| Cerebral Palsy (CP) is among the most prevalent of neuromotor conditions. CP has been defined as a disorder of the central nervous system (CNS), resulting from lesions in the brain. Clinical experience indicates that CP patients have altered sensitivities to neuromuscular blocking agents suggesting the involvement of the peripheral nervous system (PNS), specifically neuromuscular junctions (NMJs), in the muscular defects seen in CP patients. Previous findings in our lab had suggested that the synaptic basal lamina (BL) is aberrant in CP patients. Specifically, certain components of the BL, such as acetylcholine esterase (AChE) and laminin beta2, that are known to co-localize and tightly appose with acetylcholine receptors (AChR) in the synapse, were found as non-apposed or highly dysmorphic. I hypothesized that the CP NMJs are aberrant with respect to the gene expression and localization of synaptic BL proteins. Since, several components of the BL play an important role in formation, maturation, and maintenance of structure and function of the NMJs and muscle, aberrant BL may lead to immature muscle in CP patients. I therefore hypothesized that muscle, as well as NMJs in CP are immature. I further hypothesize that aberrant BL has an impact on the structure of NMJs in CP. The present study tests the above two hypotheses by first investigating whether CP muscle are immature. Muscle sample from CP and control patients were analyzed for immaturity markers by real time Q-PCR and western blotting. I did not find the presence of any immaturity markers in CP muscle samples arguing against the presence of immature muscles and NMJs in CP muscle. Global analysis for differential expression of abundant proteins by proteomics also did not reveal any difference between CP and control muscles. Since, dysmorphic NMJ could also result from aberrant expression of genes by the synaptic nuclei I analyzed gene expression patterns of the dysmorphic and non-dysmorphic junctional and extrajunctional nuclei in CP and compared them to junctional and extrajunctional nuclei of control muscles. RNA from laser capture microdissected junctional and extra junctional nuclei were isolated and subjected to gene expression analysis using a focused array of genes that are enriched in the NMJ. Our analysis revealed several genes that play key role in NMJ structure and function, including several components of BL, are differentially expressed in CP NMJs as compared to controls. Further, our analysis revealed that there exist a differential expression of NMJ enriched genes in non-dysmorphic and highly dysmorphic CP NMJs. This suggests that CP patient's NMJs harbor a mosaic of dysmorphic and non-dysmorphic NMJs which differ in gene expression. Since these genes function in maintenance of structure and function of NMJs, I investigated whether there is a change in structure of NMJ in CP through ultrastuctural analysis. For this, we first devised a method to localize NMJs in human muscle biopsy specimen, which involved tile mapping and localization of NMJs by confocal microscopy, for efficient localization of NMJ in subsequent ultrastructral analysis. Our data indicates that CP NMJs have increase length of primary folds, an increase in distance between the primary folds, a decrease in number of aligned active zones, and a decrease in synaptic mitochondria in the nerve terminal. All these structural changes could potentially have an impact in neurotransmission at the NMJ in CP, which might be responsible for some of the clinical features seen in CP muscle. |
