Disruption of Purkinje cell function prior to huntingtin accumulation and cell loss in an animal model of Huntington Disease

Abstract

Huntington Disease (HD) is a devastating neurological disorder characterized by progressive deterioration of psychiatric, motor, and cognitive function. Purkinje cells (PCs), the output neurons of the cerebellar cortex, have been found to be vulnerable in multiple CAG repeat disorders, but little is known about the involvement of PC dysfunction in HD. To investigate possible PC abnormalities, we performed quantitative real time PCR, western blot analysis, and immunohistochemistry experiments to explore the changes in PC markers in the R6/2 mouse model of severe HD. Interestingly, there were reductions in the transcript and protein levels of the calcium-binding proteins parvalbumin and calbindin, as well as the enzyme glutamic acid decarboxylase 67. Immunohistochemistry supported these results, with the most substantial changes occurring in the PC layer. To determine whether the reductions in PC marker expression were due to cell loss, we performed stereology on both presymptomatic and end-stage R6/2 mice. Stereological counts indicated a significant reduction in PC number by end-stage but no change in presymptomatic animals (4 weeks of age). To assess cellular function prior to cell loss and symptom onset, we measured spontaneous firing in PCs from 4-week old animals and found a striking deficit in PC firing as indicated by a 57% decrease in spike rate. Interestingly, huntingtin inclusions were not widely observed in PCs until 12 weeks of age, indicating that soluble huntingtin and/or abnormalities in other cell types may contribute to PC dysfunction. Considering the roles for PCs in motor control, these data suggest that early PC dysfunction potentially contributes to motor impairment in this model of HD.

Purkinje cell dysfunction and loss in a knock-in mouse model of Huntington Disease

“…Additionally, as evidenced by the PV staining, interneurons of the molecular layer were still present (arrows; Fig 2J). This staining pattern is strikingly similar to the one we previously observed in the R6/2 model (Dougherty, Reeves et al 2012) as well as to the pattern reported in a model of spinocerebellar ataxia-1 (Vig, Subramony et al 1998). Importantly, at this age, mutant htt inclusions were present within PCs of HdhQ200/Q200 mice (Fig 3).…”

“…Given that a change in firing frequency was an early robust measure of PC dysfunction in the R6/2 mice (Dougherty, Reeves et al 2012), cell-attached loose patch electrophysiology was performed in 50 week old HdhQ200/Q200 and HdhQ200/+ mice (n=3–6 mice/group; 10–12 cells/group). Loose patch recordings revealed a significant reduction in spike frequency in the HdhQ200/Q200 mice as compared to age-matched WT controls (Fig 5; p<0.05), while there was no significant difference in firing rate in the HdhQ200/+ mice as compared to age-matched WT controls.…”

“…We recently provided evidence for PC abnormalities in the R6/2 model of HD (Dougherty, Reeves et al 2012); changes include a reduction in transcript and protein levels of GABAergic cell population components (glutamic acid decarboxylase 67 and parvalbumin) and a specific purkinje cell marker (calbindin). Furthermore, we found that PC loss was preceded by a significant reduction in PC spike rate.…”

Rapid generation of sub-type, region-specific neurons and neural networks from human pluripotent stem cell-derived neurospheres

“…However, SHH and FGF8 were added after neuronal induction to generate these neurons. Cerebellar Purkinje neurons, which are known to have a crucial role in NPD 36 and HD 50,51 , appeared in our culture between 45–48 days, which is relatively longer and is a similar pattern as that observed in the developing cerebellum in vivo 29,30 . Thus, our method can rapidly generate subtype-specific or region-specific neurons compared to the other currently available neurodifferentiation methods 10,11,21,22 .…”

The Role for Alterations in Neuronal Activity in the Pathogenesis of Polyglutamine Repeat Disorders

“…In the R6/2 model of HD, there is a striking reduction in Purkinje neuron firing frequency by postnatal day 28. It should be noted that there are no huntingtin inclusions in the Purkinje neurons until 12 weeks [73]. In the HdhQ200 model of HD, recordings were performed at a time where there was prominent Purkinje neuron loss, and loose patch electrophysiology of remaining Purkinje neurons identified a similar reduction in Purkinje neuron firing frequency in the HD mice compared with control littermates [74].…”