DNA methylome, R-loop and clinical exome profiling of patients with sporadic amyotrophic lateral sclerosis.

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disorder characterized by the death of motor neurons, the aetiology of which is essentially unknown. Here, we present an integrative epigenomic study in blood samples from seven clinically characterised sporadic ALS patients to elucidate molecular factors associated with the disease. We used clinical exome sequencing (CES) to study DNA variants, DNA-RNA hybrid immunoprecipitation sequencing (DRIP-seq) to assess R-loop distribution, and reduced representation bisulfite sequencing (RRBS) to examine DNA methylation changes. The above datasets were combined to create a comprehensive repository of genetic and epigenetic changes associated with the ALS cases studied. This repository is well-suited to unveil new correlations within individual patients and across the entire patient cohort. The molecular attributes described here are expected to guide further mechanistic studies on ALS, shedding light on the underlying genetic causes and facilitating the development of new epigenetic therapies to combat this life-threatening disease.

Beyond C9orf72: repeat expansions and copy number variations as risk factors of amyotrophic lateral sclerosis across various populations.

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disorder which is characterized by the loss of both upper and lower motor neurons in the central nervous system. In a significant fraction of ALS cases - irrespective of family history- a genetic background may be identified. The genetic background of ALS shows a high variability from one ethnicity to another. The most frequent genetic cause of ALS is the repeat expansion of the C9orf72 gene. With the emergence of next-generation sequencing techniques and copy number alteration calling tools the focus in ALS genetics has shifted from disease causing genes and mutations towards genetic susceptibility and risk factors.In this review we aimed to summarize the most widely recognized and studied ALS linked repeat expansions and copy number variations other than the hexanucleotide repeat expansion in the C9orf72 gene. We compare and contrast their involvement and phenotype modifying roles in ALS among different populations.

Passive Transfer of Blood Sera from ALS Patients with Identified Mutations Results in Elevated Motoneuronal Calcium Level and Loss of Motor Neurons in the Spinal Cord of Mice.

Introduction: Previously, we demonstrated the degeneration of axon terminals in mice after repeated injections of blood sera from amyotrophic lateral sclerosis (ALS) patients with identified mutations. However, whether a similar treatment affects the cell body of motor neurons (MNs) remained unresolved. Methods: Sera from healthy individuals or ALS patients with a mutation in different ALS-related genes were intraperitoneally injected into ten-week-old male Balb/c mice (n = 3/serum) for two days. Afterward, the perikaryal calcium level was measured using electron microscopy. Furthermore, the optical disector method was used to evaluate the number of lumbar MNs. Results: The cytoplasmic calcium level of the lumbar MNs of the ALS-serum-treated mice, compared to untreated and healthy-serum-treated controls, was significantly elevated. While injections of the healthy serum did not reduce the number of MNs compared to the untreated control group, ALS sera induced a remarkable loss of MNs. Discussion: Similarly to the distant motor axon terminals, the injection of blood sera of ALS patients has a rapid degenerative effect on MNs. Analogously, the magnitude of the evoked changes was specific to the type of mutation; furthermore, the degeneration was most pronounced in the group treated with sera from ALS patients with a mutation in the chromosome 9 open reading frame 72 gene.

Passive Transfer of Sera from ALS Patients with Identified Mutations Evokes an Increased Synaptic Vesicle Number and Elevation of Calcium Levels in Motor Axon Terminals, Similar to Sera from Sporadic Patients.

Previously, we demonstrated increased calcium levels and synaptic vesicle densities in the motor axon terminals (MATs) of sporadic amyotrophic lateral sclerosis (ALS) patients. Such alterations could be conferred to mice with an intraperitoneal injection of sera from these patients or with purified immunoglobulin G. Later, we confirmed the presence of similar alterations in the superoxide dismutase 1 G93A transgenic mouse strain model of familial ALS. These consistent observations suggested that calcium plays a central role in the pathomechanism of ALS. This may be further reinforced by completing a similar analytical study of the MATs of ALS patients with identified mutations. However, due to the low yield of muscle biopsy samples containing MATs, and the low incidence of ALS patients with the identified mutations, these examinations are not technically feasible. Alternatively, a passive transfer of sera from ALS patients with known mutations was used, and the MATs of the inoculated mice were tested for alterations in their calcium homeostasis and synaptic activity. Patients with 11 different ALS-related mutations participated in the study. Intraperitoneal injection of sera from these patients on two consecutive days resulted in elevated intracellular calcium levels and increased vesicle densities in the MATs of mice, which is comparable to the effect of the passive transfer from sporadic patients. Our results support the idea that the pathomechanism underlying the identical manifestation of the disease with or without identified mutations is based on a common final pathway, in which increasing calcium levels play a central role.

Comprehensive Genetic Analysis of a Hungarian Amyotrophic Lateral Sclerosis Cohort.

Amyotrophic lateral sclerosis (ALS) is a progressive neurodegenerative disease characterized by the degeneration of motor neurons. Genetic factors play a key role in ALS, and identifying variants that contribute to ALS susceptibility is an important step toward understanding the etiology of the disease. The frequency of protein altering variants in ALS patients has been extensively investigated in populations of different ethnic origin. To further delineate the genetic architecture of the Hungarian ALS patients, we aimed to detect potentially damaging variants in major and minor ALS genes and in genes related to other neurogenetic disorders. A combination of repeat-sizing of C9orf72 and next-generation sequencing (NGS) was used to comprehensively assess genetic variations in 107 Hungarian patients with ALS. Variants in major ALS genes were detected in 36.45% of patients. As a result of repeat sizing, pathogenic repeat expansions in the C9orf72 gene were detected in 10 patients (9.3%). According to the NGS results, the most frequently mutated genes were NEK1 (5.6%), NEFH, SQSTM1 (3.7%), KIF5A, SPG11 (2.8%), ALS2, CCNF, FUS, MATR3, TBK1, and UBQLN2 (1.9%). Furthermore, potentially pathogenic variants were found in GRN and SIGMAR1 genes in single patients. Additional 33 novel or rare known variants were detected in minor ALS genes, as well as 48 variants in genes previously linked to other neurogenetic disorders. The latter finding supports the hypothesis that common pathways in different neurodegenerative diseases may contribute to the development of ALS. While the disease-causing role of several variants identified in this study has previously been established, other variants may show reduced penetrance or may be rare benign variants. Our findings highlight the necessity for large-scale multicenter studies on ALS patients to gain a more accurate view of the genetic pattern of ALS.

Experimental Motor Neuron Disease Induced in Mice with Long-Term Repeated Intraperitoneal Injections of Serum from ALS Patients.

In an earlier study, signs of commencing degeneration of spinal motor neurons were induced in mice with short-term intraperitoneal injections of immunoglobulin G (IgG) taken from patients with amyotrophic lateral sclerosis (ALS). Since in that study, neither weakness nor loss of motor neurons was noted, to test whether the ALS IgG in this paradigm has the potential to evoke relentless degeneration of motor neurons, treatment with repeated injections over a longer period was carried out. Mice were systematically injected intraperitoneally with serum taken from ALS patients over a 75-day period. At selected time points, the isometric force of the limbs, number of spinal motor neurons and their intracellular calcium levels were determined. Furthermore, markers of glial activation and the motoneuronal uptake of human IgG were monitored. During this period, gliosis and progressive motoneuronal degeneration developed, which led to gradual loss of spinal motor neurons, more than 40% at day 21, along with decreasing muscle strength in the limbs. The inclusion-like accumulation of IgG appeared in the perikarya with the increase of intracellular calcium in the cell bodies and motor nerve terminals. Our results demonstrate that ALS serum can transfer motor neuron disease to mice.

Angiogenin mutations in Hungarian patients with amyotrophic lateral sclerosis: Clinical, genetic, computational, and functional analyses.

INTRODUCTION: Mutations in the angiogenin (ANG) gene are known to be associated with both familial and sporadic amyotrophic lateral sclerosis (ALS). The majority of disease-causing mutations of ANG result in loss of either ribonucleolytic activity, nuclear translocation activity or both. METHODS: We sequenced ANG gene from a total of 136 sporadic ALS patients and 112 healthy controls of Hungarian origin. To elucidate the role of the R33W mutation in the disease mechanism, computational, and functional analyses were performed. RESULTS: Mutation screening revealed a mutation located in the signal peptide (M-24I) and two mutations that affect the mature protein (R33W, V103I). The R33W mutation, which has not been previously detected in ALS patients, affects the key amino acid of the nuclear translocation signal of the ANG protein. Molecular dynamics simulations suggested that the R33W mutation results in partial loss of ribonucleolytic activity and reduced nuclear translocation activity. The ribonucleolytic assay and nuclear translocation assay of the R33W ANG protein confirmed the molecular dynamics results. CONCLUSIONS: In the Hungarian ALS population, the observed frequency of ANG mutations was 2.9%, which is higher than previously reported for sporadic cohorts. The evidence from computational and functional analyses support the deleterious effect of the novel R33W variant detected in this study.

The rs13388259 Intergenic Polymorphism in the Genomic Context of the BCYRN1 Gene Is Associated with Parkinson's Disease in the Hungarian Population.

Parkinson's disease (PD) is a common neurodegenerative disorder characterized by bradykinesia, resting tremor, and muscle rigidity. To date, approximately 50 genes have been implicated in PD pathogenesis, including both Mendelian genes with rare mutations and low-penetrance genes with common polymorphisms. Previous studies of low-penetrance genes focused on protein-coding genes, and less attention was given to long noncoding RNAs (lncRNAs). In this study, we aimed to investigate the susceptibility roles of lncRNA gene polymorphisms in the development of PD. Therefore, polymorphisms (n=15) of the PINK1-AS, UCHL1-AS, BCYRN1, SOX2-OT, ANRIL and HAR1A lncRNAs genes were genotyped in Hungarian PD patients (n=160) and age- and sex-matched controls (n=167). The rare allele of the rs13388259 intergenic polymorphism, located downstream of the BCYRN1 gene, was significantly more frequent among PD patients than control individuals (OR = 2.31; p=0.0015). In silico prediction suggested that this polymorphism is located in a noncoding region close to the binding site of the transcription factor HNF4A, which is a central regulatory hub gene that has been shown to be upregulated in the peripheral blood of PD patients. The rs13388259 polymorphism may interfere with the binding affinity of transcription factor HNF4A, potentially resulting in abnormal expression of target genes, such as BCYRN1.

Presymptomatically applied AMPA receptor antagonist prevents calcium increase in vulnerable type of motor axon terminals of mice modeling amyotrophic lateral sclerosis.

Increased intracellular calcium (Ca), which might be the consequence of an excess influx through Ca-permeable α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors, plays a crucial role in degeneration of motor neurons. Previously we demonstrated that the presymptomatic application of AMPA receptor antagonist, talampanel, could reduce Ca elevation in spinal motor neurons of mice carrying the G93A mutation of superoxide dismutase 1 (SOD1), modeling amyotrophic lateral sclerosis (ALS). It remained to be examined whether the remote, functionally semi-autonomous motor axon terminals could be rescued from the Ca overload, or if the terminals, where the degeneration possibly starts, already experience intractable changes at early time points. Thus using electron microscopic techniques, we measured the Ca level of motor axon terminals in the interosseus muscle of the SOD1 mutant animals, which are prototypes of vulnerable nerve endings in ALS. In line with the results obtained in the perikarya, talampanel treatment could reduce Ca increase evoked by the presence of mutant SOD1 in the axon terminals if the treatment was started presymptomatically but not at an early symptomatic stage. We also tested the Ca level in the cell bodies and axon terminals of the oculomotor neurons, which are resistant to the disease. Neither Ca increase, nor talampanel effect could be demonstrated at either time point. This is consistent with the observations that oculomotor neurons contain increased level of Ca buffer, which could reduce excess Ca load, and they also express glutamate receptor subunit type 2, which renders AMPA receptors impermeable to Ca.

High-throughput sequencing revealed a novel SETX mutation in a Hungarian patient with amyotrophic lateral sclerosis.

BACKGROUND: Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease characterized by the degeneration of the motor neurons. To date, 126 genes have been implicated in ALS. Therefore, the heterogenous genetic background of ALS requires comprehensive genetic investigative approaches. METHODS: In this study, DNA from 28 Hungarian ALS patients was subjected to targeted high-throughput sequencing of the coding regions of three Mendelian ALS genes: FUS, SETX, and C9ORF72. RESULTS: A novel heterozygous missense mutation (c.791A>G, p.N264S) of the SETX gene was identified in a female patient presenting an atypical ALS phenotype, including adult onset and lower motor neuron impairment. No further mutations were detected in the other Mendelian ALS genes investigated. CONCLUSION: Our study contributes to the understanding of the genetic and phenotypic diversity of motor neuron diseases (MNDs). Our results also suggest that the elucidation of the genetic background of MNDs requires a complex approach, including the screening of both Mendelian and non-Mendelian genes.

Genetic analysis of the SOD1 and C9ORF72 genes in Hungarian patients with amyotrophic lateral sclerosis.

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease characterized by the death of motor neurons. To date, more than 20 genes have been implicated in ALS, and of these, the 2 most frequently mutated are the superoxide dismutase 1 (SOD1) gene and the chromosome 9 open reading frame 72 (C9ORF72) gene. In this study, we aimed to investigate the contribution of these 2 Mendelian genes to the development of the disease in Hungarian ALS patients (n = 66). Direct sequencing of the SOD1 gene revealed a novel (p.Lys91ArgfsTer8) and 3 recurrent heterozygous mutations (p.Val14Met, p.Asp90Ala, and p.Leu144Phe) in 5 patients. The novel p.Lys91ArgfsTer8 mutation led to a frameshift causing the addition of 8 new amino acids, including a premature stop codon at position 99. The GGGGCC hexanucleotide repeat expansion of the C9ORF72 gene was present in 1 ALS patient. This study represents the first genetic analysis of 2 major ALS causative genes in a cohort of Hungarian ALS patients and contributes to the further understanding of the genetic and phenotypic diversity of ALS.

[Calcium ion is a common denominator in the pathophysiological processes of amyotrophic lateral sclerosis]. / Az amyotrophiás lateralsclerosis patofiziológiai tényezoinek központi kapcsolóeleme, a kalcium.

Amyotrophic lateral sclerosis (ALS), the most frequent motor neuron disease is characterized by progressive muscle weakness caused by the degeneration of the motor neurons in the spinal cord and motor cortex. However, according to the recent observations, ALS is a rather complex syndrome which frequently involves symptoms of cognitive impairment. Therefore, ALS cases can be interpreted in a clinico-pathological spectrum spanning from the classical ALS involving only the motor system to the fronto-temporal dementia. The progression of the disease, however, manifested in the degeneration of the upper and lower motor neurons, is based on the same complex pathobiology. The main elements of the pathomechanism, such as oxidative stress, excitotoxicity, immune/inflammatory processes and mitochondrial dysfunction are well described already, which operate in orchestrated way and amplify the deleterious effect of each other. It is assumed that calcium ions act as a catalyst in this interaction, hence each of the individual mechanisms has strong, positive and reciprocal calcium dependence thus may combine the individual pathological processes into a unified escalating mechanism of neuronal destruction. This review provides an overview of the role of calcium in connecting and amplifying the major mechanisms which lead to degeneration of the motor neurons in ALS.

Axotomy Leads to Reduced Calcium Increase and Earlier Termination of CCL2 Release in Spinal Motoneurons with Upregulated Parvalbumin Followed by Decreased Neighboring Microglial Activation.

BACKGROUND: Motoneurons with naturally elevated calcium binding protein content, such as parvalbumin, are more resistant against injury. Furthermore, increase of intracellular calcium, which plays a pivotal role in injury of neurons, could be moderated by elevating their calcium binding proteins. OBJECTIVE: To test whether by elevating parvalbumin content of motoneurons, activation of neighboring microglial cells, a robust component of the inflammatory reaction after injury, could be influenced. METHODS: Mice overexpressing neuronal parvalbumin were derived and the spinal motoneurons were challenged by cutting the sciatic nerve. At postoperative days 1, 4, 7, 14 and 21 the change of the chemokine ligand 2 immunostaining in the motoneurons and the activation of microglial cells, measured as alterations in CD11b immunostaining were determined. Calcium level of motoneurons was tested electron microscopically at postoperative day 7. RESULTS: After axotomy, increased level of chemokine ligand 2 was detected in the lumbar motoneurons. The staining intensity reached its maximum at day 7 and decayed faster in transgenic mice compared to controls. Microglial activation around motoneurons attenuated faster in parvalbumin overexpressing mice, too, but the decrease of microglial activation was delayed compared to the decline of the chemokine ligand 2 signal. At the time when the microglial reaction peaked, no intracellular calcium increase was detected in the motoneurons of transgenic mice, in contrast to the twofold increase in wild type animals. CONCLUSION: Increased calcium buffering capacity, which augments resistance of motoneurons against calcium-mediated injury, leads to earlier termination of motoneuronal emission of CCL2 followed by a reduction of neighboring microglial activation after axotomy.

Novel AARS2 gene mutation producing leukodystrophy: a case report.

AARS2 gene (NM_020745.3) mutations result in two different phenotypic diseases: infantile mitochondrial cardiomyopathy and late-onset leukoencephalopathy. The patient's first symptoms appeared at the age of 18 years with behavioral changes and psychiatric problems. Some years later, extrapyramidal symptoms, cognitive impairment, nystagmus, dysarthria and pyramidal symptoms also developed. The brain magnetic resonance imaging (MRI) indicated extensive white matter abnormalities. The diagnosis of AARS2 gene mutations causing leukodystrophy was confirmed by genetic testing. Segregation analysis confirmed the compound heterozygous state of the patient. Histological examination of the biopsy did not prove specific pathological alterations. The clinical phenotype of our patient was compared with seven previously described patients suffering from leukoencephalopathy caused by AARS2 mutations. We have documented a new, nonsense AARS2 gene mutation (c.578T>G, p.Leu193*) and a known missense mutation (c.595C>T, p.Arg199Cys) associated with leukoencephalopathy in a male patient. Clinical features, imaging characteristics and genetic testing are presented, and histological data from an AARS2-related leukodystrophy patient are described for the first time.

Calcium in the pathomechanism of amyotrophic lateral sclerosis - Taking center stage?

Amyotrophic lateral sclerosis is an incurable, relentlessly progressive disease primarily affecting motor neurons. The cause of the disease, except for the mutations identified in a small fraction of patients, is unknown. The major mechanisms contributing to the degeneration of motor neurons have already been disclosed and characterized, including excitotoxicity, oxidative stress, mitochondrial dysfunction, and immune/inflammatory processes. During the progression of the disease these toxic processes are not discrete, but each facilitates the deleterious effect of the other. However, due to their common reciprocal calcium dependence, calcium ions may act as a common denominator and through a positive feedback loop may combine the individual pathological processes into a unified escalating mechanism of neuronal destruction. This mini-review provides an overview of the mutual calcium dependence of the major toxic mechanisms associated with amyotrophic lateral sclerosis.

Intraperitoneally administered IgG from patients with amyotrophic lateral sclerosis or from an immune-mediated goat model increase the levels of TNF-α, IL-6, and IL-10 in the spinal cord and serum of mice.

BACKGROUND: Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease that involves the selective loss of the upper and lower motor neurons (MNs). Neuroinflammation has been implicated in the pathogenesis of the sporadic form of the disease. We earlier developed immune-mediated animal models of ALS and demonstrated humoral and cellular immune reactions in the nervous system and in the sera of patients and animals. The accumulation of immunoglobulin G (IgG), an elevated intracellular level of calcium, ultrastructural alterations in the MNs, and activation of the microglia were noted in the spinal cord of ALS patients. Similar alterations developed in mice inoculated intraperitoneally with IgG from ALS patients or from an immune-mediated goat model. METHODS: We have now examined whether the intraperitoneal injection of mice with IgG from sporadic ALS patients or from immunized goats with the homogenate of the anterior horn of the bovine spinal cord is associated with changes in the pro-inflammatory (TNF-α and IL-6) and anti-inflammatory (IL-10) cytokines in the spinal cord and serum of the mice. The levels of cytokines were measured by ELISA. RESULTS: Intraperitoneally administered IgG from the ALS patients induced subclinical signs of MN disease, while the injection of IgG from immunized goats resulted in a severe respiratory dysfunction and limb paralysis 24 h after the injections. Significantly increased levels of TNF-α and IL-10 were detected in the spinal cord of the mice injected with the human ALS IgG. The level of IL-6 increased primarily in the serum. The IgG from the immunized goats induced highly significant increases in the levels of all three cytokines in the serum and the spinal cord of mice. CONCLUSIONS: Our earlier experiments had proved that when ALS IgG or IgG from immune-mediated animal models was inoculated into mice, it was taken up in the MNs and had the ability to initiate damage in them. The pathological process was paralleled by microglia recruitment and activation in the spinal cord. The present experiment revealed that these forms of IgG cause significant increases in certain cytokine levels locally in the spinal cord and in the serum of the inoculated mice. These results suggest that IgG directed to the MNs may be an initial element in the damage to the MNs both in human ALS and in its immune-mediated animal models.

The role of cognitive training in the neurorehabilitation of a patient who survived a lightning strike. A case study.

BACKGROUND: Lightning-related injuries most often involve impairment of the functions of the central and peripheral nervous systems, usually including cognitive dysfunctions. We evaluated the cognitive deficit of a patient who had survived a lightning strike and measured the improvement after her cognitive training. This therapeutic method appears to be a powerful tool in the neurorehabilitation treatment. OBJECTIVE: The aim of this case study was to prove the beneficial effects of cognitive training as part of the neurorehabilitation after a lightning strike. METHODS: Six neuropsychological functions were examined in order to test the cognitive status of the patient before and after the 2-month cognitive training: phonological short-term memory (digit span test and word repetitions test), visuo-spatial short-term memory (Corsi Block Tapping Test), working memory (backward digit span test and listening span test), executive functions (letter and semantic fluencies), language functions (non-word repetition test, Pléh-Palotás-Lörik (PPL) test and sentence repetition test) and episodic memory (Rivermead Behavioral Memory Test and Mini Mental State Examination). We also utilized these tests in aged-matched healthy individuals so as to be able to characterize the domains of the observed improvements more precisely. RESULTS: The patient exhibited a considerable improvement in the backward digit span, semantic fluency, non-word repetition, PPL, sentence repetition and Rivermead Behavioral Memory tests. CONCLUSIONS: The cognitive training played an important role in the neurorehabilitation treatment of this lightning injury patient. It considerably improved her quality of life through the functional recovery.

Highly immunoreactive IgG antibodies directed against a set of twenty human proteins in the sera of patients with amyotrophic lateral sclerosis identified by protein array.

Amyotrophic lateral sclerosis (ALS), the most common adult-onset motor neuron disorder, is characterized by the progressive and selective loss of upper and lower motor neurons. Diagnosis of this disorder is based on clinical assessment, and the average survival time is less than 3 years. Injections of IgG from ALS patients into mice are known to specifically mark motor neurons. Moreover, IgG has been found in upper and lower motor neurons in ALS patients. These results led us to perform a case-control study using human protein microarrays to identify the antibody profiles of serum samples from 20 ALS patients and 20 healthy controls. We demonstrated high levels of 20 IgG antibodies that distinguished the patients from the controls. These findings suggest that a panel of antibodies may serve as a potential diagnostic biomarker for ALS.

[Hashimoto encephalopathy]. / Hashimoto-encephalopathia.

The authors present a case report and review the literature on Hashimoto encephalopathy. The onset of the disease may be marked by focal and then progressively generalized seizures or other neurological symptoms, but a cognitive decline or various psychiatric symptoms may also emerge. High levels of anti-thyroid peroxidase antibodies and/or anti-thyroglobulin antibodies are present in the serum. Corticosteroid treatment usually results in an improvement of symptoms. The syndrome is frequently overlooked and, therefore, the authors strongly recommend testing serum thyroid autoantibodies in cases with encephalopathy of unknown origin independently on the presence of thyroid disease in the patient or family history. The importance of long-term immunosuppressive treatment should also be stressed.