ALS-linked mutations induce aberrant conformations within the SOD1 protein that are thought to underlie the pathogenic mechanism of SOD1-mediated ALS. Although clinical trials are underway for gene silencing of SOD1, these approaches reduce both wild-type and mutated forms of SOD1. Here, we sought to develop anti-SOD1 nanobodies with selectivity for mutant and misfolded forms of human SOD1 over wild-type SOD1. Characterization of two anti-SOD1 nanobodies revealed that these biologics stabilize mutant SOD1 in vitro. Further, SOD1 expression levels were enhanced and the physiological subcellular localization of mutant SOD1 was restored upon co-expression of anti-SOD1 nanobodies in immortalized cells. In human motor neurons harboring the SOD1 A4V mutation, anti-SOD1 nanobody expression promoted neurite outgrowth, demonstrating a protective effect of anti-SOD1 nanobodies in otherwise unhealthy cells. In vitro assays revealed that an anti-SOD1 nanobody exhibited selectivity for human mutant SOD1 over endogenous murine SOD1, thus supporting the preclinical utility of anti-SOD1 nanobodies for testing in animal models of ALS. In sum, the anti-SOD1 nanobodies developed and presented herein represent viable biologics for further preclinical testing in human and mouse models of ALS.
Amyotrophic lateral sclerosis (ALS) is an incurable neurodegenerative
disease characterized by the death of upper and lower motor neurons.
While causative genes have been identified, 90% of ALS cases are not
inherited and are hypothesized to result from the accumulation of
genetic and environmental risk factors. While no specific causative
environmental toxin has been identified, previous work has indicated
that the presence of the organochlorine pesticide cis-chlordane in the blood is highly correlated with ALS incidence. Never
before tested on the motor system, here, we show that cis-chlordane is especially toxic to motor neurons in vitro- and in vivo-independent
of its known antagonism of the GABAA receptor. We find
that human stem-cell-derived motor neurons are more sensitive to cis-chlordane than other cell types and their action potential
dynamics are altered. Utilizing zebrafish larvae, we show that cis-chlordane induces motor neuron and neuromuscular junction
degeneration and subsequent motor deficits in a touch-evoked escape
response. Together, our work points to cis-chlordane
as a potential sporadic ALS exacerbating environmental pollutant.
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