Journal: Acta Neuropathologica Communications

Article Title: Ischemic axonal injury up-regulates MARK4 in cortical neurons and primes tau phosphorylation and aggregation

doi: 10.1186/s40478-019-0783-6

Figure Lengend Snippet: Mark4 potentiates tau phosphorylation in vivo and aggregation in vitro. Immunolabeling for pTau-Ser262 (purple) in stroke-injured FR+ (red) cells with uninjured NeuN+ cortical neurons (green) (upper panels, a ). Immunolabeling for Mark4 (green) and 12E8 (white) in FR+ (red) cells (lower panels, a ). Subcortical stroke with retrograde tracing highlighting stroke-injured cortical neurons 7d after stroke (left, b ). Cortical tissue overlying stroke enriched for stroke-injured FR+ cells is selectively isolated (middle, b ). ECLIA for pTau-Thr231 (pg/mL) in ipsilateral cortex of sham and stroke ( b ) ( n = 8/grp, p = 0.012). Schematic of FRET-based tau biosensor assay used to measure tau aggregation in presence of human Mark4 (upper, c ). Representative images of FRET signal induced by tau aggregation in presence of varying concentrations of transfected human Mark4 protein (pM) with or without Mark enzymatic inhibitor (left, c ). Tau aggregation quantified by integrated FRET density in tau-biosensor cells in presence of 20 nM of tau repeat domains and increasing concentrations of human Mark4 (1–250 pM) and Mark enzymatic inhibition (10 μM) (right, c ) ( p < 0.0001 by ANOVA) with specific statistical comparison shown with brackets and p- values. Scale bars = 10 μm ( a ); 500 μm ( b ). Mean ± S.E.M

Article Snippet: The membrane was incubated overnight with the primary antibody to p-tau Ser262 (ThermoFisher Scientific, #OPA1–03142) diluted 1:1000 in 2.5% milk) at 4 °C.

Techniques: Phospho-proteomics, In Vivo, In Vitro, Immunolabeling, Retrograde Tracing, Isolation, Biosensor Assay, Transfection, Inhibition, Comparison