Hyperphosphorylation, cytoskeleton and cell‐surface binding aptamers: Developing markers and understanding molecular changes in early stage Alzheimer’s disease

Abstract

AbstractBackgroundHyperphosphorylated tau (pTau) is a characteristic of AD, leading to intracellular “tau tangles” followed by dementia. However, intracellular tau is difficult to measure in vivo, requiring a probe to enter the cell. Proteomic changes on the diseased cell‐surface however, provide a diagnostic target that eliminates the need for a probe to enter the cell. We therefore sought cell surface markers of pathological neurons in the process of hyperphosphorylating tau.MethodSH‐SY5Y cells differentiated to a neuronal phenotype were rendered hyperphosphorylative by okadaic acid, and aptamers specifically binding these cells were captured using a modified cell‐SELEX methodology. Mass spectrometric proteomics identified the binding targets. Target presence was validated on SH‐SY5Y cells, P301S mouse brain tissue and human AD brain tissue by immunofluorescence. Gene‐expression data were analysed to identify the utility of the target as a diagnostic marker. A Gd‐bearing nanoparticle targeted using the lead aptamers was injected i.v. in 2‐month P301S mice and T1‐weighted MRI imaging was used to assess binding as a predictor of future tau pathology.ResultAptamers identified by sequencing of the enriched aptamer pools were demonstrated to specifically bind hyperphosphorylative SH‐SY5Y cells . Proteomic analysis suggested Vimentin and keratin6A/6B as potential binding targets. Analysis of hyperphosphorylative SH‐SY5Ycells, human AD tissue and P301S mouse brain tissue confirmed increased levels of cell‐surface Vimentin. Meta‐analysis revealed that Vimentin is expressed at higher levels in the diseased state. Further pathway analysis identified alternative targets related to desmosomes. Two month old P301S mice had no tau pathology, but the targeted MRI particles were still ∼80% accurate in identifying the transgenic mice and predicting future tau pathology.ConclusionOur results suggest that Vimentin and other related desmosomal molecules could be prognostic markers of future tauopathy, and also suggest a need to investigate cell‐surface changes under conditions that cause tauopathies. Targeted MRI particles are promising as an indicator of future tau pathology.