RESUMEN
Protein kinase C (PKC) functions are essential for synaptic plasticity, learning, and memory. However, the roles of specific members of the PKC family in synaptic function, learning, and memory are poorly understood. Here, we investigated the role of individual PKC homologs for synaptic plasticity in Caenorhabditis elegans and found a differential role for pkc-2 and tpa-1, but not pkc-1 and pkc-3 in associative olfactory learning and memory. More specifically we show that PKC-2 is essential for associative learning and TPA-1 for short-term associative memory (STAM). Using endogenous labeling and cell-specific rescues, we show that TPA-1 and PKC-2 are required in AVA for their functions. Previous studies demonstrated that olfactory learning and memory in C. elegans are tied to proper synaptic content and trafficking of AMPA-type ionotropic glutamate receptor homolog GLR-1 in the AVA command interneurons. Therefore, we quantified synaptic content, transport, and delivery of GLR-1 in AVA and showed that loss of pkc-2 and tpa-1 leads to decreased transport and delivery but only a subtle decrease in GLR-1 levels at synapses. AVA-specific expression of both PKC-2 and TPA-1 rescued these defects. Finally, genetic epistasis showed that PKC-2 and TPA-1 likely act in the same pathway to control GLR-1 transport and delivery, while regulating different aspects of olfactory learning and STAM. Thus, our data tie together cell-specific functions of 2 PKCs to neuronal and behavioral outcomes in C. elegans, enabling comparative approaches to understand the evolutionarily conserved role of PKC in synaptic plasticity, learning, and memory.
Asunto(s)
Proteínas de Caenorhabditis elegans , Caenorhabditis elegans , Animales , Caenorhabditis elegans/fisiología , Proteínas de Caenorhabditis elegans/metabolismo , Aprendizaje , Proteína Quinasa C/metabolismo , Receptores AMPA/metabolismo , Sinapsis/metabolismoRESUMEN
Cardiac dysfunction is a common phenotypic manifestation of primary mitochondrial disease with multiple nuclear and mitochondrial DNA pathogenic variants as a cause, including disorders of mitochondrial translation. To date, five patients have been described with pathogenic variants in MRPL44, encoding the ml44 protein which is part of the large subunit of the mitochondrial ribosome (mitoribosome). Three presented as infants with hypertrophic cardiomyopathy, mild lactic acidosis, and easy fatigue and muscle weakness, whereas two presented in adolescence with myopathy and neurological symptoms. We describe two infants who presented with cardiomyopathy from the neonatal period, failure to thrive, hypoglycemia and in one infant lactic acidosis. A decompensation of the cardiac function in the first year resulted in demise. Exome sequencing identified compound heterozygous variants in the MRPL44 gene including the known pathogenic variant c.467 T > G and two novel pathogenic variants. We document a combined respiratory chain enzyme deficiency with emphasis on complex I and IV, affecting heart muscle tissue more than skeletal muscle or fibroblasts. We show this to be caused by reduced mitochondrial DNA encoded protein synthesis affecting all subunits, and resulting in dysfunction of complex I and IV assembly. The degree of oxidative phosphorylation dysfunction correlated with the impairment of mitochondrial protein synthesis due to different pathogenic variants. These functional studies allow for improved understanding of the pathogenesis of MRPL44-associated mitochondrial disorder.
Asunto(s)
Cardiomiopatías/etiología , Cardiomiopatías/genética , ADN Mitocondrial/genética , Enfermedades Mitocondriales/complicaciones , Proteínas Mitocondriales/genética , Biosíntesis de Proteínas , Proteínas Ribosómicas/genética , Femenino , Variación Genética , Humanos , Lactante , Recién Nacido , Mitocondrias/metabolismo , Mitocondrias/patología , Proteínas Mitocondriales/clasificación , Mutación , Fosforilación Oxidativa , Proteínas Ribosómicas/clasificaciónRESUMEN
Genetic defects in mitochondrial DNA encoded tRNA genes impair mitochondrial translation with resultant defects in the mitochondrial respiratory chain and oxidative phosphorylation system. The phenotypic spectrum of disease seen in mitochondrial tRNA defects is variable and proving pathogenicity of new variants is challenging. Only three pathogenic variants have been described previously in the mitochondrial tRNATyr gene MT-TY, with the reported phenotypes consisting largely of adult onset myopathy and ptosis. We report a patient with a novel MT-TY acceptor stem variant m.5889A>G at high heteroplasmy in muscle, low in blood, and absent in the mother's blood. The phenotype consisted of a childhood-onset severe multi-system disorder characterized by a neurodegenerative course including ataxia and seizures, failure-to-thrive, combined myopathy and neuropathy, and hearing and vision loss. Brain imaging showed progressive atrophy and basal ganglia calcifications. Mitochondrial biomarkers lactate and GDF15 were increased. Functional studies showed a deficient activity of the respiratory chain enzyme complexes containing mtDNA-encoded subunits I, III and IV. There were decreased steady state levels of these mitochondrial complex proteins, and presence of incompletely assembled complex V forms in muscle. These changes are typical of a mitochondrial translational defect. These data support the pathogenicity of this novel variant. Careful review of variants in MT-TY additionally identified two other pathogenic variants, one likely pathogenic variant, nine variants of unknown significance, five likely benign and four benign variants.
Asunto(s)
ADN Mitocondrial/genética , Enfermedades Musculares/genética , ARN de Transferencia/genética , Tirosina/genética , Complejo IV de Transporte de Electrones/genética , Complejo IV de Transporte de Electrones/metabolismo , Humanos , Mitocondrias/genética , Mitocondrias/patología , Músculo Esquelético/metabolismo , Músculo Esquelético/patología , Enfermedades Musculares/metabolismo , Enfermedades Musculares/patología , Mutación/genética , Fosforilación Oxidativa , FenotipoRESUMEN
Disorders of the white matter are genetically very heterogeneous including several genes involved in mitochondrial bioenergetics. Diagnosis of the underlying cause is aided by pattern recognition on neuroimaging and by next-generation sequencing. Recently, genetic changes in the complex I assembly factor NUBPL have been characterized by a consistent recognizable pattern of leukoencephalopathy affecting deep white matter including the corpus callosum and cerebellum. Here, we report twin boys with biallelic variants in NUBPL, an unreported c.351 G > A; p.(Met117Ile) and a previously reported pathological variant c. 693 + 1 G > A. Brain magnetic resonance imaging showed abnormal T2 hyperintense signal involving the periventricular white matter, external capsule, corpus callosum, and, prominently, the bilateral thalami. The neuroimaging pattern evolved over 18 months with marked diffuse white matter signal abnormality, volume loss, and new areas of signal abnormality in the cerebellar folia and vermis. Magnetic resonance spectroscopy showed elevated lactate. Functional studies in cultured fibroblasts confirmed pathogenicity of the genetic variants. Complex I activity of the respiratory chain was deficient spectrophotometrically and on blue native gel with in-gel activity staining. There was absent assembly and loss of proteins of the matrix arm of complex I when traced with an antibody to NDUFS2, and incomplete assembly of the membrane arm when traced with an NDUFB6 antibody. There was decreased NUBPL protein on Western blot in patient fibroblasts compared to controls. Compromised NUBPL activity impairs assembly of the matrix arm of complex I and produces a severe, rapidly-progressive leukoencephalopathy with thalamic involvement on MRI, further expanding the neuroimaging phenotype.
Asunto(s)
Enfermedades en Gemelos/genética , Complejo I de Transporte de Electrón/metabolismo , Leucoencefalopatías/genética , Mitocondrias/metabolismo , Proteínas Mitocondriales/genética , Tálamo/diagnóstico por imagen , Línea Celular , Cuerpo Calloso/diagnóstico por imagen , Cuerpo Calloso/patología , Enfermedades en Gemelos/diagnóstico por imagen , Enfermedades en Gemelos/metabolismo , Enfermedades en Gemelos/fisiopatología , Complejo I de Transporte de Electrón/deficiencia , Complejo I de Transporte de Electrón/genética , Cápsula Externa/diagnóstico por imagen , Cápsula Externa/patología , Ojo/fisiopatología , Fibroblastos/metabolismo , Humanos , Lactante , Ácido Láctico/metabolismo , Leucoencefalopatías/diagnóstico por imagen , Leucoencefalopatías/metabolismo , Leucoencefalopatías/fisiopatología , Imagen por Resonancia Magnética , Espectroscopía de Resonancia Magnética , Masculino , Mitocondrias/genética , Proteínas Mitocondriales/metabolismo , Mutación , NADH Deshidrogenasa/metabolismo , Gemelos Monocigóticos/genética , Sustancia Blanca/diagnóstico por imagen , Sustancia Blanca/patología , Secuenciación del ExomaRESUMEN
Our understanding of mitochondrial signaling in the nervous system has been limited by the technical challenge of analyzing mitochondrial function in vivo. In the transparent genetic model Caenorhabditis elegans, we were able to manipulate and measure mitochondrial reactive oxygen species (mitoROS) signaling of individual mitochondria as well as neuronal activity of single neurons in vivo. Using this approach, we provide evidence supporting a novel role for mitoROS signaling in dendrites of excitatory glutamatergic C. elegans interneurons. Specifically, we show that following neuronal activity, dendritic mitochondria take up calcium (Ca2+) via the mitochondrial Ca2+ uniporter (MCU-1) that results in an upregulation of mitoROS production. We also observed that mitochondria are positioned in close proximity to synaptic clusters of GLR-1, the C. elegans ortholog of the AMPA subtype of glutamate receptors that mediate neuronal excitation. We show that synaptic recruitment of GLR-1 is upregulated when MCU-1 function is pharmacologically or genetically impaired but is downregulated by mitoROS signaling. Thus, signaling from postsynaptic mitochondria may regulate excitatory synapse function to maintain neuronal homeostasis by preventing excitotoxicity and energy depletion.
Asunto(s)
Caenorhabditis elegans , Receptores de Glutamato , Animales , Caenorhabditis elegans/genética , Especies Reactivas de Oxígeno , Neuronas , Sinapsis , CalcioRESUMEN
Calcium (Ca2+) imaging has been largely used to examine neuronal activity, but it is becoming increasingly clear that subcellular Ca2+ handling is a crucial component of intracellular signaling. The visualization of subcellular Ca2+ dynamics in vivo, where neurons can be studied in their native, intact circuitry, has proven technically challenging in complex nervous systems. The transparency and relatively simple nervous system of the nematode Caenorhabditis elegans enable the cell-specific expression and in vivo visualization of fluorescent tags and indicators. Among these are fluorescent indicators that have been modified for use in the cytoplasm as well as various subcellular compartments, such as the mitochondria. This protocol enables non-ratiometric Ca2+ imaging in vivo with a subcellular resolution that permits the analysis of Ca2+ dynamics down to the level of individual dendritic spines and mitochondria. Here, two available genetically encoded indicators with different Ca2+ affinities are used to demonstrate the use of this protocol for measuring relative Ca2+ levels within the cytoplasm or mitochondrial matrix in a single pair of excitatory interneurons (AVA). Together with the genetic manipulations and longitudinal observations possible in C. elegans, this imaging protocol may be useful for answering questions regarding how Ca2+ handling regulates neuronal function and plasticity.
Asunto(s)
Caenorhabditis elegans , Calcio , Animales , Calcio/metabolismo , Caenorhabditis elegans/metabolismo , Mitocondrias/metabolismo , Diagnóstico por Imagen , Neuronas/metabolismo , Señalización del CalcioRESUMEN
Diagnosing complex V deficiencies caused by new variants in mitochondrial DNA is challenging due to the rarity, phenotypic diversity, and limited functional assessments. We describe a child with the m.9032T > C variant in MT-ATP6 encoding p.(Leu169Pro), with primary presentation of microcephaly, ataxia, hearing loss, and lactic acidosis. Functional studies reveal abnormal fragment F1 of complex V on blue native gel electrophoresis. Respirometry showed excessively tight coupling through complex V depressing oxygen consumption upon ADP stimulation and an excessive increase following uncoupling, in the presence of upregulation of mitochondrial biogenesis. These data add evidence about pathogenicity and functional impact of this variant.