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.

Apart from the well-defined factors in neuronal cells 1 , only few reports consider that variability of sporadic ALS progression can depend on the less-defined contributions from glia 2,3 and blood vessels 4 .In this study we use an expression weighted cell-type enrichment method to infer cell activity in spinal cord samples from sporadic ALS patients and mouse models of this disease. Here we report that sporadic ALS patients present cell activity patterns consistent with two mouse models in which enrichments of vascular cell genes preceded the microglial response. Notably, during the presymptomatic stage, perivascular fibroblast cells showed the strongest gene enrichments and their marker proteins SPP1 and COL6A1 accumulated in enlarged perivascular spaces in sporadic ALS patients. Moreover, in plasma of 574 ALS patients from four independent cohorts, increased levels of SPP1 at disease diagnosis repeatedly predicted shorter survival with stronger effect than the established risk factors of bulbar onset or neurofilament levels in cerebrospinal fluid. We propose that the activity of the recently-discovered perivascular fibroblast can predict ALS patient survival and provide a novel conceptual framework to re-evaluate definitions of ALS etiology.

Mutations in superoxide dismutase 1 ( SOD1 ) cause amyotrophic lateral sclerosis (ALS). Disease pathogenesis is linked to destabilization, disorder and aggregation of the SOD1 protein. However, the non-genetic factors that promote disorder and the subsequent aggregation of SOD1 have not been studied. Mainly located to the reducing cytosol, mature SOD1 contains an oxidized disulfide bond that is important for its stability. Since O 2 is required for formation of the bond, we reasoned that low O 2 tension might be a risk factor for the pathological changes associated with ALS development. By combining biochemical approaches in an extensive range of genetically distinct patient-derived cell lines, we show that the disulfide bond is an Achilles heel of the SOD1 protein. Culture of patient-derived fibroblasts, astrocytes, and induced pluripotent stem cell-derived mixed motor neuron and astrocyte cultures (MNACs) under low O 2 tensions caused reductive bond cleavage and increases in disordered SOD1. The effects were greatest in cells derived from patients carrying ALS-linked mutations in SOD1 . However, significant increases also occurred in wild-type SOD1 in cultures derived from non-disease controls, and patients carrying mutations in other common ALS-linked genes. Compared to fibroblasts, MNACs showed far greater increases in SOD1 disorder and even aggregation of mutant SOD1s, in line with the vulnerability of the motor system to SOD1-mediated neurotoxicity. Our results show for the first time that O 2 tension is a principal determinant of SOD1 stability in human patient-derived cells. Furthermore, we provide a mechanism by which non-genetic risk factors for ALS, such as aging and other conditions causing reduced vascular perfusion, could promote disease initiation and progression. Electronic supplementary material The online version of this article (10.1007/s00401-019-01986-1) contains supplementary material, which is available to authorized users.

24Mutations that destabilize superoxide dismutase 1 (SOD1) are a cause of amyotrophic lateral 25 sclerosis (ALS). SOD1, which is located in the reducing cytosol, contains an oxidized 26 disulfide bond required for stability. We show that the bond is an Achilles heel of the protein 27 because it is sensitive to the oxygen tension. Culture of ALS patient-derived fibroblasts, 28 astrocytes and induced pluripotent stem cell-derived mixed motor neuron and astrocyte 29cultures ( (which was not peer-reviewed) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity.

Increasing evidence suggests that propagation of the motor neuron disease amyotrophic lateral sclerosis (ALS) involves the pathogenic aggregation of disease-associated proteins that spread in a prion-like manner. We have identified two aggregate strains of human superoxide dismutase 1 (hSOD1) that arise in the CNS of transgenic mouse models of SOD1-mediated ALS. Both strains transmit template-directed aggregation and premature fatal paralysis when inoculated into the spinal cord of adult hSOD1 transgenic mice. This spread of pathogenic aggregation could be a potential target for immunotherapeutic intervention. Here we generated mouse monoclonal antibodies (mAbs) directed to exposed epitopes in hSOD1 aggregate strains and identified an aggregate selective mAb that targets the aa 143–153 C-terminal extremity of hSOD1 (αSOD1143–153). Both pre-incubation of seeds with αSOD1143–153 prior to inoculation, and weekly intraperitoneal (i.p.) administration attenuated transmission of pathogenic aggregation and prolonged the survival of seed-inoculated hSOD1G85R Tg mice. In contrast, administration of a mAb targeting aa 65–72 (αSOD165–72), which exhibits high affinity towards monomeric disordered hSOD1, had an adverse effect and aggravated seed induced premature ALS-like disease. Although the mAbs reached similar concentrations in CSF, only αSOD1143–153 was found in association with aggregated hSOD1 in spinal cord homogenates. Our results suggest that an aggregate-selective immunotherapeutic approach may suppress seeded transmission of pathogenic aggregation in ALS. However, long-term administration of αSOD1143–153 was unable to prolong the lifespan of non-inoculated hSOD1G85R Tg mice. Thus, spontaneously initiated hSOD1 aggregation in spinal motor neurons may be poorly accessible to therapeutic antibodies.