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Non-cell-autonomous mechanisms contribute to neurodegenerative diseases such as amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD), in which astrocytes release unidentified factors that are toxic to motoneurons (MNs). We report here that mouse and patient iPSC-derived astrocytes with diverse ALS/FTD-linked mutations (SOD1, TARDBP, and C9ORF72) display elevated levels of intracellular inorganic polyphosphate (polyP), a ubiquitous, negatively charged biopolymer. PolyP levels are also increased in astrocyte-conditioned media (ACM) from ALS/FTD astrocytes. ACM-mediated MN death is prevented by degrading or neutralizing polyP in ALS/FTD astrocytes or ACM. Studies further reveal that postmortem familial and sporadic ALS spinal cord sections display enriched polyP staining signals and that ALS cerebrospinal fluid (CSF) exhibits increased polyP concentrations. Our in vitro results establish excessive astrocyte-derived polyP as a critical factor in non-cell-autonomous MN degeneration and a potential therapeutic target for ALS/FTD. The CSF data indicate that polyP might serve as a new biomarker for ALS/FTD.

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease in which pathogenesis and death of motor neurons are triggered by non-cell-autonomous mechanisms. We showed earlier that exposing primary rat spinal cord cultures to conditioned media derived from primary mouse astrocyte conditioned media (ACM) that express human SOD1(G93A) (ACM-hSOD1(G93A)) quickly enhances Nav channel-mediated excitability and calcium influx, generates intracellular reactive oxygen species (ROS), and leads to death of motoneurons within days. Here we examined the role of mitochondrial structure and physiology and of the activation of c-Abl, a tyrosine kinase that induces apoptosis. We show that ACM-hSOD1(G93A), but not ACM-hSOD1(WT), increases c-Abl activity in motoneurons, interneurons and glial cells, starting at 60 min; the c-Abl inhibitor STI571 (imatinib) prevents this ACM-hSOD1(G93A)-mediated motoneuron death. Interestingly, similar results were obtained with ACM derived from astrocytes expressing SOD1(G86R) or TDP43(A315T). We further find that co-application of ACM-SOD1(G93A) with blockers of Nav channels (spermidine, mexiletine, or riluzole) or anti-oxidants (Trolox, esculetin, or tiron) effectively prevent c-Abl activation and motoneuron death. In addition, ACM-SOD1(G93A) induces alterations in the morphology of neuronal mitochondria that are related with their membrane depolarization. Finally, we find that blocking the opening of the mitochondrial permeability transition pore with cyclosporine A, or inhibiting mitochondrial calcium uptake with Ru360, reduces ROS production and c-Abl activation. Together, our data point to a sequence of events in which a toxic factor(s) released by ALS-expressing astrocytes rapidly induces hyper-excitability, which in turn increases calcium influx and affects mitochondrial structure and physiology. ROS production, mediated at least in part through mitochondrial alterations, trigger c-Abl signaling and lead to motoneuron death.

Amyotrophic lateral sclerosis (ALS) is a devastating paralytic disorder caused by dysfunction and degeneration of motoneurons starting in adulthood. Recent studies using cell or animal models document that astrocytes expressing disease-causing mutations of human superoxide dismutase 1 (hSOD1) contribute to the pathogenesis of ALS by releasing a neurotoxic factor(s). Neither the mechanism by which this neurotoxic factor induces motoneuron death nor its cellular site of action has been elucidated. Here we show that acute exposure of primary wild-type spinal cord cultures to conditioned medium derived from astrocytes expressing mutant SOD1 (ACM-hSOD1G93A) increases persistent sodium inward currents (PCNa), repetitive firing, and intracellular calcium transients, leading to specific motoneuron death days later. In contrast to TTX, which paradoxically increased twofold the amplitude of calcium transients and killed motoneurons, reduction of hyperexcitability by other specific (mexiletine) and nonspecific (spermidine and riluzole) blockers of voltage-sensitive sodium (Nav) channels restored basal calcium transients and prevented motoneuron death induced by ACM-hSOD1G93A. These findings suggest that riluzole, the only FDA-approved drug with known benefits for ALS patients, acts by inhibiting hyperexcitability. Together, our data document that a critical element mediating the non-cell-autonomous toxicity of ACM-hSOD1G93A on motoneurons is increased excitability, an observation with direct implications for therapy of ALS.

Patients with amyotrophic lateral sclerosis (ALS), Alzheimer’s disease (AD), Parkinson’s disease (PD), Huntington’s disease (HD), and other neurodegenerative disorders typically start to display clinical symptoms during adulthood. Groundbreaking genetic studies in humans, rodents, Caenorhabditis elegans, Drosophila, and yeast have identified mutations in genes such as SOD1, FUS/TLS and TDP-43 (for ALS), APP and presenilin (for AD), parkin and alpha-synuclein (for PD), and huntingtin (for HD), as being responsible for causing these diseases in adults, even though the mutation is present throughout life. Despite remarkable advances in understanding the diseases, no mechanism-based cures are currently available. This unfortunate situation is mainly due to the fact that the primary target(s) of the mutant proteins are not known, in part because early and ubiquitous expression of the aberrant genes in the developing CNS leads to many secondary effects and triggers compensatory mechanisms that mask the primary pathological event. Thus, although the primary disease process might be present throughout life, onset of symptoms coincides with the saturation of protection and compensation mechanisms, either by accumulation of dysfunction or via other pathogenic events such as trauma and environmental factors, which may be aggravated by aging [DeKosky and Marek, 2003; Palop et al., 2006]. To enable identification of mutant gene-driven mechanisms that trigger the cascade of events that culminate in degeneration and death later in life, a first step is to establish exactly when the neuropathology is initiated. The fact that the expression of the disease-causative proteins starts during embryonic stages raises the intriguing possibility that pathological changes in patients with adult-onset neurodegenerative disorders are triggered much earlier—in humans, such alterations may occur decades, and in mouse models months—prior to the manifestation of symptoms. Recent support for this contention derives from findings that alterations are found in many CNS regions in pre-symptomatic transgenic mouse models (and even in pre-symptomatic human patients) of ALS (see below), AD [Santos et al., 2010], PK [Obeso et al., 2010], and HD [Raymond et al., 2011]. Based on evidence that is discussed more fully below, we propose a model (Fig. 1) in which mutant SOD1 initiates synaptic pathogenesis very early in development, causing alterations in neural circuits and network activity, and resulting in a vicious cycle that leads to neurological impairment. The onset of symptoms of ALS in humans may take years and even decades following these early events, and may become apparent only when compensatory mechanisms breaks down. Thus, it is imperative that the primary target(s) of disease-causing proteins be identified, which can then be used to establish pre-symptomatic diagnostic tools, and to develop novel therapies that can effectively prevent the onset of irreversible neuronal damage. Fig. 1 Model of how mutant SOD1 may underlie the etiology of ALS. Subtle synaptic dysfunction may be initiated by mutant SOD1 during development: either at early postnatal stages, or even during embryogenesis, when expression of wild-type and mutant SOD1 is ...