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210th ENMC International Workshop: Research and clinical management of patients with spinal and bulbar muscular atrophy, 27–29 March, 2015, Naarden, The Netherlands Maria Pennuto *, Linda Greensmith , Pierre-Francois Pradat , Gianni Soraru ** on behalf of the European SBMA Consortium 1 a Dulbecco Telethon Institute Lab of Neurodegenerative Diseases, Centre for Integrative Biology, University of Trento, Italy b The Graham Watts Laboratories for Research into Motor Neuron Disease, UCL Institute of Neurology, London, UK c Departement des Maladies du Systeme Nerveux, AP-HP, Groupe hospitalier Pitie-Salpetriere, F-75013 Paris, France d UPMC Univ Paris 06, CNRS, INSERM, Laboratoire d’Imagerie Biomedicale (LIB), Sorbonne Universites, 75013 Paris, France e Department of Neurosciences, University of Padova, Padova, Italy

Background: The mechanism by which astrocytes contribute to disease progression in mutant SOD1 mouse models of ALS is not known. Results: Mutant SOD1 astrocytes release mutant SOD1-containing exosomes that are toxic for motor neurons. Conclusion: Astrocyte-derived exosomes may have a role in disease spreading and motor neuron pathology. Significance: New therapeutic approaches should target exosomes to contain disease progression., Amyotrophic lateral sclerosis is the most common motor neuron disease and is still incurable. The mechanisms leading to the selective motor neuron vulnerability are still not known. The interplay between motor neurons and astrocytes is crucial in the outcome of the disease. We show that mutant copper-zinc superoxide dismutase (SOD1) overexpression in primary astrocyte cultures is associated with decreased levels of proteins involved in secretory pathways. This is linked to a general reduction of total secreted proteins, except for specific enrichment in a number of proteins in the media, such as mutant SOD1 and valosin-containing protein (VCP)/p97. Because there was also an increase in exosome release, we can deduce that astrocytes expressing mutant SOD1 activate unconventional secretory pathways, possibly as a protective mechanism. This may help limit the formation of intracellular aggregates and overcome mutant SOD1 toxicity. We also found that astrocyte-derived exosomes efficiently transfer mutant SOD1 to spinal neurons and induce selective motor neuron death. We conclude that the expression of mutant SOD1 has a substantial impact on astrocyte protein secretion pathways, contributing to motor neuron pathology and disease spread.

none 14 si Alzheimer's disease (AD) is a multifactorial pathology that requires multifaceted agents able to address its peculiar nature. In recent years, a plethora of proteins and biochemical pathways has been proposed as possible targets to counteract neurotoxicity. Although the complex scenario is not completely elucidated, close relationships are emerging among some of these actors. In particular, increasing evidence has shown that aggregation of amyloid beta (Aβ), glycogen synthase kinase 3β (GSK-3β) and oxidative stress are strictly interconnected and their concomitant modulation may have a positive and synergic effect in contrasting AD-related impairments. We designed compound 3 which demonstrated the ability to inhibit both GSK-3β (IC50 = 24.36 ± 0.01 μM) and Aβ42 self-aggregation (IC50 = 9.0 ± 1.4 μM), to chelate copper (II) and to act as exceptionally strong radical scavenger (kinh = 6.8 ± 0.5 · 105 M−1s−1) even in phosphate buffer at pH 7.4 (kinh = 3.2 ± 0.5 · 105 M−1s−1). Importantly, compound 3 showed high-predicted blood-brain barrier permeability, did not exert any significant cytotoxic effects in immature cortical neurons up to 50 μM and showed neuroprotective properties at micromolar concentration against toxic insult induced by glutamate. De Simone, Angela; Bartolini, Manuela; Baschieri, Andrea; Apperley, Kim Y.P.; Chen, Huan Huan; Guardigni, Melissa; Montanari, Serena; Kobrlova, Tereza; Soukup, Ondrej; Valgimigli, Luca; Andrisano, Vincenza; Keillor, Jeffrey W.; Basso, Manuela; Milelli, Andrea De Simone, Angela; Bartolini, Manuela; Baschieri, Andrea; Apperley, Kim Y.P.; Chen, Huan Huan; Guardigni, Melissa; Montanari, Serena; Kobrlova, Tereza; Soukup, Ondrej; Valgimigli, Luca; Andrisano, Vincenza; Keillor, Jeffrey W.; Basso, Manuela; Milelli, Andrea

Neuroinflammation is a major hallmark of amyotrophic lateral sclerosis (ALS), which is currently untreatable. Several anti-inflammatory compounds have been evaluated in patients and in animal models of ALS, but have been proven disappointing in part because effective targets have not yet been identified. Cyclophilin A, also known as peptidylprolyl cis-/trans-isomerase A (PPIA), as a foldase is beneficial intracellularly, but extracellularly has detrimental functions. We found that extracellular PPIA is a mediator of neuroinflammation in ALS. It is a major inducer of matrix metalloproteinase 9 and is selectively toxic for motor neurons. High levels of PPIA were found in the CSF of SOD1G93Amice and rats and sporadic ALS patients, suggesting that our findings may be relevant for familial and sporadic cases. A specific inhibitor of extracellular PPIA, MM218, given at symptom onset, rescued motor neurons and extended survival in the SOD1G93Amouse model of familial ALS by 11 d. The treatment resulted in the polarization of glia toward a prohealing phenotype associated with reduced NF-κB activation, proinflammatory markers, endoplasmic reticulum stress, and insoluble phosphorylated TDP-43. Our results indicates that extracellular PPIA is a promising druggable target for ALS and support further studies to develop a therapy to arrest or slow the progression of the disease in patients.SIGNIFICANCE STATEMENTWe provide evidence that extracellular cyclophilin A, also known as peptidylprolyl cis-/trans-isomerase A (PPIA), is a mediator of the neuroinflammatory reaction in amyotrophic lateral sclerosis (ALS) and is toxic for motor neurons. Supporting this, a specific extracellular PPIA inhibitor reduced neuroinflammation, rescued motor neurons, and extended survival in the SOD1G93Amouse model of familial ALS. Our findings suggest selective pharmacological inhibition of extracellular PPIA as a novel therapeutic strategy, not only for SOD1-linked ALS, but possibly also for sporadic ALS. This approach aims to address the neuroinflammatory reaction that is a major hallmark of ALS. However, given the complexity of the disease, a combination of therapeutic approaches may be necessary.

Amyotrophic lateral sclerosis (ALS) is a fatal motor neuron disease, whose primary mechanisms or causes are still not defined and for which no effective treatment is available. We have recently reported that before disease onset the level of tyrosine nitrated proteins is increased in the G93A SOD1 transgenic mouse model of ALS. In the present investigation, we carried out a protcomic analysis of spinal cord extracts from G93A SOD1 mice at the presymptomatic stage of the disease to further unravel primary events in the pathogenesis and tentatively screen for potential pharmacological targets. Using a robust two-dimensional get electrophoresis-based proteomic approach, we detected a number of proteins differentially represented in presymptomatic mice in comparison with controls. Alterations of these proteins correlate with mitochondrial dysfunction, aggregation, and stress response. Moreover, we found a variation in the isoform pattern of cyclophilin A, a molecular chaperone that protects cells from the oxidative stress. (c) 2006 Elsevier Inc. All rights reserved.