RESUMEN
The dynamicity of the mitochondrial network is crucial for meeting the ever-changing metabolic and energy needs of the cell. Mitochondrial fission promotes the degradation and distribution of mitochondria, while mitochondrial fusion maintains mitochondrial function through the complementation of mitochondrial components. Previously, we have reported that mitochondrial networks are tubular, interconnected, and well-organized in young, healthy C. elegans, but become fragmented and disorganized with advancing age and in models of age-associated neurodegenerative disease. In this work, we examine the effects of increasing mitochondrial fission or mitochondrial fusion capacity by ubiquitously overexpressing the mitochondrial fission gene drp-1 or the mitochondrial fusion genes fzo-1 and eat-3, individually or in combination. We then measured mitochondrial function, mitochondrial network morphology, physiologic rates, stress resistance, and lifespan. Surprisingly, we found that overexpression of either mitochondrial fission or fusion machinery both resulted in an increase in mitochondrial fragmentation. Similarly, both mitochondrial fission and mitochondrial fusion overexpression strains have extended lifespans and increased stress resistance, which in the case of the mitochondrial fusion overexpression strains appears to be at least partially due to the upregulation of multiple pathways of cellular resilience in these strains. Overall, our work demonstrates that increasing the expression of mitochondrial fission or fusion genes extends lifespan and improves biological resilience without promoting the maintenance of a youthful mitochondrial network morphology. This work highlights the importance of the mitochondria for both resilience and longevity.
RESUMEN
Protein misfolding and mislocalization are common themes in neurodegenerative disorders, including motor neuron disease, and amyotrophic lateral sclerosis (ALS). Maintaining proteostasis is a crosscutting therapeutic target, including the upregulation of heat shock proteins (HSP) to increase chaperoning capacity. Motor neurons have a high threshold for upregulating stress-inducible HSPA1A, but constitutively express high levels of HSPA8. This study compared the expression of these HSPs in cultured motor neurons expressing three variants linked to familial ALS: TAR DNA binding protein 43 kDa (TDP-43)G348C, fused in sarcoma (FUS)R521G, or superoxide dismutase I (SOD1)G93A. All variants were poor inducers of Hspa1a, and reduced levels of Hspa8 mRNA and protein, indicating multiple compromises in chaperoning capacity. To promote HSP expression, cultures were treated with the putative HSP coinducer, arimoclomol, and class I histone deacetylase inhibitors, to promote active chromatin for transcription, and with the combination. Treatments had variable, often different effects on the expression of Hspa1a and Hspa8, depending on the ALS variant expressed, mRNA distribution (somata and dendrites), and biomarker of toxicity measured (histone acetylation, maintaining nuclear TDP-43 and the neuronal Brm/Brg-associated factor chromatin remodeling complex component Brg1, mitochondrial transport, FUS aggregation). Overall, histone deacetylase inhibition alone was effective on more measures than arimoclomol. As in the FUS model, arimoclomol failed to induce HSPA1A or preserve Hspa8 mRNA in the TDP-43 model, despite preserving nuclear TDP-43 and Brg1, indicating neuroprotective properties other than HSP induction. The data speak to the complexity of drug mechanisms against multiple biomarkers of ALS pathogenesis, as well as to the importance of HSPA8 for neuronal proteostasis in both somata and dendrites.
Asunto(s)
Esclerosis Amiotrófica Lateral , Biomarcadores , Proteínas de Unión al ADN , Inhibidores de Histona Desacetilasas , Neuronas Motoras , Esclerosis Amiotrófica Lateral/metabolismo , Esclerosis Amiotrófica Lateral/genética , Esclerosis Amiotrófica Lateral/tratamiento farmacológico , Inhibidores de Histona Desacetilasas/farmacología , Biomarcadores/metabolismo , Proteínas de Unión al ADN/metabolismo , Proteínas de Unión al ADN/genética , Humanos , Neuronas Motoras/metabolismo , Neuronas Motoras/efectos de los fármacos , Neuronas Motoras/patología , Animales , Proteínas HSP70 de Choque Térmico/metabolismo , Proteínas HSP70 de Choque Térmico/genética , Proteínas del Choque Térmico HSC70/metabolismo , Proteínas del Choque Térmico HSC70/genética , Hidroxilaminas/farmacología , Células Cultivadas , Proteína FUS de Unión a ARN/metabolismo , Proteína FUS de Unión a ARN/genética , Superóxido Dismutasa-1/metabolismo , Superóxido Dismutasa-1/genéticaRESUMEN
Neurons are challenged to maintain proteostasis in neuronal projections, particularly with the physiological stress at synapses to support intercellular communication underlying important functions such as memory and movement control. Proteostasis is maintained through regulated protein synthesis and degradation and chaperone-assisted protein folding. Using high-resolution fluorescent microscopy, we discovered that neurons localize a subset of chaperone mRNAs to their dendrites, particularly more proximal regions, and increase this asymmetric localization following proteotoxic stress through microtubule-based transport from the soma. The most abundant chaperone mRNA in dendrites encodes the constitutive heat shock protein 70, HSPA8. Proteotoxic stress in cultured neurons, induced by inhibiting proteasome activity or inducing oxidative stress, enhanced transport of Hspa8 mRNAs to dendrites and the percentage of mRNAs engaged in translation on mono and polyribosomes. Knocking down the ALS-related protein Fused in Sarcoma (FUS) and a dominant mutation in the heterogenous nuclear ribonucleoprotein A2/B1 (HNRNPA2B1) impaired stress-mediated localization of Hspa8 mRNA to dendrites in cultured murine motor neurons and human iPSC-derived neurons, respectively, revealing the importance of these RNA-binding proteins in maintaining proteostasis. These results reveal the increased dendritic localization and translation of the constitutive HSP70 Hspa8 mRNA as a crucial neuronal stress response to uphold proteostasis and prevent neurodegeneration.
RESUMEN
Proteostasis is maintained through regulated protein synthesis and degradation and chaperone-assisted protein folding. However, this is challenging in neuronal projections because of their polarized morphology and constant synaptic proteome remodeling. Using high-resolution fluorescence microscopy, we discovered that neurons localize a subset of chaperone mRNAs to their dendrites and use microtubule-based transport to increase this asymmetric localization following proteotoxic stress. The most abundant dendritic chaperone mRNA encodes a constitutive heat shock protein 70 family member (HSPA8). Proteotoxic stress also enhanced HSPA8 mRNA translation efficiency in dendrites. Stress-mediated HSPA8 mRNA localization to the dendrites was impaired by depleting fused in sarcoma-an amyotrophic lateral sclerosis-related protein-in cultured mouse motor neurons and expressing a pathogenic variant of heterogenous nuclear ribonucleoprotein A2/B1 in neurons derived from human induced pluripotent stem cells. These results reveal a crucial and unexpected neuronal stress response in which RNA-binding proteins increase the dendritic localization of HSPA8 mRNA to maintain proteostasis and prevent neurodegeneration.
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Neurodegenerative disorders (NDs) are diverse age-related conditions also described as "conformational diseases." The hallmark of NDs is the accumulation of disease-specific proteins as toxic misfolded aggregates in some areas of the brain. They lead to the loss of protein homeostasis (proteostasis) that causes neuronal dysfunction and death. A potential therapeutic strategy for NDs is to prevent the accumulation of misfolded proteins by activating the heat shock response (HSR). The HSR maintains proteostasis through the upregulation of heat shock proteins (HSPs), molecular chaperones that recognize misfolded proteins, and either refold them to their functional conformations and/or target them for degradation. However, how to manipulate the expression of HSPs to obtain a therapeutic effect in neurons remains unclear. Furthermore, the regulation of the HSR in neurons is more complex than what we have learned from culturing somatic nonneuronal cells. This chapter describes a method to investigate the induction of HSP70 in primary hippocampal neurons using single-molecule fluorescence in situ hybridization (smFISH). Quantification of smFISH provides the means to analyze neuron-to-neuron variability in the activation of the HSR and enables us to study the transcriptional induction and localization of HSP70 mRNA in primary neurons. This information might be critical to find the druggable steps for developing effective therapies to treat age-related NDs.
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Proteínas de Choque Térmico , Enfermedades Neurodegenerativas , Proteínas HSP70 de Choque Térmico/metabolismo , Proteínas de Choque Térmico/metabolismo , Respuesta al Choque Térmico/genética , Humanos , Hibridación Fluorescente in Situ , Microscopía Fluorescente , Enfermedades Neurodegenerativas/metabolismo , Neuronas/metabolismoRESUMEN
Neurodevelopment is accompanied by a precise change in the expression of the translation elongation factor 1A variants from eEF1A1 to eEF1A2. These are paralogue genes that encode 92% identical proteins in mammals. The switch in the expression of eEF1A variants has been well studied in mouse motor neurons, which solely express eEF1A2 by four weeks of postnatal development. However, changes in the subcellular localization of eEF1A variants during neurodevelopment have not been studied in detail in other neuronal types because antibodies lack perfect specificity, and immunofluorescence has a low sensitivity. In hippocampal neurons, eEF1A is related to synaptic plasticity and memory consolidation, and decreased eEF1A expression is observed in the hippocampus of Alzheimer's patients. However, the specific variant involved in these functions is unknown. To distinguish eEF1A1 from eEF1A2 expression, we have designed single-molecule fluorescence in-situ hybridization probes to detect either eEF1A1 or eEF1A2 mRNAs in cultured primary hippocampal neurons and brain tissues. We have developed a computational framework, ARLIN (analysis of RNA localization in neurons), to analyze and compare the subcellular distribution of eEF1A1 and eEF1A2 mRNAs at specific developmental stages and in mature neurons. We found that eEF1A1 and eEF1A2 mRNAs differ in expression and subcellular localization over neurodevelopment, and eEF1A1 mRNAs localize in dendrites and synapses during dendritogenesis and synaptogenesis. Interestingly, mature hippocampal neurons coexpress both variant mRNAs, and eEF1A1 remains the predominant variant in dendrites.
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Neuronas , Factor 1 de Elongación Peptídica , Biosíntesis de Proteínas , Animales , Hipocampo , Humanos , Mamíferos , Ratones , Factor 1 de Elongación Peptídica/genética , ARN Mensajero/genéticaRESUMEN
All cells possess an internal stress response to cope with environmental and pathophysiological challenges. Upon stress, cells reprogram their molecular functions to activate a survival mechanism known as the heat shock response, which mediates the rapid induction of molecular chaperones such as the heat shock proteins (HSPs). This potent production overcomes the general suppression of gene expression and results in high levels of HSPs to subsequently refold or degrade misfolded proteins. Once the damage or stress is repaired or removed, cells terminate the production of HSPs and resume regular functions. Thus, fulfillment of the stress response requires swift and robust coordination between stress response activation and completion that is determined by the status of the cell. In recent years, single-cell fluorescence microscopy techniques have begun to be used in unravelling HSP-gene expression pathways, from DNA transcription to mRNA degradation. In this review, we will address the molecular mechanisms in different organisms and cell types that coordinate the expression of HSPs with signaling networks that act to reprogram gene transcription, mRNA translation, and decay and ensure protein quality control.
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Proteínas de Choque Térmico , Proteostasis , Proteínas de Choque Térmico/metabolismo , Respuesta al Choque Térmico/fisiología , Chaperonas Moleculares/genética , Chaperonas Moleculares/metabolismo , Transducción de SeñalRESUMEN
Magnocellular neurosecretory cells (MNCs) are intrinsically osmosensitive and can be activated by increases in blood osmolality, triggering the release of antidiuretic hormone vasopressin (VP) to promote water retention. Hence, the activity of magnocellular VP neurons is one of the key elements contributing to the regulation of body fluid homeostasis in healthy organisms. Chronic exposure to high dietary salt leads to excessive activation of VP neurons, thereby elevating levels of circulating VP, which can cause increases in blood pressure contributing to salt-dependent hypertension. However, the molecular basis underlying high-salt diet-induced hyperactivation of magnocellular VP neurons remains not fully understood. Previous studies suggest that magnocellular neurosecretory neurons contain a subcortical layer of actin filaments and pharmacological stabilization of this actin network potentiates osmotically-induced activation of magnocellular neurons. Using super-resolution imaging in situ, we investigated the organization of the actin cytoskeleton in rat MNCs under normal physiological conditions and after a chronic increase in blood osmolality following 7 d of salt-loading (SL). We found that, in addition to the subcortical layer of actin filaments, magnocellular VP neurons are endowed with a unique network of cytoplasmic actin filaments throughout their somata. Moreover, we revealed that the density of both subcortical and cytoplasmic actin networks in magnocellular VP neurons is dramatically increased following SL. These results suggest that increased osmo-responsiveness of VP neurons following chronic exposure to high dietary salt may be mediated by the modulation of unique actin networks in magnocellular VP neurons, possibly contributing to elevated blood pressure in this condition.
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Cloruro de Sodio Dietético , Núcleo Supraóptico , Citoesqueleto de Actina/metabolismo , Animales , Neuronas/metabolismo , Ratas , Núcleo Supraóptico/metabolismo , Vasopresinas/metabolismoRESUMEN
Magnocellular vasopressin (VP) neurones are activated by increases in blood osmolality, leading to the secretion of VP into the circulation to promote water retention in the kidney, thus constituting a key mechanism for the regulation of body fluid homeostasis. However, chronic high salt intake can lead to excessive activation of VP neurones and increased circulating levels of VP, contributing to an elevation in blood pressure. Multiple extrinsic factors, such as synaptic inputs and glial cells, modulate the activity of VP neurones. Moreover, magnocellular neurones are intrinsically osmosensitive, and are activated by hypertonicity in the absence of neighbouring cells or synaptic contacts. Hypertonicity triggers cell shrinking, leading to the activation of VP neurones. This cell-autonomous activation is mediated by a scaffold of dense somatic microtubules, uniquely present in VP magnocellular neurones. Treating isolated magnocellular neurones with drugs modulating microtubule stability modifies the sensitivity of neuronal activation in response to acute hypertonic stimuli. However, whether the microtubule network is altered in conditions associated with enhanced neuronal activation and increased VP release, such as chronic high salt intake, remains unknown. We examined the organisation of microtubules in VP neurones of the supraoptic and paraventricular hypothalamic nuclei (SON and PVN, respectively) of rats subjected to salt-loading (drinking 2% NaCl for 7 days). Using super-resolution imaging, we found that the density of microtubules in magnocellular VP neurones from the SON and PVN was significantly increased, whereas the density and organisation of microtubules remain unchanged in other hypothalamic neurones, as well as in neurones from other brain areas (e.g., hippocampus, cortex). We propose that the increase in microtubule density in magnocellular VP neurones in salt-loading promotes their enhanced activation, possibly contributing to elevated blood pressure in this condition.