Characterization of exosome-mediated propagation of systemic inflammatory responses into the Central Nervous System.

The mechanisms through which systemic inflammation exerts its effect on the CNS are still not completely understood. Exosomes are small (30 to 100 nanometers) membrane-bound extracellular vesicles released by most of the mammalian cells. Exosomes play a vital role in cell-to-cell communication. This includes regulation of inflammatory responses by shuttling mRNAs, miRNAs, and cytokines both locally and systemically to the neighboring as well as distant cells to further modulate their transcriptional and/or translational states and affect the functional phenotype of those cells that have taken up these exosomes. The role of circulating blood exosomes leading to neuroinflammation during systemic inflammatory conditions was further characterized. Serum-derived exosomes from LPS-challenged mice (SDEL) were freshly isolated from the sera of the mice that were earlier treated with LPS and used to study SDEL effects on neuroinflammation. Exosomes isolated from the sera of the mice injected with saline were used as a control. In-vitro studies showed that the SDEL upregulate pro-inflammatory cytokine gene expression in the murine cell lines of microglia (BV-2), astrocytes (C8-D1A), and cerebral microvascular endothelial cells (bEnd.3). To further study their effects in-vivo, SDEL were intravenously injected into normal adult mice. Elevated mRNA expression of pro-inflammatory cytokines was observed in the brains of SDEL recipient mice. Proteomic analysis of the SDEL confirmed the increased expression of inflammatory cytokines in them. Together, these results further demonstrate and strengthen the novel role of peripheral circulating exosomes in causing neuroinflammation during systemic inflammatory conditions.

Dopaminergic inhibition of the inwardly rectifying potassium current in direct pathway medium spiny neurons in normal and parkinsonian striatum.

Despite the profound behavioral effects of the striatal dopamine (DA) activity and the inwardly rectifying potassium channel ( Kir ) being a key determinant of striatal medium spiny neuron (MSN) activity that also profoundly affects behavior, previously reported DA regulations of Kir are conflicting and incompatible with MSN function in behavior. Here we show that in normal mice with an intact striatal DA system, the predominant effect of DA activation of D1Rs in D1-MSNs is to cause a modest depolarization and increase in input resistance by inhibiting Kir, thus moderately increasing the spike outputs from behavior-promoting D1-MSNs. In parkinsonian (DA-depleted) striatum, DA increases D1-MSN intrinsic excitability more strongly than in normal striatum, consequently strongly increasing D1-MSN spike firing that is behavior-promoting; this DA excitation of D1-MSNs is stronger when the DA depletion is more severe. The DA inhibition of Kir is occluded by the Kir blocker barium chloride (BaCl 2 ). In behaving parkinsonian mice, BaCl 2 microinjection into the dorsal striatum stimulates movement but occludes the motor stimulation of D1R agonism. Taken together, our results resolve the long-standing question about what D1R agonism does to D1-MSN excitability in normal and parkinsonian striatum and strongly indicate that D1R inhibition of Kir is a key ion channel mechanism that mediates D1R agonistic behavioral stimulation in normal and parkinsonian animals.

Dopamine-independent development and maintenance of mouse striatal medium spiny neuron dendritic spines.

Striatal medium spiny neurons (MSNs) and striatal dopamine (DA) innervation are profoundly important for brain function such as motor control and cognition. A widely accepted theory posits that striatal DA loss causes (or leads to) MSN dendritic atrophy. However, examination of the literature indicates that the data from Parkinson's disease (PD) patients and animal PD models were contradictory among studies and hard to interpret. Here we have re-examined the potential effects of DA activity on MSN morphology or lack thereof. We found that in 15-day, 4- and 12-month old Pitx3 null mutant mice that have severe DA denervation in the dorsal striatum while having substantial residual DA innervation in the ventral striatum, MSN dendrites and spine numbers were similar in dorsal and ventral striatum, and also similar to those in normal mice. In 15-day, 4- and 12-month old tyrosine hydroxylase knockout mice that cannot synthesize L-dopa and thus have no endogenous DA in the entire brain, MSN dendrites and spine numbers were also indistinguishable from age-matched wild-type (WT) mice. Furthermore, in adult WT mice, unilateral 6-OHDA lesion at 12 months of age caused an almost complete striatal DA denervation in the lesioned side, but MSN dendrites and spine numbers were similar in the lesioned and control sides. Taken together, our data indicate that in mice, the development and maintenance of MSN dendrites and spines are DA-independent such that DA depletion does not trigger MSN dendritic atrophy; our data also suggest that the reported MSN dendritic atrophy in PD may be a component of neurodegeneration in PD rather than a consequence of DA denervation.

Heat shock factor HSF1 regulates BDNF gene promoters upon acute stress in the hippocampus, together with pCREB.

Heat shock factor 1 (HSF1) is a master stress-responsive transcriptional factor, protecting cells from death. However, its gene regulation in vivo in the brain in response to neuronal stimuli remains elusive. Here, we investigated its direct regulation of the brain-derived neurotrophic factor (BDNF) gene (Bdnf) in response to acute neuronal stress stimuli in the brain. The results of immunohistochemistry and chromatin immunoprecipitation quantitative PCR (ChIP-qPCR) showed that administration of kainic acid (a glutamate receptor agonist inducing excitotoxity) to young adult mice induced HSF1 nuclear translocation and its binding to multiple Bdnf promoters in the hippocampus. Footshock, a physical stressor used for learning, also induced HSF1 binding to selected Bdnf promoters I and IV. This is, to our knowledge, the first demonstration of HSF1 gene regulation in response to neuronal stimuli in the hippocampus in vivo. HSF1 binding sites (HSEs) in Bdnf promoters I and IV were also detected when immunoprecipitated by an antibody of phosphorylated (p)CREB (cAMP-responsive element-binding protein), suggesting their possible interplay in acute stress-induced Bdnf transcription. Interestingly, their promoter binding patterns differed by KA and footshock, suggesting that HSF1 and pCREB orchestrate to render fine-tuned promoter control depending on the types of stress. Further, HSF1 overexpression increased Bdnf promoter activity in a luciferase assay, while virus infection of constitutively active-form HSF1 increased levels of BDNF mRNA and protein in vitro in primary cultured neurons. These results indicated that HSF1 activation of Bdnf promoter was sufficient to induce BDNF expression. Taken together, these results suggest that HSF1 promoter-specific control of Bdnf gene regulation plays an important role in neuronal protection and plasticity in the hippocampus in response to acute stress, possibly interplaying with pCREB.

White matter damage as a consequence of vascular dysfunction in a spontaneous mouse model of chronic mild chronic hypoperfusion with eNOS deficiency.

Vascular cognitive impairment and dementia (VCID) is the second most common form of dementia after Alzheimer's disease (AD). Currently, the mechanistic insights into the evolution and progression of VCID remain elusive. White matter change represents an invariant feature. Compelling clinical neuroimaging and pathological evidence suggest a link between white matter changes and neurodegeneration. Our prior study detected hypoperfused lesions in mice with partial deficiency of endothelial nitric oxide (eNOS) at very young age, precisely matching to those hypoperfused areas identified in preclinical AD patients. White matter tracts are particularly susceptible to the vascular damage induced by chronic hypoperfusion. Using immunohistochemistry, we detected severe demyelination in the middle-aged eNOS-deficient mice. The demyelinated areas were confined to cortical and subcortical areas including the corpus callosum and hippocampus. The intensity of demyelination correlated with behavioral deficits of gait and associative recognition memory performances. By Evans blue angiography, we detected blood-brain barrier (BBB) leakage as another early pathological change affecting frontal and parietal cortex in eNOS-deficient mice. Sodium nitrate fortified drinking water provided to young and middle-aged eNOS-deficient mice completely prevented non-perfusion, BBB leakage, and white matter pathology, indicating that impaired endothelium-derived NO signaling may have caused these pathological events. Furthermore, genome-wide transcriptomic analysis revealed altered gene clusters most related to mitochondrial respiratory pathways selectively in the white matter of young eNOS-deficient mice. Using eNOS-deficient mice, we identified BBB breakdown and hypoperfusion as the two earliest pathological events, resulting from insufficient vascular NO signaling. We speculate that the compromised BBB and mild chronic hypoperfusion trigger vascular damage, along with oxidative stress and astrogliosis, accounting for the white matter pathological changes in the eNOS-deficient mouse model. We conclude that eNOS-deficient mice represent an ideal spontaneous evolving model for studying the earliest events leading to white matter changes, which will be instrumental to future therapeutic testing of drug candidates and for targeting novel/specific vascular mechanisms contributing to VCID and AD.

Reciprocal interplay between OTULIN-LUBAC determines genotoxic and inflammatory NF-κB signal responses.

Targeting nuclear factor-kappa B (NF-κB) represents a highly viable strategy against chemoresistance in cancers as well as cell death. Ubiquitination, including linear ubiquitination mediated by the linear ubiquitin chain assembly complex (LUBAC), is emerging as a crucial mechanism of overactivated NF-κB signaling. Ovarian tumor family deubiquitinase OTULIN is the only linear linkage-specific deubiquitinase; however, the molecular mechanisms of how it counteracts LUBAC-mediated NF-κB activation have been largely unknown. Here, we identify Lys64/66 of OTULIN for linear ubiquitination facilitated in a LUBAC-dependent manner as a necessary event required for OTULIN-LUBAC interaction under unstressed conditions, which becomes deubiquitinated by OTULIN itself in response to genotoxic stress. Furthermore, this self-deubiquitination of OTULIN occurs intermolecularly, mediated by OTULIN dimerization, resulting in the subsequent dissociation of OTULIN from the LUBAC complex and NF-κB overactivation. Oxidative stress induces OTULIN dimerization via cysteine-mediated covalent disulfide bonds. Our study reveals that the status of the physical interaction between OTULIN and LUBAC is a crucial determining factor for the genotoxic NF-κB signaling, as measured by cell survival and proliferation, while OTULIN loss of function resulting from its dimerization and deubiquitination leads to a dissociation of OTULIN from the LUBAC complex. Of note, similar molecular mechanisms apply to the inflammatory NF-κB signaling in response to tumor necrosis factor α. Hence, a fuller understanding of the detailed molecular mechanisms underlying the disruption of the OTULIN-LUBAC interaction will be instrumental for developing future therapeutic strategies against cancer chemoresistance and necroptotic processes pertinent to numerous human diseases.

Direct AT2R Stimulation Slows Post-stroke Cognitive Decline in the 5XFAD Alzheimer's Disease Mice.

Alzheimer's disease (AD), currently the single leading cause of death still on the rise, almost always coexists alongside vascular cognitive impairment (VCI). In fact, the ischemic disease affects up to 90% of AD patients, with strokes and major infarctions representing over a third of vascular lesions. Studies also confirmed that amyloid plaques, typical of AD, are much more likely to cause dementia if strokes or cerebrovascular damage also exist, leading to the term "mixed pathology" cognitive impairment. Although its incidence is expected to grow, there are no satisfactory treatments. There is hence an urgent need for safe and effective therapies that preserve cognition, maintain function, and prevent the clinical deterioration that results from the progression of this irreversible, neurodegenerative disease. To our knowledge, this is the first study to investigate the effects of long-term treatment with C21, a novel angiotensin II type 2 receptor (AT2R) agonist, on the development of "mixed pathology" cognitive impairment. This was accomplished using a unique model that employs the fundamental elements of both AD and VCI. Treatment with C21/vehicle was started 1 h post-stroke and continued for 5 weeks in mice with concurrent AD pathology. Efficacy was established through a series of functional tests assessing various aspects of cognition, including spatial learning, short-term/working memory, long-term/reference memory, and cognitive flexibility, in addition to the molecular markers characteristic of AD. Our findings demonstrate that C21 treatment preserves cognitive function, maintains cerebral blood flow, and reduces Aß accumulation and toxic tau phosphorylation in AD animals post-stroke.

ER stress associated TXNIP-NLRP3 inflammasome activation in hippocampus of human Alzheimer's disease.

Although the exact etiology of Alzheimer's disease (AD) is poorly understood, experimental and clinical evidences suggest the contribution of neuroinflammation in the pathogenesis of AD. Pathologically, AD brain is characterized by an imbalance in redox status, elevated endoplasmic reticulum (ER) stress, synaptic dysfunction, inflammation, and progressive neurodegeneration. It has been noted that continuous accumulation of amyloid-beta (Aß) and intracellular neurofibrillary tangles (NFTs) in AD brain trigger ER stress, which contributes to neurodegeneration. Similarly, experimental evidences supports the hypothesis that thioredoxin-interacting protein (TXNIP), an endogenous regulator of redox regulator thioredoxin (TRX), is activated by ER stress and contributes to activation of NLRP3 (NOD-like receptor protein 3) inflammatory cascade in hippocampus of the AD brain. Hippocampus of postmortem human AD and aged matched non-AD controls were analyzed for the expression ER stress markers and TXNIP-NLRP3 inflammasome at cellular and molecular levels. We found higher expression of TXNIP at protein and transcript levels in close association with pathological markers of AD such as Aß and NFTs in AD hippocampus. In addition, our results demonstrated that TXNIP was co-localized in neurons and microglia. Moreover, expression of binding immunoglobulin protein (BiP), activated eukaryotic initiation factor-2α (eIf2α) and C/EBP homology protein (CHOP), proteins involved the development of ER stress, were elevated in AD hippocampus. Further, elevated expression of effector molecules of NLRP3 inflammasome activation such as apoptosis associated speck-like protein (ASC), cleaved caspase-1 and cleaved interleukin-1ß were observed in the AD hippocampus. The study suggests that TXNIP could be a link that connect ER stress with neuroinflammation. Thus, TXNIP can be a possible therapeutic target to mitigate the progression of neuroinflammation in the pathogenesis of AD.

Thioredoxin interacting protein regulates age-associated neuroinflammation.

Immune system hypersensitivity is believed to contribute to mental frailty in the elderly. Solid evidence indicates NOD-like receptor pyrin domain containing-3 (NLRP3)-inflammasome activation intimately connects aging-associated chronic inflammation (inflammaging) to senile cognitive decline. Thioredoxin interacting protein (TXNIP), an inducible protein involved in oxidative stress, is essential for NLRP3 inflammasome activity. This study aims to find whether TXNIP/NLRP3 inflammasome pathway is involved in senile dementia. According to our studies on sex-matched mice, TXNIP was significantly upregulated in aged animals, paralleled by the NLRP3-inflammasome over-activity leading to enhanced caspase-1 cleavage and IL-1ß maturation, in both sexes. This was closely associated with depletion of the anti-aging and cognition enhancing protein klotho, in aged males. Txnip knockout reversed age-related NLRP3-hyperactivity and enhanced thioredoxin (TRX) levels. Further, TXNIP inhibition along with verapamil replicated TXNIP/NLRP3-inflammasome downregulation in aged animals, with FOXO-1 and mTOR upregulation. These alterations concurred with substantial improvements in both cognitive and sensorimotor abilities. Together, these findings substantiate the pivotal role of TXNIP to drive inflammaging in parallel with klotho depletion and functional decline, and delineate thioredoxin system as a potential target to decelerate senile dementia.

Discovery of a Highly Selective and Potent TRPC3 Inhibitor with High Metabolic Stability and Low Toxicity.

The overactivation of transient receptor potential canonical 3 (TRPC3) is associated with neurodegenerative diseases and hypertension. Pyrazole 3 (Pyr3) is reported as the most selective TRPC3 inhibitor, but it has two inherent structural limitations: (1) the labile ester moiety leads to its rapid hydrolysis to the inactive Pyr8 in vivo, and (2) the alkylating trichloroacrylic amide moiety is known to be toxic. To circumvent these limitations, we designed a series of conformationally restricted Pyr3 analogues and reported that compound 20 maintains high potency and selectivity for human TRPC3 over its closely related TRP channels. It has significantly improved metabolic stability compared with Pyr3 and has a good safety profile. Preliminary evaluation of 20 demonstrated its ability to rescue Aß-induced neuron damage with similar potency to that of Pyr3 in vitro. Collectively, these results suggest that 20 represents a promising scaffold to potentially ameliorate the symptoms associated with TRPC3-mediated neurological and cardiovascular disorders.

Endothelial Nitric Oxide Synthase-Deficient Mice: A Model of Spontaneous Cerebral Small-Vessel Disease.

Age-related cerebral small-vessel disease (CSVD) is a major cause of stroke and dementia. Despite a widespread acceptance of small-vessel arteriopathy, lacunar infarction, diffuse white matter injury, and cognitive impairment as four cardinal features of CSVD, a unifying pathologic mechanism of CSVD remains elusive. Herein, we introduce partial endothelial nitric oxide synthase (eNOS)-deficient mice as a model of age-dependent, spontaneous CSVD. These mice developed cerebral hypoperfusion and blood-brain barrier leakage at a young age, which progressively worsened with advanced age. Their brains exhibited elevated oxidative stress, astrogliosis, cerebral amyloid angiopathy, microbleeds, microinfarction, and white matter pathology. Partial eNOS-deficient mice developed gait disturbances at middle age, and hippocampus-dependent memory deficits at older ages. These mice also showed enhanced expression of bone morphogenetic protein 4 (BMP4) in brain pericytes before myelin loss and white matter pathology. Because BMP4 signaling not only promotes astrogliogenesis but also blocks oligodendrocyte differentiation, we posit that paracrine actions of BMP4, localized within the neurovascular unit, promote white matter disorganization and neurodegeneration. These observations point to BMP4 signaling pathway in the aging brain vasculature as a potential therapeutic target. Finally, because studies in partial eNOS-deficient mice corroborated recent clinical evidence that blood-brain barrier disruption is a primary cause of white matter pathology, the mechanism of impaired nitric oxide signaling-mediated CSVD warrants further investigation.

Temporal unsnarling of brain's acute neuroinflammatory transcriptional profiles reveals panendothelitis as the earliest event preceding microgliosis.

Sepsis-associated encephalopathy (SAE) is an acutely progressing brain dysfunction induced by systemic inflammation. The mechanism of initiation of neuroinflammation during SAE, which ultimately leads to delirium and cognitive dysfunction, remains elusive. We aimed to study the molecular events of SAE to capture its onset and progression into the central nervous system (CNS), and further identify the cellular players involved in mediating acute inflammatory signaling. Gene expression profiling on the cerebral vessels isolated from the brains of the mice treated with peripheral lipopolysaccharide (LPS) revealed that the cerebral vasculature responds within minutes to acute systemic inflammation by upregulating the expression of immediate early response genes, followed by activation of the nuclear factor-κB pathway. To identify the earliest responding cell type, we used fluorescence-activated cell sorting (FACS) to sort the glial and vascular cells from the brains of the mice treated with LPS at different time points, and RNA-seq was performed on microglia and cerebral endothelial cells (CECs). Bioinformatic analysis followed by further validation in all the cell types revealed that panendothelitis. i.e., the activation of CECs is the earliest event in the CNS during the inception of acute neuroinflammation. Microglial activation occurs later than that of CECs, suggesting that CECs are the most likely initial source of proinflammatory mediators, which could further initiate glial cell activation. This is then followed by the activation of apoptotic signaling in the CECs, which is known to lead to the blood-brain barrier disruption and allow peripheral cytokines to leak into the CNS, exacerbate the gliosis, and result in the vicious neuroinflammatory cascade. Together, our results model the earliest sequential events during the advancement of systemic inflammation into the CNS and facilitate to understand the interplay between the vascular and glial cells in initiating and driving acute neuroinflammation during SAE.

Regulation of BACE1 expression after injury is linked to the p75 neurotrophin receptor.

BACE1 is a transmembrane aspartic protease that cleaves various substrates and it is required for normal brain function. BACE1 expression is high during early development, but it is reduced in adulthood. Under conditions of stress and injury, BACE1 levels are increased; however, the underlying mechanisms that drive BACE1 elevation are not well understood. One mechanism associated with brain injury is the activation of injurious p75 neurotrophin receptor (p75), which can trigger pathological signals. Here we report that within 72 h after controlled cortical impact (CCI) or laser injury, BACE1 and p75 are increased and tightly co-expressed in cortical neurons of mouse brain. Additionally, BACE1 is not up-regulated in p75 null mice in response to focal cortical injury, while p75 over-expression results in BACE1 augmentation in HEK-293 and SY5Y cell lines. A luciferase assay conducted in SY5Y cell line revealed that BACE1 expression is regulated at the transcriptional level in response to p75 transfection. Interestingly, this effect does not appear to be dependent upon p75 ligands including mature and pro-neurotrophins. In addition, BACE1 activity on amyloid precursor protein (APP) is enhanced in SY5Y-APP cells transfected with a p75 construct. Lastly, we found that the activation of c-jun n-terminal kinase (JNK) by p75 contributes to BACE1 up-regulation. This study explores how two injury-induced molecules are intimately connected and suggests a potential link between p75 signaling and the expression of BACE1 after brain injury.

Thioredoxin-Interacting Protein (TXNIP) Associated NLRP3 Inflammasome Activation in Human Alzheimer's Disease Brain.

Alzheimer's disease (AD) is the most common form of age-associated dementia characterized by amyloid-ß plaques and neurofibrillary tangles. Recent studies have demonstrated that thioredoxin-interacting protein (TXNIP), an endogenous regulator of redox/glucose induced stress and inflammation, is now known to be upregulated in stroke, traumatic brain injury, diabetes and AD. We hypothesized that TXNIP overexpression sustains neurodegeneration through activation of the nucleotide binding and oligomerization domain-like receptor protein 3 in human AD brains. We analyzed TXNIP and the components of the NLRP3 inflammasome in the cortex of postmortem human brain samples by western blotting, real-time PCR, and immunohistochemical techniques in comparison with age-matched non-demented controls. Our results demonstrate that TXNIP protein as well as its mRNA levels in the cortex was significantly upregulated in AD compared to control brains. Moreover, using double immunofluorescence staining, TXNIP and interlukin-1ß (IL-1ß) were co-localized near Aß plaques and p-tau. These results suggest an association between TXNIP overexpression levels and AD pathogenesis. Further, a significant increased expression of cleaved caspase-1 and IL-1ß, the products of inflammasome activation, was detected in the cortex of AD brains. Together, these findings suggest that TXNIP, an upstream promising new therapeutic target, is a molecular link between inflammation and AD. The significant contribution of TXNIP to AD pathology suggests that strategies focusing on specific targeting of the TXNIP-NLRP3 inflammasome may lead to novel therapies for the management of AD and other age-related dementias.

In vivo evidence for the contribution of peripheral circulating inflammatory exosomes to neuroinflammation.

BACKGROUND: Neuroinflammation is implicated in the development and progression of many neurodegenerative diseases. Conditions that lead to a peripheral immune response are often associated with inflammation in the central nervous system (CNS), suggesting a communication between the peripheral immune system and the neuroimmune system. The underlying mechanism of this relationship remains largely unknown; however, experimental studies have demonstrated that exposure to infectious stimuli, such as lipopolysaccharide (LPS) or high-fat diet (HFD) feeding, result in profound peripheral- and neuro-inflammation. METHODS: Using the model of endotoxemia with LPS, we studied the role of serum-derived exosomes in mediating neuroinflammation. We purified circulating exosomes from the sera of LPS-challenged mice, which were then intravenously injected into normal adult mice. RESULTS: We found that the recipient mice that received serum-derived exosomes from LPS-challenged mice exhibited elevated microglial activation. Moreover, we observed astrogliosis, increased systemic pro-inflammatory cytokine production, and elevated CNS expression of pro-inflammatory cytokine mRNA and the inflammation-associated microRNA (miR-155) in these recipient mice. Gene expression analysis confirmed that many inflammatory microRNAs were significantly upregulated in the purified exosomes under LPS-challenged conditions. We observed accumulated signaling within the microglia of mice that received tail-vein injections of fluorescently labeled exosomes though the percentage of those microglial cells was found low. Finally, purified LPS-stimulated exosomes from blood when infused directly into the cerebral ventricles provoked significant microgliosis and, to a lesser extent, astrogliosis. CONCLUSIONS: The experimental results suggest that circulating exosomes may act as a neuroinflammatory mediator in systemic inflammation.

Brain Cytosolic Phospholipase A2α Mediates Angiotensin II-Induced Hypertension and Reactive Oxygen Species Production in Male Mice.

BACKGROUND: Recently, we reported that angiotensin II (Ang II)-induced hypertension is mediated by group IV cytosolic phospholipase A2α (cPLA2α) via production of prohypertensive eicosanoids. Since Ang II increases blood pressure (BP) via its action in the subfornical organ (SFO), it led us to investigate the expression and possible contribution of cPLA2α to oxidative stress and development of hypertension in this brain area. METHODS: Adenovirus (Ad)-green fluorescence protein (GFP) cPLA2α short hairpin (sh) RNA (Ad-cPLA2α shRNA) and its control Ad-scrambled shRNA (Ad-Scr shRNA) or Ad-enhanced cyan fluorescence protein cPLA2α DNA (Ad-cPLA2α DNA) and its control Ad-GFP DNA were transduced into SFO of cPLA2α+/+ and cPLA2α-/- male mice, respectively. Ang II (700 ng/kg/min) was infused for 14 days in these mice, and BP was measured by tail-cuff and radio telemetry. cPLA2 activity, reactive oxygen species production, and endoplasmic reticulum stress were measured in the SFO. RESULTS: Transduction of SFO with Ad-cPLA2α shRNA, but not Ad-Scr shRNA in cPLA2α+/+ mice, minimized expression of cPLA2α, Ang II-induced cPLA2α activity and oxidative stress in the SFO, BP, and cardiac and renal fibrosis. In contrast, Ad-cPLA2α DNA, but not its control Ad-GFP DNA in cPLA2α-/- mice, restored the expression of cPLA2α, and Ang II-induced increase in cPLA2 activity and oxidative stress in the SFO, BP, cardiac, and renal fibrosis. CONCLUSIONS: These data suggest that cPLA2α in the SFO is crucial in mediating Ang II-induced hypertension and associated pathogenesis. Therefore, development of selective cPLA2α inhibitors could be useful in treating hypertension and its pathogenesis.

Does pathology of small venules contribute to cerebral microinfarcts and dementia?

Microinfarcts are small, but strikingly common, ischemic brain lesions in the aging human brain. There is mounting evidence that microinfarcts contribute to vascular cognitive impairment and dementia, but the origins of microinfarcts are unclear. Understanding the vascular pathologies that cause microinfarcts may yield strategies to prevent their occurrence and reduce their deleterious effects on brain function. Current thinking suggests that cortical microinfarcts arise from the occlusion of penetrating arterioles, which are responsible for delivering oxygenated blood to small volumes of tissue. Unexpectedly, pre-clinical studies have shown that the occlusion of penetrating venules, which drain deoxygenated blood from cortex, lead to microinfarcts that appear identical to those resulting from arteriole occlusion. Here we discuss the idea that cerebral venule pathology could be an overlooked source for brain microinfarcts in humans. This article is part of the Special Issue "Vascular Dementia". Cover Image for this Issue: doi: 10.1111/jnc.14167.

Bidirectional interplay of HSF1 degradation and UPR activation promotes tau hyperphosphorylation.

The unfolded protein response (UPR) in the endoplasmic reticulum (ER) and the cytoplasmic heat stress response are two major stress response systems necessary for maintaining proteostasis for cellular health. Failure of either of these systems, such as in sustained UPR activation or in insufficient heat shock response activation, can lead to the development of neurodegeneration. Alleviation of ER stress and enhancement of heat shock response through heat shock factor 1 (HSF1) activation have previously been considered as attractive potential therapeutic targets for Alzheimer's disease (AD)-a prevalent and devastating tauopathy. Understanding the interplay of the two aforementioned systems and their cooperative role in AD remain elusive. Here we report studies in human brain and tau pathogenic mouse models (rTg4510, PS19, and rTg21221), identifying HSF1 degradation and UPR activation as precursors of aberrant tau pathogenesis. We demonstrate that chemical ER stress inducers caused autophagy-lysosomal HSF1 degradation, resulting in tau hyperphosphorylation in rat primary neurons. In addition, permanent HSF1 loss reversely causes chronic UPR activation, leading to aberrant tau phosphorylation and aggregation in the hippocampus of aged HSF1 heterozygous knock-out mice. The deleterious interplay of UPR activation and HSF1 loss is exacerbated in N2a cells stably overexpressing a pro-aggregation mutant TauRD ΔK280 (N2a-TauRD ΔK280). We provide evidence of how these two stress response systems are intrinsically interweaved by showing that the gene encoding C/EBP-homologous protein (CHOP) activation in the UPR apoptotic pathway facilitates HSF1 degradation, which likely further contributes to prolonged UPR via ER chaperone HSP70 a5 (BiP/GRP78) suppression. Upregulating HSF1 relieves the tau toxicity in N2a-TauRD ΔK280 by reducing CHOP and increasing HSP70 a5 (BiP/GRP78). Our work reveals how the bidirectional crosstalk between the two stress response systems promotes early tau pathology and identifies HSF1 being one likely key player in both systems.

Ubiquitin Ligase NEDD4 Regulates PPARγ Stability and Adipocyte Differentiation in 3T3-L1 Cells.

Peroxisome proliferator-activated receptor-γ (PPARγ) is a ligand-activated nuclear receptor which controls lipid and glucose metabolism. It is also the master regulator of adipogenesis. In adipocytes, ligand-dependent PPARγ activation is associated with proteasomal degradation; therefore, regulation of PPARγ degradation may modulate its transcriptional activity. Here, we show that neural precursor cell expressed developmentally down-regulated protein 4 (NEDD4), an E3 ubiquitin ligase, interacts with the hinge and ligand binding domains of PPARγ and is a bona fide E3 ligase for PPARγ. NEDD4 increases PPARγ stability through the inhibition of its proteasomal degradation. Knockdown of NEDD4 in 3T3-L1 adipocytes reduces PPARγ protein levels and suppresses adipocyte conversion. PPARγ correlates positively with NEDD4 in obese adipose tissue. Together, these findings support NEDD4 as a novel regulator of adipogenesis by modulating the stability of PPARγ.

NEDD4-mediated HSF1 degradation underlies α-synucleinopathy.

Cellular protein homeostasis is achieved by a delicate network of molecular chaperones and various proteolytic processes such as ubiquitin-proteasome system (UPS) to avoid a build-up of misfolded protein aggregates. The latter is a common denominator of neurodegeneration. Neurons are found to be particularly vulnerable to toxic stress from aggregation-prone proteins such as α-synuclein. Induction of heat-shock proteins (HSPs), such as through activated heat shock transcription factor 1 (HSF1) via Hsp90 inhibition, is being investigated as a therapeutic option for proteinopathic diseases. HSF1 is a master stress-protective transcription factor which activates genes encoding protein chaperones (e.g. iHsp70) and anti-apoptotic proteins. However, whether and how HSF1 is dysregulated during neurodegeneration has not been studied. Here, we discover aberrant HSF1 degradation by aggregated α-synuclein (or α-synuclein-induced proteotoxic stress) in transfected neuroblastoma cells. HSF1 dysregulation via α-synuclein was confirmed by in vivo assessment of mouse and in situ studies of human specimens with α-synucleinopathy. We demonstrate that elevated NEDD4 is implicated as the responsible ubiquitin E3 ligase for HSF1 degradation through UPS. Furthermore, pharmacologically induced SIRT1-mediated deacetylation can attenuate aberrant NEDD4-mediated HSF1 degradation. Indeed, we define the acetylation status of the Lys 80 residue located in the DNA-binding domain of HSF1 as a critical factor in modulating HSF1 protein stability in addition to its previously identified role in the transcriptional activity. Together with the finding that preserving HSF1 can alleviate α-synuclein toxicity, this study strongly suggests that aberrant HSF1 degradation is a key neurodegenerative mechanism underlying α-synucleinopathy.