Conference Agenda

Amyotrophic Lateral Sclerosis (ALS) is a fatal neurodegenerative disease, in which upper and lower motorneurons are differentially affected and are selectively lost during during the course of the disease. This results in muscle atrophy and gradually to death of the patients mainly for respiratory failure.

ALS mainly occurs as sporadic (s)ALS form. Only 10% of cases are familial (f)ALS; of these, 40% involve the Chromosome 9 Open Reading Frame 72 (C9orf72) gene; other fALS associate to mutations in genes encoding for the proteins SOD1, TDP-43, FUS, SQSTM1/p62, TIA-1, optineurin, ubiquilin, and others, that are causative of motorneuronal death by different pathological mechanisms. One of the most studied is the proteotoxic stress triggered by potentially toxic misfolded proteins that accumulate perturbing several fundamental cell processes. In fact, both as wild type, but particularly when mutated, most of the protein products of these fALS genes (including the aberrant dipeptides (DPRs) arising from unconventional (RAN) translation of the G9ORF72 transcripts) are prone to misfold and to aggregate.

The intracellular proteotoxic response to these misfolded proteins and DPRs is based on the coordinate action of chaperones and the degradative systems, mainly the proteasome and the autophagy. Modulators of both the chaperones and of the degradative systems have been found able to modulate misfolded protein toxicity in a variety of preclinical models of fALS.

We tested compounds that are able to enhance the autophagy activity and/or to ameliorate the capability of chaperone assisted selective autophagy (CASA) in the removal of ALS associated misfolded proteins including C9ORF72 RAN translated neurotoxic DPRs and their aggregated species. Some of these have been recently clinically tested as potential therapeutic approach to treat different type of neurodegenerative diseases.

Fondazione Telethon; Fondazione Cariplo; Fondazione AriSLA; AFM-Telethon, France; Kennedy’s Disease Association, USA; MIUR (PRIN/PNRR; CN3; Dipartimenti Eccellenza); Agenzia Italiana del Farmaco (AIFA).

The intronic hexanucleotide expansion in the C9ORF72 gene represent the most frequent mutation associated to the gentic forms of frontotemporal dementia (FTD) and amyotrophic lateral sclerosis (ALS; C9ORF72-FTD/ALS).

This presentation will cover an update on the clinical features of the 'spectrum' of C9 manifestations. Recent discoveries in C9ORF72-FTD/ALS disease begin to unrevel its pathogenetic mechanisms, and new therapeutic approaches are in the pipeline of different research groups.
The clinical trials designs are critical challenges in this field, and are based of the avaiability of reliable biomarkers for diagnosis and prognosis, which are the main focus of this topic.

ALS substantial heterogeneity mirrors its biological complexity and has implications for patients’ counselling and individual prognosis assessment, participant stratification in clinical trials and developing of new therapeutic strategies, and, not least, timing of treatment interventions and care management. Currently, in absence of established pharmacodynamics or predictive biomarkers to be used in clinical settings, the identification of biological indicators of disease state and progression represents an urgent unmet need.

We recently completed two multicenter, randomized, double, blind, placebo controlled clinical trials, where patients were treated with different drugs and biological samples were collected at baseline and longitudinally.

In the RAP-ALS trial, 63 patients were randomized in 1:1:1 ratio to treatment with rapamycin 1 mg/m2/day, 2 mg/m2/day or placebo. Patients treated with the lower dose of rapamycin showed a significant reduction in IL18, and had a lower monthly decline at ALSFRS-R, although primary outcome (an increase of at least 30% of Treg cells in treated versus untreated patients) was not met. In the Co-ALS trial, 54 patients were randomized in 1:1:1 ratio to treatment with colchicine 0.01 mg/kg/day, 0.005 mg/kg/day or placebo. Patients treated with the lower dose of colchicine showed a significant reduction in the monthly decline of the ALSFRS-R, although primary outcome was not met. Interestingly, both drugs have different actions (on immune system, autophagy and metabolism) at different dosages.

On post-hoc analysis patients classification according to combined immune and metabolic profile, independently of BMI, allowed to identify subgroups of ALS patients with distinct disease progressions and response to treatment.

While taking into account all the limitations of post-hoc analyses, it should be acknowledged that the stratification of patients on a biological basis can now represent a significant step forward for improving the design of clinical trials and overcoming possible drawbacks due to the clinical heterogeneity of the disease.

Background. ADAMs comprise a large family of transmembrane metalloproteases responsible for ectodomain proteolytic cleavage (shedding) of membrane-tethered proteins. ADAM17 was originally identified as the major enzyme for TNF-alpha release, and ADAM10 could compensate for this function. ADAM 10/17 activity is increased in proinflammatory conditions and has already been implicated in the pathogenesis of Alzheimer’s disease and Multiple Sclerosis. However, information on potential involvement of ADAM10/17 in ALS is still scarce.

Aims. Our goal is to elucidate whether alterations of ADAM10/17 protein expression, distribution and/or enzymatic activity could play a role in ALS. Moreover, ADAM10/17 can be released in circulation, and we explored CSF/blood ADAM10/17 activity as a potential ALS biomarker.

Methods. We performed immunohistochemistry and western blot analyses in spinal cord districts at different disease stages in the SOD1.G93A transgenic (TG) rat model of ALS. In parallel, we measured ADAM10/17 activity in spinal cord homogenates, in CSF and blood.

Results. We highlighted a selective increase of ADAM10/17 immunoreactivity in motor neurons of TG animals at the onset of the disease; as the disease progresses to the late stages, ADAMs are upregulated in glial cells in the white and gray matter. ADAM10/17 enzymatic activity, measured in tissue homogenats, was increased at the symptomatic stage of the disease. Finally, we measured increased levels of ADAM10/17 activity only in the CSF of TG at the symptomatic stage, whereas in the blood was negligible.

Discussion. Although our data are still preliminary, we highlighted alterations of ADAM10/17 distribution at the early symptomatic stage of the disease. We are currently investigating the correlation between ADAMs and some of their substrates, such as TNF-alpha or GPNMB implicated in the disease; in parallel, we are exploring selective pharmacological ADAMs inhibitors as a potential therapeutic approach.