28 research outputs found

    Morphologic and functional correlates of synaptic pathology in the cathepsin D knockout mouse model of congenital neuronal ceroid lipofuscinosis

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    Mutations in the cathepsin D (CTSD) gene cause an aggressive neurodegenerative disease (congenital neuronal ceroid lipofuscinosis) that leads to early death. Recent evidence suggests that presynaptic abnormalities play a major role in the pathogenesis of CTSD deficiencies. To identify the early events that lead to synaptic alterations, we investigated synaptic ultrastructure and function in pre-symptomatic CTSD knock-out (Ctsd(−/−)) mice. Electron microscopy revealed that there were significantly greater numbers of readily releasable synaptic vesicles present in Ctsd(−/−) mice than in wild-type control mice as early as postnatal day 16. The size of this synaptic vesicle pool continued to increase with disease progression in the hippocampus and thalamus of the Ctsd(−/−) mice. Electrophysiology revealed a markedly decreased frequency of miniature excitatory postsynaptic currents (EPSCs) with no effect on pair-pulse modulation of the evoked EPSPs in the hippocampus of Ctsd(−/−) mice. The reduced miniature EPSC frequency was observed before the appearance of epilepsy or any morphological sign of synaptic degeneration. Taken together, the data indicate that CTSD is required for normal synaptic function, and that a failure in synaptic trafficking or recycling may be an early and important pathological mechanism in Ctsd(−/−) mice; these presynaptic abnormalities may initiate synaptic degeneration in advance of subsequent neuronal loss

    ALS and Parkinson's disease genes CHCHD10 and CHCHD2 modify synaptic transcriptomes in human iPSC-derived motor neurons

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    Mitochondrial intermembrane space proteins CHCHD2 and CHCHD10 have roles in motor neuron diseases such as amyotrophic lateral sclerosis, spinal muscular atrophy and axonal neuropathy and in Parkinson's disease. They form a complex of unknown function. Here we address the importance of these two proteins in human motor neurons. We show that gene edited human induced pluripotent stem cells (iPSC) lacking either CHCHD2 or CHCHD10 are viable and can be differentiated into functional motor neurons that fire spontaneous and evoked action potentials. Mitochondria in knockout iPSC and motor neurons sustain ultrastructure but show increased proton leakage and respiration, and reciprocal compensatory increases in CHCHD2 or CHCHD10. Knockout motor neurons have largely overlapping transcriptome profiles compared to isogenic control line, in particular for synaptic gene expression. Our results show that the absence of either CHCHD2 or CHCHD10 alters mitochondrial respiration in human motor neurons, inducing similar compensatory responses. Thus, pathogenic mechanisms may involve loss of synaptic function resulting from defective energy metabolism.Peer reviewe

    Neurofilament Light Regulates Axon Caliber, Synaptic Activity, and Organelle Trafficking in Cultured Human Motor Neurons

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    Neurofilament light (NFL) is one of the proteins forming multimeric neuron-specific intermediate filaments, neurofilaments, which fill the axonal cytoplasm, establish caliber growth, and provide structural support. Dominant missense mutations and recessive nonsense mutations in the neurofilament light gene (NEFL) are among the causes of Charcot-Marie-Tooth (CMT) neuropathy, which affects the peripheral nerves with the longest axons. We previously demonstrated that a neuropathy-causing homozygous nonsense mutation in NEFL led to the absence of NFL in patient-specific neurons. To understand the disease-causing mechanisms, we investigate here the functional effects of NFL loss in human motor neurons differentiated from induced pluripotent stem cells (iPSC). We used genome editing to generate NEFL knockouts and compared them to patient-specific nonsense mutants and isogenic controls. iPSC lacking NFL differentiated efficiently into motor neurons with normal axon growth and regrowth after mechanical axotomy and contained neurofilaments. Electrophysiological analysis revealed that motor neurons without NFL fired spontaneous and evoked action potentials with similar characteristics as controls. However, we found that, in the absence of NFL, human motor neurons 1) had reduced axonal caliber, 2) the amplitude of miniature excitatory postsynaptic currents (mEPSC) was decreased, 3) neurofilament heavy (NFH) levels were reduced and no compensatory increases in other filament subunits were observed, and 4) the movement of mitochondria and to a lesser extent lysosomes was increased. Our findings elaborate the functional roles of NFL in human motor neurons. NFL is not only a structural protein forming neurofilaments and filling the axonal cytoplasm, but our study supports the role of NFL in the regulation of synaptic transmission and organelle trafficking. To rescue the NFL deficiency in the patient-specific nonsense mutant motor neurons, we used three drugs, amlexanox, ataluren (PTC-124), and gentamicin to induce translational read-through or inhibit nonsense-mediated decay. However, the drugs failed to increase the amount of NFL protein to detectable levels and were toxic to iPSC-derived motor neurons.Peer reviewe

    Dominant mutations in ITPR3 cause Charcot-Marie-Tooth disease

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    Objective ITPR3, encoding inositol 1,4,5-trisphosphate receptor type 3, was previously reported as a potential candidate disease gene for Charcot-Marie-Tooth neuropathy. Here, we present genetic and functional evidence thatITPR3is a Charcot-Marie-Tooth disease gene. Methods Whole-exome sequencing of four affected individuals in an autosomal dominant family and one individual who was the only affected individual in his family was used to identify disease-causing variants. Skin fibroblasts from two individuals of the autosomal dominant family were analyzed functionally by western blotting, quantitative reverse transcription PCR, and Ca(2+)imaging. Results Affected individuals in the autosomal dominant family had onset of symmetrical neuropathy with demyelinating and secondary axonal features at around age 30, showing signs of gradual progression with severe distal leg weakness and hand involvement in the proband at age 64. Exome sequencing identified a heterozygousITPR3p.Val615Met variant segregating with the disease. The individual who was the only affected in his family had disease onset at age 4 with demyelinating neuropathy. His condition was progressive, leading to severe muscle atrophy below knees and atrophy of proximal leg and hand muscles by age 16. Trio exome sequencing identified ade novo ITPR3variant p.Arg2524Cys. Altered Ca2+-transients in p.Val615Met patient fibroblasts suggested that the variant has a dominant-negative effect on inositol 1,4,5-trisphosphate receptor type 3 function. Interpretation Together with two previously identified variants, our report adds further evidence thatITPR3is a disease-causing gene for CMT and indicates altered Ca(2+)homeostasis in disease pathogenesis.Peer reviewe

    Alpha2-Containing Glycine Receptors Promote Neonatal Spontaneous Activity of Striatal Medium Spiny Neurons and Support Maturation of Glutamatergic Inputs

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    Glycine receptors (GlyRs) containing the α2 subunit are highly expressed in the developing brain, where they regulate neuronal migration and maturation, promote spontaneous network activity and subsequent development of synaptic connections. Mutations in GLRA2 are associated with autism spectrum disorder, but the underlying pathophysiology is not described yet. Here, using Glra2-knockout mice, we found a GlyR-dependent effect on neonatal spontaneous activity of dorsal striatum medium spiny neurons (MSNs) and maturation of the incoming glutamatergic innervation. Our data demonstrate that functional GlyRs are highly expressed in MSNs of one-week-old mice, but they do not generate endogenous chloride-mediated tonic or phasic current. Despite of that, knocking out the Glra2 severely affects the shape of action potentials and impairs spontaneous activity and the frequency of miniature AMPA receptor-mediated currents in MSNs. This reduction in spontaneous activity and glutamatergic signaling can attribute to the observed changes in neonatal behavioral phenotypes as seen in ultrasonic vocalizations and righting reflex. In adult Glra2-knockout animals, the glutamatergic synapses in MSNs remain functionally underdeveloped. The number of glutamatergic synapses and release probability at presynaptic site remain unaffected, but the amount of postsynaptic AMPA receptors is decreased. This deficit is a consequence of impaired development of the neuronal circuitry since acute inhibition of GlyRs by strychnine in adult MSNs does not affect the properties of glutamatergic synapses. Altogether, these results demonstrate that GlyR-mediated signaling supports neonatal spontaneous MSN activity and, in consequence, promotes the functional maturation of glutamatergic synapses on MSNs. The described mechanism might shed light on the pathophysiological mechanisms in GLRA2-linked autism spectrum disorder cases

    Tonically active α2 subunit-containing glycine receptors regulate the excitability of striatal medium spiny neurons

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    Medium spiny neurons (MSNs) of the dorsal striatum represent the first relay of cortico- striato-thalamic loop, responsible for the initiation of voluntary movements and motor learning. GABAergic transmission exerts the main inhibitory control of MSNs. However, MSNs also express chloride-permeable glycine receptors (GlyRs) although their subunit composition and functional significance in the striatum is unknown. Here, we studied the function of GlyRs in MSNs of young adult mice. We show that MSNs express functional GlyRs, with α2 being the main agonist binding subunit. These receptors are extrasynaptic and depolarizing at resting state. The pharmacological inhibition of GlyRs, as well as inactivation of the GlyR α2 subunit gene hyperpolarize the membrane potential of MSNs and increase their action potential firing offset. Mice lacking GlyR α2 showed impaired motor memory consolidation without any changes in the initial motor performance. Taken together, these results demonstrate that tonically active GlyRs regulate the firing properties of MSNs and may thus affect the function of basal ganglia.SCOPUS: ar.jinfo:eu-repo/semantics/publishe

    Alpha2-Containing Glycine Receptors Promote Neonatal Spontaneous Activity of Striatal Medium Spiny Neurons and Support Maturation of Glutamatergic Inputs

    No full text
    Glycine receptors (GlyRs) containing the α2 subunit are highly expressed in the developing brain, where they regulate neuronal migration and maturation, promote spontaneous network activity and subsequent development of synaptic connections. Mutations in GLRA2 are associated with autism spectrum disorder, but the underlying pathophysiology is not described yet. Here, using Glra2-knockout mice, we found a GlyR-dependent effect on neonatal spontaneous activity of dorsal striatum medium spiny neurons (MSNs) and maturation of the incoming glutamatergic innervation. Our data demonstrate that functional GlyRs are highly expressed in MSNs of one-week-old mice, but they do not generate endogenous chloride-mediated tonic or phasic current. Despite of that, knocking out the Glra2 severely affects the shape of action potentials and impairs spontaneous activity and the frequency of miniature AMPA receptor-mediated currents in MSNs. This reduction in spontaneous activity and glutamatergic signaling can attribute to the observed changes in neonatal behavioral phenotypes as seen in ultrasonic vocalizations and righting reflex. In adult Glra2-knockout animals, the glutamatergic synapses in MSNs remain functionally underdeveloped. The number of glutamatergic synapses and release probability at presynaptic site remain unaffected, but the amount of postsynaptic AMPA receptors is decreased. This deficit is a consequence of impaired development of the neuronal circuitry since acute inhibition of GlyRs by strychnine in adult MSNs does not affect the properties of glutamatergic synapses. Altogether, these results demonstrate that GlyR-mediated signaling supports neonatal spontaneous MSN activity and, in consequence, promotes the functional maturation of glutamatergic synapses on MSNs. The described mechanism might shed light on the pathophysiological mechanisms in GLRA2-linked autism spectrum disorder cases.status: publishe

    Dominant mutations in ITPR3 cause Charcot-Marie-Tooth disease

    No full text
    OBJECTIVE: ITPR3, encoding inositol 1,4,5-trisphosphate receptor type 3, was previously reported as a potential candidate disease gene for Charcot-Marie-Tooth neuropathy. Here, we present genetic and functional evidence that ITPR3 is a Charcot-Marie-Tooth disease gene. METHODS: Whole-exome sequencing of four affected individuals in an autosomal dominant family and one individual who was the only affected individual in his family was used to identify disease-causing variants. Skin fibroblasts from two individuals of the autosomal dominant family were analyzed functionally by western blotting, quantitative reverse transcription PCR, and Ca2+ imaging. RESULTS: Affected individuals in the autosomal dominant family had onset of symmetrical neuropathy with demyelinating and secondary axonal features at around age 30, showing signs of gradual progression with severe distal leg weakness and hand involvement in the proband at age 64. Exome sequencing identified a heterozygous ITPR3 p.Val615Met variant segregating with the disease. The individual who was the only affected in his family had disease onset at age 4 with demyelinating neuropathy. His condition was progressive, leading to severe muscle atrophy below knees and atrophy of proximal leg and hand muscles by age 16. Trio exome sequencing identified a de novo ITPR3 variant p.Arg2524Cys. Altered Ca2+ -transients in p.Val615Met patient fibroblasts suggested that the variant has a dominant-negative effect on inositol 1,4,5-trisphosphate receptor type 3 function. INTERPRETATION: Together with two previously identified variants, our report adds further evidence that ITPR3 is a disease-causing gene for CMT and indicates altered Ca2+ homeostasis in disease pathogenesis.status: publishe
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