Abstract
Amyotrophic lateral sclerosis (ALS) is a progressive, fatal, and incurable neurodegenerative disease. Recent studies suggest that dysregulation of gene expression by microRNAs (miRNAs) may play an important role in ALS pathogenesis. The reversible nature of this dysregulation makes miRNAs attractive pharmacological targets and a potential therapeutic avenue. Under physiological conditions, miRNA biogenesis, which begins in the nucleus and includes further maturation in the cytoplasm, involves trans-activation response element DNA/RNA-binding protein of 43 kDa (TDP43). However, TDP43 mutations or stress trigger TDP43 mislocalization and inclusion formation, a hallmark of most ALS cases, that may lead to aberrant protein/miRNA interactions in the cytoplasm. Herein, we demonstrated that TDP43 exhibits differential binding affinity for select miRNAs, which prompted us to profile miRNAs that preferentially bind cytoplasmic TDP43. Using cellular models expressing TDP43 variants and miRNA profiling analyses, we identified differential levels of 65 cytoplasmic TDP43-associated miRNAs. Of these, approximately 30% exhibited levels that differed by more than 3-fold in the cytoplasmic TDP43 models relative to our control model. The hits included both novel miRNAs and miRNAs previously associated with ALS that potentially regulate several predicted genes and pathways that may be important for pathogenesis. Accordingly, these findings highlight specific miRNAs that may shed light on relevant disease pathways and could represent potential biomarkers and reversible treatment targets for ALS.
Highlights
Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease characterized by cortical, brainstem, and spinal cord motor neuron loss that results in progressive skeletal muscle weakness and atrophy (Brown and Al-Chalabi, 2017)
To gain insight into TDP43 s ability to interact with specific miRNAs, we first used a native gel mobility shift assay to evaluate binding of denatured TDP43 to miR-132, miR-143, and miR-574, miRNAs all previously reported to associate with TDP43 (Buratti et al, 2010; Kawahara and Mieda-Sato, 2012; Freischmidt et al, 2013)
Since TDP43 binding often relies on associations with UG repeats (Ayala et al, 2005; Bhardwaj et al, 2013), the apparent binding affinity of TDP43 for miR-574 (9 UG repeats), miR-652 (3 UG repeats), and miR-204 (2 UG repeats) was evaluated using a colorimetric ELISA to detect natively folded TDP43 bound to surface-captured biotinylated miRNAs (Rumora et al, 2010, 2013)
Summary
Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease characterized by cortical, brainstem, and spinal cord motor neuron loss that results in progressive skeletal muscle weakness and atrophy (Brown and Al-Chalabi, 2017). The disease can occur as familial ALS (fALS), which constitutes around 15% of ALS incidence, or the more frequent sporadic ALS (sALS; Brown and Al-Chalabi, 2017; Chia et al, 2018; Goutman et al, 2018; Oskarsson et al, 2018). FALS and sALS share similarities in histopathological features, with over 90% of cases exhibiting TDP43 protein inclusions in the cytoplasm of the diseased brain and spinal cord neurons and glia (Hardiman et al, 2017). Aberrant protein and RNA metabolism and altered epigenetic mechanisms, such as those involving microRNAs (miRNAs), are recurrent themes among the dysregulated pathways (Vucic et al, 2014; Paez-Colasante et al, 2015; Brown and Al-Chalabi, 2017; Jimenez-Pacheco et al, 2017)
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