Abstract
BackgroundThe progression of Alzheimer’s disease (AD) is associated with an increase of phosphorylated tau in the brain. One of the earliest phosphorylated sites on tau is Ser262 that is preferentially phosphorylated by microtubule affinity regulating kinase (MARK), of which four isoforms exist. Herein we investigated the expression of MARK1-4 in the hippocampus of non-demented elderly (NDE) and AD cases.ResultsIn situ hybridization revealed a uniform, neuronal distribution of all four isoform mRNAs in NDE and AD. Immunohistochemical analyses using isoform-selective antibodies demonstrated that MARK4 in a phosphorylated form colocalizes with p-tau Ser262 in granulovacuolar degeneration bodies (GVDs) that progressively accumulate in AD. In contrast MARK4 is largely absent in the neuronal cytoplasm. MARK3 was localized to a subset of the GVD-containing neurons and also had a weak general cytoplasmic neuronal staining in both NDE and AD. These results suggest that in AD, phosphorylated MARK3 and MARK4 are sequestered and proteolysed in GVDs. MARK1 and MARK2 were absent in GVDs and exhibited relatively uniform neuronal expressions with no apparent differences between NDE and AD.ConclusionWe found that the phosphorylated and fragmented forms of MARK4 and to some extent MARK3 are present in GVDs in AD, and that this expression is highly correlated with phosphorylation of tau at Ser262. This may represent a cellular defense mechanism to remove activated MARK and p-tau Ser262 from the cytosol, thereby reducing the phosphorylating effect on tau Ser262 that appears to be a critical step for subsequent neurodegeneration.
Highlights
The progression of Alzheimer’s disease (AD) is associated with an increase of phosphorylated tau in the brain
In situ hybridization In situ hybridization was performed on 2 non-demented elderly (NDE) and 4 AD cases. 35S-UTP labeled cRNA probes were synthesized by in vitro transcription with the MAXIscript Kit (Ambion) from a synthetic DNA fragment corresponding to part of the coding sequence of human MARK1, human MARK2 (1629–2228 of NM_001039469), human MARK3 (1823–2412 of NM_001128918) or human MARK4 (1181– 1781 of NM_031417) cloned into a pGEM-5Z (+) vector (GeneART)
MARK1 and MARK2 immunoreactivity was recorded in the cytoplasm of cornu ammonis (CA) neurons and in the neuropil (Figure 3A-D), the staining
Summary
The progression of Alzheimer’s disease (AD) is associated with an increase of phosphorylated tau in the brain. Tau protein was identified as the principal component of Alzheimer’s disease (AD) neurofibrillary tangles (NFTs) in the 1980s [1,2,3]. This finding led to the identification of many kinases that have the ability to phosphorylate tau and the description of more than 45 phosphorylation sites present on paired helical filament (PHF) tau [4,5,6]. Using these specific antibodies and a monoclonal antibody towards unphosphorylated tau we were able to demonstrate an increased interaction of MARK2 and MARK4 in AD hippocampal tissue compared to controls using the in situ proximity ligation assay [13,21]
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