Influence of kidney function and CSF/serum albumin ratio on plasma Aß42 and Aß40 levels measured on a fully automated platform in patients with Alzheimer's disease.

INTRODUCTION: Alzheimer's disease  (AD) is characterized by decreased cerebrospinal fluid (CSF) Aß42 and Aß42/Aß40 ratio. Aß peptides can now be measured also in plasma and are promising peripheral biomarkers for AD. We evaluated the relationships of plasma Aß species with their CSF counterparts, kidney function, and serum/CSF albumin ratio (Q-Alb) in AD patients. MATERIALS AND METHODS: We measured plasma Aß42 and Aß40, as well as CSF AD biomarkers, with the fully automated Lumipulse platform in a cohort of N = 30 patients with clinical and neurochemical diagnosis of AD. RESULTS: The two plasma Aß peptides correlated strongly with each other (r = 0.7449), as did the corresponding CSF biomarkers (r = 0.7670). On the contrary, the positive correlations of plasma Aß42, Aß40, and Aß42/Aß40 ratio with their CSF counterparts and the negative correlation of plasma Aß42/Aß40 ratio with CSF P-tau181 were not statistically significant. Plasma levels of both Aß species negatively correlated with estimated glomerular filtration rate (eGFR) (Aß42: r = -0.4138; Aß40: r = -0.6015), but plasma Aß42/Aß40 ratio did not. Q-Alb did not correlate with any plasma Aß parameter. DISCUSSION: Plasma Aß42 and Aß40 are critically influenced by kidney function; however, their ratio is advantageously spared from this effect. The lack of significant correlations between plasma Aß species and their CSF counterparts is probably mainly due to small sample size and inclusion of only Aß + individuals. Q-Alb is not a major determinant of plasma Aß concentrations, highlighting the uncertainties about mechanisms of Aß transfer between CNS and periphery.

Phosphorylated tau in plasma could be a biomarker of lower motor neuron impairment in amyotrophic lateral sclerosis.

INTRODUCTION: Plasma levels of phosphorylated tau (P-tau181) have been recently reported to be increased in amyotrophic lateral sclerosis (ALS) and associated with lower motor neuron (LMN) impairment. PATIENTS AND METHODS: We quantified plasma P-tau181 (pP-tau181) in a cohort of 29 deeply phenotyped ALS patients using the new fully automated Lumipulse assay and analysed phenotype-biomarker correlations. RESULTS: pP-tau181 levels correlated positively with a clinical LMN score (r = 0.3803) and negatively, albeit not significantly, with a composite index of muscle strength (r = - 0.3416; p = 0.0811), but not with Penn Upper Motor Neuron (UMN) Score. Accordingly, pP-tau181 correlated with electromyographic indices of spinal active and chronic denervation (r = 0.4507 and r = 0.3864, respectively) but not with transcranial magnetic stimulation parameters of UMN dysfunction. pP-tau181 levels did not correlate with those in the cerebrospinal fluid (CSF), serum NFL, serum GFAP, CSF/serum albumin ratio, or estimated glomerular filtration rate, but correlated with plasma creatine kinase levels (r = 0.4661). Finally, while not being associated with neuropsychological phenotype, pP-tau181 correlated negatively with pH (r = - 0.5632) and positively with partial pressure of carbon dioxide (PaCO2; r = 0.7092), bicarbonate (sHCO3-; r = 0.6667) and base excess (r = 0.6611) on arterial blood gas analysis. DISCUSSION: pP-tau181 has potential as ALS biomarker and could be associated with LMN impairment. Its raised levels might reflect pathophysiological processes (tau hyperphosphorylation and/or release) occurring in the axons of LMNs distantly from the CNS and the CSF. pP-tau181 could also be associated with respiratory dysfunction.

C9ORF72 Repeat Expansion Affects the Proteome of Primary Skin Fibroblasts in ALS.

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease characterized by progressive degeneration of the corticospinal motor neurons, which ultimately leads to death. The repeat expansion in chromosome 9 open reading frame 72 (C9ORF72) represents the most common genetic cause of ALS and it is also involved in the pathogenesis of other neurodegenerative disorders. To offer insights into C9ORF72-mediated pathogenesis, we quantitatively analyzed the proteome of patient-derived primary skin fibroblasts from ALS patients carrying the C9ORF72 mutation compared with ALS patients who tested negative for it. Differentially expressed proteins were identified, used to generate a protein-protein interaction network and subjected to a functional enrichment analysis to unveil altered molecular pathways. ALS patients were also compared with patients affected by frontotemporal dementia carrying the C9ORF72 repeat expansion. As a result, we demonstrated that the molecular pathways mainly altered in fibroblasts (e.g., protein homeostasis) mirror the alterations observed in C9ORF72-mutated neurons. Moreover, we highlighted novel molecular pathways (nuclear and mitochondrial transports, vesicle trafficking, mitochondrial bioenergetics, glucose metabolism, ER-phagosome crosstalk and Slit/Robo signaling pathway) which might be further investigated as C9ORF72-specific pathogenetic mechanisms. Data are available via ProteomeXchange with the identifier PXD023866.

Reprogramming fibroblasts and peripheral blood cells from a C9ORF72 patient: A proof-of-principle study.

As for the majority of neurodegenerative diseases, pathological mechanisms of amyotrophic lateral sclerosis (ALS) have been challenging to study due to the difficult access to alive patients' cells. Induced pluripotent stem cells (iPSCs) offer a useful in vitro system for modelling human diseases. iPSCs can be theoretically obtained by reprogramming any somatic tissue although fibroblasts (FB) remain the most used cells. However, reprogramming peripheral blood cells (PB) may offer significant advantages. In order to investigate whether the choice of starting cells may affect reprogramming and motor neuron (MNs) differentiation potential, we used both FB and PB from a same C9ORF72-mutated ALS patient to obtain iPSCs and compared several hallmarks of the pathology. We found that both iPSCs and MNs derived from the two tissues showed identical properties and features and can therefore be used interchangeably, giving the opportunity to easily obtain iPSCs from a more manageable source of cells, such as PB.

Chronic stress induces formation of stress granules and pathological TDP-43 aggregates in human ALS fibroblasts and iPSC-motoneurons.

Amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) are fatal neurodegenerative diseases characterized by the presence of neuropathological aggregates of phosphorylated TDP-43 (P-TDP-43) protein. The RNA-binding protein TDP-43 participates also to cell stress response by forming stress granules (SG) in the cytoplasm to temporarily arrest translation. The hypothesis that TDP-43 pathology directly arises from SG has been proposed but is still under debate because only sub-lethal stress conditions have been tested experimentally so far. In this study we reproduced a mild and chronic oxidative stress by sodium arsenite to better mimic the persistent and subtle alterations occurring during the neurodegenerative process in primary fibroblasts and induced pluripotent stem cell-derived motoneurons (iPSC-MN) from ALS patients carrying mutations in TARDBP and C9ORF72 genes. We found that not only the acute sub-lethal stress usually used in literature, but also the chronic oxidative insult was able to induce SG formation in both primary fibroblasts and iPSC-MN. We also observed the recruitment of TDP-43 into SG only upon chronic stress in association to the formation of distinct cytoplasmic P-TDP-43 aggregates and a significant increase of the autophagy marker p62. A quantitative analysis revealed differences in both the number of cells forming SG in mutant ALS and healthy control fibroblasts, suggesting a specific genetic contribution to cell stress response, and in SG size, suggesting a different composition of these cytoplasmic foci in the two stress conditions. Upon removal of arsenite, the recovery from chronic stress was complete for SG and P-TDP-43 aggregates at 72 h with the exception of p62, which was reduced but still persistent, supporting the hypothesis that autophagy impairment may drive pathological TDP-43 aggregates formation. The gene-specific differences observed in fibroblasts in response to oxidative stress were not present in iPSC-MN, which showed a similar formation of SG and P-TDP-43 aggregates regardless their genotype. Our results show that SG and P-TDP-43 aggregates may be recapitulated in patient-derived neuronal and non-neuronal cells exposed to prolonged oxidative stress, which may be therefore exploited to study TDP-43 pathology and to develop individualized therapeutic strategies for ALS/FTD.

Inter-Species Differences in Regulation of the Progranulin-Sortilin Axis in TDP-43 Cell Models of Neurodegeneration.

Cytoplasmic aggregates and nuclear depletion of the ubiquitous RNA-binding protein TDP-43 have been described in the autoptic brain tissues of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTLD) patients and both TDP-43 loss-of-function and gain-of-function mechanisms seem to contribute to the neurodegenerative process. Among the wide array of RNA targets, TDP-43 regulates progranulin (GRN) mRNA stability and sortilin (SORT1) splicing. Progranulin is a secreted neurotrophic and neuro-immunomodulatory factor whose endocytosis and delivery to the lysosomes are regulated by the neuronal receptor sortilin. Moreover, GRN loss-of-function mutations are causative of a subset of FTLD cases showing TDP-43 pathological aggregates. Here we show that TDP-43 loss-of-function differently affects the progranulin-sortilin axis in murine and human neuronal cell models. We demonstrated that although TDP-43 binding to GRN mRNA occurs similarly in human and murine cells, upon TDP-43 depletion, a different control of sortilin splicing and protein content may determine changes in extracellular progranulin uptake that account for increased or unchanged secreted protein in murine and human cells, respectively. As targeting the progranulin-sortilin axis has been proposed as a therapeutic approach for GRN-FTLD patients, the inter-species differences in TDP-43-mediated regulation of this pathway must be considered when translating studies from animal models to patients.

From transcriptomic to protein level changes in TDP-43 and FUS loss-of-function cell models.

The full definition of the physiological RNA targets regulated by TDP-43 and FUS RNA-binding proteins (RBPs) represents an important issue in understanding the pathogenic mechanisms associated to these two proteins in amyotrophic lateral sclerosis and frontotemporal dementia. In the last few years several high-throughput screenings have generated a plethora of data, which are difficult to compare due to the different experimental designs and models explored. In this study by using the Affymetrix Exon Arrays, we were able to assess and compare the effects of both TDP-43 and FUS loss-of-function on the whole transcriptome using the same human neuronal SK-N-BE cell model. We showed that TDP-43 and FUS depletion induces splicing and gene expression changes mainly distinct for the two RBPs, although they may regulate common pathways, including neuron differentiation and cytoskeleton organization as evidenced by functional annotation analysis. In particular, TDP-43 and FUS were found to regulate splicing and expression of genes related to neuronal (SEPT6, SULT4A1, TNIK) and RNA metabolism (DICER, ELAVL3/HuC, POLDIP3). Our extended analysis at protein level revealed that these changes have also impact on the protein isoform ratio and content, not always in a direct correlation with transcriptomic data. Contrarily to a loss-of-function mechanism, we showed that mutant TDP-43 proteins maintained their splicing activity in human ALS fibroblasts and experimental cell lines. Our findings further contribute to define the biological functions of these two RBPs in physiological and disease state, strongly encouraging the evaluation of the identified transcriptomic changes at protein level in neuronal experimental models.

hnRNPA2/B1 and nELAV proteins bind to a specific U-rich element in CDK5R1 3'-UTR and oppositely regulate its expression.

Cyclin-dependent kinase 5 regulatory subunit 1 (CDK5R1) encodes p35, a specific activator of cyclin-dependent kinase 5 (CDK5). CDK5 and p35 have a fundamental role in neuronal migration and differentiation during CNS development. Both the CDK5R1 3'-UTR's remarkable size and its conservation during evolution strongly indicate an important role in post-transcriptional regulation. We previously validated different regulatory elements in the 3'-UTR of CDK5R1, which affect transcript stability, p35 levels and cellular migration through the binding with nELAV proteins and miR-103/7 miRNAs. Interestingly, a 138 bp-long region, named C2.1, was identified as the most mRNA destabilizing portion within CDK5R1 3'-UTR. This feature was maintained by a shorter region of 73 bp, characterized by two poly-U stretches. UV-CL experiments showed that this region interacts with protein factors. UV-CLIP assays and pull-down experiments followed by mass spectrometry analysis demonstrated that nELAV and hnRNPA2/B1 proteins bind to the same U-rich element. These RNA-binding proteins (RBPs) were shown to oppositely control CDK5R1 mRNA stability and p35 protein content at post-trascriptional level. While nELAV proteins have a positive regulatory effect, hnRNPA2/B1 has a negative action that is responsible for the mRNA destabilizing activity both of the C2.1 region and of the full-length 3'-UTR. In co-expression experiments of hnRNPA2/B1 and nELAV RBPs we observed an overall decrease of p35 content. We also demonstrated that hnRNPA2/B1 can downregulate nELAV protein content but not vice versa. This study, by providing new insights on the combined action of different regulatory factors, contributes to clarify the complex post-transcriptional control of CDK5R1 gene expression.

ELAV proteins along evolution: back to the nucleus?

The complex interplay of post-transcriptional regulatory mechanisms mediated by RNA-binding proteins (RBP) at different steps of RNA metabolism is pivotal for the development of the nervous system and the maintenance of adult brain activities. In this review, we will focus on the highly conserved ELAV gene family encoding for neuronal-specific RBPs which are necessary for proper neuronal differentiation and important for synaptic plasticity process. In the evolution from Drosophila to man, ELAV proteins seem to have changed their biological functions in relation to their different subcellular localization. While in Drosophila, they are localized in the nuclear compartment of neuronal cells and regulate splicing and polyadenylation, in mammals, the neuronal ELAV proteins are mainly present in the cytoplasm where they participate in regulating mRNA target stability, translation and transport into neurites. However, recent data indicate that the mammalian ELAV RBPs also have nuclear activities, similarly to their fly counterpart, being them able to continuously shuttle between the cytoplasm and the nucleus. Here, we will review and comment on all the biological functions associated with neuronal ELAV proteins along evolution and will show that the post-transcriptional regulatory network mediated by these RBPs in the brain is highly complex and only at an initial stage of being fully understood. This article is part of a Special Issue entitled 'RNA and splicing regulation in neurodegeneration'.

The value of routine blood work-up in clinical stratification and prognosis of patients with amyotrophic lateral sclerosis.

BACKGROUND: There is an unmet need in amyotrophic lateral sclerosis (ALS) to provide specific biomarkers for the disease. Due to their easy availability, we aimed to investigate whether routine blood parameters provide useful clues for phenotypic classification and disease prognosis. METHODS: We analyzed a large inpatient cohort of 836 ALS patients who underwent deep phenotyping with evaluation of the clinical and neurophysiological burden of upper (UMN) and lower (LMN) motor neuron signs. Disability and progression rate were measured through the revised ALS Functional Rating Scale (ALSFRS-R) and its changes during time. Cox regression analysis was performed to assess survival associations. RESULTS: Creatinine significantly correlated with LMN damage (r = 0.38), active (r = 0.18) and chronic (r = 0.24) denervation and baseline ALSFRS-R (r = 0.33). Creatine kinase (CK), alanine (ALT) and aspartate (AST) transaminases correlated with active (r = 0.35, r = 0.27, r = 0.24) and chronic (r = 0.37, r = 0.20, r = 0.19) denervation, while albumin and C-reactive protein significantly correlated with LMN score (r = 0.20 and r = 0.17). Disease progression rate showed correlations with chloride (r = -0.19) and potassium levels (r = -0.16). After adjustment for known prognostic factors, total protein [HR 0.70 (95% CI 0.57-0.86)], creatinine [HR 0.86 (95% CI 0.81-0.92)], chloride [HR 0.95 (95% CI 0.92-0.99)], lactate dehydrogenase [HR 0.99 (95% CI 0.99-0.99)], and AST [HR 1.02 (95% CI 1.01-1.02)] were independently associated with survival. CONCLUSIONS: Creatinine is a reliable biomarker for ALS, associated with clinical features, disability and survival. Markers of nutrition/inflammation may offer additional prognostic information and partially correlate with clinical features. AST and chloride could further assist in predicting progression rate and survival.

TDP-43 and FUS RNA-binding proteins bind distinct sets of cytoplasmic messenger RNAs and differently regulate their post-transcriptional fate in motoneuron-like cells.

The RNA-binding proteins TDP-43 and FUS form abnormal cytoplasmic aggregates in affected tissues of patients with amyotrophic lateral sclerosis and frontotemporal lobar dementia. TDP-43 and FUS localize mainly in the nucleus where they regulate pre-mRNA splicing, but they are also involved in mRNA transport, stability, and translation. To better investigate their cytoplasmic activities, we applied an RNA immunoprecipitation and chip analysis to define the mRNAs associated to TDP-43 and FUS in the cytoplasmic ribonucleoprotein complexes from motoneuronal NSC-34 cells. We found that they bind different sets of mRNAs although converging on common cellular pathways. Bioinformatics analyses identified the (UG)(n) consensus motif in 80% of 3'-UTR sequences of TDP-43 targets, whereas for FUS the binding motif was less evident. By in vitro assays we validated binding to selected target 3'-UTRs, including Vegfa and Grn for TDP-43, and Vps54, Nvl, and Taf15 for FUS. We showed that TDP-43 has a destabilizing activity on Vegfa and Grn mRNAs and may ultimately affect progranulin protein content, whereas FUS does not affect mRNA stability/translation of its targets. We also demonstrated that three different point mutations in TDP-43 did not change the binding affinity for Vegfa and Grn mRNAs or their protein level. Our data indicate that TDP-43 and FUS recognize distinct sets of mRNAs and differently regulate their fate in the cytoplasm of motoneuron-like cells, therefore suggesting complementary roles in neuronal RNA metabolism and neurodegeneration.

Generation of an iPSC line from a patient with spastic paraplegia type 10 carrying a novel mutation in KIF5A gene.

We generated an iPSC line from a patient with spastic paraplegia type 10 (SPG10) carrying the novel missense variant c.50G > A (p.R17Q) in the N-terminal motor domain of the kinesin family member 5A (KIF5A) gene. This patient-derived in vitro cell model will help to investigate the role of different KIF5A mutations in inducing neurodegeneration in spastic paraplegia and in other KIF5A-related disorders, including Charcot-Marie-Tooth type 2 (CMT2) and amyotrophic lateral sclerosis (ALS).

Motor, cognitive and behavioural profiles of C9orf72 expansion-related amyotrophic lateral sclerosis.

INTRODUCTION: Amyotrophic lateral sclerosis (ALS) individuals carrying the hexanucleotide repeat expansion (HRE) in the C9orf72 gene (C9Pos) have been described as presenting distinct features compared to the general ALS population (C9Neg). We aim to identify the phenotypic traits more closely associated with the HRE and analyse the role of the repeat length as a modifier factor. METHODS: We studied a cohort of 960 ALS patients (101 familial and 859 sporadic cases). Motor phenotype was determined using the MRC scale, the lower motor neuron score (LMNS) and the Penn upper motor neuron score (PUMNS). Neuropsychological profile was studied using the Italian version of the Edinburgh Cognitive and Behavioral ALS Screen (ECAS), the Frontal Behavioral Inventory (FBI), the Beck Depression Inventory-II (BDI-II) and the State-Trait Anxiety Inventory (STAI). A two-step PCR protocol and Southern blotting were performed to determine the presence and the size of C9orf72 HRE, respectively. RESULTS: C9orf72 HRE was detected in 55/960 ALS patients. C9Pos patients showed a younger onset, higher odds of bulbar onset, increased burden of UMN signs, reduced survival and higher frequency of concurrent dementia. We found an inverse correlation between the HRE length and the performance at ECAS ALS-specific tasks (P = 0.031). Patients also showed higher burden of behavioural disinhibition (P = 1.6 × 10-4), lower degrees of depression (P = 0.015) and anxiety (P = 0.008) compared to C9Neg cases. CONCLUSIONS: Our study provides an extensive characterization of motor, cognitive and behavioural features of C9orf72-related ALS, indicating that the C9orf72 HRE size may represent a modifier of the cognitive phenotype.

Human motor neurons derived from induced pluripotent stem cells are susceptible to SARS-CoV-2 infection.

Introduction: COVID-19 typically causes Q7 respiratory disorders, but a high proportion of patients also reports neurological and neuromuscular symptoms during and after SARSCoV-2 infection. Despite a number of studies documenting SARS-CoV-2 infection of various neuronal cell populations, the impact of SARS-CoV-2 exposure on motor neuronal cells specifically has not been investigated so far. Methods: Thus, by using human iPSC-derived motor neurons (iPSC-MNs) we assessed: (i) the expression of SARS-CoV-2 main receptors; (ii) iPSC-MN infectability by SARS-CoV-2; and (iii) the effect of SARS-CoV-2 exposure on iPSC-MN transcriptome. Results: Gene expression profiling and immunofluorescence (IF) analysis of the main host cell receptors recognized by SARS-CoV-2 revealed that all of them are expressed in iPSC-MNs, with CD147 and NRP1 being the most represented ones. By analyzing SARS-CoV-2 N1 and N2 gene expression over time, we observed that human iPSC-MNs were productively infected by SARS-CoV-2 in the absence of cytopathic effect. Supernatants collected from SARS-CoV-2-infected iPSC-MNs were able to re-infect VeroE6 cells. Image analyses of SARS-CoV-2 nucleocapsid proteins by IF confirmed iPSC-MN infectability. Furthermore, SARS-CoV-2 infection in iPSCMNs significantly altered the expression of genes (IL-6, ANG, S1PR1, BCL2, BAX, Casp8, HLA-A, ERAP1, CD147, MX1) associated with cell survival and metabolism, as well as antiviral and inflammatory response. Discussion: These results suggest for the very first time that SARS-CoV-2 can productively infect human iPSC-derived MNs probably by binding CD147 and NRP1 receptors. Such information will be important to unveil the biological bases of neuromuscular disorders characterizing SARS-CoV-2 infection and the so called long-COVID symptoms.

Genetic and epigenetic disease modifiers in an Italian C9orf72 family expressing ALS, FTD or PD clinical phenotypes.

Objective: The presence of the hexanucleotide repeat expansion (HRE) in C9orf72 gene is associated to the ALS/FTD spectrum, but also to parkinsonisms. We here describe an Italian family with the father diagnosed with Parkinson disease (PD) at the age of 67 and the two daughters developing FTD and ALS at 45 years of age. We searched for C9orf72 HRE with possible genetic and epigenetic modifiers to account for the intrafamilial phenotypic variability. Methods: C9orf72 mutational analysis was performed by fragment length analysis, Repeat-primed PCR and Southern blot. Targeted next generation sequencing was used to analyze 48 genes associated to neurodegenerative diseases. Promoter methylation was analyzed by bisulfite sequencing. Results: Genetic analysis identified C9orf72 HRE in all the affected members with a similar repeat expansion size. Both the father and the FTD daughter also carried the heterozygous p.Ile946Phe variant in ATP13A2 gene, associated to PD. In addition, the father also showed a heterozygous EIF4G1 variant (p.Ala13Pro), that might increase his susceptibility to develop PD. The DNA methylation analysis showed that all the 26 CpG sites within C9orf72 promoter were unmethylated in all family members. Conclusions: Neither C9orf72 HRE size nor promoter methylation act as disease modifiers within this family, at least in blood, not excluding HRE mosaicism and a different methylation pattern in the brain. However, the presence of rare genetic variants in PD genes suggests that they may influence the clinical manifestation in the father. Other genetic and/or epigenetic modifiers must be responsible for disease variability in this C9orf72 family case.

Serum neurofilament light chain levels in Covid-19 patients without major neurological manifestations.

BACKGROUND: Increased serum levels of neurofilament light chain (sNFL), a biomarker of neuroaxonal damage, have been reported in patients with Covid-19. We aimed at investigating whether sNFL is increased in Covid-19 patients without major neurological manifestations, is associated with disease severity, respiratory and routine blood parameters, and changes longitudinally in the short term. METHODS: sNFL levels were measured with single molecule array (Simoa) technology in 57 hospitalized Covid-19 patients without major neurological manifestations and in 30 neurologically healthy controls. Patients were evaluated for PaO2/FiO2 ratio on arterial blood gas, Brescia Respiratory Covid Severity Scale (BRCSS), white blood cell counts, serum C-reactive protein (CRP), plasma D-dimer, plasma fibrinogen, and serum creatinine at admission. In 20 patients, NFL was also measured on serum samples obtained at a later timepoint during the hospital stay. RESULTS: Covid-19 patients had higher baseline sNFL levels compared to controls, regardless of disease severity. Baseline sNFL correlated with serum CRP and plasma D-dimer in patients with mild disease, but was not associated with measures of respiratory impairment. Longitudinal sNFL levels tended to be higher than baseline ones, albeit not significantly, and correlated with serum CRP and plasma D-dimer. The PaO2/FiO2 ratio was not associated with longitudinal sNFL, whereas BRCSS only correlated with longitudinal sNFL variation. CONCLUSIONS: We provide neurochemical evidence of subclinical axonal damage in Covid-19 also in the absence of major neurological manifestations. This is apparently not fully explained by hypoxic injury; rather, systemic inflammation might promote this damage. However, a direct neurotoxic effect of SARS-CoV-2 cannot be excluded.

Mutations of FUS gene in sporadic amyotrophic lateral sclerosis.

BACKGROUND: Mutations in the FUS gene have recently been discovered to be a major cause of familial amyotrophic lateral sclerosis (FALS). OBJECTIVE: To determine the identity and frequency of FUS gene mutations in a large cohort of Italian patients enriched in sporadic cases (SALS). METHODS: Exons 5, 6, 14 and 15 of the FUS gene were screened for mutations in 1009 patients (45 FALS and 964 SALS). The genetic analysis was extended to the entire coding sequence of FUS in all the FALS and 293 of the SALS patients. RESULTS: Seven missense mutations (p.G191S, p.R216C, p.G225V, p.G230C, p.R234C, p.G507D and p.R521C) were identified in nine patients (seven SALS and two FALS), and none in 500 healthy Italian controls. All mutations are novel except for the p.R521C mutation identified in one SALS and one FALS case. Both patients showed a similar unusual presentation, with proximal, mostly symmetrical, upper limb weakness, with neck and axial involvement. With the exception of p.G507D and p.R521C, the mutations identified in SALS patients are all localised in the glycine-rich region encoded by exon 6. In addition, eight different in-frame deletions in two polyglycine motifs were detected, the frequency of which was not significantly different in patients and controls. CONCLUSIONS: The results show that FUS missense mutations are present in 0.7% of Italian SALS cases, and confirm the previous mutational frequency reported in FALS (4.4%). An unusual proximal and axial clinical presentation seems to be associated with the presence of the p.R521C mutation.

RNA-binding proteins and RNA metabolism: a new scenario in the pathogenesis of Amyotrophic lateral sclerosis.

Several RNA-processing genes have been implicated in the pathogenesis of Amyotrophic lateral sclerosis (ALS). In particular, causative mutations in the genes encoding for two DNA/RNA binding proteins, TAR DNA binding protein-43 (TDP-43) and fused in sarcoma/translocated in liposarcoma (FUS/TLS), were recently identified in ALS patients. These genetic findings and the presence of abnormal aggregates of these two RNA-binding proteins in ALS affected tissues suggest that molecular mechanisms regulating RNA metabolism are implicated in ALS pathogenesis through common pathways. In this review similarities and differences between TDP-43 and FUS/TLS proteins and their activities in physiological and pathological conditions will be discussed.

Genetics of familial Amyotrophic lateral sclerosis.

Amyotrophic lateral sclerosis (ALS) is a late onset, rapidly progressive and ultimately fatal neurodegenerative disease, caused by the loss of motor neurons in the brain and spinal cord. About 10% of all ALS cases are familial (FALS), and constitute a clinically and genetically heterogeneous entity. To date, FALS has been linked to mutations in 10 different genes and to four additional chromosomal loci. Research on FALS genetics, and in particular the discoveries of mutations in the SOD1, TARDBP, and FUS genes, has provided essential information toward the understanding of the pathogenesis of ALS in general. This review presents a tentative classification of all FALS-associated genes identified so far.