Piera Pasinelli

Molecular biology of amyotrophic lateral sclerosis: insights from genetics

Amyotrophic lateral sclerosis (ALS) is a paralytic disorder caused by motor neuron degeneration. Mutations in more than 50 human genes cause diverse types of motor neuron pathology. Moreover, defects in five Mendelian genes lead to motor neuron disease, with two mutations reproducing the ALS phenotype. Analyses of these genetic effects have generated new insights into the diverse molecular pathways involved in ALS pathogenesis. Here, we present an overview of the mechanisms for motor neuron death and of the role of non-neuronal cells in ALS.

Wild-type and mutant SOD1 share an aberrant conformation and a common pathogenic pathway in ALS

Many mutations confer one or more toxic function(s) on copper/zinc superoxide dismutase 1 (SOD1) that impair motor neuron viability and cause familial amyotrophic lateral sclerosis (FALS). Using a conformation-specific antibody that detects misfolded SOD1 (C4F6), we found that oxidized wild-type SOD1 and mutant SOD1 share a conformational epitope that is not present in normal wild-type SOD1. In a subset of human sporadic ALS (SALS) cases, motor neurons in the lumbosacral spinal cord were markedly C4F6 immunoreactive, indicating that an aberrant wild-type SOD1 species was present. Recombinant, oxidized wild-type SOD1 and wild-type SOD1 immunopurified from SALS tissues inhibited kinesin-based fast axonal transport in a manner similar to that of FALS-linked mutant SOD1. Our findings suggest that wild-type SOD1 can be pathogenic in SALS and identify an SOD1-dependent pathogenic mechanism common to FALS and SALS.

Amyotrophic Lateral Sclerosis-Associated SOD1 Mutant Proteins Bind and Aggregate with Bcl-2 in Spinal Cord Mitochondria

Familial amyotrophic lateral sclerosis (ALS)-linked mutations in the copper-zinc superoxide dismutase (SOD1) gene cause motor neuron death in about 3% of ALS cases. While the wild-type (wt) protein is anti-apoptotic, mutant SOD1 promotes apoptosis. We now demonstrate that both wt and mutant SOD1 bind the anti-apoptotic protein Bcl-2, providing evidence of a direct link between SOD1 and an apoptotic pathway. This interaction is evident in vitro and in vivo in mouse and human spinal cord. We also demonstrate that in mice and humans, Bcl-2 binds to high molecular weight SDS-resistant mutant SOD1 containing aggregates that are present in mitochondria from spinal cord but not liver. These findings provide new insights into the anti-apoptotic function of SOD1 and suggest that entrapment of Bcl-2 by large SOD1 aggregates may deplete motor neurons of this anti-apoptotic protein.

Antisense proline-arginine RAN dipeptides linked to C9ORF72-ALS/FTD form toxic nuclear aggregates that initiate in vitro and in vivo neuronal death.

Expanded GGGGCC (G4C2) nucleotide repeats within the C9ORF72 gene are the most common genetic mutation associated with both amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). Sense and antisense transcripts of these expansions are translated to form five dipeptide repeat proteins (DRPs). We employed primary cortical and motor neuron cultures, live-cell imaging, and transgenic fly models and found that the arginine-rich dipeptides, in particular Proline-Arginine (PR), are potently neurotoxic. Factors that anticipated their neurotoxicity included aggregation in nucleoli, decreased number of processing bodies, and stress granule formation, implying global translational dysregulation as path accountable for toxicity. Nuclear PR aggregates were also found in human induced motor neurons and postmortem spinal cord tissues from C9ORF72 ALS and ALS/FTD patients. Intronic G4C2 transcripts, but not loss of C9ORF72 protein, are also toxic to motor and cortical neurons. Interestingly, G4C2 transcript-mediated neurotoxicity synergizes with that of PR aggregates, suggesting convergence of mechanisms.

Prophylactic Creatine Administration Mediates Neuroprotection in Cerebral Ischemia in Mice

Creatine mediates remarkable neuroprotection in experimental models of amyotrophic lateral sclerosis, Huntingtons disease, Parkinsons disease, and traumatic brain injury. Because caspase-mediated pathways are shared functional mechanistic components in these diseases, as well as in ischemia, we evaluated the effect of creatine supplementation on an experimental stroke model. Oral creatine administration resulted in a remarkable reduction in ischemic brain infarction and neuroprotection after cerebral ischemia in mice. Postischemic caspase-3 activation and cytochrome c release were significantly reduced in creatine-treated mice. Creatine administration buffered ischemia-mediated cerebral ATP depletion. These data provide the first direct correlation between the preservation of bioenergetic cellular status and the inhibition of activation of caspase cell-death pathways in vivo. An alternative explanation to our findings is that creatine is neuroprotective through other mechanisms that are independent of mitochondrial cell-death pathways, and therefore postischemic ATP preservation is the result of tissue spearing. Given its safety record, creatine might be considered as a novel therapeutic agent for inhibition of ischemic brain injury in humans. Prophylactic creatine supplementation, similar to what is recommended for an agent such as aspirin, may be considered for patients in high stroke-risk categories.

An over-oxidized form of superoxide dismutase found in sporadic amyotrophic lateral sclerosis with bulbar onset shares a toxic mechanism with mutant SOD1

Recent studies suggest that Cu/Zn superoxide dismutase (SOD1) could be pathogenic in both familial and sporadic amyotrophic lateral sclerosis (ALS) through either inheritable or nonheritable modifications. The presence of a misfolded WT SOD1 in patients with sporadic ALS, along with the recently reported evidence that reducing SOD1 levels in astrocytes derived from sporadic patients inhibits astrocyte-mediated toxicity on motor neurons, suggest that WT SOD1 may acquire toxic properties similar to familial ALS-linked mutant SOD1, perhaps through posttranslational modifications. Using patients’ lymphoblasts, we show here that indeed WT SOD1 is modified posttranslationally in sporadic ALS and is iper-oxidized (i.e., above baseline oxidation levels) in a subset of patients with bulbar onset. Derivatization analysis of oxidized carbonyl compounds performed on immunoprecipitated SOD1 identified an iper-oxidized SOD1 that recapitulates mutant SOD1-like properties and damages mitochondria by forming a toxic complex with mitochondrial Bcl-2. This study conclusively demonstrates the existence of an iper-oxidized SOD1 with toxic properties in patient-derived cells and identifies a common SOD1-dependent toxicity between mutant SOD1-linked familial ALS and a subset of sporadic ALS, providing an opportunity to develop biomarkers to subclassify ALS and devise SOD1-based therapies that go beyond the small group of patients with mutant SOD1.

Caspase-3 Cleaves and Inactivates the Glutamate Transporter EAAT2

EAAT2 is a high affinity, Na+-dependent glutamate transporter with predominant astroglial localization. It accounts for the clearance of the bulk of glutamate released at central nervous system synapses and therefore has a crucial role in shaping glutamatergic neurotransmission and limiting excitotoxicity. Caspase-3 activation and impairment in expression and activity of EAAT2 are two distinct molecular mechanisms occurring in human amyotrophic lateral sclerosis (ALS) and in the transgenic rodent model of the disease. Excitotoxicity caused by down-regulation of EAAT2 is thought to be a contributing factor to motor neuron death in ALS. In this study, we report the novel evidence that caspase-3 cleaves EAAT2 at a unique site located in the cytosolic C-terminal domain of the transporter, a finding that links excitotoxicity and activation of caspase-3 as converging mechanisms in the pathogenesis of ALS. Caspase-3 cleavage of EAAT2 leads to a drastic and selective inhibition of this transporter. Heterologous expression of mutant SOD1 proteins linked to the familial form of ALS leads to inhibition of EAAT2 through a mechanism that largely involves activation of caspase-3 and cleavage of the transporter. In addition, we found evidence in spinal cord homogenates of mutant SOD1 ALS mice of a truncated form of EAAT2, likely deriving from caspase-3-mediated proteolytic cleavage, which appeared concurrently to the loss of EAAT2 immunoreactivity and to increased expression of activated caspase-3. Taken together, our findings suggest that caspase-3 cleavage of EAAT2 is one mechanism responsible for the impairment of glutamate uptake in mutant SOD1-linked ALS.

ALS-linked mutant SOD1 damages mitochondria by promoting conformational changes in Bcl-2

In mutant superoxide dismutase (SOD1)-linked amyotrophic lateral sclerosis (ALS), accumulation of misfolded mutant SOD1 in spinal cord mitochondria is thought to cause mitochondrial dysfunction. Whether mutant SOD1 is toxic per se or whether it damages the mitochondria through interactions with other mitochondrial proteins is not known. We previously identified Bcl-2 as an interacting partner of mutant SOD1 specifically in spinal cord, but not in liver, mitochondria of SOD1 mice and patients. We now show that mutant SOD1 toxicity relies on this interaction. Mutant SOD1 induces mitochondrial morphological changes and compromises mitochondrial membrane integrity leading to release of Cytochrome C only in the presence of Bcl-2. In cells, mouse and human spinal cord with SOD1 mutations, the binding to mutant SOD1 triggers a conformational change in Bcl-2 that results in the uncovering of its toxic BH3 domain and conversion of Bcl-2 into a toxic protein. Bcl-2 carrying a mutagenized, non-toxic BH3 domain fails to support mutant SOD1 mitochondrial toxicity. The identification of Bcl-2 as a specific target and active partner in mutant SOD1 mitochondrial toxicity suggests new therapeutic strategies to inhibit the formation of the toxic mutant SOD1/Bcl-2 complex and to prevent mitochondrial damage in ALS.

Protein kinase C in synaptic plasticity: changes in the in situ phosphorylation state of identified pre- and postsynaptic substrates

1. Long-term potentiation and its counterpart long-term depression are two forms of activity dependent synaptic plasticity, in which protein kinases and protein phosphatases are essential. 2. B-50/GAP-43 and RC3/neurogranin are two defined neuronal PKC substrates with different synaptic localization. B-50/GAP-43 is a presynaptic protein and RC3/neurogranin is only found at the postsynaptic site. Measuring their phosphorylation state in hippocampal slices, allows us to simultaneously monitor changes in pre- and postsynaptic PKC mediated phosphorylation. 3. Induction of LTP in the CA1 field of the hippocampus is accompanied with an increase in the in situ phosphorylation of both B-50/GAP-43 and RC3/neurogranin, during narrow, partially overlapping, time windows. 4. Pharmacological data show that mGluR stimulation results in an increase in the in situ phosphorylation of B-50/GAP-43 and RC3/neurogranin.

A Caspase-3-cleaved Fragment of the Glial Glutamate Transporter EAAT2 Is Sumoylated and Targeted to Promyelocytic Leukemia Nuclear Bodies in Mutant SOD1-linked Amyotrophic Lateral Sclerosis

EAAT2 (excitatory amino acid transporter 2) is a high affinity, Na+-dependent glutamate transporter of glial origin that is essential for the clearance of synaptically released glutamate and prevention of excitotoxicity. During the course of human amyotrophic lateral sclerosis (ALS) and in a transgenic mutant SOD1 mouse model of the disease, expression and activity of EAAT2 is remarkably reduced. We previously showed that some of the mutant SOD1 proteins exposed to oxidative stress inhibit EAAT2 by triggering caspase-3 cleavage of EAAT2 at a single defined locus. This gives rise to two fragments that we termed truncated EAAT2 and COOH terminus of EAAT2 (CTE). In this study, we report that analysis of spinal cord homogenates prepared from mutant G93A-SOD1 mice reveals CTE to be of a higher molecular weight than expected because it is conjugated with SUMO-1. The sumoylated CTE fragment (CTE-SUMO-1) accumulates in the spinal cord of these mice as early as presymptomatic stage (70 days of age) and not in other central nervous system areas unaffected by the disease. The presence and accumulation of CTE-SUMO-1 is specific to ALS mice, since it does not occur in the R6/2 mouse model for Huntington disease. Furthermore, using an astroglial cell line, primary culture of astrocytes, and tissue samples from G93A-SOD1 mice, we show that CTE-SUMO-1 is targeted to promyelocytic leukemia nuclear bodies. Since one of the proposed functions of promyelocytic leukemia nuclear bodies is regulation of gene transcription, we suggest a possible novel mechanism by which the glial glutamate transporter EAAT2 could contribute to the pathology of ALS.