Concurrent AFG3L2 and SPG7 mutations associated with syndromic parkinsonism and optic atrophy with aberrant OPA1 processing and mitochondrial network fragmentation

Mitochondrial dynamics and quality control are crucial for neuronal survival and their perturbation is a major cause of neurodegeneration. m-AAA complex is an ATP-dependent metalloprotease located in the inner mitochondrial membrane and involved in protein quality control. Mutations in the m-AAA subunits AFG3L2 and paraplegin are associated with autosomal dominant spinocerebellar ataxia (SCA28) and autosomal recessive hereditary spastic paraplegia (SPG7), respectively. We report a novel m-AAA-associated phenotype characterized by early-onset optic atrophy with spastic ataxia and L-Dopa-responsive parkinsonism. The proband carried a de-novo AFG3L2 heterozygous mutation (p.R468C) along with a heterozygous maternally-inherited intragenic deletion of SPG7. Functional analysis in yeast demonstrated the pathogenic role of AFG3L2 p.R468C mutation shedding light on its pathogenic mechanism. Analysis of patient's fibroblasts showed an abnormal processing pattern of OPA1, a dynamin-related protein essential for mitochondrial fusion and responsible for most cases of hereditary optic atrophy. Consistently, assessment of mitochondrial morphology revealed a severe fragmentation of the mitochondrial network, not observed in SCA28 and SPG7 patients\u2019 cells. This case suggests that coincidental mutations in both components of the mitochondrial m-AAA protease may result in a complex phenotype and reveals a crucial role for OPA1 processing in the pathogenesis of neurodegenerative disease caused by m-AAA defects

Circulating MyomiRs as Potential Biomarkers to Monitor Response to Nusinersen in Pediatric SMA Patients

Spinal muscular atrophy (SMA) is an autosomal recessive disorder caused by mutations in survival motor neuron (SMN) 1 gene, resulting in a truncated SMN protein responsible for degeneration of brain stem and spinal motor neurons. The paralogous SMN2 gene partially compensates full-length SMN protein production, mitigating the phenotype. Antisense oligonucleotide nusinersen (Spinraza®) enhances SMN2 gene expression. SMN is involved in RNA metabolism and biogenesis of microRNA (miRNA), key gene expression modulators, whose dysregulation contributes to neuromuscular diseases. They are stable in body fluids and may reflect distinct pathophysiological states, thus acting as promising biomarkers. Muscle-specific miRNAs (myomiRs) as biomarkers for clinical use in SMA have not been investigated yet. Here, we analyzed the expression of miR-133a, -133b, -206 and -1, in serum of 21 infantile SMA patients at baseline and after 6 months of nusinersen treatment, and correlated molecular data with response to therapy evaluated by the Hammersmith Functional Motor Scale Expanded (HFMSE). Our results demonstrate that myomiR serological levels decrease over disease course upon nusinersen treatment. Notably, miR-133a reduction predicted patients’ response to therapy. Our findings identify myomiRs as potential biomarkers to monitor disease progression and therapeutic response in SMA patients

Glutamine Synthetase 1 Increases Autophagy Lysosomal Degradation of Mutant Huntingtin Aggregates in Neurons, Ameliorating Motility in a Drosophila Model for Huntington's Disease

Glutamine Synthetase 1 (GS1) is a key enzyme that catalyzes the ATP-dependent synthesis of l-glutamine from l-glutamate and is also member of the Glutamate Glutamine Cycle, a complex physiological process between glia and neurons that controls glutamate homeostasis and is often found compromised in neurodegenerative diseases including Huntington's disease (HD). Here we report that the expression of GS1 in neurons ameliorates the motility defects induced by the expression of the mutant Htt, using a Drosophila model for HD. This phenotype is associated with the ability of GS1 to favor the autophagy that we associate with the presence of reduced Htt toxic protein aggregates in neurons expressing mutant Htt. Expression of GS1 prevents the TOR activation and phosphorylation of S6K, a mechanism that we associate with the reduced levels of essential amino acids, particularly of arginine and asparagine important for TOR activation. This study reveals a novel function for GS1 to ameliorate neuronal survival by changing amino acids' levels that induce a "starvation-like" condition responsible to induce autophagy. The identification of novel targets that inhibit TOR in neurons is of particular interest for the beneficial role that autophagy has in preserving physiological neuronal health and in the mechanisms that eliminate the formation of toxic aggregates in proteinopathies

Functional Characterization of Two Variants at the Intron 6-Exon 7 Boundary of the KCNQ2 Potassium Channel Gene Causing Distinct Epileptic Phenotypes

Pathogenic variants in KCNQ2 encoding for Kv7.2 potassium channel subunits have been found in patients affected by widely diverging epileptic phenotypes, ranging from Self-Limiting Familial Neonatal Epilepsy (SLFNE) to severe Developmental and Epileptic Encephalopathy (DEE). Thus, understanding the pathogenic molecular mechanisms of KCNQ2 variants and their correlation with clinical phenotypes has a relevant impact on the clinical management of these patients. In the present study, the genetic, biochemical, and functional effects prompted by two variants, each found in a non-familial SLNE or a DEE patient but both affecting nucleotides at the KCNQ2 intron 6-exon 7 boundary, have been investigated to test whether and how they affected the splicing process and to clarify whether such mechanism might play a pathogenetic role in these patients. Analysis of KCNQ2 mRNA splicing in patient-derived lymphoblasts revealed that the SLNE-causing intronic variant (c.928-1G > C) impeded the use of the natural splice site, but lead to a 10-aa Kv7.2 in frame deletion (Kv7.2 p.G310Δ10); by contrast, the DEE-causing exonic variant (c.928G > A) only had subtle effects on the splicing process at this site, thus leading to the synthesis of a full-length subunit carrying the G310S missense variant (Kv7.2 p.G310S). Patch-clamp recordings in transiently-transfected CHO cells and primary neurons revealed that both variants fully impeded Kv7.2 channel function, and exerted strong dominant-negative effects when co-expressed with Kv7.2 and/or Kv7.3 subunits. Notably, Kv7.2 p.G310S, but not Kv7.2 p.G310Δ10, currents were recovered upon overexpression of the PIP2-synthesizing enzyme PIP5K, and/or CaM; moreover, currents from heteromeric Kv7.2/Kv7.3 channels incorporating either Kv7.2 mutant subunits were differentially regulated by changes in PIP2 availability, with Kv7.2/Kv7.2 G310S/Kv7.3 currents showing a greater sensitivity to PIP2 depletion when compared to those from Kv7.2/Kv7.2 G310Δ10/Kv7.3 channels. Altogether, these results suggest that the two variants investigated differentially affected the splicing process at the intron 6-exon 7 boundary, and led to the synthesis of Kv7.2 subunits showing a differential sensitivity to PIP2 and CaM regulation; more studies are needed to clarify how such different functional properties contribute to the widely-divergent clinical phenotypes

Progressive myoclonus epilepsies due to SEMA6B mutations. New variants and appraisal of published phenotypes

Variants of SEMA6B have been identified in an increasing number of patients, often presenting with progressive myoclonus epilepsy (PME), and to lesser extent developmental encephalopathy, with or without epilepsy. The exon 17 is mainly involved, with truncating mutations causing the production of aberrant proteins with toxic gain of function. Herein, we describe three adjunctive patients carrying de novo truncating SEMA6B variants in this exon (c.1976delC and c.2086C > T novel; c.1978delC previously reported). These subjects presented with PME preceded by developmental delay, motor and cognitive impairment, worsening myoclonus, and epilepsy with polymorphic features, including focal to bilateral seizures in two, and non-convulsive status epilepticus in one. The evidence of developmental delay in these cases suggests their inclusion in the “PME plus developmental delay” nosological group. This work further expands our knowledge of SEMA6B variants causing PMEs. However, the data to date available confirms that phenotypic features do not correlate with the type or location of variants, aspects that need to be further clarified by future studie

Cortical markers of cognitive syndromes in amyotrophic lateral sclerosis

Amyotrophic lateral sclerosis (ALS) can be associated with a spectrum of cognitive and behavioural symptoms, but the related patterns of focal cortical atrophy in non-demented ALS patients remain largely unknown. We enrolled 48 non-demented ALS patients and 26 healthy controls for a comprehensive neuropsychological assessment and a magnetic resonance exam. Behavioural and cognitive impairment was defined on the basis of a data-driven multi-domain approach in 21 ALS patients. Averaged cortical thickness of 74 bilateral brain regions was used as a measure of cortical atrophy. Cortical thinning in a fronto-parietal network, suggesting a disease-specific pattern of neurodegeneration, was present in all patients, independent of cognitive and behavioural status. Between-group and correlational analyses revealed that inferior frontal, temporal, cingular and insular thinning are markers for cognitive and behavioural deficits, with language impairment mainly related to left temporal pole and insular involvement. These specific correlates support the concept of a spectrum of deficits, with an overlap between the ALS cognitive phenotypes and the syndromes of frontotemporal dementia

A novel de novo HCN1 loss-of-function mutation in genetic generalized epilepsy causing increased neuronal excitability

Abstract The causes of genetic epilepsies are unknown in the majority of patients. HCN ion channels have a widespread expression in neurons and increasing evidence demonstrates their functional involvement in human epilepsies. Among the four known isoforms, HCN1 is the most expressed in the neocortex and hippocampus and de novo HCN1 point mutations have been recently associated with early infantile epileptic encephalopathy. So far, HCN1 mutations have not been reported in patients with idiopathic epilepsy. Using a Next Generation Sequencing approach, we identified the de novo heterozygous p.Leu157Val (c.469C > G) novel mutation in HCN1 in an adult male patient affected by genetic generalized epilepsy (GGE), with normal cognitive development. Electrophysiological analysis in heterologous expression model (CHO cells) and in neurons revealed that L157V is a loss-of-function, dominant negative mutation causing reduced HCN1 contribution to net inward current and responsible for an increased neuronal firing rate and excitability, potentially predisposing to epilepsy. These data represent the first evidence that autosomal dominant missense mutations of HCN1 can also be involved in GGE, without the characteristics of epileptic encephalopathy reported previously. It will be important to include HCN1 screening in patients with GGE, in order to extend the knowledge of the genetic causes of idiopathic epilepsies, thus paving the way for the identification of innovative therapeutic strategies

Gabapentin treatment in a patient with KCNQ2 developmental epileptic encephalopathy

De novo variants in KCNQ2 encoding for Kv7.2 voltage-dependent neuronal potassium (K+) channel subunits are associated with developmental epileptic encephalopathy (DEE). We herein describe a the clinical and electroencephalographic (EEG) features of a child with early-onset DEE caused by the novel KCNQ2 p.G310S variant. In vitro experiments demonstrated that the mutation induces loss-of-function effects on the currents produced by channels incorporating mutant subunits; these effects were counteracted by the selective Kv7 opener retigabine and by gabapentin, a recently described Kv7 activator. Given these data, the patient started treatment with gabapentin, showing a rapid and sustained clinical and EEG improvement over the following months. Overall, these results suggest that gabapentin can be regarded as a precision therapy for DEEs due to KCNQ2 loss-of-function mutations

Cognitive Syndromes and C9orf72 Mutation Are Not Related to Cerebellar Degeneration in Amyotrophic Lateral Sclerosis

Objective: The notion that cerebellar pathology may contribute to cognitive impairment in ALS, especially in patients with C9orf72 repeated expansion, has been inconsistently reported. This study aimed exploring the relationship between cerebellar involvement, cognitive impairment and C9orf72 repeated expansion of patients with ALS. Methods: Quantitative in vivo assessment of cerebellar lobules has been investigated in 66 non-demented patients with ALS and 28 healthy controls (HCs). Pathologic C9orf72 repeated expansion was found in 13 patients. Mild cognitive and/or behavioral impairment was diagnosed in 22 C9orf72 negative ALS patients. Measures of cortical volume (CV} and cortical thickness (CT) of cerebellar lobules of all participants were used for Principal Component Analysis (PCA) to identify clusters of lobular measures highly correlated with each other. PCA outcomes were used for between group comparisons and correlation analyses with neuropsychological and clinical features. Results: Disease severity measured with ALS functional rating scale and index of disease progression rate significantly correlated with CV reduction of the second PCA cluster loading CV measures of anterior lobules. In all patients, cognitive impairment, measured with verbal fluency, was related to CV reduction of the third cluster comprising posterior lobules. No specific cortical thinning or volume reduction of cerebellar clustering patterns could be detected in ALS subgroups. Conclusion: Our data show that specific patterns of subregional cerebellar involvement are associated with physical disability or cognitive impairment in ALS, in line with the topographic organization of the cerebellum. However, there was no specific correlation between cerebellar degeneration and cognitive syndromes or C9orf72 mutations

Evoluzione del gene Hdh nei primati

Il gene Hdh si origina senza la tripletta CAG in Dictyostelium discoideum (Dd), circa 800 milioni di anni fa, prima della divergenza dei protostomi-deuterostomi (Zuccato et al., Physiol Rev 2010). La tripletta CAG compare in seguito ed ? esclusiva della branca dei deuterostomi (Tartari et al., Mol Biol Evol 2008). Due ripetizioni CAGs si trovano nel gene Hdh del riccio di mare (Strongylocentrotus purpuratus, Sp), la prima specie ad avere un sistema nervoso primitivo, e due ripetizioni si trovano anche nell\u27anfiosso (Branchiostoma floridae, Bf), che presenta un rudimentale tubo nervoso e un primo tentativo di cefalizzazione. Quattro ripetizioni CAG si riscontrano nei pesci pi? evoluti, negli anfibi e negli uccelli. Il numero di triplette CAG aumenta ulteriormente nei mammiferi e raggiunge la lunghezza massima nella nostra specie (Tartari et al., Mol Biol Evol 2008). In Homo sapiens il gene Hdh si trova sul braccio corto del cromosoma 4 e presenta la sequenza trinucleotidica ripetuta da 11 a 35 volte. Un recente studio su 278 individui normali ha rivelato che chi ha pi? CAG nel range sano ha pi? materia grigia (Muhlau et al., PlosOne 2012), indicazione del fatto che il numero di CAG potrebbe influire sulla normale struttura del cervello. Infatti, un numero di triplette CAG superiore a 35 causa la Corea di Huntington, una malattia neurodegenerativa che insorge tanto pi? precocemente quanto maggiore ? il numero delle ripetizioni CAG. Al fine di verificare se il progressivo incremento del numero di ripetizioni CAG nel gene Hdh durante l\u27evoluzione avesse un possibile ruolo nelle funzioni cognitive emergenti del cervello dei mammiferi, abbiamo analizzato il gene Hdh in diverse specie di primati non umani per analizzare la variabilit? interspecifica e intraspecifica. I risultati sperimentali e le ricostruzioni filogenetiche sostengono che il numero di ripetizioni CAG aumenta nel corso dell\u27evoluzione dei deuterostomi e sembra essere correlato con l\u27aspetto e/o l\u27evoluzione dei sistemi nervosi progressivamente pi? complessi