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., Cowley SA., Wade-Martins R., Caffrey TM.
OBJECTIVES • To investigate allele-specific regulation of tau expression. • To investigate the physiological role of tau in axonal transport within healthy dopamine neurons. BACKGROUND Microtubule-associated protein tau, encoded by the gene MAPT, is involved in neurodegenerative disease by forming hyperphosphorylated aggregates. However, genome-wide association studies for Parkinson’s implicate common variation at the MAPT locus with Parkinson’s despite a general lack of tau neuropathology. Two common haplotypes exist at the MAPT locus, with haplotype H1 increasing risk for Parkinson’s. Haplotype differences do not affect tau protein sequence, suggesting that risk must lie in altered protein expression. We have previously described haplotype-specific differences in the inclusion of exons 3 and 10 in transcripts from H1 and H2 alleles in post-mortem human brain. METHODS To examine MAPT in the midbrain dopamine neurons notably lost in Parkinson’s, 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 followed by fluorescence-activated cell sorting. RNA was extracted to examine MAPT transcripts on the Sequenom platform. To further investigate regulation of tau expression, candidate microRNAs were expressed in H1/H2 neuroblastoma cells with quantification of tau protein knockdown. The function of tau protein isoforms in axonal transport within human dopamine neurons was examined using live axonal imaging of fluorescent amyloid precursor protein with selective siRNA knockdown of specific tau isoforms or total tau. CONCLUSIONS Understanding how the regulation of MAPT expression can result in altered functionality will help identify therapeutic targets to help prevent Parkinson’s progression from its earliest signs.