Sulfhydration of AKT triggers Tau-phosphorylation by activating glycogen synthase kinase 3ß in Alzheimer's disease.

In Alzheimer's disease (AD), human Tau is phosphorylated at S199 (hTau-S199-P) by the protein kinase glycogen synthase kinase 3ß (GSK3ß). HTau-S199-P mislocalizes to dendritic spines, which induces synaptic dysfunction at the early stage of AD. The AKT kinase, once phosphorylated, inhibits GSK3ß by phosphorylating it at S9. In AD patients, the abundance of phosphorylated AKT with active GSK3ß implies that phosphorylated AKT was unable to inactivate GSK3ß. However, the underlying mechanism of the inability of phosphorylated AKT to phosphorylate GSK3ß remains unknown. Here, we show that total AKT and phosphorylated AKT was sulfhydrated at C77 due to the induction of intracellular hydrogen sulfide (H2S). The increase in intracellular H2S levels resulted from the induction of the proinflammatory cytokine, IL-1ß, which is a pathological hallmark of AD. Sulfhydrated AKT does not interact with GSK3ß, and therefore does not phosphorylate GSK3ß. Thus, active GSK3ß phosphorylates Tau aberrantly. In a transgenic knockin mouse (AKT-KI+/+) that lacked sulfhydrated AKT, the interaction between AKT or phospho-AKT with GSK3ß was restored, and GSK3ß became phosphorylated. In AKT-KI+/+ mice, expressing the pathogenic human Tau mutant (hTau-P301L), the hTau S199 phosphorylation was ameliorated as GSK3ß 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.

Microglial-oligodendrocyte interactions in myelination and neurological function recovery after traumatic brain injury.

Differential microglial inflammatory responses play a role in regulation of differentiation and maturation of oligodendrocytes (OLs) in brain white matter. How microglia-OL crosstalk is altered by traumatic brain injury (TBI) and its impact on axonal myelination and neurological function impairment remain poorly understood. In this study, we investigated roles of a Na+/H+ exchanger (NHE1), an essential microglial pH regulatory protein, in microglial proinflammatory activation and OL survival and differentiation in a murine TBI model induced by controlled cortical impact. Similar TBI-induced contusion volumes were detected in the Cx3cr1-CreERT2 control (Ctrl) mice and selective microglial Nhe1 knockout (Cx3cr1-CreERT2;Nhe1flox/flox, Nhe1 cKO) mice. Compared to the Ctrl mice, the Nhe1 cKO mice displayed increased resistance to initial TBI-induced white matter damage and accelerated chronic phase of OL regeneration at 30 days post-TBI. The cKO brains presented increased anti-inflammatory phenotypes of microglia and infiltrated myeloid cells, with reduced proinflammatory transcriptome profiles. Moreover, the cKO mice exhibited accelerated post-TBI sensorimotor and cognitive functional recovery than the Ctrl mice. These phenotypic outcomes in cKO mice were recapitulated in C57BL6J wild-type TBI mice receiving treatment of a potent NHE1 inhibitor HOE642 for 1-7 days post-TBI. Taken together, these findings collectively demonstrated that blocking NHE1 protein stimulates restorative microglial activation in oligodendrogenesis and neuroprotection, which contributes to accelerated brain repair and neurological function recovery after TBI.

Cytokine-induced GAPDH sulfhydration affects PSD95 degradation and memory.

Induction of a proinflammatory cytokine, interleukin-1ß (IL-1ß) plays a role in memory impairment associated with various neurological disorders and brain injury. Here we show that IL-1ß-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-1ß-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-1ß-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.

Aberrant ER Stress Induced Neuronal-IFNß Elicits White Matter Injury Due to Microglial Activation and T-Cell Infiltration after TBI.

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)ß due to atypical activation of the neuronal-STING signaling pathway. IFNß 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 IFNß, 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 STATEMENT A 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 IFNß and whether induction of IFNß 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 IFNß, 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.

Hydrogen sulfide-linked sulfhydration of NF-κB mediates its antiapoptotic actions.

Nuclear factor κB (NF-κB) is an antiapoptotic transcription factor. We show that the antiapoptotic actions of NF-κB are mediated by hydrogen sulfide (H(2)S) synthesized by cystathionine gamma-lyase (CSE). TNF-α treatment triples H(2)S generation by stimulating binding of SP1 to the CSE promoter. H(2)S 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. H(2)S acts by sulfhydrating the p65 subunit of NF-κB at cysteine-38, which promotes its binding to the coactivator ribosomal protein S3 (RPS3). Sulfhydration of p65 predominates early after TNF-α treatment, then declines and is succeeded by a reciprocal enhancement of p65 nitrosylation. In CSE mutant mice, antiapoptotic influences of NF-κB are markedly diminished. Thus, sulfhydration of NF-κB appears to be a physiologic determinant of its antiapoptotic transcriptional activity.

Activation of PERK Elicits Memory Impairment through Inactivation of CREB and Downregulation of PSD95 After Traumatic Brain Injury.

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.

Activation of cyclin D1 affects mitochondrial mass following traumatic brain injury.

Cell cycle activation has been associated with varying types of neurological disorders including brain injury. Cyclin D1 is a critical modulator of cell cycle activation and upregulation of Cyclin D1 in neurons contributes to the pathology associated with traumatic brain injury (TBI). Mitochondrial mass is a critical factor to maintain the mitochondrial function, and it can be regulated by different signaling cascades and transcription factors including NRF1. However, the underlying mechanism of how TBI leads to impairment of mitochondrial mass following TBI remains obscure. Our results indicate that augmentation of CyclinD1 attenuates mitochondrial mass formation following TBI. To elucidate the molecular mechanism, we found that Cyclin D1 interacts with a transcription factor NRF1 in the nucleus and prevents NRF1's interaction with p300 in the pericontusional cortex following TBI. As a result, the acetylation level of NRF1 was decreased, and its transcriptional activity was attenuated. This event leads to a loss of mitochondrial mass in the pericontusional cortex following TBI. Intranasal delivery of Cyclin D1 RNAi immediately after TBI rescues transcriptional activation of NRF1 and recovers mitochondrial mass after TBI.

Isoflurane-induced inactivation of CREB through histone deacetylase 4 is responsible for cognitive impairment in developing brain.

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 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.

VEGF 165 b in the developing vasculatures of the fetal human eye.

VEGF(165) b is an anti-angiogenic form of VEGF(165) produced by alternative splicing. The localization of pro-angiogenic VEGF(165) and anti-angiogenic VEGF(165) b was investigated during development of the vasculatures in fetal human eyes from 7 to 21 weeks gestation (WG). The fetal vasculature of vitreous, which includes tunica vasculosa lentis (TVL), had moderate VEGF(165) immunoreactivity at 7WG and very little VEGF(165) b. Both forms were elevated at 12WG. VEGF(165) then decreased around 17WG when the TVL regresses but VEGF(165) b remained elevated. In choroid, VEGF(165) was present in forming choriocapillaris (CC) and retinal pigment epithelium (RPE) at 7WG while VEGF165b was present in CC and mesenchymal precursors within the choroidal stroma. By 21WG, both forms were elevated in RPE and choroidal blood vessels but VEGF(165) b was apical and VEGF(165) basal in RPE. Diffuse VEGF(165) immunoreactivity was prominent in 12WG innermost retina where blood vessels will form while VEGF(165) b was present in most CXCR4(+) progenitors in the inner neuroblastic layer and migrating angioblasts in the putative nerve fiber layer. By 21WG, VEGF(165) was present in nerve fibers and VEGF(165) b in the inner Muller cell process. The localization of VEGF(165) b was distinctly different from VEGF(165) both spatially and temporally and it was often associated with nucleus in progenitors.

Tumor protein p63/nuclear factor κB feedback loop in regulation of cell death.

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.

An augmentation in histone dimethylation at lysine nine residues elicits vision impairment following traumatic brain injury.

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.

Histone Deacetylase 4 Downregulation Elicits Post-Traumatic Psychiatric Disorders through Impairment of Neurogenesis.

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 GSK3ß 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.

Nitrosylation of GAPDH augments pathological tau acetylation upon exposure to amyloid-ß.

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-ß1-42 (Aß1-42), 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 Aß1-42-induced tau acetylation, memory impairment, and locomotor dysfunction in mice, suggesting that this drug might be repurposed to treat patients with AD.

Integrated transcriptomic and epigenomic analysis of ovarian cancer reveals epigenetically silenced GULP1.

Many epigenetically inactivated genes involved in ovarian cancer (OC) development and progression remain to be identified. In this study we undertook an integrated approach that consisted of identification of genome-wide expression patterns of primary OC samples and normal ovarian surface epithelium along with a pharmacologic unmasking strategy using 3 OC and 3 immortalized normal ovarian epithelial cell lines. Our filtering scheme identified 43 OC specific methylated genes and among the 5 top candidates (GULP1, CLIP4, BAMBI, NT5E, TGFß2), we performed extended studies of GULP1. In a training set, we identified GULP1 methylation in 21/61 (34%) of cases with 100% specificity. In an independent cohort, the observed methylation was 40% (146/365) in OC, 12.5% (2/16) in borderline tumors, 11% (2/18) in cystadenoma and 0% (0/13) in normal ovarian epithelium samples. GULP1 methylation was associated with clinicopathological parameters such as stage III/IV (p = 0.001), poorly differentiated grade (p = 0.033), residual disease (p < 0.0003), worse overall (p = 0.02) and disease specific survival (p = 0.01). Depletion of GULP1 in OC cells led to increased pro-survival signaling, inducing survival and colony formation, whereas reconstitution of GULP1 negated these effects, suggesting that GULP1 is required for maintaining cellular growth control.

Depolarization and cardiolipin depletion in aged rat brain mitochondria: relationship with oxidative stress and electron transport chain activity.

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.

Gene-specific oxidative lesions in aged rat brain detected by polymerase chain reaction inhibition assay.

An exposure of isolated rat brain genomic DNA to oxidative stress in the form of iron salts (Fe2+) and ascorbate results in gene-specific DNA lesions detectable by a quantitative polymerase chain reaction (PCR) based assay in which PCR amplification efficiency of the affected genes (e.g. beta-actin and p53) is grossly impaired. Such oxidative DNA lesions are prevented by hydroxyl radical scavengers like mannitol (20 mM) and sodium benzoate (20 mM) or by the antioxidant enzyme catalase (50 microg/ml) present in the incubation mixture during exposure to Fe2+ and ascorbate. When brain DNA isolated from young (4-6 months of age) and aged (20-24 months of age) rats are analyzed similarly by the PCR based method, the amplification levels of beta-actin and p53 genes are noticeably decreased in the case of aged rat indicating an accumulation of gene-specific DNA lesions during brain aging.

Diet-driven microbiota dysbiosis is associated with vagal remodeling and obesity.

Obesity is one of the major health issues in the United States. Consumption of diets rich in energy, notably from fats and sugars (high-fat/high-sugar diet: HF/HSD) is linked to the development of obesity and a popular dietary approach for weight loss is to reduce fat intake. Obesity research traditionally uses low and high fat diets and there has been limited investigation of the potential detrimental effects of a low-fat/high-sugar diet (LF/HSD) on body fat accumulation and health. Therefore, in the present study, we investigated the effects of HF/HSD and LF/HSD on microbiota composition, gut inflammation, gut-brain vagal communication and body fat accumulation. Specifically, we tested the hypothesis that LF/HSD changes the gut microbiota, induces gut inflammation and alters vagal gut-brain communication, associated with increased body fat accumulation. Sprague-Dawley rats were fed an HF/HSD, LF/HSD or control low-fat/low-sugar diet (LF/LSD) for 4weeks. Body weight, caloric intake, and body composition were monitored daily and fecal samples were collected at baseline, 1, 6 and 27days after the dietary switch. After four weeks, blood and tissues (gut, brain, liver and nodose ganglia) were sampled. Both HF/HSD and LF/HSD-fed rats displayed significant increases in body weight and body fat compared to LF/LSD-fed rats. 16S rRNA sequencing showed that both HF/HSD and LF/HSD-fed animals exhibited gut microbiota dysbiosis characterized by an overall decrease in bacterial diversity and an increase in Firmicutes/Bacteriodetes ratio. Dysbiosis was typified by a bloom in Clostridia and Bacilli and a marked decrease in Lactobacillus spp. LF/HSD-fed animals showed a specific increase in Sutterella and Bilophila, both Proteobacteria, abundances of which have been associated with liver damage. Expression of pro-inflammatory cytokines, such as IL-6, IL-1ß and TNFα, was upregulated in the cecum while levels of tight junction protein occludin were downregulated in both HF/HSD and LF/HSD fed rats. HF/HSD and LF/HSD-fed rats also exhibited an increase in cecum and serum levels of lipopolysaccharide (LPS), a pro-inflammatory bacterial product. Immunofluorescence revealed the withdrawal of vagal afferents from the gut and at their site of termination the nucleus of the solitary tract (NTS) in both the HF/HSD and LF/HSD rats. Moreover, there was significant microglia activation in the nodose ganglia, which contain the vagal afferent neuron cell bodies, of HF/HSD and LF/HSD rats. Taken together, these data indicate that, similar to HF/HSD, consumption of an LF/HSD induces dysbiosis of gut microbiota, increases gut inflammation and alters vagal gut-brain communication. These changes are associated with an increase in body fat accumulation.

Energy-dense diet triggers changes in gut microbiota, reorganization of gut­brain vagal communication and increases body fat accumulation.

Obesity is associated with consumption of energy-dense diets and development of systemic inflammation. Gut microbiota play a role in energy harvest and inflammation and can influence the change from lean to obese phenotypes. The nucleus of the solitary tract (NTS) is a brain target for gastrointestinal signals modulating satiety and alterations in gut-brain vagal pathway may promote overeating and obesity. Therefore, we tested the hypothesis that high-fat diet­induced changes in gut microbiota alter vagal gut-brain communication associated with increased body fat accumulation. Sprague-Dawley rats consumed a low energy­dense rodent diet (LFD; 3.1 kcal/g) or high energy­dense diet (HFD, 5.24 kcal/g). Minocycline was used to manipulate gut microbiota composition. 16S Sequencing was used to determine microbiota composition. Immunofluorescence against IB4 and Iba1 was used to determine NTS reorganization and microglia activation. Nodose ganglia from LFD rats were isolated and co-cultured with different bacteria strains to determine neurotoxicity. HFD altered gut microbiota with increases in Firmicutes/Bacteriodetes ratio and in pro-inflammatory Proteobacteria proliferation. HFD triggered reorganization of vagal afferents and microglia activation in the NTS, associated with weight gain. Minocycline-treated HFD rats exhibited microbiota profile comparable to LFD animals. Minocycline suppressed HFD­induced reorganization of vagal afferents and microglia activation in the NTS, and reduced body fat accumulation. Proteobacteria isolated from cecum of HFD rats were toxic to vagal afferent neurons in culture. Our findings show that diet­induced shift in gut microbiome may disrupt vagal gut­brain communication resulting in microglia activation and increased body fat accumulation.

Inhibition of rat brain mitochondrial electron transport chain activity by dopamine oxidation products during extended in vitro incubation: implications for Parkinson's disease.

Several studies on mitochondrial functions following brief exposure (5-15 min) to dopamine (DA) in vitro have produced extremely variable results. In contrast, this study demonstrates that a prolonged exposure (up to 2 h) of disrupted or lysed mitochondria to DA (0.1-0.4 mM) causes a remarkable and dose-dependent inhibition of complex I and complex IV activities. The inhibition of complex I and complex IV activities is not prevented by the antioxidant enzyme catalase (0.05 mg/ml) or the metal-chelator diethylenetriaminepentaacetic acid (0.1 mM) or the hydroxyl radical scavengers like mannitol (20 mM) and dimethyl sulphoxide (20 mM) indicating the non-involvement of *OH radicals and Fenton's chemistry in this process. However, reduced glutathione (5 mM), a quinone scavenger, almost completely abolishes the DA effect on mitochondrial complex I and complex IV activities, while tyrosinase (250 units/ml) which catalyses the conversion of DA to quinone products dramatically enhances the former effect. The results suggest the predominant involvement of quinone products instead of reactive oxygen radicals in long-term DA-mediated inactivation of complex I and complex IV. This is further indicated from the fact that significant amount of quinones and quinoprotein adducts (covalent adducts of reactive quinones with protein thiols) are formed during incubation of mitochondria with DA. Monoamine oxidase A (MAO-A) inhibitor clorgyline also provides variable but significant protection against DA induced inactivation of complex I and complex IV activities, presumably again through inhibition of quinoprotein formation. Mitochondrial ability to reduce tetrazolium dye 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) in presence of a respiratory substrate like succinate (10 mM) is also reduced by nearly 85% following 2 h incubation with 0.4 mM DA. This effect of DA on mitochondrial function is also dose-dependent and presumably mediated by quinone products of DA oxidation. The mitochondrial dysfunction induced by dopamine during extended periods of incubation as reported here have important implications in the context of dopaminergic neuronal death in Parkinson's disease (PD).

Lipid peroxidation associated cardiolipin loss and membrane depolarization in rat brain mitochondria.

Oxidative stress induced by Fe2+ (50 microM) 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 Fe2+ 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.