Aggregation of mutant superoxide dismutase 1 (SOD1) is a pathological hallmark of a subset of familial ALS patients. However, the possible role of misfolded wild type SOD1 in human ALS is highly debated. To ascertain whether or not misfolded SOD1 is a common pathological feature in non-SOD1 ALS, we performed a blinded histological and biochemical analysis of post mortem brain and spinal cord tissues from 19 sporadic ALS, compared with a SOD1 A4V patient as well as Alzheimer’s disease (AD) and non-neurological controls. Multiple conformation- or misfolded-specific antibodies for human SOD1 were compared. These were generated independently by different research groups and were compared using standardized conditions. Five different misSOD1 staining patterns were found consistently in tissue sections from SALS cases and the SOD1 A4V patient, but were essentially absent in AD and non-neurological controls. We have established clear experimental protocols and provide specific guidelines for working, with conformational/misfolded SOD1-specific antibodies. Adherence to these guidelines will aid in the comparison of the results of future studies and better interpretation of staining patterns. This blinded, standardized and unbiased approach provides further support for a possible pathological role of misSOD1 in SALS.

Amyotrophic lateral sclerosis (ALS) is an adult-onset disease characterized by the selective degeneration of motor neurons in the brain and spinal cord progressively leading to paralysis and death. Current diagnosis of ALS is based on clinical assessment of related symptoms. The clinical manifestations observed in ALS appear relatively late in the disease course after degeneration of a significant number of motor neurons. As a result, the identification and development of disease-modifying therapies is difficult. Therefore, novel strategies for early diagnosis of neurodegeneration, to monitor disease progression and to assess response to existing and future treatments are urgently needed. Factually, many neurological disorders, including ALS, are accompanied by skin changes that often precede the onset of neurological symptoms. Aiming to generate an innovative human-based model to facilitate the identification of predictive biomarkers associated with the disease, we developed a unique ALS tissue-engineered skin model (ALS-TES) derived from patient's own cells. The ALS-TES presents a number of striking features including altered epidermal differentiation, abnormal dermo-epidermal junction, delamination, keratinocyte infiltration, collagen disorganization and cytoplasmic TDP-43 inclusions. Remarkably, these abnormal skin defects, uniquely seen in the ALS-derived skins, were detected in pre-symtomatic C9orf72-linked ALS patients carrying the GGGGCC DNA repeat expansion. Consequently, our ALS skin model could represent a renewable source of human tissue, quickly and easily accessible to better understand the physiopathological mechanisms underlying this disease, to facilitate the identification of disease-specific biomarkers, and to develop innovative tools for early diagnosis and disease monitoring.

Extracellular matrix (ECM) secretion, deposition and assembly are part of a whole complex biological process influencing the microenvironment and other cellular behaviors. Emerging evidence is attributing a significant role to extracellular vesicles (EVs) and exosomes in a plethora of ECM-associated functions, but the role of dermal fibroblast-derived EVs in paracrine signalling is yet unclear. Herein, we investigated the effect of exosomes isolated from stimulated human dermal fibroblasts. We report that tridimensional (3D) cell culture of dermal fibroblasts promotes secretion of exosomes carrying a large quantity of proteins involved in the formation, organisation and remodelling of the ECM. In our 3D model, gene expression was highly modulated and linked to ECM, cellular migration and proliferation, as well as inflammatory response. Mass spectrometry analysis of exosomal proteins, isolated from 3D cultured fibroblast-conditioned media, revealed ECM protein enrichment, of which many were associated with the matrisome. We also show that the cytokine interleukin 6 (IL-6) is predicted to be central to the signalling pathways related to ECM formation and contributing to cell migration and proliferation. Overall, our data suggest that dermal fibroblast-derived EVs participate in many steps of the establishment of dermis’s ECM.

In vitro selection of remdesivir-resistant severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) revealed the emergence of a V166L substitution, located outside of the polymerase active site of the Nsp12 protein, after 9 passages of a single lineage. V166L remained the only Nsp12 substitution after 17 passages (10 μM remdesivir), conferring a 2.3-fold increase in 50% effective concentration (EC 50 ).