in drug designing and development of new intervention strategies. This review summarizes the biology of kinetoplastid topoisomerases, which are the key molecular targets in antileishmanial chemotherapy.

Emergence of the bi-subunit topoisomerase I in the kinetoplastid family (Trypanosoma and Leishmania) has brought a new twist in topoisomerase research related to evolution, functional conservation and preferential sensitivities to the specific inhibitors of type IB topoisomerase family. In the present study, we describe that naturally occurring flavones baicalein, luteolin and quercetin are potent inhibitors of the recombinant Leishmania donovani topoisomerase I. These compounds bind to the free enzyme and also intercalate into the DNA at a very high concentration (300 microM) without binding to the minor grove. Here, we show that inhibition of topoisomerase I by these flavones is due to stabilization of topoisomerase I-DNA cleavage complexes, which subsequently inhibit the religation step. Their ability to stabilize the covalent topoisomerase I-DNA complex in vitro and in living cells is similar to that of the known topoisomerase I inhibitor camptothecin (CPT). However, in contrast to CPT, baicalein and luteolin failed to inhibit the religation step when the drugs were added to pre-formed enzyme substrate binary complex. This differential mechanism to induce the stabilization of cleavable complex with topoisomerase I and DNA by these selected flavones and CPT led us to investigate the effect of baicalein and luteolin on CPT-resistant mutant enzyme LdTOP1Delta39LS lacking 1-39 amino acids of the large subunit [B. B. Das, N. Sen, S. B. Dasgupta, A. Ganguly and H. K. Majumder (2005) J. Biol. Chem. 280, 16335-16344]. Baicalein and luteolin stabilize duplex oligonucleotide cleavage with LdTOP1Delta39LS. This observation was further supported by the stabilization of in vivo cleavable complex by baicalein and luteolin with highly CPT-resistant L.donovani strain. Taken together, our data suggest that the interacting amino acid residues of topoisomerase I may be partially overlapping or different for flavones and CPT. This study illuminates new properties of the flavones and provide additional insights into the ligand binding properties of L.donovani topoisomerase I.

Oxidative stress induced by Fe 2+ (50 μM) and ascorbate (2 mM) in isolated rat brain mitochondria incubated in vitro leads to an enhanced lipid peroxidation, cardiolipin loss and an increased formation of protein carbonyls. These changes are associated with a loss of mitochondrial membrane potential (depolarization) and an impaired activity of electron transport chain (ETC) as measured by MTT reduction assay. Butylated hydroxytoluene (0.2 mM), an inhibitor of lipid peroxidation, can prevent significantly the loss of cardiolipin, the increased protein carbonyl formation and the decrease in mitochondrial membrane potential induced by Fe 2+ and ascorbate, implying that the changes are secondary to membrane lipid peroxidation. However, iron-ascorbate induced impairment of mitochondrial ETC activity is apparently independent of lipid peroxidation process. The structural and functional derangement of mitochondria induced by oxidative stress as reported here may have implications in neuronal damage associated with brain aging and neurodegenerative disorders.