RESUMO
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
Homeostatic synaptic plasticity adjusts the strength of synapses to restrain neuronal activity within a physiological range. Postsynaptic guanylate kinase-associated protein (GKAP) controls the bidirectional synaptic scaling of AMPA receptors (AMPARs); however, mechanisms by which chronic activity triggers cytoskeletal remodeling to downscale synaptic transmission are barely understood. Here, we report that the microtubule-dependent kinesin motor Kif21b binds GKAP and likewise is located in dendritic spines in a myosin Va- and neuronal-activity-dependent manner. Kif21b depletion unexpectedly alters actin dynamics in spines, and adaptation of actin turnover following chronic activity is lost in Kif21b-knockout neurons. Consistent with a role of the kinesin in regulating actin dynamics, Kif21b overexpression promotes actin polymerization. Moreover, Kif21b controls GKAP removal from spines and the decrease of GluA2-containing AMPARs from the neuronal surface, thereby inducing homeostatic synaptic downscaling. Our data highlight a critical role of Kif21b at the synaptic actin cytoskeleton underlying homeostatic scaling of neuronal firing.
Assuntos
Actinas , Cinesinas , Actinas/metabolismo , Cinesinas/metabolismo , Neurônios/metabolismo , Plasticidade Neuronal/fisiologia , Sinapses/metabolismo , Miosinas/metabolismo , Espinhas Dendríticas/metabolismoRESUMO
Prion diseases are neurodegenerative diseases that affect humans and animals. They are always fatal and, to date, no treatment exists. The hallmark of prion disease pathophysiology is the misfolding of an endogenous protein, the cellular prion protein (PrPC), into its disease-associated isoform PrPSc. Besides the aggregation and deposition of misfolded PrPSc, prion diseases are characterized by spongiform lesions and the activation of astrocytes and microglia. Microglia are the innate immune cells of the brain. Activated microglia and astrocytes represent a common pathological feature in neurodegenerative disorders. The role of activated microglia has already been studied in prion disease mouse models; however, it is still not fully clear how they contribute to disease progression. Moreover, the role of microglia in human prion diseases has not been thoroughly investigated thus far, and specific molecular pathways are still undetermined. Here, we review the current knowledge on the different roles of microglia in prion pathophysiology. We discuss microglia markers that are also dysregulated in other neurodegenerative diseases including microglia homeostasis markers. Data on murine and human brain tissues show that microglia are highly dysregulated in prion diseases. We highlight here that the loss of homeostatic markers may especially stand out.
Assuntos
Doenças Neurodegenerativas , Doenças Priônicas , Príons , Animais , Homeostase , Humanos , Camundongos , Microglia/metabolismo , Doenças Neurodegenerativas/metabolismo , Doenças Priônicas/metabolismo , Proteínas Priônicas/metabolismo , Príons/metabolismo , Isoformas de Proteínas/metabolismoRESUMO
BACKGROUND: Autopsy studies have provided valuable insights into the pathophysiology of COVID-19. Controversies remain about whether the clinical presentation is due to direct organ damage by SARS-CoV-2 or secondary effects, such as overshooting immune response. SARS-CoV-2 detection in tissues by RT-qPCR and immunohistochemistry (IHC) or electron microscopy (EM) can help answer these questions, but a comprehensive evaluation of these applications is missing. METHODS: We assessed publications using IHC and EM for SARS-CoV-2 detection in autopsy tissues. We systematically evaluated commercially available antibodies against the SARS-CoV-2 proteins in cultured cell lines and COVID-19 autopsy tissues. In a multicentre study, we evaluated specificity, reproducibility, and inter-observer variability of SARS-CoV-2 IHC. We correlated RT-qPCR viral tissue loads with semiquantitative IHC scoring. We used qualitative and quantitative EM analyses to refine criteria for ultrastructural identification of SARS-CoV-2. FINDINGS: Publications show high variability in detection and interpretation of SARS-CoV-2 abundance in autopsy tissues by IHC or EM. We show that IHC using antibodies against SARS-CoV-2 nucleocapsid yields the highest sensitivity and specificity. We found a positive correlation between presence of viral proteins by IHC and RT-qPCR-determined SARS-CoV-2 viral RNA load (N= 35; r=-0.83, p-value <0.0001). For EM, we refined criteria for virus identification and provide recommendations for optimized sampling and analysis. 135 of 144 publications misinterpret cellular structures as virus using EM or show only insufficient data. We provide publicly accessible digitized EM sections as a reference and for training purposes. INTERPRETATION: Since detection of SARS-CoV-2 in human autopsy tissues by IHC and EM is difficult and frequently incorrect, we propose criteria for a re-evaluation of available data and guidance for further investigations of direct organ effects by SARS-CoV-2. FUNDING: German Federal Ministry of Health, German Federal Ministry of Education and Research, Berlin University Alliance, German Research Foundation, German Center for Infectious Research.
Assuntos
COVID-19 , Autopsia , Humanos , RNA Viral/análise , Reprodutibilidade dos Testes , SARS-CoV-2 , Proteínas ViraisRESUMO
Muskelin (Mkln1) is implicated in neuronal function, regulating plasma membrane receptor trafficking. However, its influence on intrinsic brain activity and corresponding behavioral processes remains unclear. Here we show that murine Mkln1 knockout causes non-habituating locomotor activity, increased exploratory drive, and decreased locomotor response to amphetamine. Muskelin deficiency impairs social novelty detection while promoting the retention of spatial reference memory and fear extinction recall. This is strongly mirrored in either weaker or stronger resting-state functional connectivity between critical circuits mediating locomotor exploration and cognition. We show that Mkln1 deletion alters dendrite branching and spine structure, coinciding with enhanced AMPAR-mediated synaptic transmission but selective impairment in synaptic potentiation maintenance. We identify muskelin at excitatory synapses and highlight its role in regulating dendritic spine actin stability. Our findings point to aberrant spine actin modulation and changes in glutamatergic synaptic function as critical mechanisms that contribute to the neurobehavioral phenotype arising from Mkln1 ablation.
Assuntos
Actinas , Extinção Psicológica , Actinas/metabolismo , Animais , Encéfalo/metabolismo , Cognição , Medo , CamundongosRESUMO
Neurological complications are common in COVID-19. Although SARS-CoV-2 has been detected in patients' brain tissues, its entry routes and resulting consequences are not well understood. Here, we show a pronounced upregulation of interferon signaling pathways of the neurovascular unit in fatal COVID-19. By investigating the susceptibility of human induced pluripotent stem cell (hiPSC)-derived brain capillary endothelial-like cells (BCECs) to SARS-CoV-2 infection, we found that BCECs were infected and recapitulated transcriptional changes detected in vivo. While BCECs were not compromised in their paracellular tightness, we found SARS-CoV-2 in the basolateral compartment in transwell assays after apical infection, suggesting active replication and transcellular transport of virus across the blood-brain barrier (BBB) in vitro. Moreover, entry of SARS-CoV-2 into BCECs could be reduced by anti-spike-, anti-angiotensin-converting enzyme 2 (ACE2)-, and anti-neuropilin-1 (NRP1)-specific antibodies or the transmembrane protease serine subtype 2 (TMPRSS2) inhibitor nafamostat. Together, our data provide strong support for SARS-CoV-2 brain entry across the BBB resulting in increased interferon signaling.
Assuntos
Barreira Hematoencefálica/virologia , Sistema Nervoso Central/virologia , SARS-CoV-2/fisiologia , Internalização do Vírus , Anticorpos/farmacologia , Benzamidinas/farmacologia , COVID-19/patologia , COVID-19/virologia , Células Endoteliais/citologia , Células Endoteliais/metabolismo , Células Endoteliais/virologia , Guanidinas/farmacologia , Humanos , Células-Tronco Pluripotentes Induzidas/citologia , Células-Tronco Pluripotentes Induzidas/metabolismo , Modelos Biológicos , RNA Viral/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , SARS-CoV-2/genética , SARS-CoV-2/isolamento & purificação , Internalização do Vírus/efeitos dos fármacosRESUMO
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
Microtubule severing regulates cytoskeletal rearrangement underlying various cellular functions. Katanin, a heterodimer, consisting of catalytic (p60) and regulatory (p80) subunits severs dynamic microtubules to modulate several stages of cell division. The role of p60 katanin in the mammalian brain with respect to embryonic and adult neurogenesis is poorly understood. Here, we generated a Katna1 knockout mouse and found that consistent with a critical role of katanin in mitosis, constitutive homozygous Katna1 depletion is lethal. Katanin p60 haploinsufficiency induced an accumulation of neuronal progenitors in the subventricular zone during corticogenesis, and impaired their proliferation in the adult hippocampus dentate gyrus (DG) subgranular zone. This did not compromise DG plasticity or spatial and contextual learning and memory tasks employed in our study, consistent with the interpretation that adult neurogenesis may be associated with selective forms of hippocampal-dependent cognitive processes. Our data identify a critical role for the microtubule-severing protein katanin p60 in regulating neuronal progenitor proliferation in vivo during embryonic development and adult neurogenesis.
Assuntos
Diferenciação Celular , Katanina/genética , Microtúbulos/metabolismo , Células-Tronco Neurais/citologia , Células-Tronco Neurais/metabolismo , Neurogênese , Fatores Etários , Alelos , Animais , Diferenciação Celular/genética , Proliferação de Células , Córtex Cerebral/embriologia , Córtex Cerebral/metabolismo , Giro Denteado/embriologia , Giro Denteado/metabolismo , Marcação de Genes , Haploinsuficiência , Katanina/metabolismo , Aprendizagem , Memória , Camundongos , Camundongos Knockout , Neurogênese/genética , Neurônios/citologia , Neurônios/metabolismo , Organogênese , FenótipoRESUMO
Autism spectrum disorders (ASDs) are associated with mutations affecting synaptic components, including GluN2B-NMDA receptors (NMDARs) and neurobeachin (NBEA). NBEA participates in biosynthetic pathways to regulate synapse receptor targeting, synaptic function, cognition, and social behavior. However, the role of NBEA-mediated transport in specific trafficking routes is unclear. Here, we highlight an additional function for NBEA in the local delivery and surface re-insertion of synaptic receptors in mouse neurons. NBEA dynamically interacts with Rab4-positive recycling endosomes, transiently enters spines in an activity-dependent manner, and regulates GluN2B-NMDAR recycling. Furthermore, we show that the microtubule growth inhibitor kinesin KIF21B constrains NBEA dynamics and is present in the NBEA-recycling endosome-NMDAR complex. Notably, Kif21b knockout decreases NMDAR surface expression and alters social behavior in mice, consistent with reported social deficits in Nbea mutants. The influence of NBEA-KIF21B interactions on GluN2B-NMDAR local recycling may be relevant to mechanisms underlying ASD etiology.
Assuntos
Comportamento Animal , Proteínas de Transporte/metabolismo , Endocitose , Cinesinas/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Receptores de N-Metil-D-Aspartato/metabolismo , Comportamento Social , Animais , Células COS , Chlorocebus aethiops , Cognição , Espinhas Dendríticas/efeitos dos fármacos , Espinhas Dendríticas/metabolismo , Dineínas/metabolismo , Endocitose/efeitos dos fármacos , Endossomos/metabolismo , Ácido Glutâmico/farmacologia , Complexo de Golgi/efeitos dos fármacos , Complexo de Golgi/metabolismo , Proteínas de Membrana , Camundongos Knockout , Microtúbulos/efeitos dos fármacos , Microtúbulos/metabolismo , Nocodazol/farmacologia , Ligação Proteica/efeitos dos fármacos , Transporte Proteico/efeitos dos fármacos , Vesículas Sinápticas/efeitos dos fármacos , Vesículas Sinápticas/metabolismo , Proteínas rab4 de Ligação ao GTP/metabolismoRESUMO
The dendritic arbor is subject to continual activity-dependent remodeling, requiring a balance between directed cargo trafficking and dynamic restructuring of the underlying microtubule tracks. How cytoskeletal components are able to dynamically regulate these processes to maintain this balance remains largely unknown. By combining single-molecule assays and live imaging in rat hippocampal neurons, we have identified the kinesin-4 KIF21B as a molecular regulator of activity-dependent trafficking and microtubule dynamicity in dendrites. We find that KIF21B contributes to the retrograde trafficking of brain-derived neurotrophic factor (BDNF)-TrkB complexes and also regulates microtubule dynamics through a separable, non-motor microtubule-binding domain. Neuronal activity enhances the motility of KIF21B at the expense of its role in cytoskeletal remodeling, the first example of a kinesin whose function is directly tuned to neuronal activity state. These studies suggest a model in which KIF21B navigates the complex cytoskeletal environment of dendrites by compartmentalizing functions in an activity-dependent manner.
Assuntos
Fator Neurotrófico Derivado do Encéfalo/metabolismo , Dendritos/metabolismo , Cinesinas/genética , Microtúbulos/metabolismo , Transporte Proteico/genética , Proteínas Tirosina Quinases/metabolismo , Animais , Citoesqueleto/metabolismo , Técnicas de Inativação de Genes , Hipocampo/citologia , Cinesinas/metabolismo , Neurônios/metabolismo , Ratos , Receptor trkBRESUMO
The kinesin KIF21B is implicated in several human neurological disorders, including delayed cognitive development, yet it remains unclear how KIF21B dysfunction may contribute to pathology. One limitation is that relatively little is known about KIF21B-mediated physiological functions. Here, we generated Kif21b knockout mice and used cellular assays to investigate the relevance of KIF21B in neuronal and in vivo function. We show that KIF21B is a processive motor protein and identify an additional role for KIF21B in regulating microtubule dynamics. In neurons lacking KIF21B, microtubules grow more slowly and persistently, leading to tighter packing in dendrites. KIF21B-deficient neurons exhibit decreased dendritic arbor complexity and reduced spine density, which correlate with deficits in synaptic transmission. Consistent with these observations, Kif21b-null mice exhibit behavioral changes involving learning and memory deficits. Our study provides insight into the cellular function of KIF21B and the basis for cognitive decline resulting from KIF21B dysregulation.
Assuntos
Forma Celular , Cinesinas/metabolismo , Memória/fisiologia , Microtúbulos/metabolismo , Neurônios/citologia , Sinapses/metabolismo , Animais , Espinhas Dendríticas/metabolismo , Espinhas Dendríticas/ultraestrutura , Marcação de Genes , Células HeLa , Humanos , Cinesinas/deficiência , Transtornos da Memória/metabolismo , Transtornos da Memória/patologia , Camundongos Knockout , Microtúbulos/ultraestrutura , Neurônios/metabolismo , Neurônios/ultraestrutura , Reprodutibilidade dos TestesRESUMO
KIF21B, a kinesin family (KIF) protein, is a plus end-directed microtubule motor. The KIF21B gene is highly expressed in neuronal tissue and spleen and is a susceptibility locus for multiple sclerosis. KIF21B motility is regulated through TRIM3, a member of the cytoskeleton-associated-recycling or transport (CART) complex, involved in vesicular receptor recycling. Here we show that the GABAA receptor γ2-subunit co-precipitates and co-localizes with KIF21B in cultured hippocampal neurons. Knockdown of KIF21B gene expression through small hairpin (sh) RNA reduces the number of γ2-subunit-containing GABAA receptor (GABAARs) clusters in neurites and at the cell surface. Our data suggest that KIF21B participates in the delivery of GABAAR transport vesicles into dendrites.
Assuntos
Membrana Celular/metabolismo , Cinesinas/metabolismo , Receptores de GABA-A/metabolismo , Animais , Comunicação Celular , Técnicas de Silenciamento de Genes , Hipocampo/citologia , Cinesinas/genética , Camundongos , Neurônios/metabolismo , Subunidades Proteicas/metabolismo , RatosRESUMO
Kinesin superfamily proteins (KIFs) are molecular motors that transport cellular cargo along the microtubule cytoskeleton. KIF21B is a neuronal kinesin that is highly enriched in dendrites. The regulation and specificity of microtubule transport involves the binding of motors to individual cargo adapters and accessory proteins. Moreover, posttranslational modifications of either the motor protein, their cargos or tubulin regulate motility, cargo recognition and the binding or unloading of cargos. Here we show that the ubiquitin E3 ligase TRIM3, also known as BERP, interacts with KIF21B via its RBCC domain. TRIM3 is found at intracellular and Golgi-derived vesicles and co-localizes with the KIF21B motor in neurons. Trim3 gene deletion in mice and TRIM3 overexpression in cultured neurons both suggested that the E3-ligase function of TRIM3 is not involved in KIF21B degradation, however TRIM3 depletion reduces the motility of the motor. Together, our data suggest that TRIM3 is a regulator in the modulation of KIF21B motor function.
Assuntos
Transporte Biológico/genética , Proteínas de Transporte/metabolismo , Cinesinas/metabolismo , Neurônios/metabolismo , Animais , Proteínas de Transporte/genética , Cinesinas/genética , Camundongos , Camundongos Knockout , Microtúbulos/genética , Microtúbulos/metabolismo , Ligação Proteica/genética , Processamento de Proteína Pós-Traducional/genéticaRESUMO
In multiple sclerosis, an inflammatory disease of the central nervous system (CNS), axonal and neuronal loss are major causes for irreversible neurological disability. However, which molecules contribute to axonal and neuronal injury under inflammatory conditions remains largely unknown. Here we show that the transient receptor potential melastatin 4 (TRPM4) cation channel is crucial in this process. TRPM4 is expressed in mouse and human neuronal somata, but it is also expressed in axons in inflammatory CNS lesions in experimental autoimmune encephalomyelitis (EAE) in mice and in human multiple sclerosis tissue. Deficiency or pharmacological inhibition of TRPM4 using the antidiabetic drug glibenclamide resulted in reduced axonal and neuronal degeneration and attenuated clinical disease scores in EAE, but this occurred without altering EAE-relevant immune function. Furthermore, Trpm4(-/-) mouse neurons were protected against inflammatory effector mechanisms such as excitotoxic stress and energy deficiency in vitro. Electrophysiological recordings revealed TRPM4-dependent neuronal ion influx and oncotic cell swelling upon excitotoxic stimulation. Therefore, interference with TRPM4 could translate into a new neuroprotective treatment strategy.
Assuntos
Axônios/metabolismo , Encefalomielite Autoimune Experimental/metabolismo , Esclerose Múltipla/metabolismo , Degeneração Neural/metabolismo , Canais de Cátion TRPM/metabolismo , Análise de Variância , Animais , Axônios/efeitos dos fármacos , Western Blotting , Proliferação de Células/efeitos dos fármacos , Primers do DNA/genética , Ensaio de Imunoadsorção Enzimática , Citometria de Fluxo , Glibureto/farmacologia , Humanos , Imuno-Histoquímica , Hibridização In Situ , Camundongos , Camundongos Knockout , Técnicas de Patch-Clamp , Reação em Cadeia da Polimerase em Tempo Real , Linfócitos T/citologia , Linfócitos T/efeitos dos fármacos , Canais de Cátion TRPM/antagonistas & inibidores , Canais de Cátion TRPM/genéticaRESUMO
Aggregation of amyloid-beta (Abeta) and Tau protein are hallmarks of Alzheimer's disease (AD), and according to the Abeta-cascade hypothesis, Abeta is considered toxic for neurons and Tau a downstream target of Abeta. We have investigated differentiated primary hippocampal neurons for early localized changes following exposure to Abeta oligomers. Initial events become evident by missorting of endogenous Tau into the somatodendritic compartment, in contrast to axonal sorting in normal neurons. In missorted dendritic regions there is a depletion of spines and local increase in Ca(2+), and breakdown of microtubules. Tau in these regions shows elevated phosphorylation at certain sites diagnostic of AD-Tau (e.g., epitope of antibody 12E8, whose phosphorylation causes detachment of Tau from microtubules, and AT8 epitope), and local elevation of certain kinase activities (e.g., MARK/par-1, BRSK/SADK, p70S6K, cdk5, but not GSK3beta, JNK, MAPK). These local effects occur without global changes in Tau, tubulin, or kinase levels. Somatodendritic missorting occurs not only with Tau, but also with other axonal proteins such as neurofilaments, and correlates with pronounced depletion of microtubules and mitochondria. The Abeta-induced effects on microtubule and mitochondria depletion, Tau missorting, and loss of spines are prevented by taxol, indicating that Abeta-induced microtubule destabilization and corresponding traffic defects are key factors in incipient degeneration. By contrast, the rise in Ca(2+) levels, kinase activities, and Tau phosphorylation cannot be prevented by taxol. Incipient and local changes similar to those of Abeta oligomers can be evoked by cell stressors (e.g., H(2)O(2), glutamate, serum deprivation), suggesting some common mechanism of signaling.
Assuntos
Peptídeos beta-Amiloides/química , Cálcio/metabolismo , Microtúbulos/metabolismo , Neurônios/ultraestrutura , Fragmentos de Peptídeos/farmacologia , Proteínas tau/metabolismo , Trifosfato de Adenosina/metabolismo , Peptídeos beta-Amiloides/farmacologia , Análise de Variância , Animais , Células Cultivadas , Dendritos , Espinhas Dendríticas , Embrião de Mamíferos , Hipocampo/citologia , L-Lactato Desidrogenase/metabolismo , Microscopia Eletrônica de Transmissão/métodos , Microtúbulos/efeitos dos fármacos , Mitocôndrias/efeitos dos fármacos , Proteínas de Neurofilamentos/metabolismo , Neurônios/efeitos dos fármacos , Fosforilação/efeitos dos fármacos , Proteínas Quinases/metabolismo , Transporte Proteico/efeitos dos fármacos , Ratos , Ratos Sprague-Dawley , Fatores de TempoRESUMO
Long-haul transport along microtubules is crucial for neuronal polarity, and transport defects cause neurodegeneration. Tau protein stabilizes microtubule tracks, but in Alzheimer's disease it aggregates and becomes missorted into the somatodendritic compartment. Tau can inhibit axonal transport by obstructing motors on microtubules, yet tau itself can still move into axons. We therefore investigated tau movement by live-cell fluorescence microscopy, FRAP (fluorescence recovery after photobleaching), and FSM (fluorescence speckle microscopy). Tau is highly dynamic, with diffusion coefficients of approximately 3 microm2/s and microtubule dwell times of approximately 4 s. This facilitates the entry of tau into axons over distances of millimeters and periods of days. For longer distances and times, two mechanisms of tau transport are observed. At low near-physiological levels, tau is cotransported with microtubule fragments from cell bodies into axons, moving at instantaneous velocities approximately 1 microm/s. At high concentrations, tau forms local accumulations moving bidirectionally at approximately 0.3 microm/s. These clusters first appear at distal endings of axons and may indicate an early stage of neurite degeneration.
Assuntos
Proteínas Associadas aos Microtúbulos/metabolismo , Microtúbulos/metabolismo , Neurônios/metabolismo , Proteínas tau/metabolismo , Animais , Células Cultivadas , Chlorocebus aethiops , Proteínas Associadas aos Microtúbulos/análise , Microtúbulos/química , Neurônios/química , Transporte Proteico/fisiologia , Ratos , Células Vero , Proteínas tau/análiseAssuntos
Precursor de Proteína beta-Amiloide/metabolismo , Axônios/metabolismo , Microscopia Confocal/métodos , Vesículas Transportadoras/metabolismo , Proteínas tau/metabolismo , Animais , Transporte Axonal/fisiologia , Galinhas , Corantes Fluorescentes/análise , Proteínas de Fluorescência Verde/análise , Proteínas Luminescentes/análise , Microscopia de Vídeo , Neurônios/metabolismo , Células Ganglionares da Retina/metabolismo , Distribuição TecidualRESUMO
Early hallmarks of Alzheimer's disease include the loss of synapses, which precedes the loss of neurons and the pathological phosphorylation and aggregation of tau protein. Mitochondrial dysfunction has been suggested as a reason, but evidence on the role of tau was lacking. Here, we show that transfection of tau in mature hippocampal neurons leads to an improper distribution of tau into the somatodendritic compartment with concomitant degeneration of synapses, as seen by the disappearance of spines and of presynaptic and postsynaptic markers. This is accompanied by transport inhibition of vesicles and organelles, concomitant with an increase and bundling of microtubules. Mitochondria degenerate, thus causing ATP levels to decrease. The tau-induced synaptic decay can be relieved by the activation of the kinase MARK2 (microtubule-associated protein/microtubule affinity regulating kinase 2)/Par-1 (protease-activated receptor 1), which can remove tau from the microtubule tracks and reverses the transport block. This leads to the rescue of dendritic spines, synapses, mitochondrial transport and ATP levels.
Assuntos
Degeneração Neural/enzimologia , Neurônios/enzimologia , Proteínas Serina-Treonina Quinases/fisiologia , Receptor PAR-1/fisiologia , Sinapses/enzimologia , Proteínas tau/metabolismo , Animais , Transporte Axonal/fisiologia , Transporte Biológico Ativo/fisiologia , Linhagem Celular , Células Cultivadas , Hipocampo/química , Hipocampo/enzimologia , Hipocampo/patologia , Humanos , Microtúbulos/enzimologia , Degeneração Neural/prevenção & controle , Neurônios/química , Neurônios/patologia , Ratos , Ratos Sprague-Dawley , Sinapses/química , Sinapses/patologiaRESUMO
Amyloid-beta, a peptide derived from the precursor protein APP, accumulates in the brain and contributes to the neuropathology of Alzheimer's disease. Increased generation of amyloid-beta might be caused by axonal transport inhibition, via increased dwell time of APP vesicles and thereby higher probability of APP cleavage by secretase enzymes residing on the same vesicles. We tested this hypothesis using a neuronal cell culture model of inhibited axonal transport and by imaging vesicular transport of fluorescently tagged APP and beta-secretase (BACE1). Microtubule-associated tau protein blocks vesicle traffic by inhibiting the access of motor proteins to the microtubule tracks. In neurons co-transfected with CFP-tau, APP-YFP traffic into distal neurites was strongly reduced. However, this did not increase amyloid-beta levels. In singly transfected axons, APP-YFP was transported in large tubules and vesicles moving very fast (on average 3 microm/s) and with high fluxes in the anterograde direction (on average 8.4 vesicles/min). By contrast, BACE1-CFP movement was in smaller tubules and vesicles that were almost 2x slower (on average 1.6 microm/s) with approximately 18x lower fluxes (on average 0.5 vesicles/min). Two-colour microscopy of co-transfected axons confirmed that the two proteins were sorted into distinct carriers. The results do not support the above hypothesis. Instead, they indicate that APP is transported on vesicles distinct from the secretase components and that amyloid-beta is not generated in transit when transport is blocked by tau.
Assuntos
Peptídeos beta-Amiloides/metabolismo , Fragmentos de Peptídeos/biossíntese , Proteínas tau/metabolismo , Peptídeos beta-Amiloides/química , Peptídeos beta-Amiloides/genética , Animais , Células Cultivadas , Córtex Cerebral/metabolismo , Chlorocebus aethiops , Regulação da Expressão Gênica , Humanos , Neurônios/metabolismo , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/genética , Fragmentos de Peptídeos/metabolismo , Transporte Proteico , Ratos , Ratos Wistar , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Células Ganglionares da Retina/metabolismo , Proteínas tau/genéticaRESUMO
Microtubule-dependent transport of vesicles and organelles appears saltatory because particles switch between periods of rest, random Brownian motion, and active transport. The transport can be regulated through motor proteins, cargo adaptors, or microtubule tracks. We report here a mechanism whereby microtubule associated proteins (MAPs) represent obstacles to motors which can be regulated by microtubule affinity regulating kinase (MARK)/Par-1, a family of kinases that is known for its involvement in establishing cell polarity and in phosphorylating tau protein during Alzheimer neurodegeneration. Expression of MARK causes the phosphorylation of MAPs at their KXGS motifs, thereby detaching MAPs from the microtubules and thus facilitating the transport of particles. This occurs without impairing the intrinsic activity of motors because the velocity during active movement remains unchanged. In primary retinal ganglion cells, transfection with tau leads to the inhibition of axonal transport of mitochondria, APP vesicles, and other cell components which leads to starvation of axons and vulnerability against stress. This transport inhibition can be rescued by phosphorylating tau with MARK.