Neuron-specific knock-down of SMN1 causes neuron degeneration and death through an apoptotic mechanism

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August
14 2016

Neuron-specific knock-down of SMN1 causes neuron degeneration and death through an apoptotic mechanism

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Spinal muscular atrophy is a devastating disease that is characterized by degeneration and death of a specific subclass of motor neurons in the anterior horn of the spinal cord. Although the gene responsible, survival motor neuron 1 (SMN1), was identified 20 years ago, it has proven difficult to investigate its effects in vivo. Consequently, a number of key questions regarding the molecular and cellular functions of this molecule have remained unanswered. We developed a Caenorhabditis elegans model of smn-1 loss-of-function using a neuron-specific RNA interference strategy to knock-down smn-1 selectively in a subclass of motor neurons. The transgenic animals presented a cell-autonomous, age-dependent degeneration of motor neurons detected as locomotory defects and the disappearance of presynaptic and cytoplasmic fluorescent markers in targeted neurons. This degeneration led to neuronal death as revealed by positive reactivity to genetic and chemical cell-death markers. We show that genes of the classical apoptosis pathway are involved in the smn-1-mediated neuronal death, and that this phenotype can be rescued by the expression of human SMN1, indicating a functional conservation between the two orthologs. Finally, we determined that Plastin3/plst-1 genetically interacts with smn-1 to prevent degeneration, and that treatment with valproic acid is able to rescue the degenerative phenotype. These results provide novel insights into the cellular and molecular mechanisms that lead to the loss of motor neurons when SMN1 function is reduced.

Introduction

Spinal muscular atrophy (SMA) is an autosomal recessive neurodegenerative disease and one of the most common genetic causes of infant mortality (1). The disease, for which no effective treatment is currently available (2), is characterized by the specific loss of lower spinal cord motor neurons, leading to the atrophy of innervated muscles, paralysis and ultimately death (3). Based on age of onset and motor disabilities, SMA can be clinically subdivided into three types of varying severity (4), all of which are associated with deletions or point mutations in the survival motor neuron 1 gene [SMN1 (5)], which has been mapped to a highly unstable, telomeric region of chromosome 5 (5q13) (6). In addition to SMN1, humans have a nearly identical gene called SMN2; however, the presence of a C-to-T mutation within exon 7 of SMN2 gives rise to a truncated protein, which is rapidly degraded (7). As SMN2 is present in different copy numbers in the genome and is able to produce very low levels of functional protein (5–10% of the total SMN), it acts solely as a genetic modifier when SMN1 is altered, and the level of its expression is responsible for the varying severity of the disease (8).

SMN proteins are ubiquitously expressed both within and outside the nervous system (9). SMN is a component of the well-characterized Gemin complex, which is involved in a series of basic cellular processes, including the biogenesis and assembly of small nuclear ribonucleic proteins and pre-mRNA splicing and transcription (10,11). SMN also plays a role in axonal growth and neuromuscular junction formation, as well as axonal mRNA transport, sub-cellular localization and/or local translation (12,13). However, the specific loss of spinal cord motor neurons in SMA patients cannot be explained simply in terms of their SMN expression pattern, and it is not completely clear which of the SMN functions is responsible for the specific degeneration and death of motor neurons (14).

Modeling SMA in animal systems is therefore crucial to further understand the pathogenesis of the disease and the function played by SMN in motor neurons, as well as to identify potential therapies. Several vertebrate and invertebrate models have been developed, which have provided important information on the changes in the nervous system that occur in response to loss of SMN1 (13,15–17). However, the absence of a second SMN gene in the genome of all animal models, including Caenorhabditis elegans, makes it very difficult to investigate the in vivo role of SMN1 due to the early lethality of SMN1 knock-outs (13,15,16,18–20), hindering the ability to visualize and study this form of neuronal degeneration. In particular, in the current animal models of SMA it has not been possible to determine in vivo the steps that lead to neuron degeneration (13) and, with few exceptions (19,21), it has not been possible to observe neuronal death when SMN1 function is reduced or absent.

Caenorhabditis elegans has a single gene homologous to human SMN, smn-1, which is ubiquitously expressed during all developmental stages (18). The nematode SMN protein exhibits an overall sequence homology and a conserved domain topology with its human counterpart (18). Moreover, the basic functions of SMN, including the ability to self-associate and bind to partner proteins, are conserved in C. elegans (18,22). As observed in other species, a genetic deletion of smn-1 (null allele ok355) causes strong larval lethality (20). The very few surviving ok355 homozygous larvae (escapers) show impaired locomotion and pharyngeal pumping defects, but not neuronal degeneration (20,23). Similar phenotypes have been observed after systemic RNA interference [RNAi (18,22)]. Finally, a hypomorphic mutant allele (cb131) with a single amino acid substitution (D27N) that mimics a point mutation found in some SMA patients (D44V) has been generated (24). Although this mutation overcomes the viability problems of the null mutant, the muscle and nervous system architecture are not affected and there is little change in motility.

Here, we report a novel SMA model in C. elegans. Using cell-specific RNAi knock-down (25), we successfully generated viable transgenic strains in which the function of smn-1 is specifically reduced in a subclass of motor neurons. These transgenic animals mimic some key features of SMA, including impaired locomotion, neuronal degeneration and neuronal death. We report, for the first time, the early steps in neuron degeneration and the loss of neurons due to smn-1 depletion in vivo. We reveal that the neuronal degeneration and death induced by smn-1 reduction are cell-autonomous and proceed in part through an apoptotic mechanism. Furthermore, we investigate the conserved neural protective function of SMN1 from nematodes to humans, and reveal that Plastin3/plst-1 is a genetic interactor of SMN1 in neuronal death. Finally, we show that valproic acid (VPA) is able to rescue the degeneration of C. elegans smn-1-depleted neurons.

Full Article
http://hmg.oxfordj.../09/hmg.ddw119.full

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