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Amyotrophic lateral sclerosis (ALS) is an incurable and fatal neurodegenerative disease. Increasing the chances of success for future clinical strategies requires more in-depth knowledge of the molecular basis underlying disease heterogeneity. We recently laid the foundation for a molecular taxonomy of ALS by whole-genome expression profiling of motor cortex from sporadic ALS (SALS) patients. Here, we analyzed copy number variants (CNVs) occurring in the same patients, by using a customized exon-centered comparative genomic hybridization array (aCGH) covering a large panel of ALS-related genes. A large number of novel and known disease-associated CNVs were detected in SALS samples, including several subgroup-specific loci, suggestive of a great divergence of two subgroups at the molecular level. Integrative analysis of copy number profiles with their associated transcriptomic data revealed subtype-specific genomic perturbations and candidate driver genes positively correlated with transcriptional signatures, suggesting a strong interaction between genomic and transcriptomic events in ALS pathogenesis. The functional analysis confirmed our previous pathway-based characterization of SALS subtypes and identified 24 potential candidates for genomic-based patient stratification. To our knowledge, this is the first comprehensive “omics” analysis of molecular events characterizing SALS pathology, providing a road map to facilitate genome-guided personalized diagnosis and treatments for this devastating disease.

Amyotrophic lateral sclerosis (ALS) is the most common adult-onset motor neuron disease. It is characterized by the neurodegeneration of motor neurons in the brain and spinal cord. The result for patients is the inability to coordinate the voluntary muscular movements and this culminates in the death due to respiratory insufficiency within 2-5 years from the diagnosis (Taylor et al., 2016). Approximately 10% of cases of the disease are classified as familial, while 90% are sporadic, although the two forms are clinically indistinguishable. Many genetic factors have been associated with ALS, but their impact on the pathology has been better characterized for the genes C9ORF72, TARDBP (TDP-43), SOD1 and FUS which, together, represent the cause of about 70% of familial cases. The main molecular consequences linked to these predisposing factors are many; they vary from protein aggregation to alterations of RNA metabolism. In particular, the discovery of a GGGGCC hexanucleotide repeat expansion within chromosome 9 open reading frame 72 (C9ORF72) - the most common genetic mutation responsible for the development of ALS - represents a major advance in ALS disease research because it provided strong support to the concept that RNA metabolism defects are critical and causative factors in the development of the disease (Ling et al., 2013). In normal individuals, the GGGGCC sequence is repeated mostly two-five times (range 0-20), while in patients with C9ORF72-linked ALS, the numbers of repeats exceed 30, and it has been estimated that it can reach several hundred or even thousands of repeats (Rohrer et al., 2015). Multiple pathogenic mechanisms are thought to underlie the pathology associated with the G4C2 expansion: haploinsufficiency due to the loss of protein expression from the mutated allele; RNA toxicity caused by the accumulation of repeat-containing transcripts; and generation of toxic protein species (poly-dipeptides, DPRs) by an unconventional mechanism of protein translation, which is known as repeat-associated non-ATG (RAN) translation (Heutink et al., 2014). Recently, in our lab we observed that the expression of an expanded GGGGCC repeat in cultured cells causes translational repression and a marked nuclear accumulation of poly-adenylated mRNA (Rossi et al., 2015). This suggests that defective trafficking of mRNAs - as a consequence of impaired nuclear mRNA export - might contribute to the pathogenesis of C9ORF72-linked ALS. On this basis, the general aim of this study was to evaluate whether the altered mRNA export characterizing C9ORF72 cells might impact on specific mRNAs with ALS-relevant functions by systematically identify, by RNA sequencing (RNAseq), those mRNAs that are specifically altered in their nucleus/cytosol distribution in cellular models expressing a C9ORF72 mutation. To this aim, we extracted nuclear and cytoplasmic RNAs from cultured cells which express an 31 GGGGCC repeats (G4C2)31 ad from control cells. After checking fraction purity and RNA integrity, RNAs prepared from samples in triplicate were analyzed through mRNA sequencing. After assessing the nucleus/cytosol ratio (N/C) of each identified RNA target for each sample, we compared the N/C ratios of cells expressing expanded repeats to those of controls cells, as a measure of the shift in nuclear vs cytosolic localization of mRNAs. Overall, this analysis led to the identification of 60 mRNAs targets that are significantly accumulated in the nucleus of cells expressing (G4C2)31 repeats, which further supports the concept that mRNA export defects might be major feature of C9ORF72 cells. Moreover, although preliminarily, these results confirms the idea that defects in a general mechanism, such as the nuclear export of mRNAs, might have selective effects on a distinct set of target genes, and that the alteration of these genes might explain the specific degeneration of motor neurons, that is the most typical feature of ALS. However, these data do not clarify whether the observed phenotypes depend upon the toxic action of the accumulated expanded RNA, or if the toxic RNA is assisted by a second pathogenic mechanism linked to its translation into poly-dipeptides (DPRs) within the cell. To clarify this point, the second part of our research focused on the characterization of a human cell model, namely HeLa cells, that transiently express the five individual DPRs, poly-glycine/arginine (GR), poly-proline/arginine (PR), poly-glycine/alanine (GA), poly-glycine/proline (GP), poly-proline/alanine (PA), from synthetic cDNAs encoding 100 repeats of DPR without a GGGGCC repeat. Immunofluorescence analysis showed that all five DPRs, even if in different proportions, are able to induce translational repression associated with the formation of stress granules and, especially in cells expressing poly-GR and poly-PR, this is accompanied by a significant accumulation of polyadenylated mRNAs and associated proteins such as PABPc in cells nuclei. The obtained data indicate that the expression of DPRs is sufficient to generate the molecular phenotypes characterizing G4C2 repeat-expressing cells, suggesting that the presence and accumulation of expanded RNAs in cells might be dispensable for C9ORF72 toxicity, or at least that DPRs co-operate with G4C2 RNA in impairing the proper nucleus/cytosol trafficking of mRNAs, thus contributing to the pathogenicity C9ORF72 mutation. In conclusion, we have collected further evidence that alterations in nucleus/cytosol transport play a crucial role in the pathogenesis of SLA, and further studies will be needed to understand the details closely associated to this mechanism.

La Sclerosi Laterale Amiotrofica (SLA) è una malattia neurodegenerativa che colpisce i motoneuroni (MN). Evidenze sperimentali indicano che fattori rilasciati dalla glia reattiva possono contribuire alla morte dei MN. Il nostro gruppo ha recentemente dimostrato i) che l'espressione dell'endotelina-1 (ET-1), un vasopeptide prodotto nel SNC da astrociti e microglia, è aumentata negli astrociti reattivi nel midollo spinale di pazienti affetti da SLA e di topi che sovra-esprimono la forma mutata della superossido-dismutasi 1, e ii) che l'ET-1 è tossica per i MN in coltura (Ranno et al., Neurobiol Dis 2014; 65:160-71). Abbiamo pertanto studiato, in un modello in vitro, i possibili meccanismi coinvolti nella tossicità selettiva di ET-1 per i MN, focalizzando su vie di trasduzione coinvolte nella sopravvivenza cellulare, stress ossidativo e processi infiammatori. I nostri dati evidenziano che l'effetto tossico di ET-1 non è mediato dallo stress ossidativo, ma è associato ad una ridotta attivazione della via di sopravvivenza fosfatidil-inositolo 3 chinasi e ad una concomitante inibizione dell'enzima nitrossido-sintasi. Abbiamo inoltre osservato che ET-1 è tossica anche per la microglia, ma il suo effetto sui MN non dipende da questo tipo di cellule gliali. Il nostro studio conferma che ET-1 può concorrere alla morte dei MN nella SLA, e ne identifica i meccanismi responsabili, suggerendo che la modulazione del signaling di ET-1 potrebbe essere una strategia terapeutica per la SLA.

Amyotrophic lateral sclerosis (ALS) is an adult-onset neurodegenerative disease that affects both lower motor neurons i n the brainstem and spinal cord, and the upper motor neurons i n the motor cortex. Loss of these neurons leads to muscle atrophy and weakness, fasciculations and spasticity and death from respiratory failure, typically within 2 to 3 years from symptom onset. About 10% of ALS is classified as familial (fALS), whereas the remaini ng 90% of cases are considered sporadic (sALS), as they appear to occur randomly throughout the community (Renton et al., 2014). fALS is associated with a wide range of genes that are autosomal dominant or recessive mutations.The recent discovery of mutations in TARDBPand FUSgenes, responsible for about 10% of casesof fSLA, focused the attention on a possible pathogenic role of RNA metabolism, which was observedto bealtered also i n other neurodegenerati ve diseases. Moreover, a non-coding G4C2 repeat expansion in the C9orf72 gene has been identified in 2011 as the major genetic determi nant of ALS. In normal individuals the G4C2 sequence is repeated mostly two-to-fi ve time (range 0-20), whi le in patients with ALS associated to the gene C9orf72the number of repeats exceeds 30, and it has been estimated that it can reach several hundred or even thousands of repeats (Robberecht and Philips, 2013). The understanding of the disease mechanisms caused by the repeat expansion is still i n an early stage. Three main hypotheses are currently being considered to explai n the disease process, including: haploinsufficiency due to the loss of protei n expression from the mutated allele, RNA toxicity caused by the accumulation of repeat containing transcripts,and toxic protei n species generated by repeat-associated non-ATG (RAN) translation (Heuti nk et al., 2014). RNAs containi ng the repeat, which accumulate as RNA foci in the nucleus and/or cytoplasm of affected cells, are able to bind to various RNA-binding proteins, possibly impairing their function. Recently, in our lab we observed that the expression of pathological repeats, which induces the formation of nuclear RNA foci, causes stress granules formation, reduction of protein translation and a marked nuclear accumulation of poly-adenylated mRNAs in cultured cells.So the aim of this research was to i nvestigate how the expansion of the hexanucleotide G4C2 repeat might induce neurodegeneration and, in particular, the functional consequences of (G4C2)31expression on nuclear mRNA export pathway and protei n translation in cultured cells. 6As a cellular model we choseHeLa cells transfected with plasmids contai ning pathological (G4C2)31or non-pathological (G4C2)10repeat.The G4C2 sequence is located within an intron,so in this study wedecided tointroduce the intronic sequences of ?-actin gene upstream (i ntron 5') and downstream (i ntron 3') of G4C2 repetitions to make our study model as close as possible to the physiologicalcondition. Fluorescence in situ hybridization (FISH) analysis showed that the expression of the pathological expanded repeatwithi n an i ntronic sequenceleads to the formation of nuclear RNA foci similar to those found i n ALS-C9orf72 patients and that are able to sequester some RNA binding protei ns (RBP). Furthermore, as previously observed, the expression of the intronic(G4C2)31iis able to induce a translational repression i n HeLa cells associated with the formation of stress granules, and accompanied by a considerable accumulation of polyadenylated mRNA in the cell nuclei. We then analyzed in vitro, using pull down assays, andin cells, using fluorescent in situhybridization and immunofluorescence analysis, the ability of the expanded repeat to interact with some key proteins involved in the mRNA nuclear export pathway, specifically NXF1 and ALY/REF. This analysis shows that the (G4C2)31irepeat is able to bind both these protei ns and sequester them into RNA foci,thus possibly i nterfering with their functions and leading to the nuclear retention of poly-adenylated mRNAs.To test this possibility we analyzed the effects of the expanded repeat on the mRNA export mechanism and the role of NXF1 through the use of plasmids coding for a dominant-negati ve mutant form NXF1 (?NXF1) and NXF1 wild type. Interestingly, the overexpression of?NXF1, which causes nuclearmRNA accumulation, reproduces key phenotypes characteri zing C9orf72 cells, including stress granules formation and translational repression. Moreover, the overexpression of wild-type NXF1 is able to reduce G4C2-induced stress granules.In conclusion, this research i ndicates that the expanded repeat G4C2 interferes withthe NXF1-mediated mRNA export pathway, and the resulti ng nuclear mRNA retention might affect translation efficiency and contribute to the pathogenesis of C9orf72-ALS.

Il recente perfezionamento delle tecnologie di caratterizzazione molecolare ha permesso di monitorare i diversi livelli del flusso di informazione biologica. Tali tecnologie, dette -omiche, hanno consentito lo sviluppo di una tassonomia molecolare delle patologie, che permette di complementare la tradizionale classificazione basata su segni e sintomi con le informazioni ottenute dallo studio dei meccanismi molecolari e dei fattori ambientali coinvolti nei processi ezio-patogenetici. L'oncologia rappresenta l'esempio più interessante di come le scienze -omiche hanno rivoluzionato la comprensione, la diagnosi ed il trattamento delle patologie. Progresso tuttavia modesto se applicato alla caratterizzazione di malattie complesse, quali le patologie neurodegenerative. Il nostro gruppo di ricerca ha recentemente caratterizzato i profili trascrizionali di corteccia motoria di pazienti con SLA di tipo sporadico, differenziando questi ultimi in due sottogruppi associati a specifici geni e pathway deregolate ed identificando potenziali biomarcatori e target farmacologici. In questo studio ci proponiamo di integrare e comparare tra loro dati provenienti da pazienti SLA ottenuti utilizzando differenti piattaforme e discipline "-omiche". L'analisi simultanea di tali dati potrebbe consentire una migliore caratterizzazione molecolare ed una più precisa ed accurata classificazione dei pazienti SLA, favorendo lo sviluppo di una migliore diagnosi e terapie sempre più efficaci e personalizzate.

La Sclerosi Laterale Amiotrofica (SLA) è una malattia neurodegenerativa progressiva ed incurabile, caratterizzata da un'ezio-patogenesi complessa ed ancora oggi sconosciuta. Il perfezionamento di tecnologie ad alta efficienza, quali il sequenziamento genomico e tecnologia microarray ha permesso lo sviluppo di un approccio di "system biology", nel quale ad assumere importanza non è più il singolo gene ma l'interazione tra tutti i componenti costituenti le diverse pathway cellulari. Il nostro gruppo di ricerca ha recentemente caratterizzato i profili trascrizionali di corteccia motoria di pazienti con SLA di tipo sporadico, differenziando questi ultimi in due sottogruppi associati a specifici pathway e geni differenzialmente espressi. Molti di questi geni deregolati codificano per potenziali target farmacologici attualmente in corso di studio preclinico o clinico per diverse patologie neurodegenerative. Al fine di consentire una più accurata selezione dei target da sottoporre a validazione preclinica su modelli animali SLA, in questo studio sono stati comparati i profili di espressione dei pazienti SLA con quelli di campioni di midollo spinale ottenuti da topi transgenici SOD1G93A. Questo lavoro ha permesso di identificare geni e processi biologici comunemente deregolati nell'uomo e nel modello animale che potrebbero costituire target farmacologici promettenti per la messa a punto di efficaci studi preclinici e la successiva traslazione nei trials clinici.

Summary Background The cause of sporadic ALS is currently unknown. Despite evidence for a role for genetics, no common genetic variants have been unequivocally linked to sporadic ALS. We sought to identify genetic variants associated with an increased or decreased risk for developing ALS in a cohort of American sporadic cases. Methods We undertook a genome-wide association study using publicly available samples from 276 patients with sporadic ALS and 271 neurologically normal controls. 555 352 unique SNPs were assayed in each sample using the Illumina Infinium II HumanHap550 SNP chip. Findings More than 300 million genotypes were produced in 547 participants. These raw genotype data are freely available on the internet and represent the first publicly accessible SNP data for ALS cases. 34 SNPs with a p value less than 0·0001 (two degrees of freedom) were found, although none of these reached significance after Bonferroni correction. Interpretation We generated publicly available genotype data for sporadic ALS patients and controls. No single locus was definitively associated with increased risk of developing disease, although potentially associated candidate SNPs were identified.

Motor neuron disease (MND) is a heterogeneous family of neurodegenerative diseases, rare, characterized by an early involvement of motor neurons and can be hereditary or sporadic, including Amyotrophic lateral sclerosis (ALS) and X-linked spinal-bulbar muscular atrophy (SBMA). Three studies will be briefly presented below, with the aim of developing an understanding of psychological and neuropsychological aspects in these diseases. In Chapter 3 is presented a retrospective study with the aim to verify whether the presence of cognitive impairment in ALS patients (according to the classification proposed by Phukan et al., 2012) may have an impact on the degree of disease progression in terms of motor decline. Up to date, in literature the debate about the influence of cognitive impairment on survival in ALS patients is still ongoing and results are conflicting. In summary, after 6 or 12 months follow up we observed the same degree of disease progression both in patients with presence or absence of cognitive impairment. In Chapter 4 it is presented a pilot study with the aim to investigate the applicability and efficacy of a hypnotic treatment on the physical and psychological well-being perceived by ALS patients, considering also the indirect effect on the well-being on caregivers. ALS implies an inevitable and devastating psychological impact and this study represent, even if at a preliminary level, the first application of a protocol of psychological intervention with evidence of efficacy in ALS patients. In Chapter 6, it is presented a study with the aim to investigate the neuropsychological and psychological profile in patients with SBMA, poorly characterized in the recent literature. In summary, in contrast with the previous literature, executive functioning seems to be apparently preserved in patients with SBMA; nevertheless patients seem to have a specific dysfunction on the cognitive component of the Theory of Mind (ToM), while the affective component seems to be preserved.

Summary The cause of amyotrophic lateral sclerosis (ALS) is still unknown. A possible relationship between ALS and sport participation has been supposed, but never definitely demonstrated. We studied a cohort of 7325 male professional football players engaged by a football team from the Italian First or Second Division in the period 1970‐2001. ALS cases were identified using different concurrent sources. Standardized morbidity ratios (SMRs) were calculated. During the 137 078 person-years of followup, five ALS cases were identified (mean age of onset, 43.4 years). Three cases had a bulbar onset, significantly more than expected (P = 0.003). Since the number of expected cases was 0.77, the overall SMR was 6.5 [95% confidence interval (CI), 2.1‐15.1]. The SMR was significantly increased for an ALS onset before 49 years, but not for older subjects. A significant increase of the SMR was found in the periods 1980‐1989 and 1990‐ 2001, whereas no ALS case was found in the 1970‐1979 period. A dose‐response relationship between the duration of professional football activity and the risk of ALS was found (>5 years, 15.2, 95% CI, 3.1‐44.4

Background: Understanding the determinants of quality of life (QoL) in amyotrophic lateral sclerosis (ALS) has become increasingly important with the recent emphasis on the comprehensive management of patients. Objective: To evaluate the determinants of QoL in ALS using two scales with different theoretical constructs: the Schedule for the Evaluation of QoL-Direct Weighting (SEIQoL-DW), which evaluates subjective aspects of QoL, and the McGill QoL Questionnaire (MQOL), which evaluates both health related and non-health related factors of QoL. Methods: Eighty consecutive patients with ALS underwent a battery of tests evaluating QoL and a series of physical, emotional, psychological, and socioeconomic predictor variables. A stepwise linear regression model was used to compare QoL scores and explicatory variables. Results: SEIQoL-DW score was related to social support, depression, religiosity, and socioeconomic status. Total MQOL score was related to social support, socioeconomic status, and clinical status. MQOL single item score (MQOL-SIS) was related to social support, depression, social withdrawal, and socioeconomic status. SEIQoL-DW score was not related to total MQOL score. Conversely, a significant correlation was found between SEIQoL-DW and MQOL-SIS. Conclusions: With both QoL scales, the most important explicatory variable of QoL was the self perceived quality of social support. Physical status was not relevant in determining QoL. This study indicates that health related QoL measures are not adequate to assess QoL in patients with ALS, because their appreciation of QoL mainly relies on psychological, supportive, and spiritual factors. Therapeutic interventions should consider the psychological needs of patients and pay greater attention to caregivers' issues.

A 34-years-old floor-layer developed optic neuropathy and motor neuron disease after being accidentally exposed to a solvent mixture containing methanol and other substances. Optic neuropathy is a complication of methanol poisoning, but the onset of a motor neuron disorder resembling amyotrophic lateral sclerosis after the exposure to these substances has not been previously described. The temporal onset of the clinical symptoms, biological plausibility, young age of the patient and absence of neurological disorders in the family history raises suspicion of a possible causative relationship.

Significant changes in the epidemiology of amyotrophic lateral sclerosis (ALS) have been observed over the last few years. Based on reports of studies from the 1970s and early 1980s, the incidence of ALS in Europe has been considered to be constant, with only slight differences in the incidence of ALS between different countries. However, flaws in the methodologies of these studies, such as case definition, case ascertainment, selection bias, study design and difficulties in the early diagnosis of cases, have been identified. More recent studies reveal a trend towards an increase in the ALS mortality rate over the past two decades, but the significance of this trend remains unclear. Recent ALS incidence studies, particularly those based on the 'register model', have given new insight into ALS epidemiology. ALS seems to be a uniform problem across Europe, with age-specific incidence rates showing a progressive increase up to the 70-79 age group. False-negative cases are frequent, ranging from 26-42% of cases. The rate of false-positive cases is approximately 10%. In approximately half of the false-positive cases, the possible diagnosis of ALS was withdrawn because the initial symptoms did not progress. No consistent epidemiological clues concerning the cause of ALS have been found in epidemiological studies. The only possible associations that perhaps warrant further investigation include farming, mechanical and electrical traumas, heavy labour and toxins or chemicals.

Questo studio ha riguardato l'analisi di alcune variabili prognostiche e di sopravvivenza nella sclerosi laterale amiotrofica, con particolare riferimento alla caratterizzazione di nuovi geni ed al ruolo di interventi di management.

While the physiologic functions of the RNA-binding protein FUS still await thorough characterization, the pathonegetic role of FUS mutations in amyotrophic lateral sclerosis (ALS) is clearly established. Here we find that a human FUS mutation that leads to increased protein expression, and was identified in two ALS patients with severe outcome, maps to the seed sequence recognized by miR-141 and miR-200a in the 3'-UTR of FUS. We demonstrate that FUS and these microRNAs are linked by a feed-forward regulatory loop where FUS upregulates miR-141/200a, which in turn impact FUS protein synthesis. We also show that Zeb1, a target of miR-141/200a and transcriptional repressor of these two microRNAs, is part of the circuitry and reinforces it. Our results reveal a possible correlation between deregulation of this regulatory circuit and ALS pathogenesis, and open interesting perspectives in the treatment of these mutations through ad hoc-modified microRNAs.

Enteral nutrition may be required in amyotrophic lateral sclerosis (ALS), and is usually achieved by percutaneous endoscopic gastrostomy (PEG). As PEG is not indicated in patients with severe respiratory impairment, an alternative is percutaneous radiological gastrostomy (PRG), involving air insufflation into the stomach under fluoroscopic guidance for tube insertion.To evaluate the safety of PRG and its effect on survival and respiratory function in ALS patients with respiratory failure.25 consecutive ALS patients with severe dysphagia and forced vital capacity (FVC)50% underwent PRG after October 2000. They were compared with 25 consecutive ALS patients with FVC50% who underwent PEG before October 2000. Respiratory function was evaluated before and after the procedure.The two groups were similar for all relevant characteristics. PRG was successful in all cases, PEG in 23/25. One patient in each group died after the procedure. The mean survival time after the procedure was 204 days in the PRG group and 85 days in the PEG group (p0.004). Respiratory function decreased more in the PEG group than in the PRG group (p0.02).PRG appears to be safer than PEG in ALS patients with moderate or severe respiratory impairment, and is followed by a longer survival.

The exact role of environmental risk factors in the etiology of the neurodegenerative disease amyotrophic lateral sclerosis (ALS) is still unknown. Their hypothetical contribution ranges from a minimal impact to a major role. Among the environmental factors strictu sensu (i.e., not life-style factors) suspected to play a role in ALS etiology, we consider pesticides, the metalloid selenium, some heavy metals, magnetic fields and cyanobacteria. However, the possibility exists that these factors exert their activity only in genetically susceptible persons and only after long-term exposures, thus further hampering epidemiologic studies. The recent availability of powerful tools such as population-based ALS registries for case ascertainment and clustering detection, and of environmental modeling techniques and of geographical information systems, may yield unique opportunities for offering insight into the etiology of the disease.

Our objective was to correlate skeletal muscle mass (SM) with cardiopulmonary exercise testing (CPET) descriptors of exercise capacity in patients with amyotrophic lateral sclerosis (ALS) and compare ALS CPET data with those of patients with mitochondrial myopathy (MM) and normal subjects (N). Twenty-four early-stage ALS patients (63±11 years) underwent bioelectrical impedance analysis of body composition, resting spirometry, and ramp CPET. Six MM and six N were used as controls (56 ± 7 and 63 ± 4 years, respectively). Results showed that ALS SM index was similar to that of N (9.0±2.1 kg/m(2) vs. 10.4±1.9 kg/m(2), respectively; p = n.s.), whereas peak VO(2)/kg SM was significantly lower (41.5 ± 11.6 ml/kg/min vs. 57.8 ± 7.5 ml/kg/min, respectively; p < 0.01). However, the heart rate/VO(2) slope did not differ between ALS and N, being significantly higher in MM than in both ALS and N (6.1 ± 1.4 beats/ml/kg/min vs. 4.2 ± 1.1 beats/ml/kg/min vs. 3.8 ± 2.0 beats/ml/kg/min, respectively; both p < 0.01), excluding a marked skeletal muscle metabolic impairment in ALS. Neither cardiovascular nor ventilatory dysfunction was detected in ALS. Early-stage ALS patients show a SM similar to N, but with a reduced peak VO(2)/kg SM. Such a reduced peripheral O(2) utilization is consistent with deconditioning as the main cause of impaired exercise capacity in this population.

FISIOTERAPIA E MALATTIA DI PARKINSON La natura degenerativa della Malattia di Parkinson prevede il progressivo declino delle abilità motorie e funzionali. L’evoluzione clinica della malattia è eterogenea, così come il conseguente grado di disabilità. La maggior parte delle persone con la Malattia di Parkinson ha crescenti difficoltà nei trasferimenti, nelle posture, nell’equilibrio, nel cammino e nelle attività di vita quotidiana. Questo porta precocemente alla perdita di indipendenza, con conseguente restrizione alla partecipazione della vita sociale. Le limitazioni motorie difficilmente si riducono mediante le terapie farmacologica o neurochirurgica ed è spesso prescritta la fisioterapia. Tuttavia, non vi sono attualmente prove sufficienti per sostenere o smentire l’efficacia delle differenti tecniche di fisioterapia nella Malattia di Parkinson. L’obiettivo della fisioterapia per le persone con Malattia di Parkinson è quello di migliorare la qualità della vita attraverso il mantenimento dell’autonomia, della sicurezza e del benessere. Questo risultato è ottenuto attraverso la prevenzione della limitazione delle attività, delle cadute, delle lesioni da decubito e dal miglioramento funzionale delle attività. FISIOTERAPIA E SCLEROSI LATERALE AMIOTROFICA A tutt’oggi non è nota alcuna cura realmente efficace che conduca alla risoluzione dei sintomi della Sclerosi Laterale Amiotrofica. Ciò che si conosce e che viene attualmente praticato è un intervento riabilitativo multidisciplinare, allo scopo di preservare la funzionalità più a lungo possibile, evitare danni secondari e terziari, migliorare la qualità della vita dei malati ed, in alcuni casi, rallentare l’evoluzione della malattia, garantendo anche una più lunga sopravvivenza. La fisioterapia ha pertanto il compito di seguire la persona durante lo sviluppo della patologia, modificando i propri obiettivi e le proposte terapeutiche in ogni sua fase, al fine di ritardare l’evoluzione dei sintomi. FISIOTERAPIA E MALATTIA DI ALZHEIMER La Malattia di Alzheimer, la più frequente causa di demenza, è un disturbo neurodegenerativo progressivo e irreversibile caratterizzato sia da deficit cognitivo-comportamentali sia neuromotori. La coesistenza di alterazioni a carico di memoria, orientamento spazio-temporale ed equilibrio, la malnutrizione e la depressione, concorrono nel determinare il progressivo declino delle caratteristiche strutturali (ipotrofia muscolare, incremento della fragilità ossea) e funzionali (riduzione della forza e resistenza muscolare, deficit nella deambulazione) a carico dell’apparato muscolo scheletrico, insieme alla riduzione della capacità di autonomia nello svolgimento delle attività della vita quotidiana (ADL), comprendendo sia le ADL di base (bagno, igiene, vestirsi, mangiare, trasferimenti) sia le ADL strumentali (pulizia casa, acquisti, preparazione pasti, lavanderia). Inoltre le alterazioni a carico di equilibrio e orientamento spazio-temporale, i deficit stenici e l’incremento della fragilità ossea, predispongono questi individui ad un aumentato rischio per cadute e fratture, rispetto ai soggetti paragonabili per caratteristiche demografiche e di salute fisica. Sulla base di quanto esposto appare evidente che un miglioramento delle condizioni fisiche nei soggetti affetti dalla Malattia di Alzheimer può contribuire ad un incremento dell’autonomia, della loro qualità di vita e riduzione del rischio per cadute e fratture, nonostante la progressione della malattia.

La sclerosi laterale amiotrofica (SLA) è una malattia ad esito invariabilmente fatale che porta alla degenerazione progressiva e selettiva dei motoneuroni. Solo nel 10-20% delle forme familiari l’eziologia è conosciuta ed è correlata alla mutazione del gene della superossido dismutasi. In tutti gli altri casi, l’eziologia è sconosciuta e questo è un limite sia per la formulazione di una diagnosi precoce che per lo sviluppo di terapie efficaci. Attualmente la diagnosi di SLA si basa su criteri clinici che nella maggior parte dei casi sono evidenti solo a malattia conclamata, non permettendo un intervento tempestivo con farmaci neuroprotettivi. È quindi necessaria l’identificazione di marcatori precoci di malattia che siano quanto più possibile specifici e facilmente misurabili. L’obiettivo di questo studio è stato quello di analizzare, mediante un approccio proteomico, campioni biologici provenienti da pazienti affetti da SLA, con l’intento di comprendere le basi molecolari della malattia, ma anche di identificare possibili indicatori diagnostici. In una prima fase del progetto di ricerca, è stata condotta un’analisi proteomica differenziale con tecniche su base fluorescente (DIGE) e redox tra i PBMC (Peripheral Blood Mononuclear Cells) ottenuti da pazienti con la SLA e individui controllo sani. I PBMC (costituiti in gran parte da monociti e linfociti T/B) sono facilmente reperibili attraverso prelievi sanguigni e a differenza del siero possiedono un complemento proteico più facilmente analizzabile. E’ stato recentemente dimostrato come i linfociti di pazienti SLA esibiscano modifiche associate allo stress ossidativo che ricalcano i meccanismi riscontrati nel sistema nervoso, rappresentando perciò un mezzo utile per lo studio dei meccanismi patogenetici nell’uomo. Inoltre l’infiltrazione di linfociti T, macrofagi e cellule dendritiche osservata a livello del CNS dei pazienti SLA farebbe supporre una comunicazione diretta tra le cellule del sistema immunitario e i neuroni e/o microglia, probabilmente coinvolta nella patogenesi della malattia. I pazienti SLA sono stati reclutati suddividendoli a seconda dello stadio di malattia sulla base dello score di funzionalità motoria. L’analisi proteomica differenziale ha consentito di identificare le proteine differenziali mediante spettrometria di massa. I risultati ottenuti sono stati validati su un numero di soggetti singoli compatibile con un’analisi statistica logistica univariata mediante saggio dot blot e la successiva combinazione di alcuni dei parametri biometrici analizzati ha consentito di discriminare tra pazienti SLA e soggetti sani con un buon grado di sensibilità e specificità. Successivamente, è risultato importante valutare l’accuratezza predittiva del set di proteine selezionate nel diagnosticare in vitro la SLA rispetto ad una popolazione di controllo costituita da pazienti affetti da PD e pazienti affetti da MS. I risultati ottenuti hanno permesso non unicamente di discriminare le tre malattie neurodegenrative reciprocamente ma anche di creare delle correlazioni funzionali tra le stesse. Lo sviluppo di tecniche proteomiche appropriate ha consentito, inoltre, di valutare il livello di nitrazione proteica in condizioni fisiologiche e patologiche. La nitrazione proteica sembra possedere un ruolo cruciale nello sviluppo e nella progressione di diverse malattie neurodegenrative. Precedentemente considerato come un epifenomeno associato alle ultime fasi di una patologia, sembra invece rappresentare un evento primario nelle lesioni di malattie come il PD, l’AD e la SLA. Mediante un approccio proteomico, sono stati identificati specifici target proteici suscettibili di nitrazione nei PBMC e nel CSF di pazienti affetti da SLA rispetto ai soggetti sani. Tali proteine potrebbero non unicamente offrire valide informazioni sullo sviluppo e la progressione della SLA ma anche rappresentare dei possibili target diagnostici e terapeutici della malattia. Il progetto è stato condotto in collaborazione con la Dott.ssa Bendotti dell’Istituto Mario Negri di Milano e con il Dr. Mora e il Dr. Dr. Callea della Fondazione Salvatore Maugeri.

A role for metal-mediated oxidative stress in the pathogenesis of amyotrophic lateral sclerosis (ALS) was proposed in 1994 in the first studies of familial ALS mutant superoxide dismutase 1, and interference with iron homoeostasis is now postulated.1 The HFE gene on chromosome 6 is a mean corpuscular haemoglobin class I-like molecule related to iron regulation. Mutations in the coding region cause hereditary haemochromatosis, a common autosomal recessive disorder of iron metabolism that leads to iron overload in adulthood. Recent reports on HFE mutations in ALS showed contradictory results. Two studies described a higher prevalence of the HFE mutations in ALS than in the control group, and one study did not find any difference between the patients with ALS and the control group.2–4 We analysed a series of Italian patients with ALS to investigate whether mutations in the HFE gene could represent a risk factor for ALS. A total of 149 sporadic Italian patients with ALS (mean (standard deviation (SD)) age 59.4 (9.7) years) according to El Escorial criteria for clinically definite or probable ALS were consecutively recruited to this study. Control samples were obtained from 168 healthy people, matched by age (difference of 5 years), sex and …

Sixteen participants fromeight countries (France,Germany, Ireland, Italy, The Netherlands, Poland, United Kingdom, and the USA) met in Naarden, The Netherlands to review the nutritional needs of patients suffering from amyotrophic lateral sclerosis and to define a protocol for interventional studies for optimal nutrition in ALS. Other aims of the workshop included improving the nutritional support given by PEG and RIG for the ALS patient and to identify any other nutritional issues relevant for the health of patients with ALS.

Background A new locus for amyotrophic lateral sclerosis – frontotemporal dementia (ALS-FTD) has recently been ascribed to chromosome 9p. Methods We identified chromosome 9p segregating haplotypes within two families with ALS-FTD (F476 and F2) and undertook mutational screening of candidate genes within this locus. Results Candidate gene sequencing at this locus revealed the presence of a disease segregating stop mutation (Q342X) in the intraflagellar transport 74 (IFT74) gene in family 476 (F476), but no mutation was detected within IFT74 in family 2 (F2). While neither family was sufficiently informative to definitively implicate or exclude IFT74 mutations as a cause of chromosome 9-linked ALS-FTD, the nature of the mutation observed within F476 (predicted to truncate the protein by 258 amino acids) led us to sequence the open reading frame of this gene in a large number of ALS and FTD cases (n = 420). An additional sequence variant (G58D) was found in a case of sporadic semantic dementia. I55L sequence variants were found in three other unrelated affected individuals, but this was also found in a single individual among 800 Human Diversity Gene Panel samples. Conclusion Confirmation of the pathogenicity of IFT74 sequence variants will require screening of other chromosome 9p-linked families.

Amyotrophic lateral sclerosis (ALS) is caused by mutations in the gene for Cu/Zn superoxide dismutase (SOD1) in 10% of familial and sporadic cases. During the SOD1 analysis of 9 FALS and 121 SALS, in only one sporadic case we found the exonic mutation N19S; in 15 SALS patients we found a 319ta variation in IVS1 sequence, at 108 bp upstream from exon 2. This variation has an unusually high frequency of 11% and is always in linkage disequilibrium with a described polymorphism in IVS3, +34ac. The 319ta variation is classified in two different public databases, HGMD and The ALS Online Database, as a splicing mutation and not as a polymorphism. The unusually high frequency of this mutation in our patients prompted us to determinate its frequency in 130 age- and gender- matched healthy controls and in 54 patients with Alzheimer's disease. We found again linkage disequilibrium with the polymorphism in intron 3, and the frequency of 11% and 7.8%, respectively. These results strongly support the idea that the IVS1 +319 ta alone is not an ALS causing mutation, and that special care must be taken in the interpretation of data from mutations databases for correct genetic counselling.

In this review we evaluate clinical neurophysiological methods, originally described for use in diagnosis that can be applied to measurement of change during the progress of amyotrophic lateral sclerosis (ALS). Such measurements are potentially important in clinical trials, and also in clinical practice. We have assessed methods for lower and upper motor neuron function, including conventional EMG, nerve conduction and F-wave studies, the derived Neurophysiological Index, motor unit counting methods (MUNE), and transcranial magnetic motor cortex stimulation. We have also addressed the validity of measurements of electromechanical coupling. Methods for measuring muscle strength are beyond the scope of this review. We conclude that MUNE, M-wave amplitude and the Neurophysiological Index are sufficiently reliable, sensitive, and relevant to the clinical problem of ALS, to be used in clinical trials in the disease. Transcranial magnetic stimulation is of limited value, but a combination of the measurements made as part of this technique may also be useful. We conclude that clinical neurophysiological techniques should now be used in measuring change in clinical trials in ALS.

Department of Neuroscience, Institute for Pharmacological Research ‘Mario Negri’, Milan, Second Division of Neurology, Department of Neuroscience, University of Turin, Turin, Directorate of Health Programming, Ministry of Health, Rome, AISLAItalian Association for Amyotrophic Lateral Sclerosis, Novara, Italian National Institute of Health, Rome, Department of Neurology, Ospedale Maggiore of Novara, Novara, Unit of Neuro-rehabilitation 2, Salvatore Maugeri Foundation, Institute of Care and Research, Pavia, Directorate of Disease Prevention, Ministry of Health, Rome, Italy, Department of Neurology, University Hospital Gasthuisberg, University of Leuven, School of Medicine, Leuven, Belgium, Department of Neurology and Laboratory of Neuroscience, University of Milan Medical School, IRCCS, Istituto Auxologico Italiano, Milano, and Molecular and Genetic Epidemiology Unit, Ospedale Policlinico IRCCS, Milano, Italy Amyotrophic Lateral Sclerosis. 2005; 6: 5–7

The determinants of ALS caregiver burden and the feeling of the patients as being a burden were assessed using the Caregiver Burden Inventory and the Self-Perceived Burden Scale in 60 caregiver-patient couples. Caregiver burden was correlated to their level of depression and quality of life and, differently from other chronic disorders, increased with the worsening of patients' disability. ALS patients have a good objective perception of their impact on caregivers.

Calcineurin (CaN) is a Ser/Thr protein phosphatase involved in a wide range of cellular responses to calcium mobilizing signals. Previous evidence supports the notion that calcineurin is oxidatively inhibited by mutant Cu, Zn superoxide dismutase (SOD1) typical of familial ALS patients in vitro and in transgenic mice. We report that calcineurin activity is markedly inhibited in lymphocytes from 37 sporadic, eight familial ALS patients and an asymptomatic subject carrying an SOD1 mutation as compared to 28 healthy controls. Two other healthy subjects, heterozygous for the D90A mutation from a recessive pedigree, have normal calcineurin activity. Immunoreactive CaN protein, age, sex and riluzole treatment are not related to calcineurin activity in samples from patients. However, we have observed a marked increase in total protein oxidation in extracts from ALS lymphocytes, as compared to extracts from control subjects. These data confirm that modification of calcineurin activity and possibly of calcineurin-mediated pathways of signal transduction (including modulation of apoptotic neuronal death) may contribute to the pathogenesis of ALS.

Disease penetrance in amyotrophic lateral sclerosis associated with mutations in the SOD1 gene.

Diagnosis communication is a mainstay of the patient-physician relationship and in ALS represents the beginning of the treatment. In Europe, the diagnosis is now communicated to most patients, but with some differences from north to south. Communication is the basis of the patient's autonomy. When adequately informed, patients are able to consider their preferences about life-extending interventions. However, this discussion is sometimes entertained too late. Both the style and the context of this discussion have profound impact on patient satisfaction. The content of the communication should include all relevant information about the disease and should be performed honestly while preserving hope. Collusion with relatives can create difficulties in the relationship with a patient and isolate them from their family. Diagnostic communication is an ongoing process and should form part of every follow-up visit. Communication with the dying patient is often reinforced by non-verbal messages, acknowledgements of patients' emotions, and by listening. Written material may increase satisfaction of patients and their relatives, but should never substitute for face-to-face discussion.

Pregnancy in women with ALS is rare and is generally considered a potentially dangerous event. We describe four ALS cases associated with pregnancy, together with a review of the literature. Three of the four women described developed ALS during pregnancy. In three cases a normal delivery was performed, with a healthy child. One patient, with severe respiratory failure, underwent an interruption of pregnancy. Seven other cases are reported in the literature, featuring a total of 11 pregnancies. The association between pregnancy and ALS is quite rare, and a pathogenic relationship cannot be excluded. The pregnancy and the delivery may be normal, but respiratory function should be carefully monitored. Generally, ALS does not have deleterious effects on fetal development. However, pregnancy in a woman with severe respiratory failure may precipitate the disease.