Reductions in Hydrogen Sulfide and Changes in Mitochondrial Quality Control Proteins Are Evident in the Early Phases of the Corneally Kindled Mouse Model of Epilepsy.

Epilepsy is a heterogenous neurological disorder characterized by recurrent unprovoked seizures, mitochondrial stress, and neurodegeneration. Hydrogen sulfide (H2S) is a gasotransmitter that promotes mitochondrial function and biogenesis, elicits neuromodulation and neuroprotection, and may acutely suppress seizures. A major gap in knowledge remains in understanding the role of mitochondrial dysfunction and progressive changes in H2S levels following acute seizures or during epileptogenesis. We thus sought to quantify changes in H2S and its methylated metabolite (MeSH) via LC-MS/MS following acute maximal electroshock and 6 Hz 44 mA seizures in mice, as well as in the early phases of the corneally kindled mouse model of chronic seizures. Plasma H2S was acutely reduced after a maximal electroshock seizure. H2S or MeSH levels and expressions of related genes in whole brain homogenates from corneally kindled mice were not altered. However, plasma H2S levels were significantly lower during kindling, but not after established kindling. Moreover, we demonstrated a time-dependent increase in expression of mitochondrial membrane integrity-related proteins, OPA1, MFN2, Drp1, and Mff during kindling, which did not correlate with changes in gene expression. Taken together, short-term reductions in plasma H2S could be a novel biomarker for seizures. Future studies should further define the role of H2S and mitochondrial stress in epilepsy.

Knock-Down of HDAC2 in Human Induced Pluripotent Stem Cell Derived Neurons Improves Neuronal Mitochondrial Dynamics, Neuronal Maturation and Reduces Amyloid Beta Peptides.

Histone deacetylase 2 (HDAC2) is a major HDAC protein in the adult brain and has been shown to regulate many neuronal genes. The aberrant expression of HDAC2 and subsequent dysregulation of neuronal gene expression is implicated in neurodegeneration and brain aging. Human induced pluripotent stem cell-derived neurons (hiPSC-Ns) are widely used models for studying neurodegenerative disease mechanisms, but the role of HDAC2 in hiPSC-N differentiation and maturation has not been explored. In this study, we show that levels of HDAC2 progressively decrease as hiPSCs are differentiated towards neurons. This suppression of HDAC2 inversely corresponds to an increase in neuron-specific isoforms of Endophilin-B1, a multifunctional protein involved in mitochondrial dynamics. Expression of neuron-specific isoforms of Endophilin-B1 is accompanied by concomitant expression of a neuron-specific alternative splicing factor, SRRM4. Manipulation of HDAC2 and Endophilin-B1 using lentiviral approaches shows that the knock-down of HDAC2 or the overexpression of a neuron-specific Endophilin-B1 isoform promotes mitochondrial elongation and protects against cytotoxic stress in hiPSC-Ns, while HDAC2 knock-down specifically influences genes regulating mitochondrial dynamics and synaptogenesis. Furthermore, HDAC2 knock-down promotes enhanced mitochondrial respiration and reduces levels of neurotoxic amyloid beta peptides. Collectively, our study demonstrates a role for HDAC2 in hiPSC-neuronal differentiation, highlights neuron-specific isoforms of Endophilin-B1 as a marker of differentiating hiPSC-Ns and demonstrates that HDAC2 regulates key neuronal and mitochondrial pathways in hiPSC-Ns.

Early-Onset Familial Alzheimer Disease Variant PSEN2 N141I Heterozygosity is Associated with Altered Microglia Phenotype.

BACKGROUND: Early-onset familial Alzheimer disease (EOFAD) is caused by heterozygous variants in the presenilin 1 (PSEN1), presenilin 2 (PSEN2), and APP genes. Decades after their discovery, the mechanisms by which these genes cause Alzheimer's disease (AD) or promote AD progression are not fully understood. While it is established that presenilin (PS) enzymatic activity produces amyloid-ß (Aß), PSs also regulate numerous other cellular functions, some of which intersect with known pathogenic drivers of neurodegeneration. Accumulating evidence suggests that microglia, resident innate immune cells in the central nervous system, play a key role in AD neurodegeneration. OBJECTIVE: Previous work has identified a regulatory role for PS2 in microglia. We hypothesized that PSEN2 variants lead to dysregulated microglia, which could further contribute to disease acceleration. To mimic the genotype of EOFAD patients, we created a transgenic mouse expressing PSEN2 N141I on a mouse background expressing one wildtype PS2 and two PS1 alleles. RESULTS: Microglial expression of PSEN2 N141I resulted in impaired γ-secretase activity as well as exaggerated inflammatory cytokine release, NFκB activity, and Aß internalization. In vivo, PS2 N141I mice showed enhanced IL-6 and TREM2 expression in brain as well as reduced branch number and length, an indication of "activated" morphology, in the absence of inflammatory stimuli. LPS intraperitoneal injection resulted in higher inflammatory gene expression in PS2 N141I mouse brain relative to controls. CONCLUSION: Our findings demonstrate that PSEN2 N141I heterozygosity is associated with disrupted innate immune homeostasis, suggesting EOFAD variants may promote disease progression through non-neuronal cells beyond canonical dysregulated Aß production.

GPR124 regulates microtubule assembly, mitotic progression, and glioblastoma cell proliferation.

GPR124 is involved in embryonic development and remains expressed by select organs. The importance of GPR124 during development suggests that its aberrant expression might participate in tumor growth. Here we show that both increases and decreases in GPR124 expression in glioblastoma cells reduce cell proliferation by differentially altering the duration mitotic progression. Using mass spectrometry-based proteomics, we discovered that GPR124 interacts with ch-TOG, a known regulator of both microtubule (MT)-plus-end assembly and mitotic progression. Accordingly, changes in GPR124 expression and ch-TOG similarly affect MT assembly measured by real-time microscopy in cells. Our study describes a novel molecular interaction involving GPR124 and ch-TOG at the plasma membrane that controls glioblastoma cell proliferation by modifying MT assembly rates and controlling the progression of distinct phases of mitosis.

Alternative splicing in a presenilin 2 variant associated with Alzheimer disease.

OBJECTIVE: Autosomal-dominant familial Alzheimer disease (AD) is caused by by variants in presenilin 1 (PSEN1), presenilin 2 (PSEN2), and amyloid precursor protein (APP). Previously, we reported a rare PSEN2 frameshift variant in an early-onset AD case (PSEN2 p.K115Efs*11). In this study, we characterize a second family with the same variant and analyze cellular transcripts from both patient fibroblasts and brain lysates. METHODS: We combined genomic, neuropathological, clinical, and molecular techniques to characterize the PSEN2 K115Efs*11 variant in two families. RESULTS: Neuropathological and clinical evaluation confirmed the AD diagnosis in two individuals carrying the PSEN2 K115Efs*11 variant. A truncated transcript from the variant allele is detectable in patient fibroblasts while levels of wild-type PSEN2 transcript and protein are reduced compared to controls. Functional studies to assess biological consequences of the variant demonstrated that PSEN2 K115Efs*11 fibroblasts secrete less Aß 1-40 compared to controls, indicating abnormal γ-secretase activity. Analysis of PSEN2 transcript levels in brain tissue revealed alternatively spliced PSEN2 products in patient brain as well as in sporadic AD and age-matched control brain. INTERPRETATION: These data suggest that PSEN2 K115Efs*11 is a likely pathogenic variant associated with AD. We uncovered novel PSEN2 alternative transcripts in addition to previously reported PSEN2 splice isoforms associated with sporadic AD. In the context of a frameshift, these alternative transcripts return to the canonical reading frame with potential to generate deleterious protein products. Our findings suggest novel potential mechanisms by which PSEN variants may influence AD pathogenesis, highlighting the complexity underlying genetic contribution to disease risk.

Metabolic and Organelle Morphology Defects in Mice and Human Patients Define Spinocerebellar Ataxia Type 7 as a Mitochondrial Disease.

Spinocerebellar ataxia type 7 (SCA7) is a retinal-cerebellar degenerative disorder caused by CAG-polyglutamine (polyQ) repeat expansions in the ataxin-7 gene. As many SCA7 clinical phenotypes occur in mitochondrial disorders, and magnetic resonance spectroscopy of patients revealed altered energy metabolism, we considered a role for mitochondrial dysfunction. Studies of SCA7 mice uncovered marked impairments in oxygen consumption and respiratory exchange. When we examined cerebellar Purkinje cells in mice, we observed mitochondrial network abnormalities, with enlarged mitochondria upon ultrastructural analysis. We developed stem cell models from patients and created stem cell knockout rescue systems, documenting mitochondrial morphology defects, impaired oxidative metabolism, and reduced expression of nicotinamide adenine dinucleotide (NAD+) production enzymes in SCA7 models. We observed NAD+ reductions in mitochondria of SCA7 patient NPCs using ratiometric fluorescent sensors and documented alterations in tryptophan-kynurenine metabolism in patients. Our results indicate that mitochondrial dysfunction, stemming from decreased NAD+, is a defining feature of SCA7.

Neuronal susceptibility to beta-amyloid toxicity and ischemic injury involves histone deacetylase-2 regulation of endophilin-B1.

Histone deacetylases (HDACs) catalyze acetyl group removal from histone proteins, leading to altered chromatin structure and gene expression. HDAC2 is highly expressed in adult brain, and HDAC2 levels are elevated in Alzheimer's disease (AD) brain. We previously reported that neuron-specific splice isoforms of Endophilin-B1 (Endo-B1) promote neuronal survival, but are reduced in human AD brain and mouse models of AD and stroke. Here, we demonstrate that HDAC2 suppresses Endo-B1 expression. HDAC2 knockdown or knockout enhances expression of Endo-B1. Conversely, HDAC2 overexpression decreases Endo-B1 expression. We also demonstrate that neurons exposed to beta-amyloid increase HDAC2 and reduce histone H3 acetylation while HDAC2 knockdown prevents Aß induced loss of histone H3 acetylation, mitochondrial dysfunction, caspase-3 activation, and neuronal death. The protective effect of HDAC2 knockdown was abrogated by Endo-B1 shRNA and in Endo-B1-null neurons, suggesting that HDAC2-induced neurotoxicity is mediated through suppression of Endo-B1. HDAC2 overexpression also modulates neuronal expression of mitofusin2 (Mfn2) and mitochondrial fission factor (MFF), recapitulating the pattern of change observed in AD. HDAC2 knockout mice demonstrate reduced injury in the middle cerebral artery occlusion with reperfusion (MCAO/R) model of cerebral ischemia demonstrating enhanced neuronal survival, minimized loss of Endo-B1, and normalized expression of Mfn2. These findings support the hypothesis that HDAC2 represses Endo-B1, sensitizing neurons to mitochondrial dysfunction and cell death in stroke and AD.

CCP1 promotes mitochondrial fusion and motility to prevent Purkinje cell neuron loss in pcd mice.

A perplexing question in neurodegeneration is why different neurons degenerate. The Purkinje cell degeneration (pcd) mouse displays a dramatic phenotype of degeneration of cerebellar Purkinje cells. Loss of CCP1/Nna1 deglutamylation of tubulin accounts for pcd neurodegeneration, but the mechanism is unknown. In this study, we modulated the dosage of fission and fusion genes in a Drosophila melanogaster loss-of-function model and found that mitochondrial fragmentation and disease phenotypes were rescued by reduced Drp1. We observed mitochondrial fragmentation in CCP1 null cells and in neurons from pcd mice, and we documented reduced mitochondrial fusion in cells lacking CCP1. We examined the effect of tubulin hyperglutamylation on microtubule-mediated mitochondrial motility in pcd neurons and noted markedly reduced retrograde axonal transport. Mitochondrial stress promoted Parkin-dependent turnover of CCP1, and CCP1 and Parkin physically interacted. Our results indicate that CCP1 regulates mitochondrial motility through deglutamylation of tubulin and that loss of CCP1-mediated mitochondrial fusion accounts for the exquisite vulnerability of Purkinje neurons in pcd mice.

Loss of endophilin-B1 exacerbates Alzheimer's disease pathology.

Endophilin-B1, also known as Bax-interacting factor 1 (Bif-1, and encoded by SH3GLB1), is a multifunctional protein involved in apoptosis, autophagy and mitochondrial function. We recently described a unique neuroprotective role for neuron-specific alternatively spliced isoforms of endophilin-B1. To examine whether endophilin-B1-mediated neuroprotection could be a novel therapeutic target for Alzheimer's disease we used a double mutant amyloid precursor protein and presenilin 1 (APPswe/PSEN1dE9) mouse model of Alzheimer's disease and observed that expression of neuron-specific endophilin-B1 isoforms declined with disease progression. To determine if this reduction in endophilin-B1 has a functional role in Alzheimer's disease pathogenesis, we crossed endophilin-B1(-/-) mice with APPswe/PSEN1dE9 mice. Deletion of endophilin-B1 accelerated disease onset and progression in 6-month-old APPswe/PSEN1dE9/endophilin-B1(-/-) mice, which showed more plaques, astrogliosis, synaptic degeneration, cognitive impairment and mortality than APPswe/PSEN1dE9 mice. In mouse primary cortical neuron cultures, overexpression of neuron-specific endophilin-B1 isoforms protected against amyloid-ß-induced apoptosis and mitochondrial dysfunction. Additionally, protein and mRNA levels of neuron-specific endophilin-B1 isoforms were also selectively decreased in the cerebral cortex and in the synaptic compartment of patients with Alzheimer's disease. Flow sorting of synaptosomes from patients with Alzheimer's disease demonstrated a negative correlation between amyloid-ß and endophilin-B1 levels. The importance of endophilin-B1 in neuronal function was further underscored by the development of synaptic degeneration and cognitive and motor impairment in endophilin-B1(-/-) mice by 12 months. Our findings suggest that endophilin-B1 is a key mediator of a feed-forward mechanism of Alzheimer's disease pathogenesis where amyloid-ß reduces neuron-specific endophilin-B1, which in turn enhances amyloid-ß accumulation and neuronal vulnerability to stress.

Mitochondrial morphology differences and mitophagy deficit in murine glaucomatous optic nerve.

PURPOSE: Decreased ATP correlates with intraocular pressure exposure in the optic nerves of mice with glaucoma. To understand what underlies this energy deficit, we examined mitochondria in the myelinated optic nerve axons of the DBA/2J mouse, a model of glaucoma secondary to iris pigment disease, and the DBA/2(wt-gpnmb) control strain. METHODS: Mitochondrial length, width, surface area, and health status were measured in 30 electron microscopic fields within the myelinated portion of optic nerves from DBA/2J and DBA/2(wt-gpnmb) mice at 3, 6, and 10 months of age. Protein was isolated from optic nerve for analysis of PINK1, Parkin, LC3-I and -II, and lysosome-associated membrane protein 1 (LAMP1) by Western blot. RESULTS: The number of mitochondria in DBA/2J optic nerve was increased, and they had significantly smaller surface area. Mitochondria in DBA/2J were closer to the axolemma, more spatially isolated, and their cristae were more disrupted at every age group as compared to DBA/2(wt-gpnmb). Autophagosomes were significantly increased in DBA/2J optic nerve at all ages. Protein analysis showed higher LC3-II to LC3-I ratio in aged DBA/2J optic nerve than in DBA/2(wt-gpnmb). PINK1 and Parkin levels were not statistically different across age groups. LAMP1 was significantly decreased in the aged DBA/2J optic nerve. CONCLUSIONS: Decreased surface area, combined with reduced oxidative capacity in mitochondria from the aged DBA/2J axon, indicate that mitochondrial pathology may contribute to the energy deficit in glaucomatous optic nerve. Though autophagosomes were increased in DBA/2J optic nerve, the increased mitochondria and decreased LAMP1 suggest deteriorating mitochondria are not being efficiently recycled by mitophagy.

Peroxisomal biogenesis in ischemic brain.

AIMS: Peroxisomes are highly adaptable and dynamic organelles, adjusting their size, number, and enzyme composition to changing environmental and metabolic demands. We determined whether peroxisomes respond to ischemia, and whether peroxisomal biogenesis is an adaptive response to cerebral ischemia. RESULTS: Focal cerebral ischemia induced peroxisomal biogenesis in peri-infarct neurons, which was associated with a corresponding increase in peroxisomal antioxidant enzyme catalase. Peroxisomal biogenesis was also observed in primary cultured cortical neurons subjected to ischemic insult induced by oxygen-glucose deprivation (OGD). A catalase inhibitor increased OGD-induced neuronal death. Moreover, preventing peroxisomal proliferation by knocking down dynamin-related protein 1 (Drp1) exacerbated neuronal death induced by OGD, whereas enhancing peroxisomal biogenesis pharmacologically using a peroxisome proliferator-activated receptor-alpha agonist protected against neuronal death induced by OGD. INNOVATION: This is the first documentation of ischemia-induced peroxisomal biogenesis in mammalian brain using a combined in vivo and in vitro approach, electron microscopy, high-resolution laser-scanning confocal microscopy, and super-resolution structured illumination microscopy. CONCLUSION: Our findings suggest that neurons respond to ischemic injury by increasing peroxisome biogenesis, which serves a protective function, likely mediated by enhanced antioxidant capacity of neurons.

Bax interacting factor-1 promotes survival and mitochondrial elongation in neurons.

Bax-interacting factor 1 (Bif-1, also known as endophilin B1) is a multifunctional protein involved in the regulation of apoptosis, mitochondrial morphology, and autophagy. Previous studies in non-neuronal cells have shown that Bif-1 is proapoptotic and promotes mitochondrial fragmentation. However, the role of Bif-1 in postmitotic neurons has not been investigated. In contrast to non-neuronal cells, we now report that in neurons Bif-1 promotes viability and mitochondrial elongation. In mouse primary cortical neurons, Bif-1 knockdown exacerbated apoptosis induced by the DNA-damaging agent camptothecin. Neurons from Bif-1-deficient mice contained fragmented mitochondria and Bif-1 knockdown in wild-type neurons also resulted in fragmented mitochondria which were more depolarized, suggesting mitochondrial dysfunction. During ischemic stroke, Bif-1 expression was downregulated in the penumbra of wild-type mice. Consistent with Bif-1 being required for neuronal viability, Bif-1-deficient mice developed larger infarcts and an exaggerated astrogliosis response following ischemic stroke. Together, these data suggest that, in contrast to non-neuronal cells, Bif-1 is essential for the maintenance of mitochondrial morphology and function in neurons, and that loss of Bif-1 renders neurons more susceptible to apoptotic stress. These unique actions may relate to the presence of longer, neuron-specific Bif-1 isoforms, because only these forms of Bif-1 were able to rescue deficiencies caused by Bif-1 suppression. This finding not only demonstrates an unexpected role for Bif-1 in the nervous system but this work also establishes Bif-1 as a potential therapeutic target for the treatment of neurological diseases, especially degenerative disorders characterized by alterations in mitochondrial dynamics.

p53 and mitochondrial function in neurons.

The p53 tumor suppressor plays a central role in dictating cell survival and death as a cellular sensor for a myriad of stresses including DNA damage, oxidative and nutritional stress, ischemia and disruption of nucleolar function. Activation of p53-dependent apoptosis leads to mitochondrial apoptotic changes via the intrinsic and extrinsic pathways triggering cell death execution most notably by release of cytochrome c and activation of the caspase cascade. Although it was previously believed that p53 induces apoptotic mitochondrial changes exclusively through transcription-dependent mechanisms, recent studies suggest that p53 also regulates apoptosis via a transcription-independent action at the mitochondria. Recent evidence further suggests that p53 can regulate necrotic cell death and autophagic activity including mitophagy. An increasing number of cytosolic and mitochondrial proteins involved in mitochondrial metabolism and respiration are regulated by p53, which influences mitochondrial ROS production as well. Cellular redox homeostasis is also directly regulated by p53 through modified expression of pro- and anti-oxidant proteins. Proper regulation of mitochondrial size and shape through fission and fusion assures optimal mitochondrial bioenergetic function while enabling adequate mitochondrial transport to accommodate local energy demands unique to neuronal architecture. Abnormal regulation of mitochondrial dynamics has been increasingly implicated in neurodegeneration, where elevated levels of p53 may have a direct contribution as the expression of some fission/fusion proteins are directly regulated by p53. Thus, p53 may have a much wider influence on mitochondrial integrity and function than one would expect from its well-established ability to transcriptionally induce mitochondrial apoptosis. However, much of the evidence demonstrating that p53 can influence mitochondria through nuclear, cytosolic or intra-mitochondrial sites of action has yet to be confirmed in neurons. Nonetheless, as mitochondria are essential for supporting normal neuronal functions and in initiating/propagating cell death signaling, it appears certain that the mitochondria-related functions of p53 will have broader implications than previously thought in acute and progressive neurological conditions, providing new therapeutic targets for treatment.

MS-275, a class I histone deacetylase inhibitor, protects the p53-deficient mouse against ischemic injury.

The administration of pan histone deacetylase (HDAC) inhibitors reduces ischemic damage to the CNS, both in vitro and in animal models of stroke, via mechanisms which we are beginning to understand. The acetylation of p53 is regulated by Class I HDACs and, because p53 appears to play a role in ischemic pathology, the purpose of this study was to discover, using an in vitro white matter ischemia model and an in vivo cerebral ischemia model, if neuroprotection mediated by HDAC inhibition depended on p53 expression. Optic nerves were excised from wild-type and p53-deficient mice, and then subjected to oxygen-glucose deprivation in the presence and absence of a specific inhibitor of Class I HDACs (MS-275, entinostat) while compound action potentials were recorded. Furthermore, transient focal ischemia was imposed on wild-type and p53-deficient mice, which were subsequently treated with MS-275. Interestingly, and in both scenarios, the beneficial effects of MS-275 were most pronounced when p53 was absent. These results suggest that modulation of p53 activity is not responsible for MS-275-mediated neuroprotection, and further illustrate how HDAC inhibitors variably influence p53 and associated apoptotic pathways. Optic nerves from wild-type and p53-deficient mice, engineered to express cyan fluorescent protein (CFP) in neuronal mitochondria, were subjected to oxygen-glucose deprivation (OGD) in the presence and absence of a specific inhibitor of Class I histone deacetylases. The protective effect of MS-275 was evidenced by mitochondrial preservation, and this was most pronounced in the absence of p53.

Novel protective effects of histone deacetylase inhibition on stroke and white matter ischemic injury.

Understanding how epigenetics influences the process and progress of a stroke could yield new targets and therapeutics for use in the clinic. Experimental evidence suggests that inhibitors of zinc-dependent histone deacetylases can protect neurons, axons, and associated glia from the devastating effects of oxygen and glucose deprivation. While the specific enzymes involved have yet to be clearly identified, there are hints from somewhat selective chemical inhibitors and also from the use of specific small hairpin RNAs to transiently knockdown protein expression. Neuroprotective mechanisms implicated thus far include the upregulation of extracellular glutamate clearance, inhibition of p53-mediated cell death, and maintenance of mitochondrial integrity. The histone deacetylases have distinct cellular and subcellular localizations, and discrete substrates. As a number of chemical inhibitors are already in clinical use for the treatment of cancer, repurposing for the stroke clinic should be expedited.

A Bcl-xL-Drp1 complex regulates synaptic vesicle membrane dynamics during endocytosis.

Following exocytosis, the rate of recovery of neurotransmitter release is determined by vesicle retrieval from the plasma membrane and by recruitment of vesicles from reserve pools within the synapse, which is dependent on mitochondrial ATP. The anti-apoptotic Bcl-2 family protein Bcl-xL also regulates neurotransmitter release and recovery in part by increasing ATP availability from mitochondria. We now find, that Bcl-xL directly regulates endocytic vesicle retrieval in hippocampal neurons through protein-protein interaction with components of the clathrin complex. Our evidence suggests that, during synaptic stimulation, Bcl-xL translocates to clathrin-coated pits in a calmodulin-dependent manner and forms a complex with the GTPase Drp1, Mff and clathrin. Depletion of Drp1 produces misformed endocytic vesicles. Mutagenesis studies suggest that formation of the Bcl-xL-Drp1 complex is necessary for the enhanced rate of vesicle endocytosis produced by Bcl-xL, thus providing a mechanism for presynaptic plasticity.

Declines in Drp1 and parkin expression underlie DNA damage-induced changes in mitochondrial length and neuronal death.

Maintaining proper mitochondrial length is essential for normal mitochondrial function in neurons. Mitochondrial fragmentation has been associated with neuronal cell death caused by a variety of experimental toxic stressors. Despite the fact that oxidative stress is a hallmark of neurodegenerative conditions and aging and the resulting activation of p53 is believed to contribute to the neuropathology, little is still known regarding changes in mitochondrial morphology in p53-dependent neuronal death. Therefore, we specifically addressed the relationship between genotoxic stress, p53 activation, and the regulation of mitochondrial morphology in neurons. In cultured postnatal mouse cortical neurons, treatment with the DNA-damaging agent camptothecin (CPT) resulted in elongated mitochondria, in contrast to fragmented mitochondria observed upon staurosporine and glutamate treatment. In fibroblasts, however, CPT resulted in fragmented mitochondria. CPT treatment in neurons suppressed expression of the mitochondrial fission protein Drp1 and the E3 ubiquitin ligase parkin. The presence of elongated mitochondria and the declines in Drp1 and parkin expression occurred before the commitment point for apoptosis. The CPT-induced changes in Drp1 and parkin were not observed in p53-deficient neurons, while p53 overexpression alone was sufficient to reduce the expression of the two proteins. Elevating Drp1 or parkin expression before CPT treatment enhanced neuronal viability and restored a normal pattern of mitochondrial morphology. The present findings demonstrate that genotoxic stress in neurons results in elongated mitochondria in contrast to fission induced by other forms of stress, and p53-dependent declines in Drp1 and parkin levels contribute to altered mitochondrial morphology and cell death.

Acute, but reversible, kainic acid-induced DNA damage in hippocampal CA1 pyramidal cells of p53-deficient mice.

p53 plays an essential role in mediating apoptotic responses to cellular stress, especially DNA damage. In a kainic acid (KA)-induced seizure model in mice, hippocampal CA1 pyramidal cells undergo delayed neuronal death at day 3-4 following systemic KA administration. We previously demonstrated that CA1 neurons in p53(-/-) animals are protected from such apoptotic neuronal loss. However, extensive morphological damage associated with DNA strand breaks in CA1 neurons was found in a fraction of p53(-/-) animals at earlier time points (8 h to 2 days). No comparable acute damage was observed in wild-type animals. Stereological counting confirmed that there was no significant loss of CA1 pyramidal cells in p53(-/-) animals at 7 days post-KA injection. These results suggest that seizure-induced DNA strand breaks are accumulated to a greater extent but do not lead to apoptosis in the absence of p53. In wild-type animals, therefore, p53 appears to stimulate DNA repair and also mediate apoptosis in CA1 neurons in this excitotoxicity model. These results also reflect remarkable plasticity of neurons in recovery from injury.

Expression of histone deacetylases in cellular compartments of the mouse brain and the effects of ischemia.

Drugs that inhibit specific histone deacetylase (HDAC) activities have enormous potential in preventing the consequences of acute injury to the nervous system and in allaying neurodegeneration. However, very little is known about the expression pattern of the HDACs in the central nervous system (CNS). Identifying the cell types that express HDACs in the CNS is important for determining therapeutic targets for HDAC inhibitors and evaluating potential side effects. We characterized the cellular expression of HDACs 1-3, and HDACs 4 and 6, in the adult mouse brain in the cingulate cortex, parietal cortex, dentate gyrus, and CA1 regions of the hippocampus and subcortical white matter. Expression of class I HDACs showed a cell-and region-specific pattern. Transient focal ischemia induced by temporary middle cerebral artery occlusion, or global ischemia induced by in vitro oxygen-glucose deprivation, altered the extent of HDAC expression in a region- and cell-specific manner. The pan-HDAC inhibitor, SAHA, reduced ischemia-induced alterations in HDACs. The results suggest that in addition to promoting epigenetic changes in transcriptional activity in the nucleus of neurons and glia, HDACs may also have non-transcriptional actions in axons and the distant processes of glial cells and may significantly modulate the response to injury in a cell- and region-specific manner.