The Power of Human Protective Modifiers: PLS3 and CORO1C Unravel Impaired Endocytosis in Spinal Muscular Atrophy and Rescue SMA Phenotype

Abstract

Homozygous loss of SMN1 causes spinal muscular atrophy (SMA), the most common and devastating childhood genetic motor-neuron disease. The copy gene SMN2 produces only ∼10% functional SMN protein, insufficient to counteract development of SMA. In contrast, the human genetic modifier plastin 3 (PLS3), an actin-binding and -bundling protein, fully protects against SMA in SMN1-deleted individuals carrying 3-4 SMN2 copies. Here, we demonstrate that the combinatorial effect of suboptimal SMN antisense oligonucleotide treatment and PLS3 overexpression-a situation resembling the human condition in asymptomatic SMN1-deleted individuals-rescues survival (from 14 to >250 days) and motoric abilities in a severe SMA mouse model. Because PLS3 knockout in yeast impairs endocytosis, we hypothesized that disturbed endocytosis might be a key cellular mechanism underlying impaired neurotransmission and neuromuscular junction maintenance in SMA. Indeed, SMN deficit dramatically reduced endocytosis, which was restored to normal levels by PLS3 overexpression. Upon low-frequency electro-stimulation, endocytotic FM1-43 (SynaptoGreen) uptake in the presynaptic terminal of neuromuscular junctions was restored to control levels in SMA-PLS3 mice. Moreover, proteomics and biochemical analysis revealed CORO1C, another F-actin binding protein, whose direct binding to PLS3 is dependent on calcium. Similar to PLS3 overexpression, CORO1C overexpression restored fluid-phase endocytosis in SMN-knockdown cells by elevating F-actin amounts and rescued the axonal truncation and branching phenotype in Smn-depleted zebrafish. Our findings emphasize the power of genetic modifiers to unravel the cellular pathomechanisms underlying SMA and the power of combinatorial therapy based on splice correction of SMN2 and endocytosis improvement to efficiently treat SMA.

Emerging therapies and challenges in spinal muscular atrophy

“…Consequently, SMN deficiency may impair targeting and local translation of axonal mRNAs essential for motor neuron development and maintenance 44, 45. Furthermore, SMN regulates several other fundamental cellular processes in the neuronal cytoplasm that are critical for maintaining axonal and synaptic health, including endocytic pathways, local translation, mitochondrial transport, and targeting to axons and ubiquitin homeostasis 46, 47, 48, 49, 50, 51…”

Evaluation of the role of an antioxidant gene in NSC-34 motor neuron-like cells as a model of a motor neuron disease

“…In agreement with this, PLS3 gene overexpression has been shown to refine axonal outgrowth in SMN-deficient MNs in a zebrafish model of SMA as well as in MNs cultured from mouse embryos [16,17]. Several studies have reported an actin-independent role of PLS3 in SMA modification [1,17]; however, investigations on the potential protective role of PLS3 against oxidative stress have not been performed.…”

“…In agreement with this, PLS3 gene overexpression has been shown to refine axonal outgrowth in SMN-deficient MNs in a zebrafish model of SMA as well as in MNs cultured from mouse embryos [16,17]. Several studies have reported an actin-independent role of PLS3 in SMA modification [1,17]; however, investigations on the potential protective role of PLS3 against oxidative stress have not been performed. Some reports have described the involvement of oxidative damage in the neurodegenerative process of SMA, and neuroprotection is considered to be an effective treatment option for many central nervous system disorders, including neurodegenerative diseases such as SMA [5,17].…”

“…Several studies have reported an actin-independent role of PLS3 in SMA modification [1,17]; however, investigations on the potential protective role of PLS3 against oxidative stress have not been performed. Some reports have described the involvement of oxidative damage in the neurodegenerative process of SMA, and neuroprotection is considered to be an effective treatment option for many central nervous system disorders, including neurodegenerative diseases such as SMA [5,17]. Therefore, a therapeutic strategy involving antioxidant gene delivery could provide significant protection for MNs in SMA [16].…”

“…The major clinical characteristic of SMA is the selective and progressive loss of MNs via apoptosis, although the factors, which trigger this loss, remain unknown [9]. Multiple interacting factors have been implicated in MN lesions, including mitochondrial dysfunction, excitotoxicity, protein aggregation and misfolding, defective axonal transport, dysregulated transcription and RNA processing, apoptosis, and oxidative stress [5,16,17].…”