An Efficient and Cost-Effective Approach to Generate Functional Human Inducible Pluripotent Stem Cell-Derived Astrocytes.

Human inducible pluripotent stem cell (hiPSC)-derived astrocytes (iAs) are critical to study astrocytes in health and disease. They provide several advantages over human fetal astrocytes in research, which include consistency, availability, disease modeling, customization, and ethical considerations. The generation of iAs is hampered by the requirement of Matrigel matrix coating for survival and proliferation. We provide a protocol demonstrating that human iAs cultured in the absence of Matrigel are viable and proliferative. Further, through a side-by-side comparison of cultures with and without Matrigel, we show significant similarities in astrocyte-specific profiling, including morphology (shape and structure), phenotype (cell-specific markers), genotype (transcriptional expression), metabolic (respiration), and functional aspects (glutamate uptake and cytokine response). In addition, we report that, unlike other CNS cell types, such as neuronal progenitor cells and neurons, iAs can withstand the absence of Matrigel coating. Our study demonstrates that Matrigel is dispensable for the culture of human iPSC-derived astrocytes, facilitating an easy, streamlined, and cost-effective method of generating these cells.

Brain autopsies of critically ill COVID-19 patients demonstrate heterogeneous profile of acute vascular injury, inflammation and age-linked chronic brain diseases.

BACKGROUND: This study examined neuropathological findings of patients who died following hospitalization in an intensive care unit with SARS-CoV-2. METHODS: Data originate from 20 decedents who underwent brain autopsy followed by ex-vivo imaging and dissection. Systematic neuropathologic examinations were performed to assess histopathologic changes including cerebrovascular disease and tissue injury, neurodegenerative diseases, and inflammatory response. Cerebrospinal fluid (CSF) and fixed tissues were evaluated for the presence of viral RNA and protein. RESULTS: The mean age-at-death was 66.2 years (range: 26-97 years) and 14 were male. The patient's medical history included cardiovascular risk factors or diseases (n = 11, 55%) and dementia (n = 5, 25%). Brain examination revealed a range of acute and chronic pathologies. Acute vascular pathologic changes were common in 16 (80%) subjects and included infarctions (n = 11, 55%) followed by acute hypoxic/ischemic injury (n = 9, 45%) and hemorrhages (n = 7, 35%). These acute pathologic changes were identified in both younger and older groups and those with and without vascular risk factors or diseases. Moderate-to-severe microglial activation were noted in 16 (80%) brains, while moderate-to-severe T lymphocyte accumulation was present in 5 (25%) brains. Encephalitis-like changes included lymphocytic cuffing (n = 6, 30%) and neuronophagia or microglial nodule (most prominent in the brainstem, n = 6, 30%) were also observed. A single brain showed vasculitis-like changes and one other exhibited foci of necrosis with ball-ring hemorrhages reminiscent of acute hemorrhagic leukoencephalopathy changes. Chronic pathologies were identified in only older decedents: 7 brains exhibited neurodegenerative diseases and 8 brains showed vascular disease pathologies. CSF and brain samples did not show evidence of viral RNA or protein. CONCLUSIONS: Acute tissue injuries and microglial activation were the most common abnormalities in COVID-19 brains. Focal evidence of encephalitis-like changes was noted despite the lack of detectable virus. The majority of older subjects showed age-related brain pathologies even in the absence of known neurologic disease. Findings of this study suggest that acute brain injury superimposed on common pre-existing brain disease may put older subjects at higher risk of post-COVID neurologic sequelae.

Human Plasma BDNF Is Associated With Amygdala-Prefrontal Cortex Functional Connectivity and Problem Drinking Behaviors.

BACKGROUND: Preclinical studies suggest that decreased levels of brain-derived neurotrophic factor in the amygdala play a role in anxiety and alcohol use disorder. The association between brain-derived neurotrophic factor levels and amygdala function in humans with alcohol use disorder is still unclear, although neuroimaging studies have also implicated the amygdala in alcohol use disorder and suggest that alcohol use disorder is associated with disrupted functional connectivity between the amygdala and prefrontal cortex during aversive states. METHODS: The current study investigated whether plasma brain-derived neurotrophic factor levels in individuals with and without alcohol use disorder (n = 57) were associated with individual differences in amygdala reactivity and amygdala-prefrontal cortex functional connectivity during 2 forms of aversive responding captured via functional magnetic resonance imaging: anxiety elicited by unpredictable threat of shock and fear elicited by predictable threat of shock. We also examined whether brain-derived neurotrophic factor and brain function were associated with binge drinking episodes and alcohol use disorder age of onset. RESULTS: During anxiety, but not fear, lower levels of plasma brain-derived neurotrophic factor were associated with less connectivity between the left amygdala and the medial prefrontal cortex and the inferior frontal gyrus. In addition, within individuals with alcohol use disorder (only), lower levels of brain-derived neurotrophic factor and amygdala-medial prefrontal cortex functional connectivity during anxiety were associated with more binge episodes within the past 60 days and a lower age of alcohol use disorder onset. There were no associations between brain-derived neurotrophic factor levels and focal amygdala task reactivity. CONCLUSIONS: Together, the results indicate that plasma brain-derived neurotrophic factor levels are related to amygdala circuit functioning in humans, particularly during anxiety, and these individual differences may relate to drinking behaviors.

Acute Ethanol Produces Ataxia and Induces Fmr1 Expression via Histone Modifications in the Rat Cerebellum.

BACKGROUND: The cerebellum is fundamental for motor coordination and therefore crucial in ethanol (EtOH)-induced ataxia. EtOH contributes to cerebellar pathophysiology. Fragile-X mental retardation protein (FMRP) is a complex regulator of RNA and synaptic plasticity implicated in fragile-X tremor and ataxia syndrome, a phenotype featuring increased Fmr1 mRNA expression. Recent studies have implicated glutamatergic targets of FMRP in hereditary cerebellar ataxias including the main cerebellar excitatory amino acid (Eaa1) transporter and a subtype of metabotropic glutamate receptor (Grm5). However, EtOH-induced changes in cerebellar Fmr1 expression and its epigenetic regulation have not been investigated. Here, we examined the effects of acute EtOH exposure on ataxic behavior, gene expression, and epigenetic regulation of the Fmr1 gene and its glutamatergic targets in the rat cerebellum. METHODS: Male adult Sprague Dawley rats received acute EtOH (2 g/kg) intraperitoneally 1 hour prior to ataxic behavioral testing on the accelerating rotarod and were sacrificed immediately thereafter. Cerebellar tissues were analyzed for gene expression and epigenetic regulation of the Fmr1 gene and its glutamatergic targets in the rat cerebellum using real-time quantitative polymerase chain reaction (PCR) and chromatin immunoprecipitation. RESULTS: Acute EtOH exposure caused marked ataxia on the accelerating rotarod test compared with saline-treated controls. This ataxic response was associated with increases in mRNA levels of Fmr1, postsynaptic density 95 (Psd95), Eaa1, and Grm5 in the cerebellum. In addition, we found increased H3K27 acetylation both at the promoter region of Fmr1 and at a proposed cyclic adenosine monophosphate (cAMP) response-element binding (CREB) site downstream of the Fmr1 transcription start site. Furthermore, acute EtOH exposure significantly increased Creb1 and the histone acetyltransferases (HAT) CREB binding protein (Cbp), and p300 mRNA transcripts. CONCLUSIONS: Overall, EtOH regulates cerebellar Fmr1 expression most likely via HAT-mediated increase in histone acetylation. We propose that FMRP regulation of glutamatergic transcripts plays an important role in disrupting the excitatory-inhibitory balance in the cerebellum underlying EtOH-induced ataxia.

The lncRNA BDNF-AS is an epigenetic regulator in the human amygdala in early onset alcohol use disorders.

Adolescent alcohol drinking is known to contribute to the development and severity of alcohol use disorders (AUDs) later in adulthood. Recent studies have shown that long non-coding RNAs (lncRNAs) are critical for brain development and synaptic plasticity. One such lncRNA is natural occurring brain-derived neurotrophic factor antisense (BDNF-AS) that has been shown to regulate BDNF expression. The role of BDNF-AS lncRNA in the molecular mechanisms of AUD is unknown. Here, we evaluated the expression and functional role of BDNF-AS in postmortem amygdala of either early onset or late onset alcoholics (individuals who began drinking before or after 21 years of age, respectively) and age-matched control subjects. BDNF-AS expression is increased in early onset but not in late onset AUD amygdala and appears to be regulated epitranscriptomically via decreased N6-methyladenosine on BDNF-AS. Upregulation of BDNF-AS is associated with a significant decrease in BDNF expression and increased recruitment of EZH2, which deposits repressive H3K27 trimethylation (H3K27me3) at regulatory regions in the BDNF gene in the early onset AUD group. Drinking during adolescence also contributed to significant decreases in activity-regulated cytoskeleton-associated protein (ARC) expression which also appeared to be mediated by increased EZH2 deposition of repressive H3K27me3 at the ARC synaptic activity response element. These results suggest an important role for BDNF-AS in the regulation of synaptic plasticity via epigenetic reprogramming in the amygdala of AUD subjects who began drinking during adolescence.

Essential Role of Histone Methyltransferase G9a in Rapid Tolerance to the Anxiolytic Effects of Ethanol.

BACKGROUND: Tolerance to ethanol-induced anxiolysis promotes alcohol intake, thus contributing to alcohol use disorder development. Recent studies implicate histone deacetylase-mediated histone H3K9 deacetylation in regulating neuropeptide Y expression during rapid ethanol tolerance to the anxiolytic effects of ethanol. Furthermore, the histone methyltransferase, G9a, and G9a-mediated H3K9 dimethylation (H3K9me2) have recently emerged as regulators of addiction and anxiety; however, their role in rapid ethanol tolerance is unknown. Therefore, we investigated the role of G9a-mediated H3K9me2 in neuropeptide Y expression during rapid ethanol tolerance. METHODS: Adult male rats were administered one injection of n-saline followed by single acute ethanol injection (1 g/kg) 24 hours later (ethanol group) or 2 injections (24 hours apart) of either n-saline (saline group) or ethanol (tolerance group). Anxiety-like behaviors and global and Npy-specific G9a and H3K9me2 levels in the amygdala were measured. Effects of G9a inhibitor (UNC0642) treatment on behavioral and epigenetic measures were also examined. RESULTS: Acute ethanol produced anxiolysis and decreased global H3K9me2 and G9a protein levels in the central and medial nucleus of the amygdala as well as decreased occupancy levels of H3K9me2 and G9a near a putative binding site for cAMP-response element binding protein on the Npy gene. Two identical doses of ethanol produced no behavioral or epigenetic changes relative to controls, indicating development of rapid ethanol tolerance. Interestingly, treatment with UNC0642, before the second ethanol dose reversed rapid ethanol tolerance, decreased global H3K9me2 and increased neuropeptide Y levels in the central and medial nucleus of the amygdala. CONCLUSIONS: These results implicate amygdaloid G9a-mediated H3K9me2 mechanisms in regulating rapid tolerance to the anxiolytic effects of ethanol via neuropeptide Y expression regulation.

Adolescent alcohol exposure epigenetically regulates CREB signaling in the adult amygdala.

Binge alcohol drinking in adolescence leads to increased risk for alcohol use and other psychiatric disorders in adulthood. The transcription factor cAMP-response element binding (CREB) protein is involved in the neuronal response to adult ethanol exposure, but its role in the enduring effects of adolescent alcohol exposure in adulthood is unknown. We exposed male rats to adolescent intermittent ethanol (AIE) or saline (AIS) during post-natal days 28-41 and evaluated the epigenetic regulation of CREB dynamics in the adult amygdala. A subset of these adult rats was exposed to an acute ethanol challenge. AIE decreased CREB, phosphorylated CREB, CREB-binding protein (CBP) and p300 protein levels in adult amygdaloid brain structures. AIE exposure also causes deficits in Creb1, Cbp, and p300 mRNA expression in the amygdala of AIE adult rats which are normalized after acute ethanol exposure. Interestingly, occupancy of acetylated histone H3K9/14 proteins at specific locations in the Creb1, Cbp, and p300 gene promoter regions was decreased in the amygdala of AIE adult rats and was normalized by acute ethanol exposure. These results suggest that AIE exposure epigenetically reduces CREB and other related transcriptional activators in the amygdala in adulthood that may be associated with the behavioral effects of adolescent alcohol exposure.

Donepezil Reverses Dendritic Spine Morphology Adaptations and Fmr1 Epigenetic Modifications in Hippocampus of Adult Rats After Adolescent Alcohol Exposure.

BACKGROUND: Adolescent intermittent ethanol (AIE) exposure produces persistent impairments in cholinergic and epigenetic signaling and alters markers of synapses in the hippocampal formation, effects that are thought to drive hippocampal dysfunction in adult rodents. Donepezil (Aricept), a cholinesterase inhibitor, is used clinically to ameliorate memory-related cognitive deficits. Given that donepezil also prevents morphological impairment in preclinical models of neuropsychiatric disorders, we investigated the ability of donepezil to reverse morphological and epigenetic adaptations in the hippocampus of adult rats exposed to AIE. Because of the known relationship between dendritic spine density and morphology with the fragile X mental retardation 1 (Fmr1) gene, we also assessed Fmr1 expression and its epigenetic regulation in hippocampus after AIE and donepezil pretreatment. METHODS: Adolescent rats were administered intermittent ethanol for 16 days starting on postnatal day 30. Rats were treated with donepezil (2.5 mg/kg) once a day for 4 days starting 20 days after the completion of AIE exposure. Brains were dissected out after the fourth donepezil dose, and spine analysis was completed in dentate gyrus granule neurons. A separate cohort of rats, treated identically, was used for molecular studies. RESULTS: AIE exposure significantly reduced dendritic spine density and altered morphological characteristics of subclasses of dendritic spines. AIE exposure also increased mRNA levels and H3-K27 acetylation occupancy of the Fmr1 gene in hippocampus. Treatment of AIE-exposed adult rats with donepezil reversed both the dendritic spine adaptations and epigenetic modifications and expression of Fmr1. CONCLUSIONS: These findings indicate that AIE produces long-lasting decreases in dendritic spine density and changes in Fmr1 gene expression in the hippocampal formation, suggesting morphological and epigenetic mechanisms underlying previously reported behavioral deficits after AIE. The reversal of these effects by subchronic, post-AIE donepezil treatment indicates that these AIE effects can be reversed by up-regulating cholinergic function.

Adolescent Alcohol Exposure: Burden of Epigenetic Reprogramming, Synaptic Remodeling, and Adult Psychopathology.

Adolescence represents a crucial phase of synaptic maturation characterized by molecular changes in the developing brain that shape normal behavioral patterns. Epigenetic mechanisms play an important role in these neuromaturation processes. Perturbations of normal epigenetic programming during adolescence by ethanol can disrupt these molecular events, leading to synaptic remodeling and abnormal adult behaviors. Repeated exposure to binge levels of alcohol increases the risk for alcohol use disorder (AUD) and comorbid psychopathology including anxiety in adulthood. Recent studies in the field clearly suggest that adolescent alcohol exposure causes widespread and persistent changes in epigenetic, neurotrophic, and neuroimmune pathways in the brain. These changes are manifested by altered synaptic remodeling and neurogenesis in key brain regions leading to adult psychopathology such as anxiety and alcoholism. This review details the molecular mechanisms underlying adolescent alcohol exposure-induced changes in synaptic plasticity and the development of alcohol addiction-related phenotypes in adulthood.

The Potential Role of Amygdaloid MicroRNA-494 in Alcohol-Induced Anxiolysis.

BACKGROUND: The antianxiety effects of ethanol appear to be a crucial factor in promoting alcohol intake. Regulation of gene expression by microRNA (miRNA) is an important epigenetic mechanism that affects neuronal pathways and behaviors. We investigated the role of miRNAs underlying the mechanisms of ethanol-induced anxiolysis. METHODS: Acute ethanol-induced anxiolysis was measured in adult rats, and amygdaloid tissues were used for miRNA profiling by microarray analysis. The expression of miR-494 and its target genes in the amygdala was measured using real-time quantitative polymerase chain reaction. The direct role of miR-494 in the anxiety phenotype was also investigated via infusion of a miR-494 antagomir into the central nucleus of amygdala. RESULTS: Microarray profiling of miRNAs in the amygdala showed significant alteration of several miRNA expression levels by acute ethanol exposure. Expression of miR-494 was significantly decreased, whereas expression of the binding protein of cyclic adenosine monophosphate response element binding protein (CBP), p300, and Cbp/p300-interacting transactivator 2 (Cited2) was increased in the amygdala during ethanol-induced anxiolysis. Inhibition of miR-494 in the central nucleus of amygdala, through infusion of a specific antagomir, provoked anxiolysis, mimicking the action of ethanol. Also, expression of Cited2, CBP, and p300 as well as histone H3-lysine 9 acetylation was significantly increased by miR-494 antagomir infusion, indicating their regulation by miR-494 in the amygdala. CONCLUSIONS: These novel results suggest that acute ethanol-induced reduction in miR-494 expression in the amygdala can serve as a key regulatory mechanism for chromatin remodeling possibly leading to anxiolysis.

Adolescent alcohol exposure alters GABAA receptor subunit expression in adult hippocampus.

BACKGROUND: The long-term consequences of adolescent alcohol abuse that persist into adulthood are poorly understood and have not been widely investigated. We have shown that intermittent exposure to alcohol during adolescence decreased the amplitude of GABAA receptor (GABAA R)-mediated tonic currents in hippocampal dentate granule cells in adulthood. The aim of this study was to investigate the enduring effects of chronic intermittent alcohol exposure during adolescence or adulthood on the expression of hippocampal GABAA Rs. METHODS: We used a previously characterized tissue fractionation method to isolate detergent resistant membranes and soluble fractions, followed by Western blots to measure GABAA R protein expression. We also measured mRNA levels of GABAA R subunits using quantitative real-time polymerase chain reaction. RESULTS: Although the protein levels of α1-, α4-, and δ-GABAA R subunits remained stable between postnatal day (PD) 30 (early adolescence) and PD71 (adulthood), the α5-GABAA R subunit was reduced across that period. In rats that were subjected to adolescent intermittent ethanol (AIE) exposure between PD30 and PD46, there was a significant reduction in the protein levels of the δ-GABAA R, in the absence of any changes in mRNA levels, at 48 hours and 26 days after the last ethanol (EtOH) exposure. Protein levels of the α4-GABAA R subunit were significantly reduced, but mRNA levels were increased, 26 days (but not 48 hours) after the last AIE exposure. Protein levels of α5-GABAA R were not changed by AIE, but mRNA levels were reduced at 48 hours but normalized 26 days after AIE. In contrast to the effects of AIE, chronic intermittent ethanol (CIE) exposure during adulthood had no effect on expression of any of the GABAA R subunits examined. CONCLUSIONS: AIE produced both short- and long-term alterations of GABAA R subunits mRNA and protein expression in the hippocampus, whereas CIE produced no long-lasting effects on those measures. The observed reduction of protein levels of the δ-GABAA R, specifically, is consistent with previously reported altered hippocampal GABAA R-mediated electrophysiological responses after AIE. The absence of effects of CIE underscores the emerging view of adolescence as a time of distinctive vulnerability to the enduring effects of repeated EtOH exposure.

The epigenetic landscape of alcoholism.

Alcoholism is a complex psychiatric disorder that has a multifactorial etiology. Epigenetic mechanisms are uniquely capable of accounting for the multifactorial nature of the disease in that they are highly stable and are affected by environmental factors, including alcohol itself. Chromatin remodeling causes changes in gene expression in specific brain regions contributing to the endophenotypes of alcoholism such as tolerance and dependence. The epigenetic mechanisms that regulate changes in gene expression observed in addictive behaviors respond not only to alcohol exposure but also to comorbid psychopathology such as the presence of anxiety and stress. This review summarizes recent developments in epigenetic research that may play a role in alcoholism. We propose that pharmacologically manipulating epigenetic targets, as demonstrated in various preclinical models, hold great therapeutic potential in the treatment and prevention of alcoholism.

Reversal of deficits in dendritic spines, BDNF and Arc expression in the amygdala during alcohol dependence by HDAC inhibitor treatment.

Development of anxiety-like behaviours during ethanol withdrawal has been correlated with increased histone deacetylase (HDAC) activity and decreased brain-derived neurotrophic factor (BDNF) and activity-regulated cytoskeleton-associated protein (Arc) gene expression in the amygdala. Furthermore, HDAC-mediated histone modifications play a role in synaptic plasticity. In this study we used the HDAC inhibitor trichostatin A (TSA) to determine whether HDAC inhibition could prevent ethanol withdrawal-induced deficits in dendritic spine density (DSD), BDNF or Arc expression in the amygdala of rats. It was found that decreased BDNF and Arc expression in the central (CeA) and medial nucleus of amygdala (MeA), observed during withdrawal after chronic ethanol exposure, were normalized following acute TSA treatment. TSA treatment was also able to attenuate anxiety-like behaviours during ethanol withdrawal and correct the observed decrease in DSD in the CeA and MeA of ethanol-withdrawn rats. Taken together, these findings demonstrate that correcting the deficits in histone acetylation through TSA treatment also amends downstream synaptic plasticity-related deficits such as BDNF and Arc expression, and DSD in the CeA and MeA as well as attenuates anxiety-like behaviours in rats during withdrawal after chronic ethanol exposure.

Hyposensitivity to gamma-aminobutyric acid in the ventral tegmental area during alcohol withdrawal: reversal by histone deacetylase inhibitors.

Putative dopaminergic (pDAergic) ventral tegmental area (VTA) neurons have an important role in alcohol addiction. Acute ethanol increases the activity of pDAergic neurons, and withdrawal from repeated ethanol administration produces a decreased sensitivity of pDAergic VTA neurons to GABA. Recent studies show that behavioral changes induced by chronic alcohol are reversed by inhibitors of histone deacetylases (HDACs). Whether HDAC-induced histone modifications regulate changes in GABA sensitivity of VTA pDAergic neurons during withdrawal is unknown. Here, we investigated modulation of withdrawal-induced changes in GABA sensitivity of pDAergic VTA neurons by HDAC inhibitors (HDACi), and also measured the levels of HDAC2, histone (H3-K9) acetylation, and GABA-Aα1 receptor (GABA (A-α1) R) subunit in VTA during ethanol withdrawal. Mice were injected intraperitoneally (ip) with either ethanol (3.5 g/kg) or saline twice daily for 3 weeks. In recordings from pDAergic VTA neurons in brain slices from ethanol-withdrawn mice, sensitivity to GABA (50-500 µM) was reduced. In brain slices from ethanol-withdrawn mice incubated with the HDACi SAHA (vorinostat) or trichostatin A (TSA) for 2 h, the hyposensitivity of pDAergic VTA neurons to GABA was significantly attenuated. There was no effect of TSA or SAHA on GABA sensitivity of pDAergic VTA neurons from saline-treated mice. In addition, ethanol withdrawal was associated with an increase in levels of HDAC2 and a decrease in histone (H3-K9) acetylation and levels of GABA (A-α1) R subunits in the VTA. Therefore, blockade of upregulation of HDAC2 by HDACi normalizes GABA hyposensitivity of pDAergic neurons developed during withdrawal after chronic ethanol treatment, which suggests the possibility that inhibition of HDACs can reverse ethanol-induced neuroadaptational changes in reward circuitry.

Cell-specific expression of neuropeptide Y Y1 receptor immunoreactivity in the rat basolateral amygdala.

Activation of neuropeptide Y (NPY) Y1 receptors (Y1r) in the rat basolateral nuclear complex of the amygdala (BLA) produces anxiolysis and interferes with the generation of conditioned fear. NPY is important in regulating the output of the BLA, yet the cell types involved in mediating this response are currently unknown. The current studies employed multiple label immunocytochemistry to determine the distribution of Y1r-immunoreactivity (-ir) in glutamatergic pyramidal and GABAergic cell populations in the BLA using scanning laser confocal stereology. Pyramidal neurons were identified by expression of calcium-calmodulin dependent kinase II (CaMKII-ir) and functionally distinct interneuron subpopulations were distinguished by peptide (cholecystokinin, somatostatin) or calcium-binding protein (parvalbumin, calretinin) content. Throughout the BLA, Y1r-ir was predominately on soma with negligible fiber staining. The high degree of coexpression of Y1r-ir (99.9%) in CaMKII-ir cells suggests that these receptors colocalize on pyramidal cells and that NPY could influence BLA output by directly regulating the activity of these projection neurons. Additionally, Y1r-ir was also colocalized with the interneuronal markers studied. Parvalbumin-ir interneurons, which participate in feedforward inhibition of BLA pyramidal cells, represented the largest number of Y1r expressing interneurons in the BLA ( approximately 4% of the total neuronal population). The anatomical localization of NPY receptors on different cell populations within the BLA provides a testable circuit whereby NPY could modulate the activity of the BLA via actions on both projection cells and interneuronal cell populations.

GSK-3beta gene expression in human postmortem brain: regional distribution, effects of age and suicide.

Glycogen synthase kinase (GSK-3beta) has been implicated in the pathophysiology of mood disorders and schizophrenia. To examine its role in suicide, we determined GSK-3beta messenger RNA (mRNA) in human postmortem brain from suicide and normal control subjects using quantitative reverse transcriptase-polymerase chain reaction (RT-PCR) technique. We found that GSK-3beta mRNA was highly abundant in almost all of the 12 brain areas we studied. We also found a significant age effect on GSK-3beta and that GSK-3beta mRNA level were significantly higher in prefrontal cortex (PFC) and hippocampus of teenage normal controls compared with adult normal controls and was significantly decreased in PFC of teenage suicide but not adult suicide victims compared with respective normal control subjects. The decrease observed in the mRNA levels in teenage suicide but not in adult suicide victims may represent a neurodevelopmentally associated decrease and may be important in the pathophysiology of teenage suicide.

Lower phosphoinositide 3-kinase (PI 3-kinase) activity and differential expression levels of selective catalytic and regulatory PI 3-kinase subunit isoforms in prefrontal cortex and hippocampus of suicide subjects.

Phosphoinositide 3 (PI 3)-kinase is one of the key signaling enzymes that participates in a myriad of physiological functions in brain and is utilized by neurotrophins to mediate neuronal plasticity, cell survival, and inhibition of apoptosis for several neuronal subtypes. Our recent demonstration that expression of neurotrophic factors and activation of the receptor tyrosine kinase B are significantly altered in postmortem brain of suicide subjects led us to examine whether suicide brain is associated with alterations in PI 3-kinase signaling. In prefrontal cortex (PFC), hippocampus, and cerebellum of suicide (n=28) and nonpsychiatric control (n=21) subjects we examined catalytic activation of PI 3-kinase, and mRNA and protein levels of regulatory (p85alpha, p85beta) and catalytic (p110alpha, p110beta) subunits of PI 3-kinase. It was observed that the catalytic activity of PI 3-kinase was significantly reduced in PFC and hippocampus of suicide subjects compared with nonpsychiatric control subjects. Competitive PCR analysis revealed significantly reduced mRNA expression of p85beta and p110alpha and increased expression of p85alpha subunit isoforms in PFC and hippocampus of suicide subjects. Alterations in these catalytic and regulatory subunits were accompanied by changes in their respective protein levels. These changes were not present in cerebellum of suicide subjects. Also, these changes were present in all suicide subjects irrespective of psychiatric diagnosis. Our findings of reduced activation and altered expression of specific PI 3-kinase regulatory and catalytic subunit isoforms demonstrate abnormalities in this signaling pathway in postmortem brain of suicide subjects and suggest possible involvement of aberrant PI 3-kinase signaling in the pathogenic mechanisms of suicide.

Aberrant extracellular signal-regulated kinase (ERK) 5 signaling in hippocampus of suicide subjects.

Extracellular signal-regulated kinase 5 (ERK5), the newest member of the mitogen-activated protein (MAP) kinase family, is regulated differently than the other MAP kinases. Emerging evidence suggest the role of ERK5 signaling in promoting cell proliferation, differentiation, neuronal survival, and neuroprotection. The present study investigates whether suicide brain is associated with alterations in components of the ERK5 signaling cascade. In the prefrontal cortex (PFC) and hippocampus of suicide subjects (n=28) and nonpsychiatric control subjects (n=21), we examined the catalytic activities and protein levels of ERK5 and upstream MAP kinase kinase MEK5 in various subcellular fractions; mRNA levels of ERK5 in total RNA; and DNA-binding activity of myocyte enhancer factor (MEF)2C, a substrate of ERK5. In the hippocampus of suicide subjects, we observed that catalytic activity of ERK5 was decreased in cytosolic and nuclear fractions, whereas catalytic activity of MEK5 was decreased in the total fraction. Further, decreased mRNA and protein levels of ERK5, but no change in protein level of MEK5 were noted. A decrease in MEF2C-DNA-binding activity in the nuclear fraction was also observed. No significant alterations were noted in the PFC of suicide subjects. The observed changes were not related to a specific psychiatric diagnosis. Our findings of reduced activation and/or expression of ERK5 and MEK5, and reduced MEF2C-DNA-binding activity demonstrate abnormalities in ERK5 signaling in hippocampus of suicide subjects and suggest possible involvement of this aberrant signaling in pathogenic mechanisms of suicide.