599 - Muscle-proteomic analysis of muscle biopsies of cerebral palsy patients

Publication Number: 4587.599

Brigette M. Romero, University of Delaware, Newark, DE, United States; Karyn Robinson, Nemours Children's Hospital, Wilmington, DE, United States; Vijay Parashar, University of Delaware, Newark, DE, United States; Wade Shrader, NemoursAlfred I. duPont Hospital for Children, Wilmington, DE, United States; Robert Akins, Nemours Children's Health, Wilmington, DE, United States; Mona Batish, University of Delaware, Newark, DE, United States

Background: Spastic Cerebral Palsy (CP) is a complex childhood-onset motor disability disorder known to involve genetic and environmental risk factors. Accumulating data further suggest that epigenetic pathways (e.g. DNA methylation, histone modifications, and non-coding RNAs) play important roles in CP, particularly in the peripheral manifestations of the movement disorder. However, the molecular and cellular mechanisms contributing to motor dysfunction in CP remain largely unknown. To address this critical gap, there is a need for more personalized and precise approaches utilizing multiomic technologies, data science, and advanced analytics. These tools will allow us to fundamentally improve our understanding of muscle development and pathology in children with CP. Given genomics and transcriptomics studies have been performed extensively but there is a lack of proteomic studies in CP field, which is essential to pinpoint the central hubs within the disease network to prioritize key pathways and genes.

Objective: We aim to compare proteomic profiles of skeletal muscle tissue from control and CP patients.

Design/Methods: Protein was isolated from erector spinae muscle biopsies (n=3) under an IRB-approved protocol. Tissue lysis was performed using 8 M urea and 5% SDS for complete protein solubilization. Protein concentration was quantified via BCA assay and then subjected to trypsin digestion in preparation for mass spectrometry analysis. Differential protein expression was validated using qRT-PCR, immunofluorescence, and Western blot. Enrichment analysis was performed to identify associated biological processes.

Results: A clear separation of proteomic profile was seen between CP and controls indicated by PCA plot (fig1). A total of 35 proteins were significantly downregulated in CP skeletal muscle relative to the control group. These proteins were mainly involved in key biological processes, including immune response, intermediate filament organization, complement activation, lipid transport, and collagen fibril formation. In contrast, 113 proteins were upregulated in CP muscle compared to controls (fig2). The functions associated with these upregulated proteins primarily included tubulin complex assembly, regulation of mRNA stability, regulation of translation, phosphorylation, and negative regulation of protein ubiquitination (fig3).

Conclusion(s): This approach will allow us to identify critical pathways involved in the onset and progression of the disease, connect it with existing data sets to get a multi-omic profile of CP to identify and validate novel diagnostic and therapeutic strategies for CP.

Principal Component Analysis (PCA) plot
Figure 1. Proteomic differences in skeletal muscle tissue between Cerebral Palsy (CP) patients and controls. Principal Component Analysis (PCA) plot showing distinct separation in the global proteomic profiles of skeletal muscle when comparing CP patients to controls, indicating significant differences in protein expression patterns.

Enrichment Analysis of Up regulated proteins
Figure 2. Functional enrichment analysis of proteins significantly upregulated in the skeletal muscle of Cerebral Palsy (CP) patients.

Enrichment Analysis of down regulated proteins
Figure 3. Functional enrichment analysis of proteins significantly downregulated in the skeletal muscle of Cerebral Palsy (CP) patients.