Screening for inhibitors of the SOD1 gene promoter: Pyrimethamine does not reduce SOD1 levels in cell and animal models
Introduction
Amyotrophic lateral sclerosis (ALS) is an age-dependent, neurodegenerative disease characterized pathologically by the selective loss of motor neurons in the brain and spinal cord. Symptoms of muscle weakness, cramp or spasticity begin in a single limb but become generalized, and death, due to respiratory failure, occurs within three to five years. ALS is uniformly fatal. The single FDA-approved treatment for ALS (Riluzole) increases lifespan by only 3–6 months and does not substantially alleviate symptoms [13]. The majority of ALS cases are sporadic but approximately 10% are familial. Mutations in the genes encoding FUS/TLS [24], [10], TDP-43 [23], angiogenin [8] and cytosolic Cu/Zn superoxide dismutase (SOD1) [21] have been shown to cause ALS. The most extensively studied of these genes is SOD1.
To date nearly 150 ALS-associated mutations have been reported in SOD1 [9]. Most are missense mutations, which occur throughout the protein. Through multiple mechanisms that remain fully to be defined, SOD1 mutations are pathogenic; data overwhelmingly supports the view that mutant SOD1 protein has acquired adverse cytotoxic properties. SOD1 knockout mice show no overt phenotype [20], whereas mice over-expressing mutant SOD1 develop progressive paralysis and death due to motor neuron loss [5]. Importantly, transgenic mice and rats expressing high levels of mutant SOD1 develop a disease phenotype but those expressing at a lower level do not [5], [14]. This evidence, along with the findings that siRNA directed against SOD1 prolong survival in mice [18] lead us to investigate the possibility that a reduction in SOD1 levels could attenuate ALS susceptibility and the rate of disease progression. To test this hypothesis, we developed a cell-based screen for small molecules capable of inhibiting the SOD1 promoter [3], thereby reducing levels of mutant SOD1 protein. Mutant SOD1 is thought to act in both a cell autonomous and a non-cell autonomous manner [7], [17], [12]. Reduction of levels of mutant SOD1 in motor neurons delays onset of paralysis in transgenic ALS mice [2] while diminished levels of mutant SOD1 in astrocytes and microglial cells delays microglial activation and slows disease progression after onset [29]. Thus, the potential benefits of compounds that suppress SOD1 expression may be mediated by motor neurons and surrounding non-neuronal cells. We note that there is a precedent for a beneficial influence of induced gene repression in a transgenic model of Huntington's disease [28].
For these reasons, we have developed screening assays to identify compounds that inhibit expression of the SOD1 gene. Our studies focused initially on pyrimethamine, several compounds currently in trials in human and murine ALS and a set of 1040 FDA-approved compounds. We elected to study pyrimethamine in detail because this compound has previously been reported to reduce SOD1 protein levels in lymphocytes of ALS patients by up to 60% [11]. Pyrimethamine (5-(4-chlorophenyl)-6-ethyl-2,4-pyrimidinediamine) is an anti-protozoal drug whose primary mode of action involves the preferential inhibition of protozoal dihydrofolate reductase [6]. It also induces peripheral blood lymphocyte apoptosis via activation of caspase 8- and caspase 10-dependent cascades, leading to mitochondrial depolarization [16]. How pyrimethamine might reduce activity of the SOD1 gene is not clear.
Section snippets
Cell culture
A PC12 cell line, stably expressing 2.2 kb of the SOD1 promoter region flanked by the gene encoding green fluorescent protein (GFP) was maintained in DMEM-F12 (Gibco, USA) with 10% (v/v) horse serum, 5% (v/v) fetal bovine serum (FBS), 1× penicillin, 1× streptomycin and 500 μg/mL G418 (Invitrogen, USA) at 37 °C with 5% CO2 [3]. HeLa cells were maintained in DMEM (Gibco, USA) with 10% (v/v) FBS, 1× penicillin and 1× streptomycin at 37 °C with 5% CO2.
Animal experiments
C57BL/6J mice (Jackson Laboratories, USA) aged 8–10
Effects of pyrimethamine on SOD1 promoter and SOD1 protein levels
To investigate the effect of pyrimethamine on the SOD1 promoter, this drug was added to our stable PC12 cell lines that express GFP under the control of the human SOD1 promoter. In these cells, fluorescence levels directly indicate promoter activity. Using a 12 point dose curve (25–0.0001 μM) we observed a 42% reduction in the level of GFP relative to the DMSO treated controls at the highest dose tested. This was associated with a 68% reduction in general cell viability, as indicated by a
Discussion
The goal of this study was to identify compounds that reduce levels of SOD1 protein without altering cellular viability; such compounds might be beneficial in mutant SOD1-mediated ALS. We elected to examine pyrimethamine, several compounds currently being tested in trials in human and murine ALS and a set of 1040 FDA-approved compounds. Of these, only pyrimethamine has previously been reported to lower levels of SOD1 without any associated toxicity [11], [31]. In our PC12 SOD1-reporter assay,
Acknowledgments
This study was supported by the National Institute for Neurological Disease and Stroke, the Angel Fund, the ALS Association, Project ALS, Pierre L. de Bourgknecht ALS Research Foundation, the Al-Athel ALS Foundation and the ALS Therapy Alliance.
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