Optineurin in patients with Amyotrophic Lateral Sclerosis associated to atypical Parkinsonism in Tunisian population.

Amyotrophic Lateral Sclerosis (ALS) is a heterogeneous disorder and the phenotypic variability goes far beyond the used clinical stratification parameter. Evidence has emerged that ALS may coexist with distinct neurodegenerative diseases in single cases. We aim to study the clinical features of two familial cases of ALS carriers of two distinct variants harbored in the Optineurin (OPTN) gene. We included definite familial ALS followed up in the Department of Neurology of Razi University Hospital, Tunisia, and selected according to Byrne criteria. Preliminary screening for the four main ALS genes (SOD1, C9ORF72, TARDBP, FUS) was conducted. Given the negative results, we proceeded to NGS target-re-sequencing with a custom panel including genes associated with ALS-FTD, Alzheimer's, and Parkinson's diseases. Both families are carriers of two different OPTN variants and they present very different ALS clinical features. The first family comprises two siblings diagnosed with ALS and Corticobasal syndrome (ALS-CBS) at an early age of onset and carriers of OPTN p.E135X in the homozygous state. The proband for the second family was diagnosed with ALS at an early age of onset presenting as progressive muscular atrophy with rapid progression. Genetic analysis revealed the presence of the homozygous variant p.R520H. Our findings highlight the peculiarity of genetic Tunisian drift. Indeed, genes with a recessive mode of inheritance may explain part of ALS diversity in clinical features. Therefore, the screening of the OPTN gene is highly recommended among inbreeding populations such as the Tunisian one.

Human salivary Raman fingerprint as biomarker for the diagnosis of Amyotrophic Lateral Sclerosis.

Amyotrophic Lateral Sclerosis (ALS) is a neurodegenerative disease leading to progressive and irreversible muscle atrophy. The diagnosis of ALS is time-consuming and complex, with the clinical and neurophysiological evaluation accompanied by monitoring of progression and a long procedure for the discrimination of similar neurodegenerative diseases. The delayed diagnosis strongly slows the potential development of adequate therapies and the time frame for a prompt intervention. The discovery of new biomarkers could improve the disease diagnosis, as well as the therapeutic and rehabilitative effectiveness and monitoring of the pathological progression. In this work saliva collected from 19 patients with ALS, 10 affected by Parkinson's disease, 10 affected by Alzheimer's disease and 10 healthy subjects, was analysed using Raman spectroscopy, optimizing the parameters for detailed and reproducible spectra. The statistical multivariate analysis of the data revealed a significant difference between the groups, allowing the discrimination of the disease onset. Correlation of Raman data revealed a direct relationship with paraclinical scores, identifying multifactorial biochemical modifications related to the pathology. The proposed approach showed a promising accuracy in ALS onset discrimination, using a fast and sensitive procedure that can make more efficient the diagnostic procedure and the monitoring of therapeutic and rehabilitative processes in ALS.

CSF angiogenin levels in amyotrophic lateral Sclerosis-Frontotemporal dementia spectrum.

Objective: Angiogenin (ANG) is a pro-angiogenic and neurotrophic factor with an important role in stress-induced injury, by promoting neovascularization and neuronal survival. Identification of loss-of-function mutations and evidence of beneficial effect of ANG administration in transgenic SOD1G93A mice have linked ANG to the pathogenesis of Amyotrophic Lateral Sclerosis (ALS), stimulating interest in considering circulating ANG levels as an ALS disease biomarker although robust evidence is still lacking. Aim of our study was to assess differences of ANG levels in the cerebrospinal fluid (CSF) of a large cohort of patients with ALS and frontotemporal dementia (FTD) compared to controls and to explore correlations between ANG content and disease-related clinical variables. Methods: ANG levels were measured in CSF samples using a commercially available ELISA kit in 88 patients affected with ALS and/or FTD and 46 unrelated individuals (control group). Results: ANG levels didn't differ significantly between cases and controls. Patients with FTD or ALS-FTD showed significantly increased CSF concentration of ANG compared to ALS patients without dementia and controls in a multivariate regression model (p < 0.001). No correlations were found in ALS/FTD patients between ANG levels and clinical parameters, including age, presence of C9orf72 repeat expansion, body mass index (BMI). Conclusions: our findings highlight a role of ANG as CSF biomarker useful to identify ALS patients with concurrent FTD and suggest that it should be further explored as potential biomarker for FTD.

Cardiovascular diseases may play a negative role in the prognosis of amyotrophic lateral sclerosis.

BACKGROUND AND PURPOSE: Only a few studies have considered the role of comorbidities in the prognosis of amyotrophic lateral sclerosis (ALS) and have provided conflicting results. METHODS: Our multicentre, retrospective study included patients diagnosed from 1 January 2009 to 31 December 2013 in 13 referral centres for ALS located in 10 Italian regions. Neurologists at these centres collected a detailed phenotypic profile and follow-up data until death in an electronic database. Comorbidities at diagnosis were recorded by main categories and single medical diagnosis, with the aim of investigating their role in ALS prognosis. RESULTS: A total of 2354 incident cases were collected, with a median survival time from onset to death/tracheostomy of 43 months. According to univariate analysis, together with well-known clinical prognostic factors (age at onset, diagnostic delay, site of onset, phenotype, Revised El Escorial Criteria and body mass index at diagnosis), the presence of dementia, hypertension, heart disease, chronic obstructive pulmonary disease, haematological and psychiatric diseases was associated with worse survival. In multivariate analysis, age at onset, diagnostic delay, phenotypes, body mass index at diagnosis, Revised El Escorial Criteria, dementia, hypertension, heart diseases (atrial fibrillation and heart failure) and haematological diseases (disorders of thrombosis and haemostasis) were independent prognostic factors of survival in ALS. CONCLUSIONS: Our large, multicentre study demonstrated that, together with the known clinical factors that are known to be prognostic for ALS survival, hypertension and heart diseases (i.e. atrial fibrillation and heart failure) as well as haematological diseases are independently associated with a shorter survival. Our findings suggest some mechanisms that are possibly involved in disease progression, giving new interesting clues that may be of value for clinical practice and ALS comorbidity management.

Novel optineurin mutations in patients with familial and sporadic amyotrophic lateral sclerosis.

BACKGROUND: Optineurin (OPTN), a causative gene of hereditary primary open-angle glaucoma, has been recently associated with amyotrophic lateral sclerosis (ALS) with mainly autosomal recessive, but also dominant, traits. To further define the contribution of OPTN gene in ALS, we performed a mutational screening in a large cohort of Italian patients. METHODS: A group of 274 ALS patients, including 161 familial (FALS) and 113 sporadic (SALS) cases, were screened for OPTN mutations by direct sequencing of its coding sequence. All patients fulfilled the El Escorial criteria for probable or definite ALS and were negative for mutations in SOD1, ANG, TARDBP and FUS/TLS genes. RESULTS: The genetic analysis revealed six novel variants in both FALS and SALS patients, all occurring in an heterozygous state. We identified three missense (c.844A→C p.T282P, c.941A→T p.Q314L, c.1670A→C p.K557T), one nonsense (c.67G→T p.G23X) and two intronic mutations (c.552+1delG, c.1401+4A→G). The intronic c.552+1delG variant determined a splicing defect as demonstrated by mRNA analysis. All mutations were absent in 280 Italian controls and over 6800 worldwide glaucoma patients and controls screened so far. The clinical phenotype of OPTN-mutated patients was heterogeneous for both age of onset and disease duration but characterised by lower-limb onset and prevalence of upper motor neuron signs. CONCLUSION: In this cohort, OPTN mutations were present both in FALS (2/161), accounting for 1.2% cases, and in SALS patients (4/113), thereby extending the spectrum of OPTN mutations associated with ALS. The study further supports the possible pathological role of optineurin protein in motor neuron disease.

Mutational analysis reveals the FUS homolog TAF15 as a candidate gene for familial amyotrophic lateral sclerosis.

FUS, EWS, and TAF15 belong to the TET family of structurally similar DNA/RNA-binding proteins. Mutations in the FUS gene have recently been discovered as a cause of familial amyotrophic lateral sclerosis (FALS). Given the structural and functional similarities between the three genes, we screened TAF15 and EWS in 263 and 94 index FALS cases, respectively. No coding variants were found in EWS, while we identified six novel changes in TAF15. Of these, two 24 bp deletions and a R388H missense variant were also found in healthy controls. A D386N substitution was shown not to segregate with the disease in the affected pedigree. A single A31T and two R395Q changes were identified in FALS cases but not in over 1,100 controls. Interestingly, one of the R395Q FALS cases also harbors a TARDBP mutation (G384R). Altogether, these results suggest that additional studies are needed to determine whether mutations in the TAF15 gene represent a cause of FALS.

Cerebral amyloid angiopathy-related inflammation: an emerging disease.

Three elderly patients with, respectively: mild cognitive impairment, severe and progressive neurologic involvement, and focal neurologic deficit, were observed. MRI showed multiple areas of white matter edema, at times partially involving the cortex, in the first two patients, and a single area in the third. Treatment with steroids determined the disappearance of the lesions and clinical amelioration. The key to the diagnosis of cerebral amyloid angiopathy-related inflammation (CAA-ri) was the demonstration, with appropriate MRI sequences, of microbleeds consistent with cerebral amyloid angiopathy (CAA). This diagnosis was supported by genetic analysis of APOE with demonstration of ε4/ε4 genotype, found in about 80% of CAA patients who develop inflammatory changes. In the appropriate clinical setting, MRI demonstration of microbleeds supported by results of genetic analysis of APOE may strongly support the diagnosis of CAA-ri thus avoiding cerebral biopsy.

Stem cells in amyotrophic lateral sclerosis: motor neuron protection or replacement?

Given the lack of effective drug treatments for amyotrophic lateral sclerosis (ALS), compelling preclinical data on stem cell research has targeted this disease as a candidate for stem cell treatment. Stem cell transplantation has been effective in several animal models, but the underlying biological pathways of restorative processes are still unresolved. Several mechanisms such as cell fusion, neurotrophic factor release, endogenous stem cell proliferation, and transdifferentiation may explain positive therapeutic results in preclinical animal models, in addition to replacement of lost motor neurons. The clinical target in ALS has shifted from being neuroncentered to focus on the interaction between motor neurons and non-neuronal cells (mainly astroglial or microglial). In fact, one of the fundamental unanswered questions in ALS is whether and how much motor neuron death depends on neighboring cells, and how wildtype non-neuronal cells may protect motor neurons expressing an ALS-causing mutation. Lately, motor neuron replacement has been successfully achieved in animal models with reinnervation of the muscle target. Even if many biological issues need to be solved in preclinical models, preliminary stem cell transplantation trials have been performed in ALS patients with conflicting results. The review discusses relevant topics regarding the application of stem cell research to ALS focusing on their therapeutic relevance and mechanisms of action.

Protein aggregation and defective RNA metabolism as mechanisms for motor neuron damage.

The presence of protein inclusions within the central nervous system is a characteristic of most neurodegenerative diseases, including amyotrophic lateral sclerosis (ALS). Aggregates may induce cell death trough several mechanisms, such as sequestration of essential cellular components, clogging of the proteasome system, and/or disruption of axonal transport. The neuropathological signature of ALS is represented by the presence of ubiquitinated inclusions immunoreactive for the protein TDP-43 in the cytoplasm of motor neurons. Recent studies demonstrated that a significant percentage of familial ALS cases are caused by pathogenic mutations in the TAR DNA binding protein and fused in sarcoma/translocated in liposarcoma genes encoding, respectively, for TDP-43 and FUS proteins. Both TDP-43 and FUS are DNA/RNA-binding proteins involved in transcriptional regulation and splicing, shuttling, maturation and transport of mRNA molecules. Mutations in the two genes seem to induce a nucleo-cytoplasmic redistribution of FUS and TDP-43, possibly promoting aggregate formation and/or disrupting their physiological nuclear functions or their interactions with specific RNA targets. Those findings collectively suggest that alterations in cellular RNA metabolism may trigger motor neuron degeneration.

Analysis of FUS gene mutation in familial amyotrophic lateral sclerosis within an Italian cohort.

OBJECTIVE: Mutations in the FUS gene on chromosome 16 have been recently discovered as a cause of familial amyotrophic lateral sclerosis (FALS). This study determined the frequency and identities of FUS gene mutations in a cohort of Italian patients with FALS. METHODS: We screened all 15 coding exons of FUS for mutations in 94 Italian patients with FALS. RESULTS: We identified 4 distinct missense mutations in 5 patients; 2 were novel. The mutations were not present in 376 healthy Italian controls and thus are likely to be pathogenic. CONCLUSIONS: Our results demonstrate that FUS mutations cause approximately 4% of familial amyotrophic lateral sclerosis cases in the Italian population.

Mutations in the FUS/TLS gene on chromosome 16 cause familial amyotrophic lateral sclerosis.

Amyotrophic lateral sclerosis (ALS) is a fatal degenerative motor neuron disorder. Ten percent of cases are inherited; most involve unidentified genes. We report here 13 mutations in the fused in sarcoma/translated in liposarcoma (FUS/TLS) gene on chromosome 16 that were specific for familial ALS. The FUS/TLS protein binds to RNA, functions in diverse processes, and is normally located predominantly in the nucleus. In contrast, the mutant forms of FUS/TLS accumulated in the cytoplasm of neurons, a pathology that is similar to that of the gene TAR DNA-binding protein 43 (TDP43), whose mutations also cause ALS. Neuronal cytoplasmic protein aggregation and defective RNA metabolism thus appear to be common pathogenic mechanisms involved in ALS and possibly in other neurodegenerative disorders.

High frequency of TARDBP gene mutations in Italian patients with amyotrophic lateral sclerosis.

Recent studies identified rare missense mutations in amyotrophic lateral sclerosis (ALS) patients in the TARDBP gene encoding TAR DNA binding protein (TDP)-43, the major protein of the ubiquitinated inclusions (UBIs) found in affected motor neurons (MNs). The aim of this study was to further define the spectrum of TARDBP mutations in a large cohort of 666 Italian ALS patients (125 familial and 541 sporadic cases). The entire coding region was sequenced in 281 patients, while in the remaining 385 cases only exon 6 was sequenced. In 18 patients, of which six are familial, we identified 12 different heterozygous missense mutations (nine novel) all locating to exon 6, which were absent in 771 matched controls. The c.1144G>A (p.A382T) variation was observed in seven patients, thus representing the most frequent TARDBP mutation in ALS. Analysis of microsatellites surrounding the TARDBP gene indicated that p.A382T was inherited from a common ancestor in 5 of the 7 patients. Altogether, the frequency of TARDBP gene mutations appears to be particularly high in Italian ALS patients compared to individuals of mainly Northern European origin (2.7% vs. 1%). Western blot analysis of lymphocyte extracts from two patients carrying the p.A382T and p.S393L TARDBP mutations showed the presence of lower molecular weight TDP-43 bands, which were more abundant than observed in healthy controls and patients negative for TARDBP mutations. In conclusion, this report contributes to the demonstration of the causative role of the TARDBP gene in ALS pathogenesis and indicates that mutations may affect the stability of the protein even in nonneuronal tissues.