Epidemiological studies of the Guamanian variants of amyotrophic lateral sclerosis (ALS) and parkinsonism, amyotrophic lateral sclerosis-parkinsonism dementia complex (ALS-PDC), have shown a positive correlation between consumption of washed cycad seed flour and disease occurrence. Previous in vivo studies by our group have shown that the same seed flour induces ALS and PDC phenotypes in out bred adult male mice. In vitro studies using isolated cycad compounds have also demonstrated that several of these are neurotoxic, specifically, a number of water insoluble phytosterol glucosides of which β-sitosterol β-D-glucoside (BSSG) forms the largest fraction. BSSG is neurotoxic to motor neurons and other neuronal populations in culture. The present study shows that an in vitro hybrid motor neuron (NSC-34) culture treated with BSSG undergoes a dose-dependent cell loss. Surviving cells show increased expression of HSP70, decreased cytosolic heavy neurofilament expression, and have various morphological abnormalities. CD-1 mice fed mouse NIH-PA Author ManuscriptNIH-PA Author Manuscript NIH-PA Author Manuscript chow pellets containing BSSG for 15 weeks showed motor deficits and motor neuron loss in the lumbar and thoracic spinal cord, along with decreased glutamate transporter labelling, and increased glial fibrillary acid protein reactivity. Other pathological outcomes included increased caspase-3 labelling in the striatum and decreased tyrosine-hydroxylase labelling in the striatum and substantia nigra. C57BL/6 mice fed BSSG-treated pellets for 10 weeks exhibited progressive loss of motor neurons in the lumbar spinal cord that continued to worsen even after the BSSG exposure ended. These results provide further support implicating sterol glucosides as one potential causal factor in the motor neuron pathology previously associated with cycad consumption and ALS-PDC.

BackgroundIn vivo animal models of familial amyotrophic lateral sclerosis (fALS) are widely used to delineate the potential role that genetic mutations play in the neurodegenerative process. While these models are extensively used for establishing the safety and efficacy of putative therapeutics during pre-clinical development, effective clinical translation of pharmacological interventions has been largely unsuccessful.ResultsIn this report we compare a recent cohort of G37R (line 29) mice generated from mating wild-type females with transgenic males obtained commercially to a previous set of offspring produced with transgenic male breeders from a colony established at a local collaborator’s facility. Commercially derived progeny presented with a tightly clustered genomic signature for the mutant human superoxide dismutase1 transgene (hSOD1) locus, and exhibited a greater than two-fold reduction in the number of transgene copies present in the genome compared to offspring derived locally. Decrease in transgene levels corresponded with delayed ALS progression and a significant increase in overall lifespan (146%).ConclusionsThese results highlight some key challenges inherent to the use of G37R (line 29) animals in pre-clinical studies for the development of ALS therapeutics. Without stringent assessment of mutant SOD1 copy number/protein levels, heterogeneity of transgene levels within cohorts may influence the behavioural and pathological presentation of disease and thus calls to question the validity of any detected therapeutic effects. Nuanced changes in mutant SOD1 copy number that currently remain unreported may undermine research endeavours, delay efforts for clinical translation, and compromise the rigor of animal studies by limiting reproducibility amongst research groups.

Amyotrophic lateral sclerosis (ALS) is a devastatingly progressive neurodegenerative disorder with multiple underlying etiological factors contributing to disease pathogenesis. Despite intensive research efforts and therapeutic development, disease presentation in ALS remains largely intractable to intervention. To date, the most common rodent model used in preclinical drug development accounts for a small proportion of the affected patient population and is predicated upon the significant overexpression of a mutant form of the human antioxidant protein, superoxide dismutase 1 (mSOD1). After more than 50 clinical trials, there is an alarming paucity of positive outcomes at the clinical level of ALS therapeutics with strong supporting pre-clinical data in mSOD1 models. Potential reasons for the negative clinical results are multifactorial in nature and include an overly reductionist model system that is heavily influenced by individual transgene level variation, as well as attempting to widely apply findings derived from a model of specific genetic causality to a patient population where the majority of cases are of unknown etiology. With such a tremendous disease burden and a lack of therapeutic options, it is critical that the research community re-evaluate the dependence on mSOD1 pre-clinical models as the gold standard prior to translating findings at the clinical level. Here we briefly review both the clinical and pre-clinical findings of select therapeutics, discuss the limitations of pre-clinical mSOD1 models, and suggest future stratagems that could aid in the clinical translation of efficacious therapeutic agents.

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