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Autophagy disorders prominently affect the brain, entailing neurodevelopmental and neurodegenerative phenotypes in adolescence or aging, respectively. Synaptic and behavioral deficits are largely recapitulated in mouse models with ablation of autophagy genes in brain cells. Yet, the nature and temporal dynamics of brain autophagic substrates remain insufficiently characterized. Here, we immunopurified LC3-positive autophagic vesicles (LC3-pAVs) from the mouse brain and proteomically profiled their content. Moreover, we characterized the LC3-pAV content that accumulates after macroautophagy impairment, validating a brain autophagic degradome. We reveal selective pathways for aggrephagy, mitophagy, and ER-phagy via selective autophagy receptors, and the turnover of numerous synaptic substrates, under basal conditions. To gain insight into the temporal dynamics of autophagic protein turnover, we quantitatively compared adolescent, adult, and aged brains, revealing critical periods of enhanced mitophagy or degradation of synaptic substrates. Overall, this resource unbiasedly characterizes the contribution of autophagy to proteostasis in the maturing, adult, and aged brain.
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Autofagia , Mitofagia , Animales , Ratones , Autofagia/genética , Macroautofagia , Envejecimiento , EncéfaloRESUMEN
Recent evidence has shown that G protein-coupled receptors (GPCRs) are direct sensors of the autophagic machinery and opioid receptors regulate neuronal plasticity and neurotransmission with an as yet unclarified mechanism. Using in vitro and in vivo experimental approaches, this study aims to clarify the potential role of autophagy and κ-opioid receptor (κ-OR) signaling in synaptic alterations. We hereby demonstrate that the selective κ-OR agonist U50,488H, induces autophagy in a time-and dose-dependent manner in Neuro-2A cells stably expressing the human κ-OR by upregulating microtubule-associated protein Light Chain 3-II (LC3-II), Beclin 1 and Autophagy Related Gene 5 (ATG5). Pretreatment of neuronal cells with pertussis toxin blocked the above κ-OR-mediated cellular responses. Our molecular analysis also revealed a κ-OR-driven upregulation of becn1 gene through ERK1,2-dependent activation of the transcription factor CREB in Neuro-2A cells. Moreover, our studies demonstrated that sub-chronic U50,488H administration in mice causes profound increases of specific autophagic markers in the hippocampus with a concomitant decrease of several pre-and post-synaptic proteins, such as spinophilin, postsynaptic density protein 95 (PSD-95) and synaptosomal associated protein 25 (SNAP25). Finally, using acute stress, a stimulus known to increase the levels of the endogenous κ-OR ligand dynorphin, we are demonstrating that administration of the κ-ΟR selective antagonist, nor-binaltorphimine (norBNI), blocks the induction of autophagy and the stress-evoked reduction of synaptic proteins in the hippocampus. These findings provide novel insights about the essential role of autophagic machinery into the mechanisms through which κ-OR signaling regulates brain plasticity.
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(Macro)autophagy is a major lysosome-dependent degradation mechanism which engulfs, removes and recycles unwanted cytoplasmic material, including damaged organelles and toxic protein aggregates. Although a few studies implicate autophagy in CNS demyelinating pathologies, its role, particularly in mature oligodendrocytes and CNS myelin, remains poorly studied. Here, using both pharmacological and genetic inhibition of the autophagic machinery, we provide evidence that autophagy is an essential mechanism for oligodendrocyte maturation in vitro. Our study reveals that two core myelin proteins, namely proteolipid protein (PLP) and myelin basic protein (MBP) are incorporated into autophagosomes in oligodendrocytes, resulting in their degradation. Furthermore, we ablated atg5, a core gene of the autophagic machinery, specifically in myelinating glial cells in vivo by tamoxifen administration (plp-Cre ERT2 ; atg5 f/f ) and showed that myelin maintenance is perturbed, leading to PLP accumulation. Significant morphological defects in myelin membrane such as decompaction accompanied with increased axonal degeneration are observed. As a result, the mice exhibit behavioral deficits. In summary, our data highlight that the maintenance of adult myelin homeostasis in the CNS requires the involvement of a fully functional autophagic machinery.
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Autophagy provides nutrients during starvation and eliminates detrimental cellular components. However, accumulating evidence indicates that autophagy is not merely a housekeeping process. Here, by combining mouse models of neuron-specific ATG5 deficiency in either excitatory or inhibitory neurons with quantitative proteomics, high-content microscopy, and live-imaging approaches, we show that autophagy protein ATG5 functions in neurons to regulate cAMP-dependent protein kinase A (PKA)-mediated phosphorylation of a synapse-confined proteome. This function of ATG5 is independent of bulk turnover of synaptic proteins and requires the targeting of PKA inhibitory R1 subunits to autophagosomes. Neuronal loss of ATG5 causes synaptic accumulation of PKA-R1, which sequesters the PKA catalytic subunit and diminishes cAMP/PKA-dependent phosphorylation of postsynaptic cytoskeletal proteins that mediate AMPAR trafficking. Furthermore, ATG5 deletion in glutamatergic neurons augments AMPAR-dependent excitatory neurotransmission and causes the appearance of spontaneous recurrent seizures in mice. Our findings identify a novel role of autophagy in regulating PKA signaling at glutamatergic synapses and suggest the PKA as a target for restoration of synaptic function in neurodegenerative conditions with autophagy dysfunction.
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Neuronas , Sinapsis , Ratones , Animales , Sinapsis/metabolismo , Neuronas/metabolismo , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Transducción de Señal , AutofagiaRESUMEN
Neurons are highly polarized and functionally compartmentalized cells. Under basal conditions, the biogenesis of autophagic vesicles (AVs) was previously shown to take place in the axon tip. As the sequestration of autophagic cargo occurs during the formation of nascent AVs, this would mean that only axonal proteins can be degraded via macroautophagy/autophagy, unless AV biogenesis can also take place on demand, in other neuronal compartments. Our work shows that indeed, activation of NMDA or group I metabotropic glutamate receptors during long-term synaptic depression (LTD) triggers the biogenesis of AVs locally in dendrites. Under these conditions, nascent dendritic AVs are required for synaptic plasticity, as they sequester postsynaptic proteins, whose removal from the postsynapse is necessary for LTD.
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Dendritas , Depresión Sináptica a Largo Plazo , Autofagia , Dendritas/metabolismo , Hipocampo/metabolismo , Depresión Sináptica a Largo Plazo/fisiología , Neuronas/metabolismo , SinapsisRESUMEN
The pruning of dendritic spines during development requires autophagy. This process is facilitated by long-term depression (LTD)-like mechanisms, which has led to speculation that LTD, a fundamental form of synaptic plasticity, also requires autophagy. Here, we show that the induction of LTD via activation of NMDA receptors or metabotropic glutamate receptors initiates autophagy in the postsynaptic dendrites in mice. Dendritic autophagic vesicles (AVs) act in parallel with the endocytic machinery to remove AMPA receptor subunits from the membrane for degradation. During NMDAR-LTD, key postsynaptic proteins are sequestered for autophagic degradation, as revealed by quantitative proteomic profiling of purified AVs. Pharmacological inhibition of AV biogenesis, or conditional ablation of atg5 in pyramidal neurons abolishes LTD and triggers sustained potentiation in the hippocampus. These deficits in synaptic plasticity are recapitulated by knockdown of atg5 specifically in postsynaptic pyramidal neurons in the CA1 area. Conducive to the role of synaptic plasticity in behavioral flexibility, mice with autophagy deficiency in excitatory neurons exhibit altered response in reversal learning. Therefore, local assembly of the autophagic machinery in dendrites ensures the degradation of postsynaptic components and facilitates LTD expression.
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Autofagia/fisiología , Espinas Dendríticas/fisiología , Depresión Sináptica a Largo Plazo/fisiología , Proteoma/metabolismo , Proteómica/métodos , Potenciales Sinápticos/fisiología , Animales , Autofagia/genética , Proteína 5 Relacionada con la Autofagia/genética , Proteína 5 Relacionada con la Autofagia/metabolismo , Células Cultivadas , Hipocampo/citología , Hipocampo/metabolismo , Hipocampo/fisiología , Masculino , Ratones Endogámicos C57BL , Ratones Noqueados , Ratones Transgénicos , Plasticidad Neuronal/fisiología , Neuronas/metabolismo , Neuronas/fisiología , Células Piramidales/metabolismo , Células Piramidales/fisiología , Receptores de Glutamato Metabotrópico/metabolismo , Receptores de N-Metil-D-Aspartato/metabolismoRESUMEN
Aging represents a cumulative form of cellular stress, which is thought to challenge many aspects of proteostasis. The non-dividing, long-lived neurons are particularly vulnerable to stress, and, not surprisingly, even normal aging is highly associated with a decline in brain function in humans, as well as in other animals. Macroautophagy is a fundamental arm of the proteostasis network, safeguarding proper protein turnover during different cellular states and against diverse cellular stressors. An intricate interplay between macroautophagy and aging is beginning to unravel, with the emergence of new tools, including those for monitoring autophagy in cultured neurons and in the nervous system of different organisms in vivo. Here, we review recent findings on the impact of aging on neuronal integrity and on neuronal macroautophagy, as they emerge from studies in invertebrate and mammalian models.
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Long-term depression (LTD) of synaptic strength can take multiple forms and contribute to circuit remodeling, memory encoding or erasure. The generic term LTD encompasses various induction pathways, including activation of NMDA, mGlu or P2X receptors. However, the associated specific molecular mechanisms and effects on synaptic physiology are still unclear. We here compare how NMDAR- or P2XR-dependent LTD affect synaptic nanoscale organization and function in rodents. While both LTDs are associated with a loss and reorganization of synaptic AMPARs, only NMDAR-dependent LTD induction triggers a profound reorganization of PSD-95. This modification, which requires the autophagy machinery to remove the T19-phosphorylated form of PSD-95 from synapses, leads to an increase in AMPAR surface mobility. We demonstrate that these post-synaptic changes that occur specifically during NMDAR-dependent LTD result in an increased short-term plasticity improving neuronal responsiveness of depressed synapses. Our results establish that P2XR- and NMDAR-mediated LTD are associated to functionally distinct forms of LTD.
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Homólogo 4 de la Proteína Discs Large/fisiología , Depresión Sináptica a Largo Plazo/fisiología , Receptores de N-Metil-D-Aspartato/fisiología , Adenosina Trifosfato/administración & dosificación , Animales , Autofagia/fisiología , Células Cultivadas , Homólogo 4 de la Proteína Discs Large/deficiencia , Femenino , Hipocampo/citología , Hipocampo/fisiología , Técnicas In Vitro , Masculino , Ratones , Ratones Endogámicos C57BL , Potenciales Postsinápticos Miniatura/fisiología , Modelos Neurológicos , N-Metilaspartato/administración & dosificación , Plasticidad Neuronal/fisiología , Neuronas/citología , Neuronas/efectos de los fármacos , Neuronas/fisiología , Ratas , Ratas Sprague-Dawley , Receptores AMPA/fisiología , Receptores Purinérgicos P2X/fisiologíaRESUMEN
Genetic studies have demonstrated that conditional ablation of core autophagy genes in the neural lineage leads to progressive neurodegeneration, indicating that this catabolic pathway is indispensable for neuronal maintenance. However, accumulating evidence also indicates that autophagy is not merely a housekeeping process. Instead, autophagy may be dynamically regulated in different neuronal compartments and dictate the turnover of selected cargo in a time- and space-dependent manner and thus contribute to specialized neuronal functions. Here, we review the mechanisms regulating autophagy in neurons and discuss recent findings on the role of autophagy in synaptic morphology and function. Moreover, we discuss relevant questions that remain open and highlight forthcoming issues in the field of neuronal autophagy.
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Autofagia , Sinapsis/metabolismo , Animales , Autofagia/genética , Humanos , Neuronas/metabolismo , Neuronas/patología , Sinapsis/patologíaRESUMEN
Proteinopathies constitute a diverse group of devastating neurodegenerative disorders, characterized by aberrant aggregation of specific proteins within neurons and in the brain parenchyma. Parkinson's disease (PD) is among the most common proteinopathies, caused by the accumulation of different species of α-synuclein and the formation of protein inclusions known as Lewy bodies. Although several mutations in the α-synuclein gene have been linked to PD, the mechanisms mediating the aggregation and toxicity of α-synuclein are not fully understood. Here, we review recent evidence that highlight an intricate interplay between α-synuclein and ionostasis, focusing on the PMR1 pump, a Golgi resident Ca2+/Mn2+ P-type ATPase, which plays a pivotal role in regulating the intracellular levels of calcium and manganese ions.
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Señalización del Calcio/fisiología , ATPasas Transportadoras de Calcio/metabolismo , Aparato de Golgi/metabolismo , Enfermedades Neurodegenerativas/metabolismo , alfa-Sinucleína/metabolismo , Animales , Aparato de Golgi/patología , Humanos , Enfermedades Neurodegenerativas/patología , alfa-Sinucleína/toxicidadRESUMEN
Autophagy is crucial for neuronal integrity. Loss of key autophagic components leads to progressive neurodegeneration and structural defects in pre- and postsynaptic morphologies. However, the molecular mechanisms regulating autophagy in the brain remain elusive. Similarly, while it is widely accepted that protein turnover is required for synaptic plasticity, the contribution of autophagy to the degradation of synaptic proteins is unknown. Here, we report that BDNF signaling via the tropomyosin receptor kinase B (TrkB) and the phosphatidylinositol-3' kinase (PI3K)/Akt pathway suppresses autophagy in vivo. In addition, we demonstrate that suppression of autophagy is required for BDNF-induced synaptic plasticity and for memory enhancement under conditions of nutritional stress. Finally, we identify three key remodelers of postsynaptic densities as cargo of autophagy. Our results establish autophagy as a pivotal component of BDNF signaling, which is essential for BDNF-induced synaptic plasticity. This molecular mechanism underlies behavioral adaptations that increase fitness in times of scarcity.
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Autofagia , Factor Neurotrófico Derivado del Encéfalo/metabolismo , Plasticidad Neuronal , Transducción de Señal , Animales , Ayuno , Masculino , Ratones Endogámicos C57BL , Neuronas/citología , Neuronas/metabolismo , Fosfatidilinositol 3-Quinasas/metabolismo , Prosencéfalo/citología , Prosencéfalo/metabolismo , Proteínas Proto-Oncogénicas c-akt/metabolismo , Receptor trkB/metabolismoRESUMEN
Nerve growth factor (NGF) holds a pivotal role in brain development and maintenance, been also involved in the pathophysiology of neurodegenerative diseases. Here, we provide evidence that a novel C17-spiroepoxy steroid derivative, BNN27, specifically interacts with and activates the TrkA receptor of NGF, inducing phosphorylation of TrkA tyrosine residues and down-stream neuronal survival-related kinase signaling. Additionally, BNN27 potentiates the efficacy of low levels of NGF, by facilitating its binding to the TrkA receptors and differentially inducing fast return of internalized TrkA receptors into neuronal cell membranes. Furthermore, BNN27 synergizes with NGF in promoting axonal outgrowth, effectively rescues from apoptosis NGF-dependent and TrkA positive sympathetic and sensory neurons, in vitro, ex vivo and in vivo in NGF null mice. Interestingly, BNN27 does not possess the hyperalgesic properties of NGF. BNN27 represents a lead molecule for the development of neuroprotective TrkA receptor agonists, with potential therapeutic applications in neurodegenerative diseases and in brain trauma.
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Diferenciación Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Deshidroepiandrosterona/farmacología , Factor de Crecimiento Nervioso/farmacología , Neuronas/efectos de los fármacos , Neuronas/metabolismo , Receptor trkA/metabolismo , Animales , Apoptosis/efectos de los fármacos , Axones/efectos de los fármacos , Axones/metabolismo , Sitios de Unión , Células CHO , Cricetulus , Deshidroepiandrosterona/química , Células HEK293 , Humanos , Hiperalgesia/inducido químicamente , Ratones , Ratones Noqueados , Modelos Moleculares , Simulación de Dinámica Molecular , Factor de Crecimiento Nervioso/genética , Neuroglía/efectos de los fármacos , Neuroglía/metabolismo , Células PC12 , Fosforilación , Ratas , Receptor trkA/agonistas , Proteínas Recombinantes/farmacología , Células Receptoras Sensoriales/efectos de los fármacos , Células Receptoras Sensoriales/metabolismo , Transducción de SeñalRESUMEN
Similar to other organisms, necrotic cell death in the nematode Caenorhabditis elegans is manifested as the catastrophic collapse of cellular homeostasis, in response to overwhelming stress that is inflicted either in the form of extreme environmental stimuli or by intrinsic insults such as the expression of proteins carrying deleterious mutations. Remarkably, necrotic cell death in C. elegans and pathological cell death in humans share multiple fundamental features and mechanistic aspects. Therefore, mechanisms mediating necrosis are also conserved across the evolutionary spectrum and render the worm a versatile tool, with the capacity to facilitate studies of human pathologies. Here, we overview necrotic paradigms that have been characterized in the nematode and outline the cellular and molecular mechanisms that mediate this mode of cell demise. In addition, we discuss experimental approaches that utilize C. elegans to elucidate the molecular underpinnings of devastating human disorders that entail necrosis.
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Apoptosis/genética , Autofagia/genética , Caenorhabditis elegans/genética , Necrosis/genética , Animales , Endocitosis/genética , Regulación de la Expresión Génica , Respuesta al Choque Térmico/genética , Humanos , Neuronas/metabolismo , Neuronas/fisiologíaRESUMEN
Human lifespan has been increasing steadily during modern times, mainly due to medical advancements that combat infant mortality and various life-threatening diseases. However, this gratifying longevity rise is accompanied by growing incidences of devastating age-related pathologies. Understanding the cellular and molecular mechanisms that underlie aging and regulate longevity is of utmost relevance towards offsetting the impact of age-associated disorders and increasing the quality of life for the elderly. Several evolutionarily conserved pathways that modulate lifespan have been identified in organisms ranging from yeast to primates. Here we survey recent findings highlighting the interplay of various genetic, epigenetic, and cell-specific factors, and also symbiotic relationships, as longevity determinants. We further discuss outstanding matters within the framework of emerging, integrative views of aging.
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Envejecimiento/fisiología , Longevidad/fisiología , Envejecimiento/genética , Humanos , Longevidad/genética , Transducción de Señal/genética , Transducción de Señal/fisiologíaAsunto(s)
Mitocondrias/metabolismo , Proteínas Mitocondriales/metabolismo , Enfermedades Neurodegenerativas/metabolismo , Neuronas/metabolismo , Animales , Humanos , Mitocondrias/genética , Proteínas Mitocondriales/genética , Mutación/genética , Enfermedades Neurodegenerativas/etiología , Enfermedades Neurodegenerativas/genéticaRESUMEN
The concept that target tissues determine the survival of neurons has inspired much of the thinking on neuronal development in vertebrates, not least because it is supported by decades of research on nerve growth factor (NGF) in the peripheral nervous system (PNS). Recent discoveries now help to understand why only some developing neurons selectively depend on NGF. They also indicate that the survival of most neurons in the central nervous system (CNS) is not simply regulated by single growth factors like in the PNS. Additionally, components of the cell death machinery have begun to be recognized as regulators of selective axonal degeneration and synaptic function, thus playing a critical role in wiring up the nervous system.
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Sistema Nervioso Central/metabolismo , Factor de Crecimiento Nervioso/metabolismo , Neurogénesis/fisiología , Sistema Nervioso Periférico/metabolismo , Receptores de Factor de Crecimiento Nervioso/metabolismo , Animales , Apoptosis , Supervivencia Celular , Sistema Nervioso Central/embriología , Humanos , Ratones , Neuronas/metabolismo , Sistema Nervioso Periférico/embriología , Receptor trkA/metabolismo , Receptor trkB/metabolismo , Receptor trkC/metabolismoRESUMEN
Apoptosis and necrosis are the two major modes of cell death, the molecular mechanisms of which have been extensively studied. Although initially thought to constitute mutually exclusive cellular states, recent findings reveal cellular contexts that require a balanced interplay between these two modes of cellular demise. Several death initiator and effector molecules, signaling pathways and subcellular sites have been identified as key mediators in both processes, either by constituting common modules or alternatively by functioning as a switch allowing cells to decide which route to take, depending on the specific situation. Importantly, autophagy, which is a predominantly cytoprotective process, has been linked to both types of cell death, serving either a pro-survival or pro-death function. Here we review the recent literature that highlights the intricate interplay between apoptosis, necrosis and autophagy, focusing on the relevance and impact of this crosstalk in normal development and in pathology. This article is part of a Special Section entitled: Cell Death Pathways.
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Apoptosis , Autofagia , Necrosis/patología , Transducción de Señal , Humanos , Modelos BiológicosAsunto(s)
Calcio/metabolismo , Fiebre/metabolismo , Proteínas de Choque Térmico/metabolismo , Respuesta al Choque Térmico , Degeneración Nerviosa , Neuronas/metabolismo , Animales , Caenorhabditis elegans , Bloqueadores de los Canales de Calcio/farmacología , Citoprotección , Fiebre/complicaciones , Fiebre/tratamiento farmacológico , Fiebre/patología , Homeostasis , Humanos , Necrosis , Neuronas/efectos de los fármacos , Neuronas/patología , Fármacos Neuroprotectores/farmacologíaRESUMEN
The nervous system becomes increasingly vulnerable to insults and prone to dysfunction during aging. Age-related decline of neuronal function is manifested by the late onset of many neurodegenerative disorders, as well as by reduced signaling and processing capacity of individual neuron populations. Recent findings indicate that impairment of Ca(2+) homeostasis underlies the increased susceptibility of neurons to damage, associated with the aging process. However, the impact of aging on Ca(2+) homeostasis in neurons remains largely unknown. Here, we survey the molecular mechanisms that mediate neuronal Ca(2+) homeostasis and discuss the impact of aging on their efficacy. To address the question of how aging impinges on Ca(2+) homeostasis, we consider potential nodes through which mechanisms regulating Ca(2+) levels interface with molecular pathways known to influence the process of aging and senescent decline. Delineation of this crosstalk would facilitate the development of interventions aiming to fortify neurons against age-associated functional deterioration and death by augmenting Ca(2+) homeostasis.
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Heat stroke is a life-threatening condition, characterized by catastrophic collapse of thermoregulation and extreme hyperthermia. In recent years, intensification of heat waves has caused a surge of heat-stroke fatalities. The mechanisms underlying heat-related pathology are poorly understood. Here we show that heat stroke triggers pervasive necrotic cell death and neurodegeneration in Caenorhabditis elegans. Preconditioning of animals at a mildly elevated temperature strongly protects from heat-induced necrosis. The heat-shock transcription factor HSF-1 and the small heat-shock protein HSP-16.1 mediate cytoprotection by preconditioning. HSP-16.1 localizes to the Golgi, where it functions with the Ca(2+)- and Mn(2+)-transporting ATPase PMR-1 to maintain Ca(2+) homeostasis under heat stroke. Preconditioning also suppresses cell death inflicted by diverse insults, and protects mammalian neurons from heat cytotoxicity. These findings reveal an evolutionarily conserved mechanism that defends against diverse necrotic stimuli, and may be relevant to heat stroke and other pathological conditions involving necrosis in humans.