Investigating the function of microtubule-associated protein tau (MAPT) and its genetic association with Parkinson’s using human iPSC-derived dopamine neurons
Beevers JE., Booth H., Zambon F., Vowles J., Robertson PA., Lai M-C., Cowley SA., Wade-Martins R., Caffrey TM.
OBJECTIVES The microtubule associated protein tau (MAPT) locus is highly associated with Parkinson's (PD); however, the mechanisms underlying susceptibility remain unclear. We propose that polymorphisms within the MAPT haplotype sequence have functional consequences on MAPT expression and tau protein function in the dopamine neurons notably lost in PD. To examine this we chose the following objectives: • To investigate allele-specific regulation of tau expression in iPSC-derived dopamine neurons. • To investigate the physiological role of tau in axonal transport by perturbing tau expression. METHODS Induced pluripotent stem cells derived from healthy heterozygous (H1/H2) donors were differentiated into midbrain-type neuronal cultures. Dopamine neurons were isolated using rapid fixation immunostaining for tyrosine hydroxylase (TH) followed by fluorescence-activated cell sorting and RNA extraction. For tau perturbation, RNAi targeting specific isoforms or total tau was designed, with initial knockdown performed in neuroblastoma cells. RESULTS Transcripts of TH were >7-fold enriched in FACS-isolated neurons and mature MAPT isoforms (exon 3+ or 10+) were detected with extended culture. siRNA-mediated specific knockdown of exon 3+ (~60%) or exon 10+ (~90%) MAPT isoforms was achieved. Isoform-specific and total MAPT shRNA sequences were incorporated into lentivirus plasmids for delivery to neuronal cultures, in addition to those expressing fluorescent amyloid precursor protein for imaging of live axonal transport. CONCLUSIONS Examining haplotype-specific tau expression and function for the first time in dopamine cultures allows us to understand the effect of haplotype on tau protein in the neurons that degenerate in PD, and to identify therapeutic targets to reduce progression beyond disease’s earliest signs.