Comparison of methods for the analysis of lysosomal enzyme activities in quality control dried blood spot specimens

Abstract

Mucopolysaccharidosis type I (MPS I) is due to deficient alpha-Liduronidase (IDUA) which leads to storage of glycosaminoglycans (GAG). The severe form of MPS I is characterized by mental retardation of unknown etiology. Trying to unveil the mechanisms of cognitive impairment, we studied alterations in the proteome from MPS I mouse hippocampus. Eight-month-old MPS I mice had GAG storage in neurons and glial cells, and impaired aversive and non-aversive memory. Shotgun proteomics was performed and 296 proteins were identified. Of those, 32 were differentially expressed (pb0.05). We found elevation in proteins such as the pro-apoptotic cathepsin D (CtsD, 7-fold normal). Glial fibrilary acid protein (GFAP) was 4.5-fold normal, and immunohistochemistry for GFAP as well as quantitative PCR for TNF-alpha and MIP-1-alpha confirmed a neuroinflammatory process that could be responsible for neuron dysfunction. We did not observe differences in ubiquitin or other proteins related to protein folding, suggesting that the ubiquitin system is working properly. We observed alterations in several proteins involved in synaptic plasticity, including overexpression of post synaptic density-95 (PSD-95) and reduction of microtubuleassociated proteins A and B. IDUA (laronidase, Genzyme, 1.2 mg/kg) was injected i.v. and was able to reduce GAG storage as well as CtsD levels in the brain, suggesting that CtsD can be used as a biomarker for efficacy of preclinical treatments and that a small fraction of laronidase can reach the brain. These results suggest that the cognitive impairment in MPS I is due to multiple processes, including neuroinflammation and alterations in synaptic plasticity.