Intracellular accumulation of alpha-synuclein (alpha-Syn) as filamentous aggregates is a pathological feature shared by Parkinson's disease, dementia with Lewy bodies and multiple system atrophy, referred to as synucleinopathies. To understand the mechanisms underlying alpha-Syn aggregation, we established a tetracycline-off inducible transfectant (3D5) of neuronal lineage overexpressing human wild-type alpha-Syn. Alpha-Syn aggregation was initiated by exposure of 3D5 cells to FeCl2. The exposure led to formation of alpha-Syn inclusions and oligomers of 34, 54, 68 kDa and higher molecular weights. The oligomers displayed immunoreactivity with antibodies to the amino-, but not to the carboxyl (C)-, terminus of alpha-Syn, indicating that C-terminally truncated alpha-Syn is a major component of oligomers. FeCl2 exposure also promoted accumulation of S129 phosphorylated monomeric alpha-Syn (P alpha-Syn) and casein kinase 2 (CK2); however, G-protein-coupled receptor kinase 2 was reduced. Treatment of FeCl2-exposed cells with CK2 inhibitors (DRB or TBB) led to decreased formation of alpha-Syn inclusions, oligomers and P alpha-Syn. FeCl2 exposure also enhanced the activity/level of cathepsin D. Treatment of the FeCl2-exposed cells with pepstatin A or NH4Cl led to reduced formation of oligomers/inclusions as well as of approximately 10 and 12 kDa truncated alpha-Syn. Our results indicate that alpha-Syn phosphorylation caused by FeCl2 is due to CK2 upregulation, and that lysosomal proteases may have a role in producing truncated alpha-Syn for oligomer assembly.
The diagnosis of idiopathic normal pressure hydrocephalus (iNPH) is sometimes complicated by concomitant Alzheimer's disease (AD) pathology. The purpose of the present study is to identify an iNPH-specific cerebrospinal fluid (CSF) biomarker dynamics and to assess its ability to differentiate iNPH from AD. Total tau (t-tau), tau phosphorylated at threonine 181 (p-tau), amyloid-β (Aβ) 42 and 40, and leucine-rich α-2-glycoprotein (LRG) were measured in 93 consecutive CSF samples consisting of 55 iNPH (46 tap test responders), 20 AD, 11 corticobasal syndrome, and 7 spinocerebeller disease. Levels of t-tau and p-tau were significantly decreased in iNPH patients especially in tap test responders compared to AD. Correlation was observed between Mini-Mental State Examination scores and Aβ42 in AD (R = 0.44) and mildly in iNPH (R = 0.28). Although Aβ42/40 ratio showed no significant difference between iNPH and AD (p = 0.08), the levels of Aβ40 and Aβ42 correlated positively with each other in iNPH (R = 0.73) but much less in AD (R = 0.26), suggesting that they have discrete amyloid clearance and pathology. LRG levels did not differ between the two. Thus, our study shows that although CSF biomarkers of iNPH patients can be affected by concomitant tau and/or amyloid pathology, CSF t-tau and p-tau are highly useful for differentiation of iNPH and AD.
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