Development of proteomic methods for studying cerebrospinal fluid proteins involved in Alzheimer´s disease

Doctoral thesis, 2003

Alzheimer's disease (AD) is the most common cause of dementia in western countries. The main neuropathological findings in the AD brain are senile plaques, neurofibrillary tangles and degeneration of neurons and synapses. Although research on AD is progressing fast, the causes and mechanisms of this disease remain to be elucidated and development of new methods is necessary to study neuron-related proteins involved in the pathophysiological mechanisms. Six low-abundance synaptic proteins in human cerebrospinal fluid (CSF), namely rab 3a, synaptotagmin, synapsin, the presynaptic protein GAP-43, the synaptosomal-associated protein 25 and the postsynaptic protein neurogranin, were detected with liquid phase isoelectric focusing and immunoblotting. An ELISA method for quantification of the phosphorylated form (Ser 9) of synapsin I was developed. Increased levels of phosphosynapsin I were demonstrated in AD patients compared to controls. These results are consistent with the hypothesis of impaired protein phosphorylation mechanisms in AD. To purify and characterise proteins in CSF, a new strategy combining two-dimensional liquid phase electrophoresis (2D-LPE) and matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry (MALDI-TOF MS) was developed. Two brain-specific proteins, cystatin C and b2 microglobulin, were isolated from CSF in sufficient quantities for analysis by MALDI-TOF MS. Special attention was needed to make 2D-LPE and mass spectrometry compatible. Chloroform/methanol/water extraction was the most efficient method for SDS removal, allowing the acquisition of good quality MALDI spectra of the tryptic digest of the proteins analysed. Two-dimensional gel electrophoresis (2-DE) and mass spectrometry have been used for clinical screening of disease-influenced CSF proteins in AD. In order to increase the detection of CSF proteins and to improve the separation of protein isoforms, micro-narrow range immobilised pH gradient strips and prefractionation prior to 2-DE of CSF were used. Previously detected protein changes by 2-DE, between AD patients and controls, such as apoliprotein E and apoliprotein A1, were confirmed. Several new protein changes were demonstrated, including kininogen, apoliprotein J, b-trace, 1 b glycoprotein, a 2-HS glycoprotein and a-1 antitrypsin. As shown in this study, different isoforms i.e. different states of glycosylated proteins, are altered in AD. Therefore, the determination of post-translational modifications such as glycosylation and phosphorylation, is of importance for an increased understanding of the neuropathology in AD. The use of complementary strategies in proteome studies of CSF offers new perspectives on the pathology in neurodegenerative diseases and also reveals new potential biomarkers for brain disorders such as AD.