Investigation of the Potential Relationship Between TDP-43 Loss and the SOCE Pathway at the Transcriptomic and Cellular Levels
Project no. 31901 · Status: Kapanmış (Yayın bekleniyor) · 2023
Summary
<p><strong style="color: black;">Aim and Scope: </strong><span style="color: black;">Loss of function of TDP-43, which regulates RNA metabolism, is associated with amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). This study aimed to investigate the relationship between TDP-43 loss and store-operated Ca²⁺ entry (SOCE) through changes in gene expression, alternative splicing, and isoform usage in Ca²⁺ homeostasis genes and cellular Ca²⁺ responses.</span></p><p><strong style="color: black;">Methods: </strong><span style="color: black;">Eight human and mouse RNA-seq comparisons were reanalyzed for differential expression and alternative splicing using DESeq2 and rMATS. Changes in genes involved in Ca²⁺ homeostasis were evaluated alongside isoform usage, mapping to protein domains, evolutionary conservation, and TDP-43 binding. Target gene mRNA levels were determined by RT-qPCR in SH-SY5Y cells following </span><em style="color: black;">TARDBP</em><span style="color: black;"> knockdown. Changes in intracellular Ca²⁺ dynamics were measured by spectrofluorometry after loading the cells with Fura-2/AM, and cell viability was determined using the WST-1 cell viability assay.</span></p><p><strong style="color: black;">Results: </strong><span style="color: black;">Alternative splicing changes were identified in the SOCE-associated genes </span><em style="color: black;">STIM1</em><span style="color: black;">, </span><em style="color: black;">STIM2</em><span style="color: black;">, and </span><em style="color: black;">STIMATE</em><span style="color: black;">; isoform usage changes were observed in </span><em style="color: black;">STIM1</em><span style="color: black;">, </span><em style="color: black;">ORAI2</em><span style="color: black;">, </span><em style="color: black;">ATP2A2</em><span style="color: black;">, and </span><em style="color: black;">ATP2A3</em><span style="color: black;">. Alternative splicing findings in </span><em style="color: black;">CBARP</em><span style="color: black;">, </span><em style="color: black;">TRPM4</em><span style="color: black;">, and </span><em style="color: black;">TPCN2</em><span style="color: black;"> recurred across datasets, while </span><em style="color: black;">P2RX6</em><span style="color: black;"> and </span><em style="color: black;">TPCN2</em><span style="color: black;"> isoform changes were supported by two analyses. Screening for cryptic exons yielded no candidates meeting the prioritization criteria. </span><em style="color: black;">TRPC1</em><span style="color: black;">, </span><em style="color: black;">STIM1</em><span style="color: black;">, </span><em style="color: black;">ORAI1</em><span style="color: black;">, and </span><em style="color: black;">ATP2A3</em><span style="color: black;"> mRNA levels increased in SH-SY5Y cells following </span><em style="color: black;">TARDBP</em><span style="color: black;"> knockdown. The SOCE response decreased relative to the control group (p = 0.0115), whereas Ca²⁺ release from the ER did not differ significantly (p = 0.299). Cell viability decreased at 48 hours.</span></p><p><strong style="color: black;">Conclusion: </strong><span style="color: black;">This study demonstrates a relationship between TDP-43 loss and intracellular Ca²⁺ homeostasis, particularly the SOCE response, through transcriptomic and cellular findings. Changes in alternative splicing, isoform usage, and mRNA levels, together with the reduced SOCE response, support a role for TDP-43 in the regulation of store-operated Ca²⁺ entry.</span></p><p><strong style="color: black;">Keywords: </strong><span style="color: black;">TDP-43, TRPC1, store-operated Ca²⁺ entry, Ca²⁺ homeostasis, alternative splicing, isoform usage, RNA-seq</span></p>