Title: RNA mediated mechanisms of motor neuron death and dysfunction in SMA
Description: Disruption of RNA homeostasis is a shared feature across multiple neurodegenerative diseases that are associated with mutations in RNA binding proteins or factors involved in RNA processing. One prime example is the neurodegenerative disease spinal muscular atrophy (SMA), which is characterized by the degeneration of spinal motor neurons and atrophy of skeletal muscle through poorly defined mechanisms. SMA is the consequence of ubiquitous deficiency in the survival motor neuron (SMN) protein, which has a well-characterized role in the assembly of small nuclear ribonucleoproteins (snRNPs). SMN-dependent dysfunction of major (U2) and minor (U12) spliceosomal snRNPs as well as U7 snRNP – which functions in histone mRNA processing – along with consequent RNA misprocessing events have been characterized in SMA. Additionally, SMN has been implicated in additional RNA pathways that may also be involved in SMA etiology. With the broad implications of multifaced roles of SMN in RNA regulation, an outstanding challenge in the SMA field has been the identification of key downstream RNA-dependent events and their contributions to pathogenesis.

While the selective loss of spinal motor neurons is a key hallmark of SMA pathology, the molecular mechanisms remain incompletely understood. Through my dissertation work, I aimed to characterize the RNA mediated pathways that underlie neurodegeneration in SMA. We previously demonstrated that SMA motor neuron death is driven by converging mechanisms of p53 activation that include upregulation and phosphorylation – the latter of which establishes the vulnerability of specific motor neuron pools – however, the upstream triggers remained unknown. Here, I show that the function of SMN in the assembly of Sm-class snRNPs of the splicing machinery regulates alternative splicing of Mdm2 and Mdm4 – two non-redundant repressors of p53 – and increased skipping of critical exons in these genes is associated with p53 stabilization. Further investigation uncovered that dysfunction of Stasimon – a U12 intron-containing gene regulated by SMN – converges on p53 upregulation to induce phosphorylation of p53 through the activation of p38α MAPK and contributes to the demise of SMA motor neurons.

Thus, this work elucidated the upstream RNA mediated mechanisms underlying multiple modes of p53 activation, implicating impairments in both the U2 and U12 snRNP pathways in SMA motor neuron death. It further established Mdm2, Mdm4, and Stasimon as effector genes that are regulated by SMN’s role in snRNP assembly and play key roles in the degeneration of SMA motor neurons. Studies in mouse models of SMA have revealed broader deficits in the motor circuit beyond motor neuron death, which include reduced excitatory drive on motor neurons brought on by a loss of proprioceptive synapses. Restoration in SMA mice revealed that Stasimon dysfunction also contributes to the deafferentation of motor neurons – a cellular defect originating in proprioceptive neurons – revealing Stasimon’s dual contribution to motor circuit pathology in SMA. However, the Stasimon-dependent molecular mechanisms that mediate synaptic loss in proprioceptive neurons, along with the pathway through which Stasimon impairment induces p38α MAPK activation in motor neurons are not well established.

Stasimon was initially identified as a novel contributor to motor circuit dysfunction in Drosophila and motor axon outgrowth deficits in zebrafish models of SMN deficiency. However its cellular roles remain poorly understood. In an effort to address this, I identify Stasimon as an endoplasmic reticulum (ER) resident protein that localizes at mitochondria-associated ER membranes (MAM) – specialized contact sites between ER and mitochondria membranes. Additionally, through characterization of novel knockout mice, I show that Stasimon is an essential gene for mouse embryonic development. These findings provide key insight into Stasimon function and set the stage for further investigation of the p53-dependent mechanisms of motor neuron degeneration as well as cellular pathways driving proprioceptive synaptic loss in SMA. This dissertation also expands beyond RNA mediated mechanisms of SMA – which occur as a consequence of SMN-deficiency – to translational efforts aimed to treat the disease by describing an unexpected gain of toxic function associated with SMN overexpression that is accompanied by RNA dysregulation and sensory-motor circuit pathology.

I further explore these surprising findings of neuronal toxicity induced by AAV9-mediated SMN overexpression that paradoxically affects sensory-motor function and reveal that they parallel features of SMA pathogenesis. Accordingly, I find that the functional basis of long-term motor toxicity of AAV9-SMN involves motor neuron deafferentation and proprioceptive neurodegeneration. At the cellular level, toxicity is associated with the accumulation of large cytoplasmic aggregates of SMN in motor circuit neurons that sequester Sm proteins, disrupt snRNP biogenesis, and induce widespread transcriptome alterations and splicing deficits. These findings identify a novel deleterious role for SMN when expressed at supraphysiological levels that acts through inhibition of SMN’s normal function in snRNP biogenesis, akin to disease mechanisms of SMA. These observations have important implications regarding the approved use of AAV9-SMN for gene therapy in humans and suggest a need for further, careful consideration of potential detrimental effects when SMN is expressed at supraphysiological levels.

Collectively, this dissertation identifies the direct involvement of key SMN-dependent splicing events in select aspects of SMA pathology in a mouse model, in particular those converging on the p53-mediated mechanisms of motor neuron death and the loss of proprioceptive synapses. This work also establishes causal links between impairments in snRNP biology and neuronal dysfunction in SMA, providing mechanistic insight into the process of motor neuron death. Lastly, it uncovers a new, clinically relevant aspect of SMN biology associated with its long-term overexpression which has shared features with the RNA mediated mechanisms of neurodegeneration in SMA.