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Caloric restriction and intermittent fasting prolong the lifespan and healthspan of model organisms and improve human health. The natural polyamine spermidine has been similarly linked to autophagy enhancement, geroprotection and reduced incidence of cardiovascular and neurodegenerative diseases across species borders. Here, we asked whether the cellular and physiological consequences of caloric restriction and fasting depend on polyamine metabolism. We report that spermidine levels increased upon distinct regimens of fasting or caloric restriction in yeast, flies, mice and human volunteers. Genetic or pharmacological blockade of endogenous spermidine synthesis reduced fasting-induced autophagy in yeast, nematodes and human cells. Furthermore, perturbing the polyamine pathway in vivo abrogated the lifespan- and healthspan-extending effects, as well as the cardioprotective and anti-arthritic consequences of fasting. Mechanistically, spermidine mediated these effects via autophagy induction and hypusination of the translation regulator eIF5A. In summary, the polyamine-hypusination axis emerges as a phylogenetically conserved metabolic control hub for fasting-mediated autophagy enhancement and longevity.
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A multitude of cellular responses to intrinsic and extrinsic signals converge on macroautophagy/autophagy, a conserved catabolic process that degrades cytoplasmic constituents and organelles in the lysosome, particularly during starvation or stress. In addition to protein degradation, autophagy is deeply interconnected with unconventional protein secretion and polarized sorting at multiple levels within eukaryotic cells. Secretory autophagy (SA) has been recognized as a novel mechanism in which autophagosomes fuse with the plasma membrane and actively participate in the secretion of a series of cytosolic proteins, ranging from tissue remodeling factors to inflammatory molecules of the IL1 family. SA is partially controlled by the glucocorticoid-responsive, HSP90 co-chaperone FKBP5 and members of the SNARE proteins, SEC22B, SNAP23, SNAP29, STX3 and STX4. SA deregulation is implicated in several inflammatory pathologies, including cancer, cell death and degeneration. However, the key molecular mechanisms governing SA and its regulation remain elusive, as does its role in neuroinflammation and neurodegeneration. To further characterize SA and pinpoint its involvement in neuroinflammatory processes, we studied SA-relevant protein interaction networks in mouse brain, microglia and human postmortem brain tissue from control subjects and Alzheimer disease cases. We demonstrate that SA regulates neuroinflammation-mediated neurodegeneration via SKA2 and FKBP5 signaling.
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The localization, number, and function of postsynaptic AMPA-type glutamate receptors (AMPARs) are crucial for synaptic plasticity, a cellular correlate for learning and memory. The Hippo pathway member WWC1 is an important component of AMPAR-containing protein complexes. However, the availability of WWC1 is constrained by its interaction with the Hippo pathway kinases LATS1 and LATS2 (LATS1/2). Here, we explored the biochemical regulation of this interaction and found that it is pharmacologically targetable in vivo. In primary hippocampal neurons, phosphorylation of LATS1/2 by the upstream kinases MST1 and MST2 (MST1/2) enhanced the interaction between WWC1 and LATS1/2, which sequestered WWC1. Pharmacologically inhibiting MST1/2 in male mice and in human brain-derived organoids promoted the dissociation of WWC1 from LATS1/2, leading to an increase in WWC1 in AMPAR-containing complexes. MST1/2 inhibition enhanced synaptic transmission in mouse hippocampal brain slices and improved cognition in healthy male mice and in male mouse models of Alzheimer's disease and aging. Thus, compounds that disrupt the interaction between WWC1 and LATS1/2 might be explored for development as cognitive enhancers.
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Hipocampo , Peptídeos e Proteínas de Sinalização Intracelular , Plasticidade Neuronal , Fosfoproteínas , Proteínas Serina-Treonina Quinases , Receptores de AMPA , Animais , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Serina-Treonina Quinases/genética , Masculino , Humanos , Receptores de AMPA/metabolismo , Receptores de AMPA/genética , Camundongos , Plasticidade Neuronal/fisiologia , Hipocampo/metabolismo , Via de Sinalização Hippo , Serina-Treonina Quinase 3 , Transdução de Sinais , Memória/fisiologia , Proteínas Supressoras de Tumor/metabolismo , Proteínas Supressoras de Tumor/genética , Fator de Crescimento de Hepatócito/metabolismo , Camundongos Endogâmicos C57BL , Doença de Alzheimer/metabolismo , Fosforilação , Neurônios/metabolismoRESUMO
High levels of proinflammatory cytokines induce neurotoxicity and catalyze inflammation-driven neurodegeneration, but the specific release mechanisms from microglia remain elusive. Here we show that secretory autophagy (SA), a non-lytic modality of autophagy for secretion of vesicular cargo, regulates neuroinflammation-mediated neurodegeneration via SKA2 and FKBP5 signaling. SKA2 inhibits SA-dependent IL-1ß release by counteracting FKBP5 function. Hippocampal Ska2 knockdown in male mice hyperactivates SA resulting in neuroinflammation, subsequent neurodegeneration and complete hippocampal atrophy within six weeks. The hyperactivation of SA increases IL-1ß release, contributing to an inflammatory feed-forward vicious cycle including NLRP3-inflammasome activation and Gasdermin D-mediated neurotoxicity, which ultimately drives neurodegeneration. Results from protein expression and co-immunoprecipitation analyses of male and female postmortem human brains demonstrate that SA is hyperactivated in Alzheimer's disease. Overall, our findings suggest that SKA2-regulated, hyperactive SA facilitates neuroinflammation and is linked to Alzheimer's disease, providing mechanistic insight into the biology of neuroinflammation.
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Doença de Alzheimer , Autofagia , Proteínas Cromossômicas não Histona , Proteína 3 que Contém Domínio de Pirina da Família NLR , Doenças Neuroinflamatórias , Animais , Feminino , Humanos , Masculino , Camundongos , Doença de Alzheimer/genética , Doença de Alzheimer/metabolismo , Autofagia/genética , Proteínas Cromossômicas não Histona/metabolismo , Citocinas/metabolismo , Inflamassomos/metabolismo , Microglia/metabolismo , Doenças Neuroinflamatórias/metabolismo , Proteína 3 que Contém Domínio de Pirina da Família NLR/metabolismoRESUMO
Neurological symptoms, including cognitive impairment and fatigue, can occur in both the acute infection phase of coronavirus disease 2019 (COVID-19) and at later stages, yet the mechanisms that contribute to this remain unclear. Here we profiled single-nucleus transcriptomes and proteomes of brainstem tissue from deceased individuals at various stages of COVID-19. We detected an inflammatory type I interferon response in acute COVID-19 cases, which resolves in the late disease phase. Integrating single-nucleus RNA sequencing and spatial transcriptomics, we could localize two patterns of reaction to severe systemic inflammation, one neuronal with a direct focus on cranial nerve nuclei and a separate diffuse pattern affecting the whole brainstem. The latter reflects a bystander effect of the respiratory infection that spreads throughout the vascular unit and alters the transcriptional state of mainly oligodendrocytes, microglia and astrocytes, while alterations of the brainstem nuclei could reflect the connection of the immune system and the central nervous system via, for example, the vagus nerve. Our results indicate that even without persistence of severe acute respiratory syndrome coronavirus 2 in the central nervous system, local immune reactions are prevailing, potentially causing functional disturbances that contribute to neurological complications of COVID-19.
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COVID-19 , Humanos , COVID-19/genética , Proteômica , Tronco Encefálico , Cerebelo , Perfilação da Expressão GênicaRESUMO
OBJECTIVE: Multidisciplinary studies of posttraumatic stress disorder (PTSD) and major depressive disorder (MDD) implicate the dorsolateral prefrontal cortex (DLPFC) in disease risk and pathophysiology. Postmortem brain studies have relied on bulk-tissue RNA sequencing (RNA-seq), but single-cell RNA-seq is needed to dissect cell-type-specific mechanisms. The authors conducted the first single-nucleus RNA-seq postmortem brain study in PTSD to elucidate disease transcriptomic pathology with cell-type-specific resolution. METHOD: Profiling of 32 DLPFC samples from 11 individuals with PTSD, 10 with MDD, and 11 control subjects was conducted (â¼415K nuclei; >13K cells per sample). A replication sample included 15 DLPFC samples (â¼160K nuclei; >11K cells per sample). RESULTS: Differential gene expression analyses identified significant single-nucleus RNA-seq differentially expressed genes (snDEGs) in excitatory (EX) and inhibitory (IN) neurons and astrocytes, but not in other cell types or bulk tissue. MDD samples had more false discovery rate-corrected significant snDEGs, and PTSD samples had a greater replication rate. In EX and IN neurons, biological pathways that were differentially enriched in PTSD compared with MDD included glucocorticoid signaling. Furthermore, glucocorticoid signaling in induced pluripotent stem cell (iPSC)-derived cortical neurons demonstrated greater relevance in PTSD and opposite direction of regulation compared with MDD, especially in EX neurons. Many snDEGs were from the 17q21.31 locus and are particularly interesting given causal roles in disease pathogenesis and DLPFC-based neuroimaging (PTSD: ARL17B, LINC02210-CRHR1, and LRRC37A2; MDD: LRRC37A and LRP4), while others were regulated by glucocorticoids in iPSC-derived neurons (PTSD: SLC16A6, TAF1C; MDD: CDH3). CONCLUSIONS: The study findings point to cell-type-specific mechanisms of brain stress response in PTSD and MDD, highlighting the importance of examining cell-type-specific gene expression and indicating promising novel biomarkers and therapeutic targets.
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Transtorno Depressivo Maior , Transtornos de Estresse Pós-Traumáticos , Humanos , Córtex Pré-Frontal Dorsolateral , Transtorno Depressivo Maior/genética , Transtorno Depressivo Maior/metabolismo , Transtornos de Estresse Pós-Traumáticos/genética , Glucocorticoides/metabolismo , Perfilação da Expressão Gênica , Transcriptoma/genética , Neurônios/metabolismo , Córtex Pré-Frontal/metabolismoRESUMO
Fatty acid amide hydrolase (FAAH) degrades the endocannabinoid anandamide. A polymorphism in FAAH (FAAH C385A) reduces FAAH expression, increases anandamide levels, and increases the risk of obesity. Nevertheless, some studies have found no association between FAAH C385A and obesity. We investigated whether the environmental context governs the impact of FAAH C385A on metabolic outcomes. Using a C385A knock-in mouse model, we found that FAAH A/A mice are more susceptible to glucocorticoid-induced hyperphagia, weight gain, and activation of hypothalamic AMP-activated protein kinase (AMPK). AMPK inhibition occluded the amplified hyperphagic response to glucocorticoids in FAAH A/A mice. FAAH knockdown exclusively in agouti-related protein (AgRP) neurons mimicked the exaggerated feeding response of FAAH A/A mice to glucocorticoids. FAAH A/A mice likewise presented exaggerated orexigenic responses to ghrelin, while FAAH knockdown in AgRP neurons blunted leptin anorectic responses. Together, the FAAH A/A genotype amplifies orexigenic responses and decreases anorexigenic responses, providing a putative mechanism explaining the diverging human findings.
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Proteínas Quinases Ativadas por AMP , Endocanabinoides , Camundongos , Humanos , Animais , Proteína Relacionada com Agouti , Endocanabinoides/metabolismo , Amidoidrolases/metabolismo , ObesidadeRESUMO
High levels of proinflammatory cytokines induce neurotoxicity and catalyze inflammation-driven neurodegeneration, but the specific release mechanisms from microglia remain elusive. We demonstrate that secretory autophagy (SA), a non-lytic modality of autophagy for secretion of vesicular cargo, regulates neuroinflammation-mediated neurodegeneration via SKA2 and FKBP5 signaling. SKA2 inhibits SA-dependent IL-1ß release by counteracting FKBP5 function. Hippocampal Ska2 knockdown in mice hyperactivates SA resulting in neuroinflammation, subsequent neurodegeneration and complete hippocampal atrophy within six weeks. The hyperactivation of SA increases IL-1ß release, initiating an inflammatory feed-forward vicious cycle including NLRP3-inflammasome activation and Gasdermin D (GSDMD)-mediated neurotoxicity, which ultimately drives neurodegeneration. Results from protein expression and co-immunoprecipitation analyses of postmortem brains demonstrate that SA is hyperactivated in Alzheimer's disease. Overall, our findings suggest that SKA2-regulated, hyperactive SA facilitates neuroinflammation and is linked to Alzheimer's disease, providing new mechanistic insight into the biology of neuroinflammation.
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Identification and characterisation of novel targets for treatment is a priority in the field of psychiatry. FKBP5 is a gene with decades of evidence suggesting its pathogenic role in a subset of psychiatric patients, with potential to be leveraged as a therapeutic target for these individuals. While it is widely reported that FKBP5/FKBP51 mRNA/protein (FKBP5/1) expression is impacted by psychiatric disease state, risk genotype and age, it is not known in which cell types and sub-anatomical areas of the human brain this occurs. This knowledge is critical to propel FKBP5/1-targeted treatment development. Here, we performed an extensive, large-scale postmortem study (n = 1024) of FKBP5/1, examining neocortical areas (BA9, BA11 and ventral BA24/BA24a) derived from subjects that lived with schizophrenia, major depression or bipolar disorder. With an extensive battery of RNA (bulk RNA sequencing, single-nucleus RNA sequencing, microarray, qPCR, RNAscope) and protein (immunoblot, immunohistochemistry) analysis approaches, we thoroughly investigated the effects of disease state, ageing and genotype on cortical FKBP5/1 expression including in a cell type-specific manner. We identified consistently heightened FKBP5/1 levels in psychopathology and with age, but not genotype, with these effects strongest in schizophrenia. Using single-nucleus RNA sequencing (snRNAseq; BA9 and BA11) and targeted histology (BA9, BA24a), we established that these disease and ageing effects on FKBP5/1 expression were most pronounced in excitatory superficial layer neurons of the neocortex, and this effect appeared to be consistent in both the granular and agranular areas examined. We then found that this increase in FKBP5 levels may impact on synaptic plasticity, as FKBP5 gex levels strongly and inversely correlated with dendritic mushroom spine density and brain-derived neurotrophic factor (BDNF) levels in superficial layer neurons in BA11. These findings pinpoint a novel cellular and molecular mechanism that has potential to open a new avenue of FKBP51 drug development to treat cognitive symptoms in psychiatric disorders.
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Transtornos Mentais , Neocórtex , Humanos , Transtornos Mentais/genética , Envelhecimento/genética , Neurônios , Genótipo , Polimorfismo de Nucleotídeo ÚnicoRESUMO
Learning and memory rely on changes in postsynaptic glutamergic α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA)-type receptor (AMPAR) number, spatial organization, and function. The Hippo pathway component WW and C2 domain-containing protein 1 (WWC1) regulates AMPAR surface expression and impacts on memory performance. However, synaptic binding partners of WWC1 and its hierarchical position in AMPAR complexes are largely unclear. Using cell-surface proteomics in hippocampal tissue of Wwc1-deficient mice and by generating a hippocampus-specific interactome, we show that WWC1 is a major regulatory platform in AMPAR signaling networks. Under basal conditions, the Hippo pathway members WWC1 and large tumor-suppressor kinase (LATS) are associated, which might prevent WWC1 effects on synaptic proteins. Reduction of WWC1/LATS binding through a point mutation at WWC1 elevates the abundance of WWC1 in AMPAR complexes and improves hippocampal-dependent learning and memory. Thus, uncoupling of WWC1 from the Hippo pathway to AMPAR-regulatory complexes provides an innovative strategy to enhance synaptic transmission.
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Proteômica , Receptores de AMPA , Animais , CamundongosRESUMO
Stress and changes in energy stores are perceived by hormone- and nutrient-sensing nuclei of the hypothalamus, which orchestrate an adaptive physiological body response to maintain homeostasis. Macroautophagy/autophagy is a fundamental lysosomal degradation system contributing to preservation of proteome balance and metabolic homeostasis. Its dysregulation is linked to diverse human pathologies, including neuropsychiatric and metabolic disorders. Autophagy is coordinated by cellular nutrient sensors, including AMPK and MTORC1 that interact with WIPI proteins. Studies suggest that WDR45/WIPI4 interacts with the stress-sensitive co-chaperone FKBP5/FKBP51, which has emerged as a key autophagy scaffold. However, the impact of FKBP5 on autophagy signaling in response to metabolic challenges, such as a high-fat diet, is elusive. Therefore, we manipulated FKBP5 in the mediobasal hypothalamus (MBH) and studied autophagy signaling and protein interactions in their physiological context. We identified FKBP5 as a scaffold of the STK11/LKB1-AMPK complex with WDR45/WIPI4 and TSC2 with WDR45B/WIPI3 in response to metabolic challenges, positioning FKBP5 in major nutrient-sensing and autophagy-regulating networks. Intriguingly, we could demonstrate that FKBP5 deletion in the MBH strongly induces obesity, whereas its overexpression protects against high-fat diet-induced obesity. Our findings suggest a crucial regulatory and adaptive function of FKBP5-regulated autophagy within the MBH in response to metabolic challenges.Abbreviations: AKT: thymoma viral proto-oncogene; AMPK: AMP-activated protein kinase; BECN1: beclin 1, autophagy related; eWAT: epididymal white adipose tissue; FKBP5/FKBP51: FK506 binding protein 5; KO, knockout; MBH, mediobasal hypothalamus; MTORC1, mechanistic target of rapamycin kinase complex 1; p: phosphorylated; PHLPP: PH domain and leucine rich repeat protein phosphatase; RPS6KB/p70S6K: ribosomal protein S6 kinase; SKP2: S-phase kinase-associated protein 2; SM: soleus muscle; SQSTM1/p62, sequestosome 1; STK11/LKB1: serine/threonine kinase 11; TSC: TSC complex; ULK1: unc-51 like kinase 1; WIPI: WD repeat domain, phosphoinositide interacting; WT: wild type.
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Proteínas Quinases Ativadas por AMP , Autofagia , Obesidade , Proteínas de Ligação a Tacrolimo , Proteínas Quinases Ativadas por AMP/metabolismo , Autofagia/fisiologia , Proteínas de Transporte , Alvo Mecanístico do Complexo 1 de Rapamicina/metabolismo , Obesidade/genética , Animais , Camundongos , Proteínas de Ligação a Tacrolimo/genéticaRESUMO
The mediobasal hypothalamus (MBH) is the central region in the physiological response to metabolic stress. The FK506-binding protein 51 (FKBP51) is a major modulator of the stress response and has recently emerged as a scaffolder regulating metabolic and autophagy pathways. However, the detailed protein-protein interactions linking FKBP51 to autophagy upon metabolic challenges remain elusive. We performed mass spectrometry-based metabolomics of FKBP51 knockout (KO) cells revealing an increased amino acid and polyamine metabolism. We identified FKBP51 as a central nexus for the recruitment of the LKB1/AMPK complex to WIPI4 and TSC2 to WIPI3, thereby regulating the balance between autophagy and mTOR signaling in response to metabolic challenges. Furthermore, we demonstrated that MBH FKBP51 deletion strongly induces obesity, while its overexpression protects against high-fat diet (HFD)-induced obesity. Our study provides an important novel regulatory function of MBH FKBP51 within the stress-adapted autophagy response to metabolic challenges.
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Hipotálamo , Proteínas de Ligação a Tacrolimo , Autofagia , Dieta Hiperlipídica/efeitos adversos , Humanos , Hipotálamo/metabolismo , Obesidade/metabolismo , Proteínas de Ligação a Tacrolimo/genética , Proteínas de Ligação a Tacrolimo/metabolismoRESUMO
FKBP51 is an important inhibitor of the glucocorticoid receptor (GR) signaling. High FKBP51 levels are associated to stress-related disorders, which are linked to GR resistance. SUMO conjugation to FKBP51 is necessary for FKBP51's inhibitory action on GR. The GR/FKBP51 pathway is target of antidepressant action. Thus we investigated if these drugs could inhibit FKBP51 SUMOylation and therefore restore GR activity. Screening cells using Ni2+ affinity and in vitro SUMOylation assays revealed that tricyclic antidepressants- particularly clomipramine- inhibited FKBP51 SUMOylation. Our data show that clomipramine binds to FKBP51 inhibiting its interaction with PIAS4 and therefore hindering its SUMOylation. The inhibition of FKBP51 SUMOylation decreased its binding to Hsp90 and GR facilitating FKBP52 recruitment, and enhancing GR activity. Reduction of PIAS4 expression in rat primary astrocytes impaired FKBP51 interaction with GR, while clomipramine could no longer exert its inhibitory action. This mechanism was verified in vivo in mice treated with clomipramine. These results describe the action of antidepressants as repressors of FKBP51 SUMOylation as a molecular switch for restoring GR sensitivity, thereby providing new potential routes of antidepressant intervention.
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Receptores de Glucocorticoides , Sumoilação , Animais , Antidepressivos Tricíclicos/farmacologia , Clomipramina , Regulação da Expressão Gênica , Camundongos , Ratos , Receptores de Glucocorticoides/metabolismo , Proteínas de Ligação a Tacrolimo/metabolismoRESUMO
Cerebral amyloid angiopathy (CAA) is an age-related condition and a major cause of intracerebral hemorrhage and cognitive decline that shows close links with Alzheimer's disease (AD). CAA is characterized by the aggregation of amyloid-ß (Aß) peptides and formation of Aß deposits in the brain vasculature resulting in a disruption of the angioarchitecture. Capillaries are a critical site of Aß pathology in CAA type 1 and become dysfunctional during disease progression. Here, applying an advanced protocol for the isolation of parenchymal microvessels from post-mortem brain tissue combined with liquid chromatography tandem mass spectrometry (LC-MS/MS), we determined the proteomes of CAA type 1 cases (n = 12) including a patient with hereditary cerebral hemorrhage with amyloidosis-Dutch type (HCHWA-D), and of AD cases without microvascular amyloid pathology (n = 13) in comparison to neurologically healthy controls (n = 12). ELISA measurements revealed microvascular Aß1-40 levels to be exclusively enriched in CAA samples (mean: > 3000-fold compared to controls). The proteomic profile of CAA type 1 was characterized by massive enrichment of multiple predominantly secreted proteins and showed significant overlap with the recently reported brain microvascular proteome of patients with cerebral autosomal-dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL), a hereditary cerebral small vessel disease (SVD) characterized by the aggregation of the Notch3 extracellular domain. We found this overlap to be largely attributable to the accumulation of high-temperature requirement protein A1 (HTRA1), a serine protease with an established role in the brain vasculature, and several of its substrates. Notably, this signature was not present in AD cases. We further show that HTRA1 co-localizes with Aß deposits in brain capillaries from CAA type 1 patients indicating a pathologic recruitment process. Together, these findings suggest a central role of HTRA1-dependent protein homeostasis in the CAA microvasculature and a molecular connection between multiple types of brain microvascular disease.
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Encéfalo/metabolismo , CADASIL/metabolismo , Angiopatia Amiloide Cerebral/metabolismo , Serina Peptidase 1 de Requerimento de Alta Temperatura A/metabolismo , Proteoma/metabolismo , Idoso , Idoso de 80 Anos ou mais , Encéfalo/patologia , CADASIL/patologia , Angiopatia Amiloide Cerebral/patologia , Cromatografia Líquida , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Proteômica , Espectrometria de Massas em TandemRESUMO
Dementia is a devastating age-related disorder. Its therapy would largely benefit from the identification of susceptible subjects at early, prodromal stages of the disease. To search for such prognostic markers of cognitive impairment, we studied spatial navigation in male BALBc vs. B6N mice in combination with in vivo magnetic resonance spectroscopy (1H-MRS). BALBc mice consistently showed higher escape latencies than B6N mice, both in the Water Cross Maze (WCM) and the Morris water maze (MWM). These performance deficits coincided with higher levels of myo-inositol (mIns) in the dorsal hippocampus before and after training. Subsequent biochemical analyses of hippocampal specimens by capillary immunodetection and liquid chromatography mass spectrometry-based (LC/MS) metabolomics revealed a higher abundance of glial markers (IBA-1, S100B, and GFAP) as well as distinct alterations in metabolites including a decrease in vitamins (pantothenic acid and nicotinamide), neurotransmitters (acetylcholine), their metabolites (glutamine), and acetyl-L-carnitine. Supplementation of low abundant acetyl-L-carnitine via the drinking water, however, failed to revert the behavioral deficits shown by BALBc mice. Based on our data we suggest (i) BALBc mice as an animal model and (ii) hippocampal mIns levels as a prognostic marker of mild cognitive impairment (MCI), due to (iii) local changes in microglia and astrocyte activity, which may (iv) result in decreased concentrations of promnesic molecules.
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Like other members of the superfamily of nuclear receptors, the peroxisome proliferator-activated receptor γ (PPARγ), is a ligand-activated transcription factor known for its insulin-sensitizing actions in the periphery. Despite only sparse evidence for PPARγ in the CNS, many reports suggest direct PPARγ-mediated actions in the brain. This study aimed to (i) map PPARγ expression in rodent brain areas, involved in the regulation of cognitive, motivational, and emotional functions, (ii) examine the regulation of central PPARγ by physiological variables (age, sex, obesity); (iii) chemotypically identify PPARγ-expressing cells in the frontal cortex (FC) and hippocampus (HP); (iv) study whether activation of PPARγ by pioglitazone (Pio) in FC and HP cells can induce target gene expression; and (v) demonstrate the impact of activated PPARγ on learning behavior and motivation. Immunoreactive PPARγ was detectable in specific sub-nuclei/subfields of the FC, HP, nucleus accumbens, amygdala, hypothalamus, thalamus, and granular layers of the cerebellum. PPARγ protein levels were upregulated during aging and in high fat diet-induced obesity. PPARγ mRNA expression was upregulated in the amygdala of females (but not males) that were made obese. Neural precursor cells, mature neurons, and astrocytes in primary FC and HP cultures were shown to express PPARγ. Pioglitazone dose-dependently upregulated PPARγ target genes in manner that was specific to the origin (FC or HP) of the cultures. Lastly, administration of Pio impaired motivation and associative learning. Collectively, we provide evidence for the presence of regulatable PPARγ in the brain and demonstrate their participation the regulation of key behaviors.
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Células-Tronco Neurais , Tiazolidinedionas , Encéfalo/metabolismo , Feminino , Humanos , Masculino , Motivação , Células-Tronco Neurais/metabolismo , PPAR gama/metabolismo , Pioglitazona/farmacologia , Tiazolidinedionas/farmacologiaRESUMO
As the cerebellar molecular stress response is understudied, we assessed protein expression levels of hypothalamic-pituitary-adrenal (HPA) axis regulators and neurostructural markers in the cerebellum of a male PTSD mouse model and of unstressed vs. stressed male FK506 binding protein 51 (Fkbp5) knockout (KO) vs. wildtype mice. We explored the translatability of our findings in the Fkbp5 KO model to the situation in humans by correlating mRNA levels of candidates with those of FKBP5 in two whole transcriptome datasets of post-mortem human cerebellum and in blood of unstressed and stressed humans. Fkbp5 deletion rescued the stress-induced loss in hippocampal, prefrontal cortical, and, possibly, also cerebellar FKBP52 expression and modulated post-stress cerebellar expression levels of the glucocorticoid receptor (GR) and possibly (trend) also of glial fibrillary acidic protein (GFAP). Accordingly, expression levels of genes encoding for these three genes correlated with those of FKBP5 in human post-mortem cerebellum, while other neurostructural markers were not related to Fkbp5 either in mouse or human cerebellum. Also, gene expression levels of the two immunophilins correlated inversely in the blood of unstressed and stressed humans. We found transient changes in FKBP52 and persistent changes in GR and GFAP in the cerebellum of PTSD-like mice. Altogether, upon elucidating the cerebellar stress response we found first evidence for a novel facet of HPA axis regulation, i.e., the ability of FKBP51 to modulate the expression of its antagonist FKBP52 in the mouse and, speculatively, also in the human brain and blood and, moreover, detected long-term single stress-induced changes in expression of cerebellar HPA axis regulators and neurostructural markers of which some might contribute to the role of the cerebellum in fear extinction.
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Delayed onset of antidepressant action is a shortcoming in depression treatment. Ketamine and its metabolite (2R,6R)-hydroxynorketamine (HNK) have emerged as promising rapid-acting antidepressants. However, their mechanism of action remains unknown. In this study, we first described the anxious and depression-prone inbred mouse strain, DBA/2J, as an animal model to assess the antidepressant-like effects of ketamine and HNK in vivo. To decode the molecular mechanisms mediating HNK's rapid antidepressant effects, a longitudinal cerebrospinal fluid (CSF) proteome profiling of its acute and sustained effects was conducted using an unbiased, hypothesis-free mass spectrometry-based proteomics approach. A total of 387 proteins were identified, with a major implication of significantly differentially expressed proteins in the glucocorticoid receptor (GR) signaling pathway, providing evidence for a link between HNK and regulation of the stress hormone system. Mechanistically, we identified HNK to repress GR-mediated transcription and reduce hormonal sensitivity of GR in vitro. In addition, mammalian target of rapamycin (mTOR) and brain-derived neurotrophic factor (BDNF) were predicted to be important upstream regulators of HNK treatment. Our results contribute to precise understanding of the temporal dynamics and molecular targets underlying HNK's rapid antidepressant-like effects, which can be used as a benchmark for improved treatment strategies for depression in future.
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The stress response is an essential mechanism for maintaining homeostasis, and its disruption is implicated in several psychiatric disorders. On the cellular level, stress activates, among other mechanisms, autophagy that regulates homeostasis through protein degradation and recycling. Secretory autophagy is a recently described pathway in which autophagosomes fuse with the plasma membrane rather than with lysosomes. Here, we demonstrate that glucocorticoid-mediated stress enhances secretory autophagy via the stress-responsive co-chaperone FK506-binding protein 51. We identify the matrix metalloproteinase 9 (MMP9) as one of the proteins secreted in response to stress. Using cellular assays and in vivo microdialysis, we further find that stress-enhanced MMP9 secretion increases the cleavage of pro-brain-derived neurotrophic factor (proBDNF) to its mature form (mBDNF). BDNF is essential for adult synaptic plasticity and its pathway is associated with major depression and posttraumatic stress disorder. These findings unravel a cellular stress adaptation mechanism that bears the potential of opening avenues for the understanding of the pathophysiology of stress-related disorders.
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Autofagia/efeitos dos fármacos , Fator Neurotrófico Derivado do Encéfalo/metabolismo , Dexametasona/farmacologia , Metaloproteinase 9 da Matriz/metabolismo , Animais , Autofagossomos/metabolismo , Linhagem Celular , Linhagem Celular Tumoral , Membrana Celular/metabolismo , Glucocorticoides/farmacologia , Células HEK293 , Humanos , Camundongos Knockout , Plasticidade Neuronal/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos , Estresse FisiológicoRESUMO
Viruses manipulate cellular metabolism and macromolecule recycling processes like autophagy. Dysregulated metabolism might lead to excessive inflammatory and autoimmune responses as observed in severe and long COVID-19 patients. Here we show that SARS-CoV-2 modulates cellular metabolism and reduces autophagy. Accordingly, compound-driven induction of autophagy limits SARS-CoV-2 propagation. In detail, SARS-CoV-2-infected cells show accumulation of key metabolites, activation of autophagy inhibitors (AKT1, SKP2) and reduction of proteins responsible for autophagy initiation (AMPK, TSC2, ULK1), membrane nucleation, and phagophore formation (BECN1, VPS34, ATG14), as well as autophagosome-lysosome fusion (BECN1, ATG14 oligomers). Consequently, phagophore-incorporated autophagy markers LC3B-II and P62 accumulate, which we confirm in a hamster model and lung samples of COVID-19 patients. Single-nucleus and single-cell sequencing of patient-derived lung and mucosal samples show differential transcriptional regulation of autophagy and immune genes depending on cell type, disease duration, and SARS-CoV-2 replication levels. Targeting of autophagic pathways by exogenous administration of the polyamines spermidine and spermine, the selective AKT1 inhibitor MK-2206, and the BECN1-stabilizing anthelmintic drug niclosamide inhibit SARS-CoV-2 propagation in vitro with IC50 values of 136.7, 7.67, 0.11, and 0.13 µM, respectively. Autophagy-inducing compounds reduce SARS-CoV-2 propagation in primary human lung cells and intestinal organoids emphasizing their potential as treatment options against COVID-19.