RESUMO
Omnivorous animals, including mice and humans, tend to prefer energy-dense nutrients rich in fat over plant-based diets, especially for short periods of time, but the health consequences of this short-term consumption of energy-dense nutrients are unclear. Here, we show that short-term reiterative switching to 'feast diets', mimicking our social eating behavior, breaches the potential buffering effect of the intestinal microbiota and reorganizes the immunological architecture of mucosa-associated lymphoid tissues. The first dietary switch was sufficient to induce transient mucosal immune depression and suppress systemic immunity, leading to higher susceptibility to Salmonella enterica serovar Typhimurium and Listeria monocytogenes infections. The ability to respond to antigenic challenges with a model antigen was also impaired. These observations could be explained by a reduction of CD4+ T cell metabolic fitness and cytokine production due to impaired mTOR activity in response to reduced microbial provision of fiber metabolites. Reintroducing dietary fiber rewired T cell metabolism and restored mucosal and systemic CD4+ T cell functions and immunity. Finally, dietary intervention with human volunteers confirmed the effect of short-term dietary switches on human CD4+ T cell functionality. Therefore, short-term nutritional changes cause a transient depression of mucosal and systemic immunity, creating a window of opportunity for pathogenic infection.
Assuntos
Mucosa , Salmonella typhimurium , Humanos , Camundongos , Animais , Linfócitos T , Imunidade nas MucosasRESUMO
Dynamic microtubules critically regulate synaptic functions, but the role of microtubule severing in these processes is barely understood. Katanin is a neuronally expressed microtubule-severing complex regulating microtubule number and length in cell division or neurogenesis; however, its potential role in synaptic functions has remained unknown. Studying mice from both sexes, we found that katanin is abundant in neuronal dendrites and can be detected at individual excitatory spine synapses. Overexpression of a dominant-negative ATPase-deficient katanin subunit to functionally inhibit severing alters the growth of microtubules in dendrites, specifically at premature but not mature neuronal stages without affecting spine density. Notably, interference with katanin function prevented structural spine remodeling following single synapse glutamate uncaging and significantly affected the potentiation of AMPA-receptor-mediated excitatory currents after chemical induction of long-term potentiation. Furthermore, katanin inhibition reduced the invasion of microtubules into fully developed spines. Our data demonstrate that katanin-mediated microtubule severing regulates structural and functional plasticity at synaptic sites.
Assuntos
Microtúbulos , Neurônios , Animais , Camundongos , Katanina/genética , Katanina/metabolismo , Microtúbulos/metabolismo , Neurônios/fisiologia , Neurogênese , Plasticidade NeuronalRESUMO
Proteins delivered by endocytosis or autophagy to lysosomes are degraded by exo- and endoproteases. In humans 15 lysosomal cathepsins (CTS) act as important physiological regulators. The cysteine proteases CTSB and CTSL and the aspartic protease CTSD are the most abundant and functional important lysosomal proteinases. Whereas their general functions in proteolysis in the lysosome, their individual substrate, cleavage specificity, and their possible sequential action on substrate proteins have been previously studied, their functional redundancy is still poorly understood. To address a possible common role of highly expressed and functional important CTS proteases, we generated CTSB-, CTSD-, CTSL-, and CTSBDL-triple deficient (KO) human neuroblastoma-derived SH-SY5Y cells and CTSB-, CTSD-, CTSL-, CTSZ and CTSBDLZ-quadruple deficient (KO) HeLa cells. These cells with a combined cathepsin deficiency exhibited enlarged lysosomes and accumulated lipofuscin-like storage material. The lack of the three (SH-SY5Y) or four (HeLa) major CTSs caused an impaired autophagic flux and reduced degradation of endocytosed albumin. Proteome analyses of parental and CTS-depleted cells revealed an enrichment of cleaved peptides, lysosome/autophagy-associated proteins, and potentially endocytosed membrane proteins like the amyloid precursor protein (APP), which can be subject to endocytic degradation. Amino- and carboxyterminal APP fragments accumulated in the multiple CTS-deficient cells, suggesting that multiple CTS-mediated cleavage events regularly process APP. In summary, our analyses support the idea that different lysosomal cathepsins act in concert, have at least partially and functionally redundant substrates, regulate protein degradation in autophagy, and control cellular proteostasis, as exemplified by their involvement in the degradation of APP fragments.
Assuntos
Autofagia , Catepsinas , Lisossomos , Proteólise , Humanos , Lisossomos/metabolismo , Catepsinas/metabolismo , Catepsinas/genética , Células HeLa , Endocitose , Catepsina L/metabolismo , Catepsina L/genética , Linhagem Celular Tumoral , Precursor de Proteína beta-Amiloide/metabolismo , Precursor de Proteína beta-Amiloide/genéticaRESUMO
Synaptic loss is an early event in the penumbra area after an ischemic stroke. Promoting synaptic preservation in this area would likely improve functional neurological recovery. We aimed to detect proteins involved in endogenous protection mechanisms of synapses in the penumbra after stroke and to analyse potential beneficial effects of these candidates for a prospective stroke treatment. For this, we performed Liquid Chromatography coupled to Mass Spectrometry (LC-MS)-based proteomics of synaptosomes isolated from the ipsilateral hemispheres of mice subjected to experimental stroke at different time points (24 h, 4 and 7 days) and compared them to sham-operated mice. Proteomic analyses indicated that, among the differentially expressed proteins between the two groups, cystatin C (CysC) was significantly increased at 24 h and 4 days following stroke, before returning to steady-state levels at 7 days, thus indicating a potential transient and intrinsic rescue mechanism attempt of neurons. When CysC was applied to primary neuronal cultures subjected to an in vitro model of ischemic damage, this treatment significantly improved the preservation of synaptic structures. Notably, similar effects were observed when CysC was loaded into brain-derived extracellular vesicles (BDEVs). Finally, when CysC contained in BDEVs was administered intracerebroventricularly to stroked mice, it significantly increased the expression of synaptic markers such as SNAP25, Homer-1, and NCAM in the penumbra area compared to the group supplied with empty BDEVs. Thus, we show that CysC-loaded BDEVs promote synaptic protection after ischemic damage in vitro and in vivo, opening the possibility of a therapeutic use in stroke patients.
Assuntos
Isquemia Encefálica , Encéfalo , Cistatina C , Vesículas Extracelulares , Camundongos Endogâmicos C57BL , Sinapses , Animais , Vesículas Extracelulares/metabolismo , Vesículas Extracelulares/transplante , Cistatina C/metabolismo , Sinapses/metabolismo , Camundongos , Masculino , Isquemia Encefálica/metabolismo , Isquemia Encefálica/patologia , Encéfalo/metabolismo , Encéfalo/patologia , Proteômica/métodos , Sinaptossomos/metabolismo , Neurônios/metabolismo , Acidente Vascular Cerebral/metabolismo , Acidente Vascular Cerebral/patologia , Acidente Vascular Cerebral/terapia , Células Cultivadas , Modelos Animais de DoençasRESUMO
A Disintegrin And Metalloproteinase 10 (ADAM10) plays a pivotal role in shaping neuronal networks by orchestrating the activity of numerous membrane proteins through the shedding of their extracellular domains. Despite its significance in the brain, the specific cellular localization of ADAM10 remains not well understood due to a lack of appropriate tools. Here, using a specific ADAM10 antibody suitable for immunostainings, we observed that ADAM10 is localized to presynapses and especially enriched at presynaptic vesicles of mossy fiber (MF)-CA3 synapses in the hippocampus. These synapses undergo pronounced frequency facilitation of neurotransmitter release, a process that play critical roles in information transfer and neural computation. We demonstrate, that in conditional ADAM10 knockout mice the ability of MF synapses to undergo this type of synaptic plasticity is greatly reduced. The loss of facilitation depends on the cytosolic domain of ADAM10 and association with the calcium sensor synaptotagmin 7 rather than ADAM10's proteolytic activity. Our findings unveil a new role of ADAM10 in the regulation of synaptic vesicle exocytosis.
Assuntos
Proteína ADAM10 , Secretases da Proteína Precursora do Amiloide , Proteínas de Membrana , Camundongos Knockout , Plasticidade Neuronal , Vesículas Sinápticas , Animais , Proteína ADAM10/metabolismo , Proteína ADAM10/genética , Plasticidade Neuronal/fisiologia , Secretases da Proteína Precursora do Amiloide/metabolismo , Secretases da Proteína Precursora do Amiloide/genética , Proteínas de Membrana/metabolismo , Proteínas de Membrana/genética , Camundongos , Vesículas Sinápticas/metabolismo , Camundongos Endogâmicos C57BL , Sinapses/metabolismo , Fibras Musgosas Hipocampais/metabolismo , Hipocampo/metabolismo , Exocitose/fisiologia , Terminações Pré-Sinápticas/metabolismo , Transmissão Sináptica , Sinaptotagminas/metabolismo , Sinaptotagminas/genéticaRESUMO
The disruption of astrocytic catabolic processes contributes to the impairment of amyloid-ß (Aß) clearance, neuroinflammatory signaling, and the loss of synaptic contacts in late-onset Alzheimer's disease (AD). While it is known that the posttranslational modifications of Aß have significant implications on biophysical properties of the peptides, their consequences for clearance impairment are not well understood. It was previously shown that N-terminally pyroglutamylated Aß3(pE)-42, a significant constituent of amyloid plaques, is efficiently taken up by astrocytes, leading to the release of pro-inflammatory cytokine tumor necrosis factor α and synapse loss. Here we report that Aß3(pE)-42, but not Aß1-42, gradually accumulates within the astrocytic endolysosomal system, disrupting this catabolic pathway and inducing the formation of heteromorphous vacuoles. This accumulation alters lysosomal kinetics, lysosome-dependent calcium signaling, and upregulates the lysosomal stress response. These changes correlate with the upregulation of glial fibrillary acidic protein (GFAP) and increased activity of nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB). Treatment with a lysosomal protease inhibitor, E-64, rescues GFAP upregulation, NF-κB activation, and synapse loss, indicating that abnormal lysosomal protease activity is upstream of pro-inflammatory signaling and related synapse loss. Collectively, our data suggest that Aß3(pE)-42-induced disruption of the astrocytic endolysosomal system leads to cytoplasmic leakage of lysosomal proteases, promoting pro-inflammatory signaling and synapse loss, hallmarks of AD-pathology.
Assuntos
Peptídeos beta-Amiloides , Astrócitos , Lisossomos , Astrócitos/metabolismo , Peptídeos beta-Amiloides/metabolismo , Animais , Lisossomos/metabolismo , Transdução de Sinais/fisiologia , Processamento de Proteína Pós-Traducional/fisiologia , Endossomos/metabolismo , Proteína Glial Fibrilar Ácida/metabolismo , Fragmentos de Peptídeos/metabolismo , Camundongos , Células Cultivadas , HumanosRESUMO
The recent outbreak of monkeypox virus (MPXV) was unprecedented in its size and distribution. Those living with uncontrolled HIV and low CD4 T cell counts might develop a fulminant clinical mpox course with increased mortality, secondary infections, and necrotizing lesions. Fatal cases display a high and widespread MPXV tissue burden. The underlying pathomechanisms are not fully understood. We report here the pathological findings of an MPXV-driven abscess in gastrocnemius muscle requiring surgery in an immunocompromised patient with severe mpox. Presence of virus particles and infectivity were confirmed by electron microscopy, expansion microscopy, and virus culture, respectively. MPXV tissue distribution by immunohistochemistry (IHC) showed a necrotic core with infection of different cell types. In contrast, at the lesion rim fibroblasts were mainly infected. Immune cells were almost absent in the necrotic core, but were abundant at the infection rim and predominantly macrophages. Further, we detected high amounts of alternatively activated GPNMB+-macrophages at the lesion border. Of note, macrophages only rarely colocalized with virus-infected cells. Insufficient clearance of infected cells and infection of lesion-associated fibroblasts sustained by the abundance of profibrotic macrophages might lead to the coalescing of lesions and the severe and persistent clinical mpox course observed in immunocompromised patients.
Assuntos
Hospedeiro Imunocomprometido , Monkeypox virus , Mpox , Músculo Esquelético , Humanos , Músculo Esquelético/virologia , Músculo Esquelético/patologia , Músculo Esquelético/imunologia , Mpox/virologia , Mpox/imunologia , Monkeypox virus/imunologia , Masculino , Macrófagos/imunologia , Macrófagos/virologia , Fibroblastos/virologia , Fibroblastos/imunologia , Imuno-Histoquímica , Abscesso/imunologia , Abscesso/virologia , Abscesso/patologia , Pessoa de Meia-IdadeRESUMO
Myasthenia gravis is a chronic antibody-mediated autoimmune disease disrupting neuromuscular synaptic transmission. Informative biomarkers remain an unmet need to stratify patients with active disease requiring intensified monitoring and therapy; their identification is the primary objective of this study. We applied mass spectrometry-based proteomic serum profiling for biomarker discovery. We studied an exploration and a prospective validation cohort consisting of 114 and 140 anti-acetylcholine receptor antibody (AChR-Ab)-positive myasthenia gravis patients, respectively. For downstream analysis, we applied a machine learning approach. Protein expression levels were confirmed by ELISA and compared to other myasthenic cohorts, in addition to myositis and neuropathy patients. Anti-AChR-Ab levels were determined by a radio receptor assay. Immunohistochemistry and immunofluorescence of intercostal muscle biopsies were employed for validation in addition to interactome studies of inter-alpha-trypsin inhibitor heavy chain H3 (ITIH3). Machine learning identified ITIH3 as potential serum biomarker reflective of disease activity. Serum levels correlated with disease activity scores in the exploration and validation cohort and were confirmed by ELISA. Lack of correlation between anti-AChR-Ab levels and clinical scores underlined the need for biomarkers. In a subgroup analysis, ITIH3 was indicative of treatment responses. Immunostaining of muscle specimens from these patients demonstrated ITIH3 localization at the neuromuscular endplates in myasthenia gravis but not in controls, thus providing a structural equivalent for our serological findings. Immunoprecipitation of ITIH3 and subsequent proteomics lead to identification of its interaction partners playing crucial roles in neuromuscular transmission. This study provides data on ITIH3 as a potential pathophysiological-relevant biomarker of disease activity in myasthenia gravis. Future studies are required to facilitate translation into clinical practice.
Assuntos
Biomarcadores , Miastenia Gravis , Humanos , Miastenia Gravis/sangue , Miastenia Gravis/diagnóstico , Miastenia Gravis/patologia , Miastenia Gravis/metabolismo , Biomarcadores/sangue , Biomarcadores/metabolismo , Masculino , Feminino , Pessoa de Meia-Idade , Adulto , Idoso , Autoanticorpos/sangue , Receptores Colinérgicos/imunologia , Receptores Colinérgicos/metabolismo , Proteômica/métodos , Estudos de Coortes , Adulto Jovem , Proteínas Secretadas Inibidoras de Proteinases/sangue , Aprendizado de MáquinaRESUMO
Polyphosphate is a procoagulant inorganic polymer of linear-linked orthophosphate residues. Multiple investigations have established the importance of platelet polyphosphate in blood coagulation; however, the mechanistic details of polyphosphate homeostasis in mammalian species remain largely undefined. In this study, xenotropic and polytropic retrovirus receptor 1 (XPR1) regulated polyphosphate in platelets and was implicated in thrombosis in vivo. We used bioinformatic analyses of omics data to identify XPR1 as a major phosphate transporter in platelets. XPR1 messenger RNA and protein expression inversely correlated with intracellular polyphosphate content and release. Pharmacological interference with XPR1 activity increased polyphosphate stores, led to enhanced platelet-driven coagulation, and amplified thrombus formation under flow via the polyphosphate/factor XII pathway. Conditional gene deletion of Xpr1 in platelets resulted in polyphosphate accumulation, accelerated arterial thrombosis, and augmented activated platelet-driven pulmonary embolism without increasing bleeding in mice. These data identify platelet XPR1 as an integral regulator of platelet polyphosphate metabolism and reveal a fundamental role for phosphate homeostasis in thrombosis.
Assuntos
Plaquetas/metabolismo , Polifosfatos/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Receptores Virais/metabolismo , Trombose/metabolismo , Animais , Transporte Biológico , Coagulação Sanguínea , Fator XII/metabolismo , Feminino , Masculino , Camundongos , Trombose/sangue , Receptor do Retrovírus Politrópico e XenotrópicoRESUMO
Mutations in the gene encoding the microtubule-severing protein spastin (spastic paraplegia 4 [SPG4]) cause hereditary spastic paraplegia (HSP), associated with neurodegeneration, spasticity, and motor impairment. Complicated forms (complicated HSP [cHSP]) further include cognitive deficits and dementia; however, the etiology and dysfunctional mechanisms of cHSP have remained unknown. Here, we report specific working and associative memory deficits upon spastin depletion in mice. Loss of spastin-mediated severing leads to reduced synapse numbers, accompanied by lower miniature excitatory postsynaptic current (mEPSC) frequencies. At the subcellular level, mutant neurons are characterized by longer microtubules with increased tubulin polyglutamylation levels. Notably, these conditions reduce kinesin-microtubule binding, impair the processivity of kinesin family protein (KIF) 5, and reduce the delivery of presynaptic vesicles and postsynaptic α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors. Rescue experiments confirm the specificity of these results by showing that wild-type spastin, but not the severing-deficient and disease-associated K388R mutant, normalizes the effects at the synaptic, microtubule, and transport levels. In addition, short hairpin RNA (shRNA)-mediated reduction of tubulin polyglutamylation on spastin knockout background normalizes KIF5 transport deficits and attenuates the loss of excitatory synapses. Our data provide a mechanism that connects spastin dysfunction with the regulation of kinesin-mediated cargo transport, synapse integrity, and cognition.
Assuntos
Ácido Glutâmico/metabolismo , Cinesinas/metabolismo , Transtornos da Memória/metabolismo , Transtornos da Memória/fisiopatologia , Memória de Curto Prazo , Neurônios/metabolismo , Espastina/deficiência , Tubulina (Proteína)/metabolismo , Potenciais de Ação , Animais , Membrana Celular/metabolismo , Espinhas Dendríticas/metabolismo , Espinhas Dendríticas/ultraestrutura , Potenciais Pós-Sinápticos Excitadores , Hipocampo/patologia , Hipocampo/fisiopatologia , Camundongos Knockout , Microtúbulos/metabolismo , Microtúbulos/ultraestrutura , Atividade Motora , Neurônios/patologia , Neurônios/ultraestrutura , Transporte Proteico , Espastina/metabolismo , Sinapses/metabolismo , Sinapses/ultraestrutura , Vesículas Sinápticas/metabolismoRESUMO
Mucopolysaccharidosis type VI (MPS-VI), caused by mutational inactivation of the glycosaminoglycan-degrading enzyme arylsulfatase B (Arsb), is a lysosomal storage disorder primarily affecting the skeleton. We have previously reported that Arsb-deficient mice display high trabecular bone mass and impaired skeletal growth. In the present study, we treated them by weekly injection of recombinant human ARSB (rhARSB) to analyze the impact of enzyme replacement therapy (ERT) on skeletal growth and bone remodeling. We found that all bone-remodeling abnormalities of Arsb-deficient mice were prevented by ERT, whereas chondrocyte defects were not. Likewise, histologic analysis of the surgically removed femoral head from an ERT-treated MPS-VI patient revealed that only chondrocytes were pathologically affected. Remarkably, a side-by-side comparison with other cell types demonstrated that chondrocytes have substantially reduced capacity to endocytose rhARSB, together with low expression of the mannose receptor. We finally took advantage of Arsb-deficient mice to establish quantification of chondroitin sulfation for treatment monitoring. Our data demonstrate that bone-remodeling cell types are accessible to systemically delivered rhARSB, whereas the uptake into chondrocytes is inefficient.
Assuntos
Remodelação Óssea , Condrócitos/patologia , Terapia de Reposição de Enzimas/métodos , Mucopolissacaridose IV/terapia , N-Acetilgalactosamina-4-Sulfatase/administração & dosagem , N-Acetilgalactosamina-4-Sulfatase/fisiologia , Adolescente , Adulto , Animais , Condrócitos/metabolismo , Feminino , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Pessoa de Meia-Idade , Mucopolissacaridose IV/enzimologia , Adulto JovemRESUMO
Ubiquitin C-terminal hydrolase L1 (UCH-L1) is one of the most abundant and enigmatic enzymes of the CNS. Based on existing UCH-L1 knockout models, UCH-L1 is thought to be required for the maintenance of axonal integrity, but not for neuronal development despite its high expression in neurons. Several lines of evidence suggest a role for UCH-L1 in mUB homeostasis, although the specific in vivo substrate remains elusive. Since the precise mechanisms underlying UCH-L1-deficient neurodegeneration remain unclear, we generated a transgenic mouse model of UCH-L1 deficiency. By performing biochemical and behavioral analyses we can show that UCH-L1 deficiency causes an acceleration of sensorimotor reflex development in the first postnatal week followed by a degeneration of motor function starting at periadolescence in the setting of normal cerebral mUB levels. In the first postnatal weeks, neuronal protein synthesis and proteasomal protein degradation are enhanced, with endoplasmic reticulum stress, and energy depletion, leading to proteasomal impairment and an accumulation of nondegraded ubiquitinated protein. Increased protein turnover is associated with enhanced mTORC1 activity restricted to the postnatal period in UCH-L1-deficient brains. Inhibition of mTORC1 with rapamycin decreases protein synthesis and ubiquitin accumulation in UCH-L1-deficient neurons. Strikingly, rapamycin treatment in the first 8 postnatal days ameliorates the neurological phenotype of UCH-L1-deficient mice up to 16 weeks, suggesting that early control of protein homeostasis is imperative for long-term neuronal survival. In summary, we identified a critical presymptomatic period during which UCH-L1-dependent enhanced protein synthesis results in neuronal strain and progressive loss of neuronal function.
Assuntos
Doenças Neurodegenerativas , Ubiquitina Tiolesterase , Animais , Modelos Animais de Doenças , Feminino , Masculino , Camundongos , Camundongos Transgênicos , Doenças Neurodegenerativas/metabolismo , Doenças Neurodegenerativas/fisiopatologia , Neurônios/metabolismo , Complexo de Endopeptidases do Proteassoma/metabolismo , Biossíntese de Proteínas , Serina-Treonina Quinases TOR/metabolismo , Ubiquitina Tiolesterase/deficiência , Ubiquitina Tiolesterase/genética , Ubiquitina Tiolesterase/fisiologiaRESUMO
BACKGROUND: DNA methylation acts as a mechanism of gene transcription regulation. It has recently gained attention as a possible therapeutic target in cardiac hypertrophy and heart failure. However, its exact role in cardiomyocytes remains controversial. Thus, we knocked out the main de novo DNA methyltransferase in cardiomyocytes, DNMT3A, in human induced pluripotent stem cells. Functional consequences of DNA methylation-deficiency under control and stress conditions were then assessed in human engineered heart tissue from knockout human induced pluripotent stem cell-derived cardiomyocytes. METHODS: DNMT3A was knocked out in human induced pluripotent stem cells by CRISPR/Cas9gene editing. Fibrin-based engineered heart tissue was generated from knockout and control human induced pluripotent stem cell-derived cardiomyocytes. Development and baseline contractility were analyzed by video-optical recording. Engineered heart tissue was subjected to different stress protocols, including serum starvation, serum variation, and restrictive feeding. Molecular, histological, and ultrastructural analyses were performed afterward. RESULTS: Knockout of DNMT3A in human cardiomyocytes had three main consequences for cardiomyocyte morphology and function: (1) Gene expression changes of contractile proteins such as higher atrial gene expression and lower MYH7/MYH6 ratio correlated with different contraction kinetics in knockout versus wild-type; (2) Aberrant activation of the glucose/lipid metabolism regulator peroxisome proliferator-activated receptor gamma was associated with accumulation of lipid vacuoles within knockout cardiomyocytes; (3) Hypoxia-inducible factor 1α protein instability was associated with impaired glucose metabolism and lower glycolytic enzyme expression, rendering knockout-engineered heart tissue sensitive to metabolic stress such as serum withdrawal and restrictive feeding. CONCLUSION: The results suggest an important role of DNA methylation in the normal homeostasis of cardiomyocytes and during cardiac stress, which could make it an interesting target for cardiac therapy.
Assuntos
DNA (Citosina-5-)-Metiltransferases/metabolismo , Metilação de DNA/genética , Epigenômica/métodos , Regulação da Expressão Gênica/genética , Miócitos Cardíacos/metabolismo , Engenharia Tecidual/métodos , Cardiomegalia/patologia , DNA Metiltransferase 3A , HumanosRESUMO
PURPOSE: Pathogenic variants in GNPTAB and GNPTG, encoding different subunits of GlcNAc-1-phosphotransferase, cause mucolipidosis (ML) II, MLIII alpha/beta, and MLIII gamma. This study aimed to investigate the cellular and molecular bases underlying skeletal abnormalities in patients with MLII and MLIII. METHODS: We analyzed bone biopsies from patients with MLIII alpha/beta or MLIII gamma by undecalcified histology and histomorphometry. The skeletal status of Gnptgko and Gnptab-deficient mice was determined and complemented by biochemical analysis of primary Gnptgko bone cells. The clinical relevance of the mouse data was underscored by systematic urinary collagen crosslinks quantification in patients with MLII, MLIII alpha/beta, and MLIII gamma. RESULTS: The analysis of iliac crest biopsies revealed that bone remodeling is impaired in patients with GNPTAB-associated MLIII alpha/beta but not with GNPTG-associated MLIII gamma. Opposed to Gnptab-deficient mice, skeletal remodeling is not affected in Gnptgko mice. Most importantly, patients with variants in GNPTAB but not in GNPTG exhibited increased bone resorption. CONCLUSION: The gene-specific impact on bone remodeling in human individuals and in mice proposes distinct molecular functions of the GlcNAc-1-phosphotransferase subunits in bone cells. We therefore appeal for the necessity to classify MLIII based on genetic in addition to clinical criteria to ensure appropriate therapy.
Assuntos
Reabsorção Óssea , Mucolipidoses , Transferases (Outros Grupos de Fosfato Substituídos) , Animais , Humanos , Camundongos , Mucolipidoses/genética , Mucolipidoses/patologia , Transferases (Outros Grupos de Fosfato Substituídos)/genéticaRESUMO
Neuroserpin is a serine protease inhibitor of the nervous system required for normal synaptic plasticity and regulating cognitive, emotional and social behavior in mice. The high expression level of neuroserpin detected at late stages of nervous system formation in most regions of the brain points to a function in neurodevelopment. In order to evaluate the contribution of neuroserpin to brain development, we investigated developmental neurogenesis and neuronal differentiation in the hippocampus of neuroserpin-deficient mice. Moreover, synaptic reorganization and composition of perineuronal net were studied during maturation and stabilization of hippocampal circuits. We showed that absence of neuroserpin results in early termination of neuronal precursor proliferation and premature neuronal differentiation in the first postnatal weeks. Additionally, at the end of the critical period neuroserpin-deficient mice had changed morphology of dendritic spines towards a more mature phenotype. This was accompanied by increased protein levels and reduced proteolytic cleavage of aggrecan, a perineuronal net core protein. These data suggest a role for neuroserpin in coordinating generation and maturation of the hippocampus, which is essential for establishment of an appropriate neuronal network.
Assuntos
Espinhas Dendríticas/metabolismo , Neurogênese , Neuropeptídeos/metabolismo , Serpinas/metabolismo , Animais , Linhagem Celular , Proliferação de Células , Células Cultivadas , Espinhas Dendríticas/fisiologia , Hipocampo/citologia , Hipocampo/embriologia , Hipocampo/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Células-Tronco Neurais/citologia , Células-Tronco Neurais/metabolismo , Células-Tronco Neurais/fisiologia , Neuropeptídeos/genética , Serpinas/genética , NeuroserpinaRESUMO
Numerous lysosomal enzymes and membrane proteins are essential for the degradation of proteins, lipids, oligosaccharides, and nucleic acids. The CLN3 gene encodes a lysosomal membrane protein of unknown function, and CLN3 mutations cause the fatal neurodegenerative lysosomal storage disorder CLN3 (Batten disease) by mechanisms that are poorly understood. To define components critical for lysosomal homeostasis that are affected by this disease, here we quantified the lysosomal proteome in cerebellar cell lines derived from a CLN3 knock-in mouse model of human Batten disease and control cells. We purified lysosomes from SILAC-labeled, and magnetite-loaded cerebellar cells by magnetic separation and analyzed them by MS. This analysis identified 70 proteins assigned to the lysosomal compartment and 3 lysosomal cargo receptors, of which most exhibited a significant differential abundance between control and CLN3-defective cells. Among these, 28 soluble lysosomal proteins catalyzing the degradation of various macromolecules had reduced levels in CLN3-defective cells. We confirmed these results by immunoblotting and selected protease and glycosidase activities. The reduction of 11 lipid-degrading lysosomal enzymes correlated with reduced capacity for lipid droplet degradation and several alterations in the distribution and composition of membrane lipids. In particular, levels of lactosylceramides and glycosphingolipids were decreased in CLN3-defective cells, which were also impaired in the recycling pathway of the exocytic transferrin receptor. Our findings suggest that CLN3 has a crucial role in regulating lysosome composition and their function, particularly in degrading of sphingolipids, and, as a consequence, in membrane transport along the recycling endosome pathway.
Assuntos
Cerebelo/metabolismo , Lipídeos/análise , Lisossomos/metabolismo , Glicoproteínas de Membrana/deficiência , Transporte Proteico , Proteínas/metabolismo , Proteoma/análise , Animais , Hidrolases/metabolismo , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/metabolismo , Camundongos , Chaperonas Moleculares/genética , Chaperonas Moleculares/metabolismo , Receptores da Transferrina/metabolismoRESUMO
Type III secretion systems (T3SSs) are essential virulence factors of numerous bacterial pathogens. Upon host cell contact the T3SS machinery-also named injectisome-assembles a pore complex/translocon within host cell membranes that serves as an entry gate for the bacterial effectors. Whether and how translocons are physically connected to injectisome needles, whether their phenotype is related to the level of effector translocation and which target cell factors trigger their formation have remained unclear. We employed the superresolution fluorescence microscopy techniques Stimulated Emission Depletion (STED) and Structured Illumination Microscopy (SIM) as well as immunogold electron microscopy to visualize Y. enterocolitica translocons during infection of different target cell types. Thereby we were able to resolve translocon and needle complex proteins within the same injectisomes and demonstrate that these fully assembled injectisomes are generated in a prevacuole, a PI(4,5)P2 enriched host cell compartment inaccessible to large extracellular proteins like antibodies. Furthermore, the operable translocons were produced by the yersiniae to a much larger degree in macrophages (up to 25% of bacteria) than in HeLa cells (2% of bacteria). However, when the Rho GTPase Rac1 was activated in the HeLa cells, uptake of the yersiniae into the prevacuole, translocon formation and effector translocation were strongly enhanced reaching the same levels as in macrophages. Our findings indicate that operable T3SS translocons can be visualized as part of fully assembled injectisomes with superresolution fluorescence microscopy techniques. By using this technology, we provide novel information about the spatiotemporal organization of T3SS translocons and their regulation by host cell factors.
Assuntos
Sistemas de Secreção Tipo III , Yersiniose/transmissão , Yersinia enterocolitica/patogenicidade , Humanos , Microscopia de FluorescênciaRESUMO
Atypical brain connectivity is a major contributor to the pathophysiology of neurodevelopmental disorders (NDDs) including autism spectrum disorders (ASDs). TAOK2 is one of several genes in the 16p11.2 microdeletion region, but whether it contributes to NDDs is unknown. We performed behavioral analysis on Taok2 heterozygous (Het) and knockout (KO) mice and found gene dosage-dependent impairments in cognition, anxiety, and social interaction. Taok2 Het and KO mice also have dosage-dependent abnormalities in brain size and neural connectivity in multiple regions, deficits in cortical layering, dendrite and synapse formation, and reduced excitatory neurotransmission. Whole-genome and -exome sequencing of ASD families identified three de novo mutations in TAOK2 and functional analysis in mice and human cells revealed that all the mutations impair protein stability, but they differentially impact kinase activity, dendrite growth, and spine/synapse development. Mechanistically, loss of Taok2 activity causes a reduction in RhoA activation, and pharmacological enhancement of RhoA activity rescues synaptic phenotypes. Together, these data provide evidence that TAOK2 is a neurodevelopmental disorder risk gene and identify RhoA signaling as a mediator of TAOK2-dependent synaptic development.
Assuntos
Transtorno do Espectro Autista/metabolismo , Transtornos do Neurodesenvolvimento/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Proteína rhoA de Ligação ao GTP/metabolismo , Adulto , Animais , Ansiedade/genética , Transtorno do Espectro Autista/genética , Transtorno do Espectro Autista/patologia , Transtorno do Espectro Autista/psicologia , Criança , Disfunção Cognitiva/genética , Disfunção Cognitiva/metabolismo , Disfunção Cognitiva/patologia , Disfunção Cognitiva/psicologia , Dendritos/metabolismo , Dendritos/patologia , Feminino , Humanos , Relações Interpessoais , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Transtornos do Neurodesenvolvimento/genética , Transtornos do Neurodesenvolvimento/patologia , Transtornos do Neurodesenvolvimento/psicologia , Neurogênese , Fenótipo , Fosforilação , Proteínas Serina-Treonina Quinases/genética , Transdução de Sinais , Transmissão Sináptica , Sequenciamento do ExomaRESUMO
Targeting of soluble lysosomal enzymes requires mannose 6-phosphate (M6P) signals whose formation is initiated by the hexameric N-acetylglucosamine (GlcNAc)-1-phosphotransferase complex (α2ß2γ2). Upon proteolytic cleavage by site-1 protease, the α/ß-subunit precursor is catalytically activated but the functions of γ-subunits (Gnptg) in M6P modification of lysosomal enzymes are unknown. To investigate this, we analyzed the Gnptg expression in mouse tissues, primary cultured cells, and in Gnptg reporter mice in vivo, and found high amounts in the brain, eye, kidney, femur, vertebra and fibroblasts. Consecutively we performed comprehensive quantitative lysosomal proteome and M6P secretome analysis in fibroblasts of wild-type and Gnptgko mice mimicking the lysosomal storage disorder mucolipidosis III. Although the cleavage of the α/ß-precursor was not affected by Gnptg deficiency, the GlcNAc-1-phosphotransferase activity was significantly reduced. We purified lysosomes and identified 29 soluble lysosomal proteins by SILAC-based mass spectrometry exhibiting differential abundance in Gnptgko fibroblasts which was confirmed by Western blotting and enzymatic activity analysis for selected proteins. A subset of these lysosomal enzymes show also reduced M6P modifications, fail to reach lysosomes and are secreted, among them α-l-fucosidase and arylsulfatase B. Low levels of these enzymes correlate with the accumulation of non-degraded fucose-containing glycostructures and sulfated glycosaminoglycans in Gnptgko lysosomes. Incubation of Gnptgko fibroblasts with arylsulfatase B partially rescued glycosaminoglycan storage. Combinatorial treatments with other here identified missorted enzymes of this degradation pathway might further correct glycosaminoglycan accumulation and will provide a useful basis to reveal mechanisms of selective, Gnptg-dependent formation of M6P residues on lysosomal proteins.
Assuntos
Enzimas/metabolismo , Lisossomos/metabolismo , Mucolipidoses/metabolismo , Mucolipidoses/patologia , Proteoma/metabolismo , Animais , Embrião de Mamíferos/citologia , Fibroblastos/metabolismo , Glicosaminoglicanos/metabolismo , Humanos , Marcação por Isótopo , Manosefosfatos/metabolismo , Camundongos Knockout , Subunidades Proteicas/metabolismo , Proteólise , Especificidade por SubstratoRESUMO
Mutations in the depalmitoylation enzyme, palmitoyl protein thioesterase (PPT1), result in the early onset neurodegenerative disease known as Infantile Neuronal Ceroid Lipofuscinosis. Here, we provide proteomic evidence suggesting that PPT1 deficiency could be considered as a ciliopathy. Analysis of membrane proteins from brain enriched for acylated proteins from neonate Ppt1 knock out and control mice revealed a list of 88 proteins with differential expression levels. Amongst them, we identified Rab3IP, which regulates ciliogenesis in concert with Rab8 and Rab11. Immunostaining analysis revealed that PPT1 is localized in the cilia. Indeed, an unbiased proteomics analysis on isolated cilia revealed 660 proteins, which differed in their abundance levels between wild type and Ppt1 knock out. We demonstrate here that Rab3IP, Rab8 and Rab11 are palmitoylated, and that palmitoylation of Rab11 is required for correct intracellular localization. Cells and brain preparations from Ppt1-/- mice exhibited fewer cells with cilia and abnormally longer cilia, with both acetylated tubulin and Rab3IP wrongly distributed along the length of cilia. Most importantly, the analysis revealed a difference in the distribution and levels of the modified proteins in cilia in the retina of mutant mice versus the wildtype, which may be important in the early neurodegenerative phenotype. Overall, our results suggest a novel link between palmitoylated proteins, cilial organization and the pathophysiology of Neuronal Ceroid Lipofuscinosis.