Spinal muscular atrophy (SMA) is a devastating disorder caused by deletions and mutations in the survival of motor neuron (SMN1) gene and is marked by motor neuron loss and muscle weakness. While its genetic basis is clear, the underlying molecular mechanisms remain elusive. Decreased levels of the survival of motor neuron (SMN) protein, encoded by the SMN1 gene, are implicated in SMA pathology. Despite splicing has been under the spotlight as a major mechanism impaired in SMA, recent evidence suggests that SMN deficiency also disrupts protein translation in vivo in a mouse model of severe SMA, complicating SMA's molecular landscape. This thesis examines the impact of SMN protein loss on translation in SMA mouse models across tissues, post-natal and pre-natal disease stages, focusing on both mild (Chapter 1) and severe forms of SMA (Chapter 2) respectively. To tackle this question, in this thesis, I took advantage of multiple cutting-edge and sequencing-based techniques (ribosome profiling and RNA-seq) coupled with biochemical and molecular biology-based assay (polysome profiling, co-sedimentation profiles, qPCR, and western blotting), which applied to study in molecular detail the translational defects in the brain, spinal cord and liver at asymptomatic, pre-symptomatic and early symptomatic stages of SMA. Polysome profiling in control mice (Smn2B/+) reveals a gradual increase in SMN association with ribosomes/polysomes during postnatal development, indicating dynamic SMN function in protein translation during post-natal development. In SMA condition, where SMN protein levels drop, this binding reveals a tissue-specific decrease in the spinal cord and liver.
Through ribosome profiling, numerous alterations in translation were identified at the pre-symptomatic stage of the disease, suggesting that translational defects are features of the early stages of SMA. Importantly these alterations are independent of transcriptional and splicing changes. Although no gene was found to be in common, I found that genes altered in at least 2 tissues are involved in the same processes. The dysregulated mRNAs exhibit rare codons at the beginning of coding sequences in all three tissues, as shown in the case of the severe model of SMA. 4 From these common processes I have identified specific mRNA targets that play key roles in the organization of the extracellular matrix I validated the presence of translational changes in Col1a1, Col1a2, and Spp1 highlighted effects of SMN deficiency on translational regulation, in the absence of transcriptional alterations. Validation studies in both mice and SMA patient-derived fibroblasts further underscored the potential of translational dysregulation and drop in Col1a1 protein expression during SMA progression.
Finally, prenatal studies have revealed distinct translational changes in embryonic tissues from Taiwanese mice. Despite no alterations in global translation, a drop in SMN association with ribosomes/polysomes and tissue-specific differences in ribosome occupancy were observed. Also, in this case, dysregulated mRNAs exhibit rare codons at the beginning of coding sequences. These findings shed light on the unique molecular landscape of prenatal development in the context of SMN deficiency. In summary, this study provides insights into translation dysregulation in SMA pathology, emphasizing tissue-specific effects and developmental stage-dependent alterations. By elucidating the complex relationship between SMN protein function and translational dynamics, it lays the groundwork for targeted therapeutic strategies and biomarkers to improve SMA management. Ongoing investigations into prenatal development and translation dynamics are crucial for a comprehensive understanding of SMA pathogenesis and effective treatment development.
| Identifer | oai:union.ndltd.org:unitn.it/oai:iris.unitn.it:11572/417910 |
| Date | 30 July 2024 |
| Creators | Sharma, Gaurav |
| Contributors | Sharma, Gaurav, Viero, Gabriella |
| Publisher | Università degli studi di Trento, place:TRENTO |
| Source Sets | Università di Trento |
| Language | English |
| Detected Language | English |
| Type | info:eu-repo/semantics/doctoralThesis |
| Rights | info:eu-repo/semantics/embargoedAccess |
| Relation | firstpage:1, lastpage:186, numberofpages:186 |
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