Background Neuronal inclusion formation is a pathognomonic feature of Huntington’s disease (HD). Recent evidence has suggested that these inclusion bodies (IBs) may comprise a heterogeneous population of huntingtin (HTT) protein species, a subset of which may lead to impaired cell viability and ultimately cell death.
Aim To investigate the formation of inclusions in human neurons derived from neural stem cells (NSCs) (ReNcell VM) stably transduced to over-express HTT exon 1 fragments with either normal (29Q) or pathogenic polyglutamine tracts (71 Q and 129Q).
Methods High-content screening of neurons stained with anti-HTT antibody S830 was carried out using a Perkin Elmer Opera LX confocal microscope. Subsequent quantitative image analysis was carried out using ImageJ and R. Individual cells were analysed in more detail using super resolution imaging (N-SIM Super Resolution System). The effect of mHTT exon 1 on neuronal viability was assessed using a variety of biochemical techniques (LDH assay, MTT and Alamar Blue).
Results Inclusion bodies form in a small proportion of (1%) of neurons, and are predominantly intra-nuclear, though a lower proportion of cells display smaller perinuclear inclusions. The formation of nuclear inclusions increases in a polyQ- length dependent manner and over time. Super resolution microscopy of individual cells has demonstrated this to be an all-or-nothing phenomenon. A diffuse cytosolic form of mHTT also accumulates in the cells over time. There is no overt effect on basal viability in these mHTT exon 1 expressing neurons.
Conclusions We have characterised the spatio-temporal profile of mHTT inclusion formation in mHTT expressing NSCs and neurons, and have demonstrated poly-Q and time-dependent accumulation of IBs. A deeper understanding of relationship between the different forms of mHTT and neuronal vulnerability is essential for the design of targeted therapeutics.
- HTT Exon 1
- Human Neurons