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Sessione Poster

Guarda tutti gli abstract sottomessi per la sessione poster. 

 

P1. Role of SUMOylation in TDP-43 nucleocytoplasmic transport and aggregation
Researchers
Anna Maria Maraschi (1), Valentina Gumina (1), Claudia Colombrita (1), Jessica Dragotto (2), Miguel Mompeán (3), Douglas Laurents (3), Vincenzo Silani (1,4), Marco Feligioni (2), Antonia Ratti (1,5)

Institutes
(1) Dept. Neurology and Lab. Neuroscience, IRCCS Istituto Auxologico Italiano, Milan, Italy; (2) European Brain Research Institute, Rita-Levi Montalcini Foundation, Rome; (3) Instituto de Química Física “Rocasolano”, Consejo Superior de Investigaciones Científicas (CSIC), Madrid, Spain; (4) Dept Pathophysiology and Transplantation, ‘Dino Ferrari’ Center, University of Milan, Milan; (5) Dept. Medical Biotechnology and Translational Medicine, University of Milan, Milan

Abstract
Post-translational modifications (PTM) of TDP-43 protein, including ubiquitinylation, phosphorylation and acetylation, influence TDP-43 aggregation. The PTM SUMOylation, targeting lysine residues, regulates a variety of biological activities, including protein stability, aggregation and nucleocytoplasmic transport, all important issues in ALS/FTD pathogenesis. Previous studies suggest that a short splicing isoform of TDP-43 can be SUMOylated and that in response to heat shock TDP-43 is a target of SUMOylation, although the biological effects of this PTM on TDP-43 protein are still unknown.
In this project we demonstrated that TDP-43 is a substrate of SUMOylation in neuronal and non-neuronal cells and that this PTM occurs within the RRMI domain at the N-terminal region as predicted by in silico analyses at Lysine 136. We also proved that TDP-43 SUMOylation levels and sub-cellular distribution between the nucleus and the cytoplasm can be modulated upon over-expression of SUMOylating (UBC9) and deSUMOylating (SENP1) enzymes, respectively. By assessing TDP-43 splicing activity on endogenous and minigene targets, the SUMO-resistant TDP-43 K136R protein showed a defective splicing activity only in exon skipping (CFTR, TNIK, STAG2), while its activity was maintained in exon inclusion (POLDIP3, MADD), although its capability to bind the pre-mRNA targets was generally reduced, regardless the consensus binding sequence. Moreover, upon exposure to Arsenite, the SUMO-resistant TDP-43 K136R protein was not able to be recruited into stress granules in contrast to TDP-43 proteins with ALS/FTD-associated mutations (Q331K, M337V, A382T).
Our results show that SUMOylation is a physiological TDP-43 PTM, potentially influencing its RNA-binding and splicing activity, as well as its sub-cellular localization, and therefore deserves further investigation as a potential mechanism regulating not only TDP-43 function, but also its aggregation, in ALS/FTD diseases.

P2. Regulation of UsnRNP trafficking by the Integrated Stress Response is compromised by mutant ALS proteins
Researchers
Simona Rossi (1,2), Valentina Rompietti (1), Ylenia Antonucci (1), Gianluca Cestra (2,4), Maria Teresa Carrì (2), Nadia D’Ambrosi (2), Mauro Cozzolino (1)

Institutes 
(1) Istituto di Farmacologia Traslazionale, CNR, Roma; (2) Dipartimento di Biologia, Università “Tor Vergata” Roma; (3) Istituto di Biologia e Patologia Molecolari, CNR, Roma; (4) Dipartimento di Biologia e Biotecnologia ‘‘Charles Darwin’’, Università ‘‘Sapienza’’, Roma

Abstract
Activation of the integrated stress response (ISR), alterations in nucleo-cytoplasmic transport and changes in alternative splicing regulation are all involved in the pathogenesis of Amyotrophic Lateral Sclerosis (ALS). However, whether these processes act independently from each other, or are part of a coordinated mechanism of gene expression regulation that is affected in pathogenic conditions, is still rather undefined. To answer these questions, in this work we set out to characterise the functional connections existing between ISR activation and nucleo-cytosol trafficking and nuclear localization of spliceosomal U-rich small nuclear ribonucleoproteins (UsnRNPs), the core constituents of the spliceosome. Activation of the ISR induces a profound reorganization of nuclear Gems and Cajal bodies, membrane-less particles where the SMN complex and assembled UsnRNPs are accumulated for further maturation or storage, and to changes in alternative splicing. Notably, these effects are reversed by both inhibiting the ISR or modulating UsnRNP import receptors, indicating that the regulation of nucleo-cytoplasmic trafficking of UsnRNPs might control alternative splicing in response to stress. Importantly, dismantling of nuclear Gems and Cajal bodies by ALS-linked mutant proteins is not halted by inhibition of the ISR. This indicates that changes in the nuclear localization of the UsnRNP complexes promoted by ALS proteins are uncoupled from ISR activation, and that defects in the nucleo-cytoplasmic trafficking of U might play a role in ALS pathogenesis.

P3. Decoding motor neuron diversity and subtype-specific vulnerability
Researchers
Emanuela Zuccaro (1,2), Antonella Emanuela Sini (1,2), Michael J. Ziller (3), Davide Cacchiarelli (4), Raffaele De Caro (5), Maria Pennuto (1,2)

Institutes
(1) Department of Biomedical Sciences, University of Padova, Padova, Italy; (2) Venetian Institute of Molecular Medicine, Padova, Italy; (3) Max-Planck Institute of Psychiatry, Munich, Germany; (4) TIGEM, Napoli, Italy; (5) Department of Neuroscience, University of Padova, Padova, Italy

Abstract
Alpha motor neurons (αMNs) are a subset of neurons residing in the brainstem and spinal cord, which extend axons into the periphery to initiate muscle contraction. Due to their central role, diseases and insults that result in loss or dysfunction of αMNs are rapidly debilitating and often fatal. Motor neuron diseases (MND), such as ALS and SBMA, are characterized by the selective vulnerability of αMNs, whilst other MN types stay functional, even at late stages of the disease. Moreover, even within the same motor pool, distinct classes of αMNs are differentially susceptible to disease, as well as to physiological aging, with the fast fatigable class of αMNs (FF-MNs) degenerating first. Several groups have investigated common patterns of selective protection or vulnerability of MNs to disease, aiming to identify transcriptional changes between susceptible and resistant MN pools. However, the molecular landscape of the distinct classes of MNs, and the molecular basis of their selective vulnerability to disease are still largely unknown, hampering the discovery of molecular candidates for treatment. This requires a more comprehensive understanding of neuronal subtype-specific molecular profiles than currently available. Our central hypothesis is that each MN class has a unique transcriptomic profile and expresses assemblies of genes that are responsible for the selective vulnerability of FF-MNs to physiological aging and MND.

P4. Nuclear and mitochondrial DNA methylation in amyotrophic lateral sclerosis
Researchers
Andrea Stoccoro (1), Lorena Mosca (2), Katie Lunnon (3), Alessandro Marocchi (2), Lucia Migliore (1), Fabio Coppedè (1)

Institutes 
(1) Department of Translational Research and of New Surgical and Medical Technologies, University of Pisa, Pisa (IT); (2) ASST Grande Ospedale Metropolitano Niguarda, Milan (IT); (3) University of Exeter Medical School, Exeter (UK)

Abstract
Recently, we detected increased global DNA methylation levels in blood DNA from patients with familial amyotrophic lateral sclerosis (ALS) with respect to their asymptomatic family members, observing that DNA methylation in blood increased with the duration of the disease. However, none of the major ALS genes, namely SOD1, C9orf72, FUS, and TARDBP, was hyper-methylated in our samples. Subsequently, we extended our investigation to the mitochondrial DNA (mtDNA), and investigated 114 members of ALS families with SOD1, C9orf72, FUS, and TARDBP mutations, including 54 ALS patients, 28 asymptomaic carriers and 32 non-carrier family members. ALS patients showed a significant increase in mtDNA copy number than the two other groups, and a significant decrease in the methylation levels of the mitochondrial D-loop region, that was inversely correlated with the mtDNA copy number, was detected in carriers of SOD1 mutations. In the present study we applied pyro-sequencing technique to further characterize mtDNA methylation levels in familial ALS and identified three CpG sites in the mtDNA regulatory region that were significantly hypo-methylated in SOD1 ALS patients, resulting in increased mtDNA copy number. We are also currently investigating mtDNA methylation in sporadic ALS, and preliminary findings in a case-control cohort of more than 100 subjects suggest differences between sporadic ALS patients and matched controls. A deeper characterization with pyrosequencing is ongoing to identify the most relevant differentially methylated CpGs. Collectively, our data suggest that both nuclear and mitochondrial DNA methylation could represent peripheral biomarkers of the disease.

P5. Identification and validation of hits modulating C9ORF72-dipeptide repeat proteins level in vitro e vivo models
Researchers
Licata NV (1,2), Cristofani R (3), D’Agostino VG (1,4), Loffredo R (1), Pancher M (5), Adami V (5), Bellosta P (6), Viero G (7), Quattrone A (2), Poletti A (3), Provenzani A (1)

Institutes 
(1) Laboratory of Genomic Screening, Department of Cellular, Computational and Integrative Biology (CIBIO), University of Trento, Trento, Italy; (2) Laboratory of Translational Genomics, Department of Cellular, Computational and Integrative Biology (CIBIO), University of Trento, Trento, Italy; (3) Laboratory of Applied Biology, Department of Pharmacological and Biomolecular Sciences (DISFEB), University of Milan, Milan, Italy; (4) Laboratory of Biotechnology and Nanomedicine, Department of Cellular, Computational and Integrative Biology (CIBIO), University of Trento, Trento, Italy; (5) HTS Core Facility, Department of Cellular, Computational and Integrative Biology (CIBIO), University of Trento, Trento, Italy; (6) Laboratory of Metabolism of Cell Growth and Neuronal Survival, Department of Cellular, Computational and Integrative Biology (CIBIO), University of Trento, Trento, Italy; (7) Institute of Biophysics, CNR Unit at Trento, Trento, Italy

Abstract
The intronic hexanucleotide repeat expansion, GGGGCC (or G4C2), in the C9ORF72 gene is the most common genetic cause of Amyotrophic Lateral Sclerosis (ALS). When it is expanded above 30 and up to hundreds repeats, the G4C2 repeat expansion is transcribed in aberrant RNAs that fold in G-quadruplex structures and generate RNA foci within motoneurons. Additionally, aberrant transcripts also shuttle in the cytoplasm and can be translated via the unconventional repeat-associated non-AUG (RAN) translation into toxic dipeptide repeat (DPR) proteins that induce neurodegeneration. However, so far, no effective pharmacological approach, to reduce the pathological load of DPRs is currently available. Here, we developed and performed a high-throughput drug-screening fluorescent assay in HEK293T cells to identify positively and negatively modulators of RAN translation. We selected and characterized 3 hits by assessing whether they affected DPRs level at transcriptional, translational or post-translational level. Finally, we tested the efficacy of one hit in vivo, using Drosophila flies carrying the G4C2X36 repeats and evaluating the hit’s capacity to rescue the climbing ability and increase life span of mutant flies.

P6. AxRibALS - Axonal translatome in mouse models of Amyotrophic Lateral Sclerosis
Researchers
Fabio Lauria (1), Marta Marchioretto (1), Alessandra Pisciottani (2), Federica Maniscalco (1), Laura Croci (2), Aurora Badaloni (2), Toma Tebaldi (3), Renato Arnese (1), Gian Giacomo Consalez (2,4), Gabriella Viero (1)

Institutes 
(1) Institute of Biophysics, CNR Unit of Trento, Trento, Italy; (2) Division of Neuroscience; San Raffaele Scienti c Institute, Milano, Italy; (3) Yale Cancer Center, Yale University School of Medicine, New Haven, USA; (4) Università Vita-Salute San Raffaele, Milano, Italy

Abstract
To date, the repertoire of treatments available to delay disease progression in ALS is severely limited, mostly due to our sketchy understanding of the underlying cellular defects. RNA-binding proteins (RBP) are major contributors to the development of ALS, which has been strongly associated to mutations affecting the RBP TDP-43. TDP43 appears to regulate translation, but the existence of TDP-43-dependent translational defects in ALS is an understudied issue. The specific aim of the project is the characterization of translational defects in axonal RNAs in mouse models of ALS.

We used in vitro and in vivo approaches to assess if overexpression of TDP-43(WT) or TDP-43(A315T) mutation dysregulate axonal translation and began to establish a comprehensive picture of ALS through a better understanding of the sub-compartment translational defects underlying ALS.

As an in vitro platform, we exploited our ability to purify axonal polysomes in cortical neurons cultured in microfluidic chambers and overexpressing TDP-43 (WT or A315T). After sequencing and qPCR validations, we identified widespread variations in sub-cellular (cell body vs axons) and sub-compartment (free RNAs vs polysomal RNAs) mRNAs levels. We found that the expression of TDP-43(A315T) results in strong alterations in RNA localization and recruitment to axonal polysomes. In particular, we observed 1) increased translation efficiency in axons and 2) cellular enrichment of free RNAs. 

To validate these results in vivo, we are developing two mouse models of ALS to study translation using the TRAP technique taking advantage of a ribo-tagged knock-in line activated selectively in the corticospinal tract. This line is being bred with two ALS mouse models, overexpressing TDP-43 WT or TDP-43(A315T).

P7. FUS-dependent phase separation initiates double strand break repair
Researchers
Marco Antonaci, Brunno R. Levone, Silvia C. Lenzken, Francesca Conte, Ҫise Kızılırmak, Fabio Biella, Marc-D. Ruepp, Silvia M. L. Barabino

Institutes 
Università degli Studi di Milano-Bicocca

Abstract
The repair of DNA lesions is essential for cell homeostasis and survival. Over the past years, studies have revealed a correlation between the accumulation of DNA damage and several types of cancer and neurodegenerative diseases, including Amyotrophic Lateral Sclerosis (ALS). Recently, RNA-binding proteins (RBPs) have emerged as important effectors of the cellular DNA Damage Response (DDR), although their precise molecular function remains poorly understood. The splicing factor FUS (Fused in Sarcoma) is an intrinsically disordered DNA/RNA-binding protein, which has been shown to play a role in multiple cellular functions, including mRNA transcription, splicing and transport, and in DNA damage repair. Here, we identify a novel molecular mechanism by which FUS contributes to DDR activation. Cells with a complete knock-out of FUS (FUS-KO) display an accumulation of RNA: DNA hybrids and of DNA double strand breaks (DSBs), as well as reduced DSB repair both by Homologous Recombination (HR) and Non-Homologous End Joining (NHEJ) pathways. FUS-KO cells also delays the recruitment of 53BP1 to DNA damage foci and alters the recruitment of NHEJ- and HR-related proteins to laser-induced DSBs. FUS itself is recruited within seconds to DSBs and is required for the retention of the apical DNA DSBs sensor KU80. Mass spectrometry analysis revealed that FUS co-purifies with RNA-binding proteins implicated in DNA damage repair, including SFPQ. We demonstrate that liquid-liquid phase separation (LLPS) occurs at DNA damage foci and is required for the efficient recruitment of key DDR factors. Finally, LLPS-deficient FUS variants impair recruitment of KU80 and of SFPQ. Overall, our findings establish for the first time that FUS-dependent LLPS contributes to the activation of the DDR and to the recruitment of DDR proteins at sites of DNA damage.

P8. DDRNALS, DNA damage response defects in cells with TPD-43 and FUS cytoplasmic inclusions
Researchers
Brandi O (1), Farina S (1,2), Gioia U (3), Riva N (4), Delia D (3), Cancellieri C (3), Pansarasa O (5), Cereda C (5), d’Adda di Fagagna F (1,3), Francia S (1,3)

Institutes 
(1) Institute of Molecular Genetics (IGM-CNR), Pavia, Italy; (2) University School for Advanced Studies (IUSS), Pavia, Italy; (3) IFOM – FIRC Institute of Molecular Oncology, Milan, Italy; 4) Department of Neurology-INSPE, San Raffaele Scientific Institute, Milan, Italy; (5) Genomic and post-Genomic Center, IRCCS Mondino Foundation, Pavia, Italy

Abstract
Predisposing mutations to Amyotrophic Lateral Sclerosis (ALS) fall in TDP-43 and FUS genes. More broadly, 90% of ALS cases are marked by the accumulation of cytoplasmic inclusions containing TDP-43 in patient motor neurons. Mutant FUS also accumulates in the cytoplasm in severe ALS cases and forms inclusions. Noteworthy, motor neurons of ALS patients show chronic DNA damage, an event that healthy cells counteract by activating the DNA damage response (DDR) pathway. Recently, a novel class of small non-coding RNAs called DNA damage response RNAs (DDRNAs) generated at damaged sites in a DROSHA-and DICER-dependent manner has been shown to regulate DDR and DNA repair. Both TDP-43 and FUS are cofactors of DICER and DROSHA complexes, acting as stimulators of their activity. Scattered evidences in the literature propose that both FUS and TDP-43 can be recruited to sites of DNA damage in physiological conditions. Instead, whether TDP-43 and FUS pathological aggregation may affect DDR activation is still unknown. Thus, we analysed genome integrity and DDR activation in cells experiencing cytoplasmic inclusions containing TDP-43 or mutant FUS-P525L. We observed that acute induction of cytoplasmic inclusions of both TDP-43 and mutant FUS, rapidly induce a strong accumulation of γH2AX signals, a well-known marker of DNA damage, which depends on the kinase activity of ATM and DNA-PK, but not ATR. Importantly, upon treatment with a radiomimetic drug, cells with TDP-43 or mutant FUS positive inclusions selectively show profound defects in the activation of DDR cascade. By chromatin immunoprecipitation (ChIP) we observed that TDP-43 inactivation by siRNA reduces the recruitment of ATM at damaged sites. In addition, we have preliminary data of features of DDR activation in skin fibroblasts from ALS patients with different TDP-43 mutations. Together our data indicate that typical ALS inclusions cause DDR defects and DNA damage accumulation and could be causative of cell death.

P9. SPLICEALS - Dissecting the functional interaction between FUS and hnRNP a2/b1 in pathogenesis of ALS
Researchers
Simona Rossi (1), Savina Apolloni (2), Michela Di Salvio (3,4), Eleonora Mammarella (2), Martina Milani (2), Ylenia Antonuccia (2), Assia De Simone (2), Serena Lattante (5,6), Mario Sabatelli (6,7), Gianluca Cestra (3,4), Nadia D’Ambrosi (2), Mauro Cozzolino (1)

Institutes 
(1) Istituto di Farmacologia Traslazionale, CNR, Roma; (2) Dipartimento di Biologia, Università “Tor Vergata, Roma; (3) Istituto di Biologia e Patologia Molecolari, Roma; (4) Dipartimento di Biologia e Biotecnologia ‘‘Charles Darwin’’, Università ‘‘Sapienza’’, Roma; (5) Fondazione Policlinico Universitario A. Gemelli IRCCS, Roma; (6) Università Cattolica del Sacro Cuore, Roma; (7) Centro Clinico NEMO, Roma

Abstract
Several genetic and experimental findings point to a crucial role of RNA dysfunction in the pathogenesis of ALS. In particular, evidence suggests that mutations in FUS, that are associated with genetic ALS, affect the regulation of alternative splicing (AS) of a selected number of target genes. Recently we have demonstrated that a major target of FUS activity is hnRNP A2/B1, an RNA binding protein with key roles in RNA metabolism, including AS regulation, which is mutated in familial ALS. Further, we have obtained compelling evidence in Drosophila showing that A2/B1 is a potent modifier of FUS toxicity. Overall, these data suggest that A2/B1 might represent a relevant target of FUS in vivo, and that functional alterations of A2/B1 induced by FUS might cause a pathogenic cascade of AS changes, eventually promoting motor neuron degeneration. The general aim of the SPLICEALS project is to validate this hypothesis. In particular, we will use mice, Drosophila and cultured cells to: i. characterize how AS deregulation promoted by FUS affects A2/B1 expression and function; ii. validate these alterations in tissues from mouse models of FUS-ALS; iii. define how modulation of A2/B1 expression impacts on ALS-relevant phenotypes in flies modelling FUS-ALS. 

Using isoform-specific antibodies, we have observed that the expression of different splice variants of A2/B1 is modified in the lumbar spinal cord of FUS transgenic mice, as well as in fibroblasts from FUS-ALS patients. Further, by using isoform‐specific A2/B1 expression constructs, we have pinpointed distinct abilities of A2/B1 variants to localise into stress granules. Finally, we have identified by bionformatics analysis a restricted number of common splicing targets of FUS and A2/B1 and verified that they are affected in diseased mice. Overall, these data support the existence of a pathological connection between FUS and A2/B1 in ALS.

 

P10. PathensTDP, Defining the role of hnRNP proteins in enhancing TDP-43 pathology
Researchers
Sara Cappelli (1), Maurizio Romano (2), Alida Spalloni (3), Patrizia Longone (3), Emanuele Buratti (1)

Institutes 
(1) Molecular Pathology, International Centre for Genetic Engineering and Biotechnology, Trieste, Italy; (2) Department of Life Sciences, University of Trieste, Trieste, Italy; (3) Molecular Neurobiology, Fondazione Santa Lucia IRCCS, Rome, Italy

Abstract
Heterogeneous ribonucleoproteins (hnRNPs) are a family of RNA-binding proteins (RBPs) implicated in several steps of RNA metabolism, including transcription, pre-mRNA splicing, mRNA transport and turnover. In particular, TDP-43 is a member of this family that was discovered in 2006 as the major component of ubiquitin-positive inclusions in brain tissues from ALS and FTLD patients. Since then, many additional RBPs have been found to play a role in neurological disorders. Unfortunately, these proteins do not represent good therapeutic targets due to the multitude of functions they play in cells. However, the identification of key transcripts regulated by their overexpression/depletion may represent a valid alternative.

In the PathensTDP project we have focused on five hnRNPs (DAZAP1, hnRNPD, hnRNPK, hnRNPQ and hnRNPU) that we previously identified as strong functional modulators of TDP-43 activity in Drosophila and human cells. Our preliminary data following whole transcriptome analysis of neuronal-like cells depleted for each hnRNP, including TDP-43, have shown the presence of several commonly regulated mRNAs that could play an important role in modulating TDP-43 pathology. 

To assess the therapeutic implications of these candidate genes, we will now examine their effects on the synaptic plasticity/death-signalling pathways both in cellular and animal models.

 

P11. RAN translated C9ORF72 arginine rich poly-dipeptides alter gene transcription in ALS/FTD cell model
Researchers
Cristofani R (1), Grilli A (2), Giulia V (1), Licata NV (3), Crippa V (1), Cicardi ME (4), Rusmini P (1), Tedesco B (1), Ferrari V (1,), Casarotto E (1), Chierichetti M (1), Galbiati M (1), Carra S (2), Bicciato S (2), Provenzani A (3), Poletti A (1)

Institutes 
(1) Dipartimento di Scienze Farmacologiche e Biomolecolari (DiSFeB), Centro di Eccellenza sulle Malattie Neurodegenerative, Università degli Studi di Milano, Milano, Italy; (2) Dipartimento di Scienze Biomediche, Metaboliche e Neuroscienze, Università degli Studi di Modena e Reggio Emilia, Modena, Italy; (3) Laboratory of Experimental Neurobiology, Department of Cellular, Computational and Integrative Biology (CIBIO), Università degli Studi di Trento, Trento, Italy; (4) Department of Neuroscience, Sidney Kimmel Medical College, Jefferson University Philadelphia, USA

Abstract
Amyotrophic Lateral Sclerosis (ALS) and frontotemporal dementia (FTD) are associated with several mutated proteins such as: mutant SOD1, TDP-43, FUS, VCP, OPTN and C9ORF72. Expanded (G4C2)n stretch of C9ORF72 give rise to poly di-peptide repeats (DPRs) that are produced by repeat-associated non-ATG (RAN) translation, a mechanism originally identified for CAG triplet repeat sequences.

DPRs misfold and aggregate into cytoplasm or nuclei of motor neuron as it has been already demonstrated in polyQ containing proteins. DPRs alter the protein quality control system which maintains protein homeostasis by re-folding (by chaperone) or by degradation (by autophagy or proteasome) and it clears misfolded proteins to counteract proteotoxicity. Chaperone assisted selective autophagy (CASA) is involved in misfolded protein degradation and is mediated by the HSPB8-BAG3-HSP70 complex. We previously demonstrated that DPRs aggregation and toxicity are prevented by autophagy facilitation through HSPB8 overexpression. 

We developed a novel inducible human neuronal model to identify aberrant mechanisms altered by RAN-DPR and PolyQ peptides. We first evaluated DPRs and polyQ stability and induced toxicity. RTqPCR show that DPRs mRNA are less expressed than polyQ. This is also recapitulated in toxicity assay where only polyQ cells shows marked cell death. We performed differential genetic profiling of neuronal transcriptional response to DPRs and polyQ, followed by bioinformatics analyses. We found a selective alteration of specific transcripts in cells expressing the two most highly aggregation prone DPRs: polyGR and Poly PR. Gene set enrichment analysis showed specific pathways modulated by polyGR and/or polyPR expression. Notably, PCSK1N related to ALS and FTD and TOMM5 related to mitophagy and protein metabolism are influenced by polyGR and/or polyPR expression.

Collectively, these data show that aggregating prone DPRs overexpression alters gene expression in our cell model.

 

P12. An aberrant interplay between RNA-binding proteins in Amyotrophic Lateral Sclerosis
Researchers
Maria Giovanna Garone (1), Riccardo De Santis (1,2), Vincenzo Alfano (1), Alessio Colantoni (1), Valeria de Turris (2), Maria Rosito (2), Nicol Birsa (3), Clarissa Braccia (2), Beatrice Salvatori (2), Emanuel Wyler (4), Giuseppe Antonacci (2), Andrea Armirotti (2), Markus Landthaler (4), Eleonora Aronica (5), R. Jeroen Pasterkamp (6), Irene Bozzoni (1,2), Pietro Fratta (3), Alessandro Rosa (1,2)

Institutes 
(1) Sapienza University of Rome, Italy; (2) Istituto Italiano di Tecnologia, Italy; (3) University College of London, UK; (4) Max-Delbruck-Center for Molecular Medicine, Berlin, Germany; (5) University of Amsterdam, the Netherlands; (6) Utrecht University, the Netherlands

Abstract
Several RNA-binding proteins (RBPs) have been genetically linked to Amyotrophic Lateral Sclerosis (ALS). We propose that an aberrant interplay between the neural RBP ELAVL4 (also known as HuD) and ALS-linked RBPs, such as FUS and TDP-43, exists in ALS patients’ motor neurons. By analyzing the RNA interactome of wild-type and mutant FUS (P525L) in human iPSC-derived motor neurons, we have identified a number of novel RNA targets. In particular, many transcripts are preferentially bound by mutant FUS in the 3’UTR and neural ELAV-like RBPs, including ELAVL4, are among those targets. Mutant FUS binding results in increased number of sites of translation, leading to aberrant increase of ELAVL4 protein levels. These findings have been confirmed in primary MNs from a FUS-ALS mouse model. Moreover, ELAVL4 and mutant FUS interact at the protein-protein level and co-localize in cytoplasmic speckles. Background-deflection Brillouin microscopy, a novel non-contact and label-free imaging method, provided insights into the peculiar biomechanical properties of such mutant FUS/ELAVL4 speckles. Interestingly, upon oxidative stress both ELAVL4 and mutant FUS are engaged in stress granules. Notably, in the spinal cord of FUS ALS patients, ELAVL4 represents a neural-specific component of FUS-positive cytoplasmic aggregates, whereas in sporadic patients it co-localizes with phosphorylated TDP-43-positive inclusions. Finally, we propose that altered ELAVL4 levels might underlie a peculiar axon branching phenotype observed in both mouse and human MNs in vitro models. 

P13. Isolation and Characterization of soluble human full-length TDP-43 associated with neurodegeneration
Researchers
Vivoli Vega M, Nigro A, Luti S, Capitini C, Fani G, Gonnelli L, Boscaro F, Chiti F

Institutes 
Università degli Studi di Firenze

Abstract
The involvement of TAR DNA-binding protein 43 (TDP-43) in neurodegenerative diseases was revealed in 2006, when it was first reported to be the main component of the intracellular inclusions in patients with amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration (FTLD). After 12 years it is not yet possible to purify to a reasonable yield and in a reproducible manner a stable full-length protein, which has limited so far, the characterization of its structure, function, molecular interactors and pathobiology. Using a novel protocol, we have achieved the purification of the full-length TDP-43, with both a PelB- and GST-tag, which consisted in its expression in bacteria, its solubilisation from inclusion bodies, purification under denaturing conditions, refolding and a final size exclusion chromatographic (SEC) step. Differential scanning fluorimetry was used to find the best buffers and combination of additives to increase both its solubility and stability. The protein is pure, as determined with electrophoresis, Western blotting and mass spectrometry, properly refolded, as revealed by circular dichroism and fluorescence spectroscopies, functional, as it binds to DNA and protein partners, and stable to degradation and aggregation in a physiological solution. Analyses with dynamic light scattering and SEC revealed that the protein is a dimer. 

P14. Circulating muscle-derived mir-206 links skeletal muscle dysfunction to heart autonomic denervation
Researchers
Valentina Prando (1,2), Silvia Bertoli (1), Giulia Favaro (1), Vittoria Di Mauro (3), Marco Ron ni (1,2), Andrea Armani (1,2), Michele Guescini (4), Anna Di Bona (1,5), Paula DaCosta Martins (6), Miriam Capri (7,8), Stefano Salvioli (7), Claudio Franceschi (9), Raffaella Mariotti (10), Christian Lunetta (11), Antonio Musarò (12,13), Daniele Catalucci (3), Marco Sandri (1,2,14), Marco Mongillo (1,2,14), Tania Zaglia (1,2,5)

Institutes 
(1) Veneto Institute of Molecular Medicine (VIMM), Padova, Italy; (2) Department of Biomedical Sciences, University of Padova, Padova, Italy; (3) Humanitas Research Hospital, Rozzano, Milan, Italy; (4) Department of Molecular Sciences, University of Urbino Carlo Bo, Urbino, Italy; (5) Department of Cardiac, Thoracic and Vascular Sciences, and Public Health, Padova, Italy; (6) Department of Cardiology, Faculty of Health, Medicine and Life Sciences, Maastricht University, Maastricht, Netherlands; (7) DIMES- Department of Experimental, Diagnostic and Specialty Medicine, University of Bologna, Bologna, Italy; (8) CIG- Interdepartmental Centre “Galvani”, University of Bologna, Bologna, Italy; (9) IRCCS, Institute of Neurological Sciences of Bologna, Bologna, Italy; (10) Department of Neurosciences, Biomedicine and Movement Science Verona, Italy; (11) NEuroMuscular Omnicentre (NEMO), Fondazione Serena Onlus, ASST Grande Ospedale Metropolitano Niguarda, Milano, Italy; (12) DAHFMO-Unit of Histology and Medical Embryology, Sapienza University of Rome, Laboratory affiliated to Istituto Pasteur Italia – Fondazione Cenci Bolognetti, Rome, Italy; (13) Center for Life Nano Science @ Sapienza, Istituto Italiano di Tecnologia, Rome, Italy; (14) CNR Institute of Neuroscience, Padova, Italy

Abstract
Rationale: ALS is a fatal neuromuscular disorder, hallmarked by motor neuron degeneration, leading to muscle weakness, paralysis and, ultimately, respiratory failure. ALS patients also show autonomic dysfunction, cardiac arrhythmias and sudden death, but the underlying mechanism is unknown and is the object of this study. 

Results: We here demonstrated that a primary block of autophagy in the mouse skeletal muscle, as that elicited by knocking-out Atg7, a key autophagy mediator, leads to increased secretion of exosomes enriched in the muscle-specific microRNA, miR206, into the bloodstream. miR206-carrying exosomes, injected via tail vein in normal mice, are taken up by the heart and cause sympathetic dysinnervation and increased arrhythmogenesis. 

In vitro assays demonstrated that miR206 directly targets sympathetic neurons (SNs), leading to reduced axonal sprouting and abnormal morphology. We showed that these effects are due to the miR206-mediated downregulation of the NGF receptor p75, as demonstrated by in vitro and ex vivo assays, which results in reduced formation of p75/TrkA complexes, thus decreasing neurotrophin responsiveness and compromising the efficiency of NFG retrograde transport. As a consequence of the impaired neurotrophic input, miR206 over-expressing SNs displayed increased death. 

Interestingly, increased plasma levels of miR206 have been described in ALS patients and SOD1G93A transgenic mice. In keeping with the effects of miR206 on cardiac sympathetic innervation, miR-206 expression was increased in the stellate ganglia of SOD1G93A mice and cardiac SNs were altered in morphology and topology. 

Conclusions: miR206 is a mediator of ‘muscle-to-heart’ communication and may be responsible for cardiac dysfunction and arrhythmias occurring in ALS. The therapeutic targeting of miR206 by, e.g. specific antagomiR, will be tested to treat disease progression, in ALS mice.

 

P15. MLOpathy, Membrane-less organelle pathology in ALS: identification of causes and rescuing factors
Researchers
L. Mediani (1), J. Vinet (1), O. Pansarasa (2), A. Poletti (3), S. Carra (1)

Institutes 
(1) Dept NeuBioMet, Univ. Modena and Reggio Emilia, Italy; (2) Genomic and post-Genomic Center, IRCCS Mondino Foundation, Pavia, Italy; (3) Center of Excellence on Neurodegenerative Diseases Univ. of Milano

Abstract
Proteostasis dysfunction, impaired stress granule (SG) dynamics, defective nucleocytoplasmic trafficking and nucleolar stress are emerging themes in ALS. We showed that interplay between protein quality control (PQC) and SG exists. In particular, misfolded proteins and defective ribosomal products (DRiPs), which include small translation products that are generated outside of protein-coding regions by pervasive and RAN translation, such as C9orf72-dipeptide repeats (DPRs), accumulate inside SGs, promoting their conversion into an aggregated-state. By enhancing the clearance of misfolded proteins, chaperones and anti-psychotics that boost autophagy can rescue SG pathology. In ALS cells, DPRs accumulate also in nucleoli, which are membrane-less organelles (MLOs) like SGs. Intriguingly, we find that, upon stress, misfolded proteins and DRiPs accumulate in PML bodies and nucleoli. Thus, similarly to SGs, misfolded proteins may compromise the dynamics of these MLOs, which are essential to enable response and adaptation to stress. Why DPRs and misfolded proteins are targeted to nucleoli and PML bodies, how cells cope with this process and its pathological significance is yet unclear. 

MLOpathy will study if: 

1) DPRs, DRiPs and misfolded proteins promote the conversion of PML bodies and nucleoli into aggregates and how they lead to cell toxicity; 

2) a cross-talk between ALS-mutated proteins and misfolded nuclear proteins exists, and how this is linked to sequestration of nucleocytoplasmic transport factors in SGs; 

3) chaperones prevent PML and nucleoli conversion into a dysfunctional state; 

4) combined treatment with drugs that rescue SG pathology and chaperone inducers can restore nuclear proteostasis and cell health.

MLOpathy will provide: 1) new insights into ALS pathomechanisms; 2) proof-of principle data for a possible repurposing of anti-psychotics combined with chaperone co-inducers to combat ALS.

 

P16. Sonic hedgehog signalling pathway may control regenerative processes in a mouse model of motoneuronal loss
Researchers
Nunzio Vicario (1), Federica M. Spitale (1), Cesarina Giallongo (2), Joshua D. Bernstock (3), Grazia Scandura (1), Graziana Spoto (1), Giovanni Li Volti (2), Michele Vecchio (4), Daniele Tibullo (2), Giampiero Leanza (5), Rosalba Parenti (1), Rosario Gulino (1)

Institutes 
(1) Dept. of Biomedical and Biotechnological Sciences, Section of Physiology, University of Catania, Italy; (2) Dept. of Biomedical and Biotechnological Sciences, Section of Biochemistry, University of Catania, Italy; (3) Medical Scientist Training Program, The University of Alabama at Birmingham, AL, USA; (4) Dept. of Biomedical and Biotechnological Sciences, Section of Pharmacology, University of Catania, Italy (5) Dept. of Drug Sciences, University of Catania, Catania, Italy

Abstract
Neuronal loss represents the major cause of persistent disability in neurodegenerative diseases, including amyotrophic lateral sclerosis. Nevertheless, we have shown that compensatory changes can take place in the spinal cord (SC) of SOD1 mice, as well as after neurotoxic removal of lumbar SC motoneurons by intramuscular injection of Cholera toxin-B conjugated to saporin (CTB-Sap). Sonic hedgehog (Shh) signalling plays a key role in development and maintenance of the central nervous system (CNS), and we found that its expression could be linked to the plastic changes observed in the CTB-Sap model, thus holding great potential in CNS repair and regeneration. 

Herein, the effects of Shh signalling modulation were evaluated in vitro on neural stem cells, finding a significant increase of growth rate and size of neurospheres upon treatment with a glucocorticoid drug (i.e. clobetasol), also acting as a smoothened (Smo) agonist. We also showed that these effects could depend, at least in part, by the activation of the canonical Shh pathway. Finally, we analysed the Shh pathway in vivo, upon CTB-Sap-induced selective ablation of lumbar SC motoneurons. Neuronal loss was accompanied by locomotor impairment, muscle atrophy and electromyographic signs of denervation at 6 weeks post-lesion, and these effects were partially restored by clobetasol treatment. We also characterized the microglial modulation upon motoneuronal ablation and clobetasol treatment, by using multicolour-assisted cytofluorimetric analysis, finding a reduced proportion of CD45+/CD11b+/GR1-/F4-80+/CX3CR1+ cells (i.e. microglia) polarized towards an M1-like pro-inflammatory phenotype, considered as CD80+ cells, as compared to CTB-Sap controls.

Our results suggest a crucial role of Shh signalling during regenerative processes and as a potential strategy to support recovery after spinal motoneuronal degeneration, thus providing new exploitable mechanisms that may contribute to tissue repair and regeneration.

 

P17. NEVALS, Neurovascular Crosstalk in ALS Pathogenesis
Researchers
Luis Martins, Ilaria Brambilla, Ganesh Bhat, Aurora Badaloni, Dario Bonanomi

Institutes 
Division of Neuroscience, San Raffaele Hospital, Milan, Italy

Abstract
The pathogenic mechanisms responsible for the selective loss of spinal motor neurons in ALS are largely unknown but the contribution of both cell autonomous and non-cell autonomous factors is well established. Elucidating the interactions between spinal motor neurons and their cellular microenvironment is critical for understanding disease mechanisms. Building on accumulating evidence of vascular abnormalities in ALS patients and animal models, the project unravels the contribution of vascular endothelial cells to ALS pathogenesis. We reason that the vulnerability of motor neurons in ALS might be at least in part attributed to specific signaling interactions with endothelial cells of the spinal cord microvasculature. Gene profiling in ALS mouse models and mass-spec proteomics are used to isolate endothelial signals that support motor neurons and to determine whether this complement of factors is affected in ALS. The translatome of the spinal cord endothelium is revealed with Translating Ribosome Affinity Purification (TRAP) in SOD-G93A mouse mutants. In parallel, we established cell-based systems to assess the effect of ALS-linked mutations on the composition of the endothelial secretome. Through this integrative approach we are identifying vascular genes and pathways deregulated in ALS with the potential to serve as diagnostic biomarkers and therapeutic targets. 

P18. TDP-43 regulates the expression levels of Disc-large in Skeletal Muscles to promote the assembly of the neuromuscular synapses in Drosophila
Researchers
Nina Strah (1,2), Giulia Romano (1,2), Clelia Introna (1), Raffaella Klima (1), Aram Megighian (2), Monica Nizzardo (3), Fabian Feiguin (1)

Institutes 
(1) International Centre for Genetic Engineering and Biotechnology, Trieste, Italy; (2) Department of Biomedical Sciences, University of Padova, Padova, Italy; (3) Department of Pathophysiology and Transplantation (DePT), Dino Ferrari Centre, University of Milan, Milan, Italy

Abstract
Alterations in the intracellular distribution of TDP-43 have been observed in skeletal muscles of patients suffering from ALS. It is not clear, however, whether these modifications play an active role in the disease or merely represent an expression of muscle homeostatic mechanisms. 

Results

We modulated the activity of TDP-43 in Drosophila muscles by means of RNA interference and observed that it is required to promote the formation and growth of neuromuscular synapses. TDP-43 regulated the expression levels of Disc-large (Dlg) and restoring Dlg expression at skeletal muscle or motoneuron level was sufficient to suppress the locomotive and synaptic defects of TDP-43-null flies. These results were validated by the observation that similar mechanisms appear to operate in human cells and cells derived from ALS patients. 

Conclusions

Our results help to unveil the physiological role of TDP-43 in skeletal muscles as well as the mechanisms responsible for the autonomous and non-autonomous behavior of this protein concerning the organization of neuromuscular synapses.

 

P19. P2X7 activation enhances skeletal muscle metabolism and regeneration in SOD1G93A mouse model of amyotrophic lateral sclerosis
Researchers
Paola Fabbrizio (1), Savina Apolloni (2), Andrea Bianchi (2), Illari Salvatori (2), Cristiana Valle (2,3), Chiara Lanzuolo (2,4), Caterina Bendotti (1), Cinzia Volonté (2,5) Giovanni Nardo (1)

Institutes 
(1) Laboratory of Molecular Neurobiology, Department of Neuroscience, Istituto di Ricerche Farmacologiche Mario Negri IRCCS, Milan, Italy; (2) IRCCS Fondazione Santa Lucia, Rome, Italy; (3) National Research Council, Institute of Translational Pharmacology, Rome, Italy; (4) National Research Council, Institute of Biomedical Technologies, Milan, Italy; (5) National Research Council, Institute of Cell Biology and Neurobiology, Rome, Italy

Abstract
Muscle weakness plays an important role in neuromuscular disorders comprising amyotrophic lateral sclerosis (ALS). However, it is not established whether muscle denervation originates from the motor neurons, the muscles or more likely both. Previous studies have shown that the expression of the SOD1G93A mutation in skeletal muscles causes denervation of the neuromuscular junctions, inability to regenerate and consequent atrophy, all clear symptoms of ALS. In this work, we used SOD1G93A mice, a model that best mimics some pathological features of both familial and sporadic ALS, and we investigated some biological effects induced by the activation of the P2X7 receptor in the skeletal muscles. The P2X7, belonging to the ionotropic family of purinergic receptors for extracellular ATP, is abundantly expressed in the healthy skeletal muscles, where it controls cell duplication, differentiation, regeneration or death. In particular, we evaluated whether an in vivo treatment in SOD1G93A mice with the P2X7 specific agonist 2′(3′)-O-(4-Benzoylbenzoyl) adenosine5′-triphosphate (BzATP) just before the onset of a pathological neuromuscular phenotype, could exert beneficial effects on the skeletal muscles. 

Our findings indicate that stimulation of P2X7 improves the innervation and metabolism of myofibers, moreover elicits the proliferation/differentiation of satellite cells, thus preventing the denervation atrophy of skeletal muscles in SOD1G93A mice. Overall, this study suggests that a P2X7-targeted and site-specific modulation might be a strategy to interfere with the complex multifactorial and multisystem nature of ALS.

 

P20. Contribution of S100A4-regulated pathways to inflammation in ALS models
Researchers
Savina Apolloni (1), Alessia Serrano (2), Simona Rossi (1,3), Eleonora Mammarella (1), Martina Milani (1), Serena Lattante (4), Mario Sabatelli (5), Pavle Andjus (6), Fabrizio Michetti (2), Maria Teresa Carrì (1), Mauro Cozzolino (3), Nadia D’Ambrosi (1)

Institutes 
(1) Department of Biology, University of Rome “Tor Vergata”, Rome, Italy; (2) Institute of Anatomy and Cell Biology, Università Cattolica del Sacro Cuore, Rome, Italy; (3) Institute of Translational Pharmacology, CNR, Rome, Italy; (4) Fondazione Policlinico Universitario A. Gemelli IRCCS, Unità Operativa Complessa di Genetica Medica, Rome, Italy; Università Cattolica del Sacro Cuore, Istituto di Medicina Genomica, Rome, Italy; (5) Fondazione Policlinico Universitario A. Gemelli IRCCS, Unità Operativa Complessa di Neurologia, Rome, Italy; Centro Clinico NEMO, Rome, Italy; Università Cattolica del Sacro Cuore, Istituto di Neurologia, Rome, Italy; (6) Institute of Physiology and Biochemistry “Ivan Djaja”, Faculty of Biology, University of Belgrade, Belgrade, Serbia

Abstract
S100A4 is a Ca2+-binding protein belonging to a large multifunctional S100 protein family. The functions of S100A4 are very well characterized in specific stromal-interstitial cell types, such as fibroblasts, immune cells, and tumor cells, where it has recently emerged as a potent factor implicated in inflammatory responses, angiogenesis, cell differentiation, apoptosis, motility, and invasion. In the nervous system, S100A4 is markedly increased in spinal glia after acute neuronal injury where it influences the formation of a non-permissive glial scar. In this study, we analyzed the expression of S100A4 and the effect of the S100A4 transcriptional inhibitor niclosamide in activated primary microglia. We found that S100A4 is strongly up-regulated in reactive microglia and that niclosamide prevents NADPH oxidase 2 (NOX2), mTOR and NF-kB increase, cytoskeletal rearrangements, migration, and phagocytosis. Interestingly, in the spinal cord of pre-symptomatic and end stage transgenic SOD1-G93A rats, we found that S100A4 is significantly up-regulated in astrocytes and microglia. Finally, since fibroblasts from ALS patients show numerous abnormalities, sharing common pathogenic pathways with CNS resident cells, we have demonstrated here that in ALS fibroblasts carrying different pathogenic mutations S100A4 is up-regulated together with an increase expression in both mTOR and NF-kB proteins.

In conclusion, we identified a new pathway that appears to be aberrantly regulated in inflammatory environments as those occurring in ALS, and niclosamide as a possible drug to be applied in the attenuation of reactive phenotypes of microglia, thus opening the way to further investigation for a new application in ALS disease.

 

P21. Heterogeneity of neuroinflammatory responses in Amyotrophic Lateral Sclerosis (ALS) revealed at single-cell resolution: a roadmap for new target discovery
Researchers
Danilo Pellin (1), Andrea Protti (2), Daniela Curti (3), Alessandra Bif (1, 4), Marco Peviani (1, 4, 3)

Institutes 
(1) Dana-Farber/Boston Children’s Cancer and Blood Disorder Center, Boston, MA, US; (2) Lurie Family Imaging Center – Dana Farber Cancer Institute, Boston, MA, US; (3) University of Pavia, Pavia, Italy; (4) Harvard Medical School, Boston, MA, US

Abstract
ALS is a complex pathology: i) the neurodegeneration is progressive; it starts focally in specific CNS areas and spreads to different districts; ii) the heterogeneous responses occurring in different CNS regions during the disease reflect not only the extent of neuronal demise but also variable engagement of astrocytes, microglia, immune cells in the attempt to cope with the neurodegeneration. 

To better investigate neuroinflammation in ALS, we ran a study in SOD1.G93A rat model where we correlated pathological alterations (highlighted by MRI) with the pattern of expression of known microgliosis markers (CD11b, TSPO and CB2) measured by flow cytometry in cells from CNS areas characterized by different extent of neurodegeneration (lumbar, thoracic and cervical spinal cord; brainstem; cortex and hippocampus). Interestingly, we highlighted different microglia phenotypes (characterized by variable combinations of the three analyzed markers) depending on the extent of region-specific neuronal demise and on type of onset, hind- vs fore-limb. To further investigate this phenomenon, we performed a single-cell RNAseq analysis: about 36’000 microglia cells retrieved from different CNS regions of WT or symptomatic SOD1.G93A (TG) rats were barcoded using a droplet-based technology. Notably, by running an unbiased analysis guided only by the genetic signature of each cell, TG microglia was very efficiently discriminated from WT cells. More importantly, when we classified each cell according to the CNS region, we identified two very different cell-clusters composed of TG microglia derived either from spinal cord or from brainstem and we found few novel gene candidates that were specifically overexpressed only in TG spinal cord. Validation on human samples is in progress. Overall, our approach unraveled new insights into the complexity of ALS and opened the way for discovery of novel markers for cell-targeting approaches or for future druggable targets for therapy.

 

P22. Protective functions of neuroinflammation in amyotrophic lateral sclerosis
Researchers
Brambilla Liliana (1), Giudotti Giulia (1), Martorana Francesca (2), Iyer Anand M (3), Aronica Eleonora (3), Valori Chiara F (4), Rossi Daniela (1)

Institutes 
(1) Laboratory for Research on Neurodegenerative Disorders, InstitutesClinici Scientifici Maugeri IRCCS, Pavia, Italy; (2) Laboratory of Neuroscience “R. Levi-Montalcini,” Department of Biotechnology and Biosciences, University of Milano-Bicocca, Milan, Italy; (3) Department of Pathology, Academic Medical Center, University of Amsterdam, Amsterdam, The Netherlands; (4) Department of Neuropathology, German Center for Neurodegenerative Diseases (DZNE), Tübingen, Germany

Abstract
Considerable evidence indicates that neurodegeneration in ALS can be conditioned by a deleterious interplay between motor neurons and astrocytes. Astrocytes are the major glial component in the central nervous system, where they fulfill several activities. In physiological and pathological conditions, astrocytes secrete a wide range of mediators, including the trophic factors GDNF and BDNF, two protective agents for motor neurons. Thus, the modulation of the endogenous mechanisms that control the production of astrocytic trophic factors may have therapeutic implications in ALS. 

We investigated the astrocytic signalling pathways driven by the two pro-inflammatory mediators TNFa and HMGB1 and controlling the astrocytic production of trophic factors.

We identified TNFa/TNFR1 signalling as a major promoter of astrocytic GDNF synthesis/release. In SOD1G93A ALS transgenic mice, where the affected tissues spontaneously exhibit high levels of TNFa and TNFR1, we verified a strict correlation in the expression of the TNFa, TNFR1 and GDNF triad at different stages of disease progression. The ablation of TNFR1 completely abolished GDNF rises in both ALS astrocytes and spinal cords, a condition that accelerated motor neuron degeneration and disease progression. In parallel, we examined the role in ALS of HMGB1, a nuclear protein typically released in the extracellular milieu by living cells experiencing physiological stress conditions or by damaged cells. We showed that the interaction of HMGB1 with its receptor RAGE and TLR4 in normal astrocytes promotes neuroprotection via the production of GDNF and BDNF. In ALS mouse spinal cords, we found that HMGB1 is significantly released from motor neurons during disease progression. We postulated that extracellularly released HMGB1 can paracrinally interact with the neighboring astrocytes to counteract the neurodegenerative process. Yet, ALS astrocytes show an impaired capacity to raise trophic factor levels upon HMGB1 stimulation.

P23. Disease course variability in ALS mouse models is driven by different mcp1 mediated neuroimmune response
Researchers
Trolese MC, Scarpa C, Mel V, Nardo G, Bendotti C

Institutes 
“Mario Negri” Institute for Pharmacological Research – IRCCS, Milan, Italy

Abstract
ALS is a clinically heterogeneous disease with high variability in the rate of symptom progression even in the familial cases, and this may explain the failure of clinical trials. 

Growing evidence suggests that immune system components and inflammatory processes are critical to disease progression in ALS. 

It has been reported an association between PNS inflammation and longer disease duration of ALS patients. Consistently, we recently found that at the disease onset, SOD1G93A mice with a slower disease progression (C57SOD1G93A) expressed higher levels of the MCP1 chemokine within motor neurons (MN) and axons compared to fast progressing ALS mice (129SvSOD1G93A). Accordingly, a higher immune cell infiltration was observed within the sciatic nerve and hind limb muscles of C57SOD1G93A mice.

Given the pivotal role of MCP1-mediated signaling in driving axonal and muscle regeneration following injury, we investigated whether a prolonged boosting of this chemokine in the early disease stages of both fast and slow ALS mouse models could delay the development and slow down the disease progression.

Surprisingly, we found that an intramuscular injection of a self-complementary (sc)AAV9 overexpressing MCP1 heightened immune cell infiltration within skeletal muscles of the slow progressing but not in the fast-progressing mice. This translated in the preservation of hindlimb muscle denervation atrophy and postponement of the disease onset in C57SOD1G93A mice, but not in 129SvSOD1G93A mice. 

These results further indicate that the immune response in the peripheral compartments is essential to maintain its regenerative capacity and slow down the ALS progression. 

The comprehension of the mechanisms underlying the different responsiveness of the two ALS models to the treatment and, thus, in regulating the recruitment of the immune cells in the peripheral compartment may provide useful biomarkers for patients’ stratification in the clinical practice.

 

P24. VAPB ER-aggregates, a possible new biomarker in ALS pathology?
Researchers
Maria Piera L Cadoni (1), Giannina Arru (2), Anand Goswami (3), Sandro Orrù (4), GianPietro Sechi (2), Roberto Manetti (1), Grazia Galleri (1)

Institutes 
(1) Department of Medical, Surgical and Experimental Sciences, Laboratory of Experimental Immunology and Cytometry, University of Sassari, Sassari, Italy; (2) Department of Medical, Surgical and Experimental Sciences, Neurologic Clinic, University of Sassari, Sassari, Italy; (3) Institute of Neuropathology, RWTH Aachen University Medical School, Aachen, Germany; (4) Department of Medical Sciences and Public Health, Laboratory of Medical Genetics, University of Cagliari, Monserrato, Italy

Abstract
A point mutation (P56S) in the gene encoding VAPB (vesicle-associated membrane protein-associated protein B) leads to autosomal-dominant form of Amyotrophic Lateral Sclerosis (ALS), classified as ALS-8. Mutant VAPB is characterized by ER-associated aggregates, leading to a complete reorganization of ER structures, activation of cellular stress and ultimately to neurodegeneration. Numerous studies demonstrated VAPB involvement also in sporadic ALS (sALS), although definite pathogenic molecular mechanisms are still unclear. Recently, the identification of new biomarkers in peripheral blood mononuclear cells (PBMCs) has been proposed as a good non-invasive option for studying ALS. 

Here we report the data obtained by the study of PBMC isolated form ALS patients, Parkinson’s disease (PD) patients and healthy control subjects (HC). We evaluated the expression of VAPB, its localisation and the presence of mutations in VAPB and in the major ALS-related genes. Immunofluorescence (IF) analysis of PBMCs revealed a characteristic ER-reorganization pattern specific for ALS patients, not evident in PD patients and HC subjects. Such ALS’s IF pattern was associated to a statistically significant reduction of medium intensity of fluorescence (MFI) in the flow cytometry assay, while we didn’t reveal a significant difference of VAPB mRNA expression between our series. Moreover, the genetic analysis did not reveal VAPB gene mutation. In conclusion our preliminary study brings further evidences that VAPB could represents a useful biomarker for ALS diagnosis and that PBMCs could serve as not invasive, simple, fast, cheap tool useful in the clinical practice and research for studying ALS pathophysiology.

 

P25. Intercellular miscommunication in the brain and periphery: characterization of extracellular vesicle in Amyotrophic Lateral Sclerosis
Researchers
Ferrara D (1), Pasetto L (2), Brunelli L (2), Corsi J (1), Corbelli A (2), Fiordaliso F (2), Cretich M (3), Pastorelli R (2), Calvo A (4), Chiò A (4), Corbo M (5), Lunetta C (6), Mora G (7), Tanel R (8), Sorarù G (9), Quattrone A (1), D’Agostino VG (1), Bonetto V (2), Basso M (1,2)

Institutes 
(1) Department of Cellular, Computational and Integrative Biology – CIBIO, University of Trento, Trento, Italy; (2) IRCCS – Istituto di Ricerche Farmacologiche Mario Negri, Milano, Italy; (3) National Research Council of Italy, Istituto di Chimica del Riconoscimento Molecolare, Italy; (4) CRESLA, Department of Neuroscience “Rita Levi Montalcini”, Università degli Studi di Torino, Torino, Italy; (5) Department of Neurorehabilitation Sciences, Casa Cura Policlinico (CCP), Milan, Italy; (6) NeuroMuscular Ominicentre, Fondazione Serena Onlus, Milano, Italy; (7) ICS Maugeri-IRCCS, Milano, Italy; (8) Neurology Unit, S. Chiara Hospital, Trento, Italy; (9) Department of Neurosciences, University of Padua, Padova, Italy

Abstract
Amyotrophic Lateral Sclerosis (ALS) is a progressive and devastating motor neuron disease that affects upper and lower motor neurons. Among several factors, the progression of the disease is mediated by altered intercellular communication in the spinal cord between neurons and glial cells. One of the possible ways in which cell-to-cell communication occurs is through extracellular vesicles (EVs) that transport proteins, lipids and nucleotides from one cell to the other. To characterize EVs in ALS, we are using a novel bead-based methodology, called NBI, allowing rapid and efficient isolation of EVs. By this approach, it is possible to recover not only a higher and purer amount of astrocyte-derived EVs but also to preserve the integrity and the stability of vesicles and analyze their cargo content. Our preliminary results suggest that EVs derived from astrocytes of a transgenic mouse model of ALS, overexpressing mutant TDP-43 (Q331K), transmit toxicity to wild type neurons. We further focused on which component of the EVs would be responsible for the propagation of toxicity. We set up novel methods to generate i) EVs unloaded of the majority of the protein cargos and ii) EVs containing ‘inactive’ RNA. At the same time, we performed an unbiased characterization of the protein and RNA cargos through a proteomic and small RNA sequencing analysis. Small RNA sequencing did not reveal notable differences between samples. Interestingly, the protein cargos differ significantly between control and disease condition. Finally, to test whether the defect is systemic or CNS-specific, we investigated the similarities between astrocyte-derived EVs and plasma EVs obtained from two different ALS mouse models and human patients. To detect whether the neuron and glia-derived EVs are present in the plasma, we set up a new methodology to define the relative amount of CNS-derived EVs in periphery. Concluding, ALS EVs could be of use for biomarker discovery and mechanistic investigations.

 

P26. The Adipose tissue and the N-acetylaspartate pathway in ALS dysmetabolism
Researchers
Fabio Ciccarone, Serena Castelli, Laura Ioannilli, Silvia Scaricamazza, Alberto Ferri, Maria Rosa Ciriolo

Institutes 
IRCCS San Raffaele Pisana, Department of Human Sciences and Promotion of the Quality of Life, San Raffaele Roma Open University; Department of Biology, University of Rome Tor Vergata; Fondazione Santa Lucia IRCCS, c/o CERC, Rome, Italy

Abstract
Metabolic dysfunction is a feature of ALS patients, whose weight loss is a strong predictor of shortened survival. Reduced body mass in ALS patients is associated with dyslipidemia and increased energy expenditure at rest, but the origin of this “hypermetabolism” is largely unknown. These features are also recapitulated in animal models of ALS, including the SOD1 G93A mice.

Adipose tissue (AT) plays pivotal roles in body energy balance at the periphery and whether its dysfunction may occur in ALS, thus contributing to bioenergetic limitations in the central nervous system (CNS), is still neglected. Regarding the metabolism of AT, we have focused the attention on the N-acetylaspartate (NAA), which contributes to enhancing energy dissipation by adipocytes.

We have data in SOD1 G93A mice showing that white AT (WAT) and Brown AT (BAT) undergo molecular/phenotypical changes that may account for increased energy expenditure and weight loss. In particular, we observed i) reduced size of white adipocytes; ii) high NAA levels in WAT and BAT; iii) WAT senescence; iv) high UCP1 levels in WAT and in BAT, indicating a brown-like phenotype and augmented thermogenic activity, respectively. Notably, all these events occur before weight loss is manifested.

The choice to focus the attention on NAA pathway also arises from the fact that i) NAA is one of the most abundant metabolites in the CNS where N-acetyltransferase 8-like (NAT8L) and aspartoacylase (ASPA), the enzymes involved in NAA turnover, are highly expressed; ii) we have evidence that NAA levels are dramatically increased in cerebrospinal fluid from ALS patients. Along with this, we also observed that the spinal cord of SOD1 G93A mice shows a reduction of NAA levels and of NAT8L expression, whereas these changes were not evidenced in the brain.

This work will provide the basis for the development of strategies targeting NAA pathway that may ameliorate the quality of life of ALS patients recovering AT homeostasis.

 

P27. Exosomes from adipose mesenchymal stem cells as innovative therapeutic approach for ALS
Researchers
Roberta Bonafede (1), Ilaria Scambi (1), Ermanna Turano (1), Lorenzo Schiaf no (1), Jessica Brandi (2), Daniela Cecconi (2), Bruno Bonetti (3), Raffaella Mariotti (1)

Institutes 
(1) Dept. of Neurological, Biomedical and Movement Science, University of Verona, Verona, Italy; (2) Dept. of Biotechnology, University of Verona, Verona, Italy; (3) Azienda Ospedaliera Universitaria Integrata, Verona, Italy

Abstract
A therapeutic approach for amyotrophic lateral sclerosis (ALS) is represented by stem cells. We have demonstrated the beneficial effect of adipose mesenchymal stem cells (ASC) in the in vivo model of ALS, the SOD1(G93A) mice. The ASC administration ameliorate motor performance and increased the number of motoneurons in the treated animals. However, a limited number of ASC were documented in the spinal cord, indicating that stem cells could exert their effect through secreted molecules and extracellular vesicles, as exosomes. Exosomes recapitulate the effect of ASC transplantation by transferring molecules to recipient cells. We demonstrated that ASC-exosomes have a neuroprotective effect on in vitro model of ALS (the NSC34 cell line), rescuing ALS motoneurons after an oxidative insult. These data support the idea that the beneficial effect of ASC is due to the release of exosomes, indicating that these extracellular vesicles can be exploited as potential therapeutic use in ALS, in alternative to stem cell-based therapy. We characterize the content of exosomes in view to understand the molecular mechanisms underlying their neuroprotection. We correlated the protein content to the anti-apoptotic effect, observing a downregulation of Bax and cleaved caspase-3 and upregulation of Bcl-2α in the in vitro model of ALS. Moreover, we investigated the neuroprotective effect of ASC-exosomes in vivo, on SOD1(G93A) mice, after intravenous or intranasal exosomes administration. We demonstrated that ASC-exosomes significantly improved the motor performance of the treated mice compared with the control group. Moreover, the count of motoneurons revealed a significantly higher number of cells in treated animals. Regarding glial cells, a decreasing trend in astrocytes activation was observed. These results indicate that ASC-exosomes exert a neuroprotective rule both in in vitro and in vivo models of ALS, underlining their possible therapeutic use in this neurodegenerative disease.

 

P28. Microvesicles and exosomes in ALS: friends or foes?
Researchers
Sproviero D (1), Gagliardi S (1), Morasso C (2), Mimmi C (1), Bordoni M (1), Crippa V (3), Giannini M (1,4), Diamanti L (4,5), Garofalo M (1,4), Pandini C (1,4), Pansarasa O (1), Corsi F (2,6), Poletti A (3), Calogero RA (7), Cereda C (1)

Institutes 
(1) Genomic and post-Genomic Centre, IRCCS Mondino Foundation, Pavia, Italy; (2) InstitutesClinici Scienti ci Maugeri IRCCS, Pavia, Italy; (3) Dipartimento di Scienze Farmacologiche e Biomolecolari (DiSFeB), Centro di Eccellenza sulle Malattie Neurodegenerative, Università degli Studi di Milano, Milan, Italy; (4) Department of Brain and Behavioural Sciences, University of Pavia, Pavia, Italy; (5) Division of General Neurology, IRCCS Mondino Foundation, Pavia, Italy; (6) Department of Biomedical and Clinical Sciences “Luigi Sacco”, University of Milan, Milano, Italy; (7) Department of Molecular Biotechnology and Health Sciences, Bioinformatics and Genomics Unit, University of Turin, Italy

Abstract
Extracellular vesicles (EVs) are spherical vesicles, which are classified mainly in microvesicles (MVs) and exosomes (EXOs) based on their biogenesis, size and surface markers. In this study, we characterized MVs and EXOs isolated from plasma of sporadic ALS patients and healthy controls and determined their number, size and their different composition (protein, lipids, nucleic acids etc.) in order to understand their neuroprotective or neurotoxic role in ALS pathogenesis. 

No variation was found in the number of EVs between ALS patients and controls. However, the mean size both for MVs and for EXOs resulted increased in ALS patients compared to controls. MVs derived from ALS patients were enriched in SOD-1, TDP-43 and FUS proteins compared to CTRLs. EXOs did not show any protein changes. In order to understand if the difference in dimension was driven by any major biological macromolecules, we analyzed MVs and EXOs by Raman Spectroscopy. EXOs showed a distinct spectral pattern from MVs. In addition, MVs of ALS patients were richer in lipids and had less intense bands relative to aromatic aminoacids compared to healthy controls. 

We also found a great presence of leukocyte derived MVs (LMVs) in ALS patients compared to AD patients and healthy donors and significant correlation with the Progression Rate of the disease. 

On the other hand, miRNA and RNA whole transcriptome sequencing identified a specific signature of miRNAs in plasma derived EXOs of ALS patients compared to a group of healthy controls and three neurological groups of control. 

In summary, these data may suggest that MVs derived from ALS patients, enriched in lipids and toxic proteins, might play a role in prion-like propagation and immunity of ALS disease, while exosomes, deriving from endosomes, might be involved in the impairment of RNA, specific feature of ALS disease.

P29. Cyclophilin A (PPIA) in amyotrophic lateral sclerosis: a TDP-43 interactor with disease modifying effect
Researchers
Pasetto L (1), Luotti S (1), Scozzari S (1), Napoli E (1), Corbo M (2), Mora G (3), Chiò A (4), Lunetta C (5), Calvo A (4), Bonetto V (1)

Institutes 
(1) Istituto di Ricerche Farmacologiche Mario Negri IRCCS, Milano; (2) Casa Cura Policlinico, Milano; (3) Department of Neurorehabilitation, Scienti c Institute of Milan, Salvatore Maugeri Foundation IRCCS, Milano; (4) “Rita Levi Montalcini” Department of Neuroscience, University of Torino; Azienda Ospedaliero-Universitaria Città della Salute e della Scienza of Torino, Torino; (5) NEuroMuscular Omnicentre (NEMO), Serena Onlus Foundation, Milano

Abstract
Amyotrophic lateral sclerosis (ALS) is a motor neuron disease with no biomarkers and no effective disease-modifying therapies available. Our team of research have provided evidence that cyclophilin A, also known as peptidyl prolyl cis-trans isomerase A (PPIA), is a disease modifier, with diagnostic and prognostic potential. PPIA is a multifunctional protein: a foldase, a molecular chaperone, and an interactor of RNA-binding proteins. We first found that PPIA levels in peripheral blood mononuclear cells (PBMCs) could distinguish ALS patients from controls. Next, we demonstrated that PPIA, in its Lys-acetylated form (acetyl-PPIA), binds TDP-43 in the low complexity domain and regulates its function. It affects TDP-43-dependent expression of genes involved in the clearance of protein aggregates and plays a key role in the assembly and dynamics of ribonucleoprotein complexes. Accordingly, absence of PPIA induces TDP-43 cytoplasmic mislocalization, aggregation and accelerates disease progression in the SOD1 mouse model of ALS. We hypothesized that decreased levels of acetyl-PPIA may be at the basis of TDP-43 mislocalization and finally aggregation of TDP-43. Indeed, we detected low acetyl-PPIA in PBMCs of ALS patients that also display TDP-43 mislocalization. We also collected evidence that restoring normal levels of Lys-acetylation reduce TDP-43 mislocalization and be a useful therapeutic target for ALS. Ongoing studies aim to verify if acetyl-PPIA and TDP-43 mislocalization in PBMCs may truly contribute to an early diagnosis of ALS and correlate with disease progression and if a therapeutic approach targeting Lys-acetylation/acetyl-PPIA reduces TDP-43 pathology in PBMCs of the patients and arrests disease progression in a TDP-43 mouse model of ALS. This project will provide important insights into the molecular mechanisms of TDP-43 pathology and could offer the rationale and tools for the development of a new therapeutic approach for ALS.

P30. HyperALS, Modulation and Hypermetabolism and Hyperexcitability as a strategy to counteract degeneration in ALS
Researchers
Scaricamazza S (1,2), Salvatori I (2), Ciriminna G (2), Valle C (2,3), Ferri A (3)

Institutes 
(1) Dipartimento di Biologia, Università degli Studi di Roma Tor Vergata, Roma; (2) IRCCS Fondazione Santa Lucia, Roma; (3) Istituto di Farmacologia Traslazionale IFT-CNR, Roma

Abstract
Background. Besides loss of motor neurons, patients with ALS show an abnormal depletion of energy stores and a parallel hypermetabolism in spite of simultaneous progression of skeletal muscle atrophy. However, malnutrition, weight loss and decrease of body mass are not only severe clinical complications that exacerbate catabolism and sarcopenia but unanswered questions about the disease’s aetiology.

Methods: We studied the evolution of the metabolic alterations in ALS mice using indirect calorimetry, molecular investigations and mitochondrial bioenergetics assessments in spinal cord and skeletal muscle.

Finally, we evaluated the effect of an FDA-approved inhibitor of fatty acid b-oxidation chronically administered to SOD1G93A mice starting from the motor symptoms onset.

Results

We highlighted bioenergetic defects in skeletal muscle of ALS mice long before the disease onset that lead to deep modifications of muscle physiology. Indeed, skeletal muscle of SOD1G93A mice shifts towards an oxidative phenotype with preferential use of lipids as fuel subsequently to bioenergetics defects. Muscle remodelling occurs in SOD1G93A mice before the activation of muscle denervation markers and parallels with a marked increase of energy expenditure unrelated to locomotor activity. Chronic treatment of symptomatic SOD1G93A mice with the inhibitor of fatty acid b-oxidation decreases energy expenditure and this correlates with a robust recovery of pathological phenotype.

Conclusion

Our investigation shed light on the early events that modify muscle physiology and energy expenditure in ALS mouse models. Our attention has been focused on the identification of precocious biomarkers closely related to hypermetabolism, a phenomenon that possesses prognostic and diagnostic relevance. Moreover, here we demonstrated, through a pharmacological intervention aimed to stem energy dissipation, that hypermetabolism inhibition is a promising strategy to counteract pathological phenotypes in ALS.

 

P31. Brain changes in the structural network organization of Amyotrophic Lateral Sclerosis patients: a graph theory analysis study
Researchers
Caterina Galandra (1), Chiara Crespi (2), Alessandra Dodich (3), Gaia Chiara Santi (1), Christian Lunetta (4), Sandro Iannaccone (5), Alessandra Marcone (5), Andrea Falini (5), Chiara Cerami (1)

Institutes 
(1) InstitutesClinici Scienti ci Maugeri IRCCS di Pavia, Pavia, Italy; (2) Istituto Universitario di Studi Superiori (IUSS), Pavia, Italy; (3) NIMTlab, Neuroimaging and Innovative Molecular Tracers Laboratory, University of Geneva, Geneva, Switzerland; (4) NEuroMuscular Omnicentre (NEMO), Niguarda Ca’ Granda Hospital, Milan, Italy; (5) San Raffaele Hospital and San Raffaele Scienti c Institute, Milan, Italy

Abstract
BACKGROUND: Despite recent neuroimaging studies highlighted changes of structural brain patterns linking the behavioral variant of frontotemporal dementia (bvFTD) and Amyotrophic Lateral Sclerosis (ALS), little is known about alterations of grey matter (GM) networks organization and efficiency. Here, we collected data on 20 bvFTD (age: 66.14±6.81), 19 ALS (age: 60.97±11) and 20 healthy control (age: 60.83±8.03) subjects and performed a graph theory analysis on GM components. 

METHODS: We used joint Independent Component Analysis (jICA) in order to find out between groups common GM components. We run ANOVA on the resulting Independent Components (ICs) mixing coefficient to highlight between group differences. Then, we performed network analysis in order to describe group differences on ICs network efficiency.

RESULTS: Multivariate analysis highlighted six ICs. Two out of six were significantly different between the three groups, specifically IC1 (F=9.910, p=0.001), encompassing frontotemporal cortex, as well as anterior cingulate cortex, and IC2 (F=6.126, p=0.004), encompassing sensorimotor, posterior insula and temporal cortex. Network analysis highlighted different patterns of ICs network organization. In particular, compared to HC, bvFTD showed reduced number of ties (bvFTD=16; HC=18), lower average degree (bvFTD=2.667; HC=3) and small-worldness (bvFTD= 1.146; HC=1.244). Interestingly, ALS patients had the opposite pattern with greater number of ties (ALS=20) and higher average degree (ALS=3.33), but a reduced small-worldness (ALS=1.119).

DISCUSSION: Our data report abnormal patterns of GM network organization and efficiency in both patient groups. However, while results support an overall pattern of weak network efficiency in bvFTD, in ALS the greater number of ties and the higher degree with reduced small-worldness suggest possible compensatory mechanisms put in place to balance a less efficient information processing.

P32. The 6-minute walk test as a promising outcome measure in Amyotrophic Lateral Sclerosis
Researchers
Russo M (1,2), Zuccarino R (3), Vita GL (2), Sframeli M (2), Lizio A (3), Becchiati S (3), La Foresta S (2), Faraone C (2), Sansone VA (4, 5), Lunetta C (4), Vita G (1,2), Messina S (1,2)

Institutes 
(1) Unit of Neurology and Neuromuscular Disorders, Department of Clinical and Experimental Medicine, University of Messina, Messina; (2) NEuroMuscular Omnicentre of Messina, University Hospital “G. Martino”, Messina; (3) NEuroMuscular Omnicentre of Arenzano, Arenzano; (4) NEuroMuscular Omnicentre of Milan, Milan; (5) Neurorehabilitation Unit, University of Milan

Abstract
OBJECTIVE: Because of the increasing therapeutic approaches entering the clinical arena, there is the burning need to identify valuable outcome measures (OM), still lacking in amyotrophic lateral sclerosis (ALS). Therefore, we assessed the 6-minute walk test (6MWT) in a large cohort of ambulant patients with ALS over a 6-month interval. We aimed to establish the spectrum of possible changes in relation to other measures related to disease severity (10 meters walking test (10 MWT),Time-up and go (TUG), ALS Functional Rating Scale-Revised (ALSFRS-R), cognitive involvement (ALS- Cognitive Behavioural Screen (CBS, Edinburgh Cognitive and Behavioral ALS Screen (ECAS)) and QoL (McGill Quality of life questionnaire). 

METHODS:

The study is a longitudinal multicentric cohort study. A specific training of the therapists from the three NEuroMuscular Omnicentres was performed. We enrolled patients with a relatively preserved motor and respiratory function. A total of 44 ambulant patients with ALS were assessed with the above mentioned outcome measures at baseline, after 5 ± 2 days (retest for 6MWT) and after 1, 3 and 6 months.  

RESULTS:

During the 6 months of the study, we observed a mean decline of 45.8 meters in the 6MWT with a SD of 104.3 meters. The retest showed changes within 5% at 6MWT. At baseline, the 6MWT correlated with all other outcome measures (10MWT: r=0.74, p<0.0001; TUG test: r=-0.80, p<0.0001; ALSFRS-R: r=0.52, p<0.0001, QoL: r=0.64, p<0.0001). Cognitive involvement did not affect the 6MWT performances. Interestingly, assessing the changes between baseline and the 6-month assessment, the 6MWT correlated with the 10MWT (r=-0.81, p<0.0001), TUG test (r=-0.78, p<0.0001), but not with ALSFRS-R (r=0.9, p<0.513) and QoL (r=0.21, p<0.142).

DISCUSSION:

The discordance between changes of 6MWT, ALSFRS-R and QoL at 6 month highlights the different content validity among these instruments.

P33. Prognostic power of psoas muscle in amyotrophic lateral sclerosis
Researchers
Alberto Miceli (1), Isabella Donegani (1), Rita Lai (2), Silvia Morbelli (1), Valentina Ceriani (1), Anna Borra (1), Stefano Raffa (1), Matteo Bauckneht (3), Selene Capitanio (3), Cristina Campi (4), Gianmario Sambuceti (1), Michele Piana (1), Cecilia Marini (3,5)

Institutes 
(1) DISSAL, University of Genoa, Genoa, Italy; (2) SPIN Institute CNR, Genoa, Italy; (3) IRCCS Policlinico San Martino, Genoa, Italy; (4) DIMED, Padova University Hospital, Padua, Italy; (5) CNR Institute of Molecular Bioimaging and Physiology (IBFM), Milan, Italy

Abstract
Purpose. Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease leading to muscle paralysis and death. Applying an artificial intelligence method to PET/CT images, we documented an increased uptake of 18F-fluoro-2-deoxyglucose (FDG) in the psoas muscle of ALS patients. 

Our study aims to evaluate whether this metabolic feature and its underlying mechanisms can represent a diagnostic/prognostic biomarker of ALS to pave the way for innovative therapies. 

Materials and Methods

We analyzed 62 ALS patients submitted to PET/CT imaging. Obtained data were compared with the corresponding findings in 36 age-and sex-matched controls. A computational 3D method was used to extract whole psoas muscle’s volumes and its average attenuation coefficient (AAC) from CT images. FDG accumulation was defined by psoas normalized standardized uptake value (N-SUV) and its distribution heterogeneity was expressed by SUV standard deviation (SUV-SD). 

Results

AAC was similar in patients and controls (39.4±8.4 vs 39.1±11.3 Hounsfield units, respectively, p=ns). By contrast, ALS was associated with a significant reduction in psoas volume normalized for IBW (3.6±1.02 vs 4.12±1.33 mL/Kg; p=0.01). Similarly, at PET imaging, N-SUV was significantly higher in patients than in controls (0.45±0.19 vs 0.29±0.09; p<0.001). Finally, heterogeneity of psoas N-SUV, expressed by SUV-SD, predicted overall survival rate at Kaplan-Meyer analysis (p<0.05) with a predictive power that was confirmed by univariate as well as by multivariate Cox analysis (p<0.02).

Conclusion

We propose that the interdependency between ALS skeletal muscle damage and second motor neuron disease further sets in a vicious cycle leading to rapid and progressive muscle paralysis and denervation.

P34. Curcumin and motor neuron disease: preliminary data from a double-blind, placebo-controlled clinical trial
Researchers
Leonardo Massoni, Lucia Chico, Annalisa Lo Gerfo, Alessandra Govoni, Erika Schirinzi, Costanza Simoncini, Gabriele Siciliano

Institutes 
Department of Clinical and Experimental Medicine, Neurological Clinic, University of Pisa, Pisa, Italy

Abstract
Increasing evidence suggests that oxidative stress plays a pivotal role in the pathogenesis of amyotrophic lateral sclerosis (ALS). Curcumin is a potent scavenger of reactive chemical species; in ALS mouse models it has been observed that curcumin interacts with TDP-43 protein improving the motoneuron membrane excitability and balancing oxidative stress and mitochondrial function. The current study was designed to determine whether curcumin oral supplementation (1500 mg/day, Alibrain Advanced) may be effective in the treatment of ALS. 

A total of 33 ALS patients were enrolled and randomized 1:1 to take experimental drug or placebo.

Clinical parameters such as ALS-Functional Rating Scale (ALS-FRS), Medical Research Council (MRC), body mass index (BMI), and biochemical hematic parameters such as oxidative stress markers including oxidative protein products (AOPPs), ferric reducing ability (FRAP), total thiols (T-SH) and lactate, were collected.  

All the patients were evaluated before the introduction of experimental therapy, while a subgroup of 10 patients were revaluated after 3 months of placebo/curcumin supplementation.

Compared to healthy controls, the whole ALS population showed at baseline greater levels of oxidative stress valued by higher AOPPs (p<0,001), and lower FRAP and t-SH (p<0,001) levels. 

Between the two patients group we observed that after 3 months of taking curcumin/placebo there was a significant difference in the lactate (with a border line p value 0,06) and thiols(p=0,028) values. 

We have also found a difference in AOPPs and FRAP levels and minimally in ALS-FRS and MRC scales, but these findings were not statistically significant. 

Further studies with a larger cohort of patients and a longer observation period are needed to confirm a possible therapeutic effect of curcumin in ALS.

P35. Rapamycin treatment for ALS: Preliminary data from a double-blind RCT
Researchers
Zucchi E, Cossarizza A, Fini N, Martinelli I, Gessani A, D’Amico R, Caponnetto C, Chiò A, Dalla Bella E, Lunetta C, Marinou K, Mazzini L, Sorarù G, Mandrioli J

Institutes 
Department of Biomedical, Metabolic, Neural Sciences – Azienda Ospedaliera Universitaria di Modena, University of Modena e Reggio Emilia, Modena

Abstract
Study Rationale and Preliminary Data: Misfolded aggregated proteins significantly contribute to ALS hence representing therapeutic targets to modify disease expression. Rapamycin inhibits mTOR pathway and enhances autophagy with demonstrated beneficial effects in neurodegeneration. Rapamycin also expands regulatory T lymphocytes (Treg) that dampen immune responses: increased Treg levels are associated with slow ALS progression. In cell lines and animal models of ALS Rapamycin reduced TDP43 accumulation and restored TDP43 localization; Rapamycin improved motor and cognitive phenotype in animal models as shown below. This is the first RCT with Rapamycin in ALS carrying the double potential effect of enhancing TDP43 autophagy and expanding Tregs.

Study design and Methods: 

Design: Phase II randomized, double-blind, placebo-controlled, multicenter, clinical trial

Subjects: 63 ALS patients enrolled in 3 groups of 21 subjects; treatment duration 18 weeks.

Rapamycin is administered at fast, in the morning, once a day. Rapamycin levels are measured at week 1,2,4,8,12,18 to avoid toxicity (>15 ng/ml). Rapamycin dosage (HPLC) is performed in the morning, before treatment assumption. Treating neurologists have no access to blood laboratory data. The Local Laboratory, blind to treatment, sends Rapamycin values for possible dosages adjustments. Sham adjustments can be performed in the placebo group too.

Expected results: We expect to obtain a biological response defined as a significant increase in Treg number of at least 30% at treatment end (W18) in Rapamycin group compared with placebo arm. This study carries the double potential effect of enhancing TDP43 autophagy and of targeting the immune system through Treg expansion with the aim to potentially modify disease expression.

Coordinating center: AOU Modena

Recruiting centers:

  • AOU Maggiore della Carità, Novara
  • IRCSS Maugeri Foundation, Milan
  • IRCSS AOU San Martino
  • IRCSS Besta Institute, Milan
  • NEMO Clinical Center”
P36. Unravelling moral reasoning in amyotrophic lateral sclerosis: How emotional detachment modifies moral judgment inclinations
Researchers
Chiara Crespi (1,2), Gaia Chiara Santi (2), Alessandra Dodich (3), Federica Lupo (4), Lucia Greco (5), Tommaso Piccoli (4) Christian Lunetta (5), Chiara Cerami (2)

Institutes 
(1) Istituto Universitario di Studi Superiori (IUSS), Pavia, Italy; (2) InstitutesClinici Scienti ci Maugeri IRCCS di Pavia, Pavia, Italy; (3) NIMTlab, Neuroimaging and Innovative Molecular Tracers Laboratory, University of Geneva, Geneva, Switzerland; (4) Department of Biomedicine and Clinical Neuroscience, University of Palermo, Palermo, Italy; (5) NEuroMuscular Omnicentre (NEMO), Niguarda Ca’ Granda Hospital, Milan, Italy

Abstract
OBJECTIVE: Recent literature proved that socio-affective deficits may characterize Amyotrophic Lateral Sclerosis (ALS) phenotype. However, little is still known about moral cognition in ALS and no study investigated socio-affective dimension of moral reasoning. In this study, we evaluated moral cognitive performances in a sample of 28 (19 spinal, 9 bulbar) patients with ALS. METHODS: Patients underwent the Edinburgh Cognitive and Behavioral ALS Screen (ECAS) battery, the Stroop, the Ekman-60 Faces and Story-based Empathy tasks. Additionally, a task of moral dilemmas including both ‘instrumental’ and ‘incidental’ conditions was administered. Thirty-six age-, gender- and education-matched healthy control subjects (HC) were also enrolled. RESULTS: A subset of ALS patients showed deficits at executive and social cognition tasks. Overall, ALS patients showed significantly different levels of moral judgment with respect to HC. The rate of yes/no response in resolution of incidental (χ2(4)=36.704, p<0.001) and instrumental (χ2(4)=14.651, p<0.05) moral dilemmas was significantly different between groups. Patients were less prone to carry out a moral transgression. While, condition x diagnosis interaction effect was significant for emotional arousal (ALS < HC; F(1,62)=22.325, p<0.001), we found no significant interactive effect for both moral permissibility (F(1,62)=0.043, p=0.837) and attribution of emotional valence (F(1,62)=3.596, p=0.63). Spinal and bulbar ALS patients showed comparable performances except for moral permissibility in which spinal patients had higher scores compared to bulbar ones. DISCUSSION: Our findings expand current literature on cognitive profiles in ALS supporting not only deficits in executive and socio-emotional domains but also altered moral judgment with poor emotional involvement. This may have relevant implications in applying moral principles in real-life situations and for the judgment of end-of-life treatments and care.

P37. Dynamic recognition of emotion in ALS patients
Researchers
Gori MC, Fiorini I, Pozzilli V, Di Bari S, Onesti E, Libonati L, Cambieri C, Ceccanti M, Frasca V, Inghilleri M

Institutes 
Università Sapienza di Roma, Dipartimento di Neurologia e Psichiatria, Centro Malattie Neuromuscolari Rare, Policlinico Universitario Umberto I

Abstract
Amyotrophic lateral sclerosis (ALS) primarily impairs motor abilities but also affects cognition and emotional processing. The aim is to study social cognition in ALS patients, ability to recognize emotions in the faces of others by administering a dynamic test, evaluate the relationship between cognitive deficit and ability to recognize emotions, and analyze correlations between non-motor symptoms of the disease and the type of clinical spinal and bulbar onset.

Methods

The cross-sectional study, carried out at the Neuromuscular Rare Diseases Center of the Policlinico Umberto I from January 2016, recruited ALS patients in according to the revised El Escorial criteria. Patients with exclusive presence of specific ALS cognitive disorders or absence of non-ALS cognitive disorders specific were excluded from ECAS test battery. Patients were subjected to dynamic GERT (Geneva Emotion Recognition Test) emotion recognition test.

Results

63 patients (20 females, 43 males), 43 spinal and 20 bulbar, with a mean age of 63.7 + 11.7 years, were recruited. 31.7% showed specific cognitive deficits for ALS, with a prevalence for bulbar patients (70% of bullets). 76% of subjects presented difficulties in decoding emotions, 23% did not present difficulties. Significant differences between the two groups for 11/14 emotions Poorly recognized emotions resulted in: relief, disgust, sadness, pleasure, fun, interest, irritation, pride, fear, anger). The difficulty in decoding emotions does not appear to be associated either with cognitive deficit or with the phenotype of spinal / bulbar onset

Conclusions

Executive dysfunctions are prevalent in bulbar patients, while impaired recognition of emotions shows no association with the phenotype. The term “executive functions” includes a heterogeneous and complex group of cognitive processes whose circuits are different from those of emotional control. So, it can be assumed that the two functions are not associated.

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