Piera Pasinelli - US grants

This is a project to study Amyotrophic Lateral Sclerosis (ALS), an age-dependent neurodegenerative disease of spinal motor neurons. Mutations in the gene encoding for copper/zinc superoxide dismutase (SOD1) cause motor neuron degeneration in about 25% of familial ALS cases (FALS). Evidence indicates that the death process involves activation of apoptotic genes. The broad goal of this project is to elucidate the mechanism(s) governing mutant SODl-mediated apoptotic death. Our investigations in mouse neuroblastoma N2A cells and transgenic ALS mice show that multiple SOD1 mutants initiate a toxic cascade that entails sequential activation of caspase-1 (a slowly initiator of cell death) and caspase-3 (the final effector). The appearance of activated caspase-1 in the ALS mice months prior to motor neurons death indicates that caspase-1 plays a role as an early mediator of cell death probably by activating other apoptotic pathways. This suggests the hypothesis that at least in mutant SODl-related ALS, apoptosis is not a secondary phenomenon that contributes to the final demise of motor neurons, but rather a slow process that develops over time. There is a gradual response to the primary insult (the inherited molecular defect in the SOD1 protein) that over time enhances motor neurons susceptibility to either an exogenous stimulus (e.g. oxidative stress) or to age-related changes in apoptotic pathways. Consistent with this view, we have found that chronic activation of caspase-1 in the N2A cells does not provoke rapid cell death, but renders the cells more sensitive to an oxidative insult. At the end of the cascade, the close temporal relationship between caspase-3 activation and cell death argues that this executioner caspase directly contributes to motor neuron death. A detailed analysis of motor neuron-related apoptotic mechanisms may have a great importance in understanding the biology of motor neuron cell death. We now plan to dissect the individual steps in the cell death cascade over time in the ALS mice using high sensitive SELDI ProteinChip Technology in GFP-labeled primary motor neurons. To this end we propose to (1) Generate ALS mice in which the GFP fluorescent protein is specifically expressed in motor neurons, and (2) use the fluorescent-sorted motor neurons from these mice for SELDI analysis of apoptotic proteins.

DESCRIPTION (provided by applicant): Amyotrophic Lateral Sclerosis (ALS) is a devastating neurodegenerative disease of the motor neurons, which leads to paralysis and death within 3-5 years from diagnosis. ALS is mainly sporadic (SALS) without a known cause. Only a small fraction of ALS is familial (FALS). Thus, one of the biggest challenges in the study of the disease is how to reconcile disease mechanisms among the small percentage of familial cases and the vast majority of sporadic cases with no known etiology. It is crucial that we identify common pathogenic mechanisms between the two forms of the disease. Mitochondrial pathology is one of these common pathways, as mitochondria defects have been found in both SALS patients and transgenic mutant SOD1 (mutSOD1) mice model of ALS. Whether similar triggers in FALS and SALS damage the mitochondria is not known. Using mutSOD1 expressing cells and transgenic mice (to mimic FALS), as well as EVB immortalized lymphoblasts from SALS patients, we identified a potentially common trigger mechanism. In mutSOD1 mice, we showed that mutSOD1 aberrantly binds and forms a toxic complex with Bcl-2 in mitochondria. Upon this aberrant binding, mutSOD1 induces a conformational change in Bcl-2 that transforms it into a harmful protein by exposing the normally hidden toxic BH3 domain. Together, mutSOD1 and conformationally modified Bcl-2 impair mitochondrial viability, eventually inducing cell death. Interestingly, in a subset (~ 30%) of SALS patients with upper motor neuron onset, an oxidized form of wild type SOD1 aberrantly binds to Bcl-2, transforming Bcl- 2 into a toxic molecule through exposure of the BH3 domain, similarly to what we have reported for mutSOD1. With this competing renewal, we intend to focus on this common pathway of mitochondrial dysfunction shared by FALS-SOD1 and a subset of SALS patients. We will test in vivo the hypothesis that the conformational change in Bcl-2 leading to exposure of the toxic BH3 domain is an important mechanism in SOD1-induced mitochondrial dysfunction (AIM 1). We will then characterize the functional implications of the toxic complex between SOD1 and Bcl-2 by identifying key downstream mitochondrial target(s) (AIM 2) and determining the cellular specificity of the SOD1/Bcl-2-mediated mitochondrial dysfunction (AIM 3). Finally, we will test the beneficial effect of SOD1-like peptides that inhibit binding to Bcl-2 against SOD1-mediated cell death (AIM 4). The ultimate goal is to identify target-based therapies whose efficacy goes beyond the limited portion of familial cases. PUBLIC HEALTH RELEVANCE: In most patients with Amyotrophic Lateral Sclerosis [(ALS)-a lethal neurodegenerative disease that leads to paralysis and death 3-5 years from diagnosis], there is no family link and only a minority of cases (10%) has a genetic link. Because all forms of ALS (with or without a genetic link) are clinically similar, the assumption is that they are also similar in their pathogenesis and the goal for scientists is to identify common pathogenic pathways. Using ALS mouse models (with a genetic mutation) and cells derived from patients without a genetic link, we will study a potentially common mechanism of disease pathogenesis that targets the powerhouse of cells (mitochondria).

ABSTRACT The goal of this project is to identify the mechanisms by which mutations in Fused in Sarcoma (FUS) induce pathological and pathogenic changes in astrocytes in FUS-linked Amyotrophic Lateral Sclerosis (FUS-ALS). Astrocytes are known to contribute to disease progression in some, but not all, forms of ALS and, until recently, not much was known about the role of astrocytes in FUS-ALS. Our lab has recently published the first in vitro evidence that astrocytes expressing mutant forms of FUS (mutFUS), but not wild-type FUS, exert toxicity on motor neurons through activation of the NFkB pathway leading to the release of secreted factor(s), and primarily TNF? (Kia & McAvoy et al., GLIA, 2018). In a TNF??dependent manner, motor neurons exposed to the conditioned medium of mutFUS astrocytes show AMPA receptor alterations that sensitize them to excitotoxic damage, leading to cell death. Activation of astrocytic NFkB and TNF? release seems to be specific to mutFUS-ALS, underscoring the importance of dissecting disease mechanisms specific to each form of ALS in order to develop tailored therapies. In this proposal, we will focus on mutFUS-ALS and will study (1) the mechanisms by which disease-casuative mutations in FUS alter astrocyte biology and (2) how mutant FUS expressing astrocyte affect the viability of other cells in the spinal cord in vivo. The ultimate goal is to identify key pathways targeted by mutFUS to eventually develop specific therapies. Ultimately, our models of FUS-ALS may serve as a platform for a comparative analysis with models mimicking other forms of ALS, so to identify genotype- specific vs. converging and more global mechanisms of disease pathogenesis. For our studies, we will use in vitro and in vivo models consisting of (1) primary rodent astrocytes transduced with different FUS mutations as well as iPSc-derived astrocytes from FUS patients; (2) an in vivo mouse model of mutFUS ALS. The in vitro systems will allow us to dissect the precise mechanisms by which mutFUS targets the astrocytes and to determine how mutFUS alters astrocyte biology in patients. The mouse model will allow us to study how mutFUS astrocytes affect the cellular environment in a complex in vivo setting.