Perinnöllisen aksonirappeumasairauden molekyylitason mallintaminen kantasoluperäisten spinaalimotoneuronien avulla

Charcot-Marie-Tooth (CMT) on yhteinen nimitys perinnöllisille ääreishermosoluja vaurioittaville neuropatioille. Kyseessä on yksi yleisimmistä neurologisista perinnöllisistä sairauksista esiintyvyyden ollessa noin 1:2500. Monimutkaisten tautimekanismien ja riittämättömien hoitomuotojen vuoksi kyseinen tauti on mielenkiintoinen tutkimuksen kohde. Tutkimusryhmämme on selvittänyt CMT-taudin geenitaustaa Suomessa. Sairauden kliiniseen kuvaan kuuluu vaihtelevin vaikeusastein motosensoriset häiriöt, jotka lievimmillään rajoittuvat distaalisten lihasten heikkouteen. Kuitenkin vakavimmissa tapauksissa sairaus voi viedä potilaan liikuntakyvyn. Tällä hetkellä mitään spesifisiä hoitoja ei ole tarjolla, mutta alati lisääntyvän molekyylitason tiedon myötä muutamia lääkekandidaatteja on otettu kliinisiin kokeisiin. Hermosolujen välikokoista filamenttia koodaava geeni NEFL (neurofilament light) on yksi CMT-tautigeeneistä (CMT1F/2E muoto). Tässä tutkimuksessa pyrin luomaan tautimallin NEFL-proteiinin puutokselle. Muokkasin indusoituja pluripotentteja kantasoluja CRISPR-Cas9 - teknologialla NEFL-geenin poistamiseksi. Sen jälkeen erilaistin muokatut kantasolut alemmiksi motoneuroneiksi. Mallin avulla pyrin selvittämään, miten muiden välikokoisten filamenttien ja sairauteen liitettyjen geenien ilmentyminen muuttuu NEFL-poistogeenisissä neuroneissa. Sain erilaistettua kolme muokkaamaamme solulinjaa alemmiksi motoneuroneiksi. Täydellinen NEFL-proteiinin poisto oli tapahtunut kahdessa näistä linjoista. Käytin tutkimuksen analyyttisessä osassa avuksi RNA- ja proteiinitason tutkimusmenetelmiä (qPCR, Western Blot, immunosytokemia). Tuloksista selvisi, että periferiinin määrä oli noussut western blotissa, kun NEFL puuttuu soluissa. Lisäksi SLITRK2 ja LYNX1 mRNA määrä oli qPCR-analyysin mukaan vähentynyt ja GRM3 mRNA oli lisääntynyt muokatuissa soluissa. Näiden tulosten perusteella CMT2E-taudin patofysiologiaan liittynee muutoksia monissa erilaisissa solunsisäisissä kompensatorisissa mekanismeissa. (202 sanaa

Serum Creatine, Not Neurofilament Light, Is Elevated in CHCHD10-Linked Spinal Muscular Atrophy

ObjectiveTo characterize serum biomarkers in mitochondrial CHCHD10-linked spinal muscular atrophy Jokela (SMAJ) type for disease monitoring and for the understanding of pathogenic mechanisms. MethodsWe collected serum samples from a cohort of 49 patients with SMAJ, all carriers of the heterozygous c.197G>T p.G66V variant in CHCHD10. As controls, we used age- and sex-matched serum samples obtained from Helsinki Biobank. Creatine kinase and creatinine were measured by standard methods. Neurofilament light (NfL) and glial fibrillary acidic protein (GFAP) were measured with single molecule array (Simoa), fibroblast growth factor 21 (FGF-21), and growth differentiation factor 15 (GDF-15) with an enzyme-linked immunosorbent assay. For non-targeted plasma metabolite profiling, samples were analyzed with liquid chromatography high-resolution mass spectrometry. Disease severity was evaluated retrospectively by calculating a symptom-based score. ResultsAxon degeneration marker, NfL, was unexpectedly not altered in the serum of patients with SMAJ, whereas astrocytic activation marker, GFAP, was slightly decreased. Creatine kinase was elevated in most patients, particularly men. We identified six metabolites that were significantly altered in serum of patients with SMAJ in comparison to controls: increased creatine and pyruvate, and decreased creatinine, taurine, N-acetyl-carnosine, and succinate. Creatine correlated with disease severity. Altered pyruvate and succinate indicated a metabolic response to mitochondrial dysfunction; however, lactate or mitochondrial myopathy markers FGF-21 or GDF-15 was not changed. ConclusionsBiomarkers of muscle mass and damage are altered in SMAJ serum, indicating a role for skeletal muscle in disease pathogenesis in addition to neurogenic damage. Despite the minimal mitochondrial pathology in skeletal muscle, signs of a metabolic shift can be detected.Peer reviewe

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

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

Serum Creatine, Not Neurofilament Light, Is Elevated in CHCHD10-Linked Spinal Muscular Atrophy

Objective: To characterize serum biomarkers in mitochondrial CHCHD10-linked spinal muscular atrophy Jokela (SMAJ) type for disease monitoring and for the understanding of pathogenic mechanisms.Methods: We collected serum samples from a cohort of 49 patients with SMAJ, all carriers of the heterozygous c.197G>T p.G66V variant in CHCHD10. As controls, we used age- and sex-matched serum samples obtained from Helsinki Biobank. Creatine kinase and creatinine were measured by standard methods. Neurofilament light (NfL) and glial fibrillary acidic protein (GFAP) were measured with single molecule array (Simoa), fibroblast growth factor 21 (FGF-21), and growth differentiation factor 15 (GDF-15) with an enzyme-linked immunosorbent assay. For non-targeted plasma metabolite profiling, samples were analyzed with liquid chromatography high-resolution mass spectrometry. Disease severity was evaluated retrospectively by calculating a symptom-based score.Results: Axon degeneration marker, NfL, was unexpectedly not altered in the serum of patients with SMAJ, whereas astrocytic activation marker, GFAP, was slightly decreased. Creatine kinase was elevated in most patients, particularly men. We identified six metabolites that were significantly altered in serum of patients with SMAJ in comparison to controls: increased creatine and pyruvate, and decreased creatinine, taurine, N-acetyl-carnosine, and succinate. Creatine correlated with disease severity. Altered pyruvate and succinate indicated a metabolic response to mitochondrial dysfunction; however, lactate or mitochondrial myopathy markers FGF-21 or GDF-15 was not changed.Conclusions: Biomarkers of muscle mass and damage are altered in SMAJ serum, indicating a role for skeletal muscle in disease pathogenesis in addition to neurogenic damage. Despite the minimal mitochondrial pathology in skeletal muscle, signs of a metabolic shift can be detected.</p