Aberrant Purkinje cell activity is the cause of dystonia in a shRNA-based mouse model of Rapid Onset Dystonia–Parkinsonism

Abstract

Loss-of-function mutations in the α3 isoform of the sodium pump are responsible for Rapid Onset Dystonia-Parkinsonism (RDP). A pharmacologic model of RDP replicates the most salient features of RDP, and implicates both the cerebellum and basal ganglia in the disorder; dystonia is associated with aberrant cerebellar output, and the parkinsonism-like features are attributable to the basal ganglia. The pharmacologic agent used to generate the model, ouabain, is selective for sodium pumps. However, close to the infusion sites in vivo it likely affects all sodium pump isoforms. Therefore, it remains to be established whether selective loss of α3-containing sodium pumps replicates the pharmacologic model. Moreover, while the pharmacologic model suggested that aberrant firing of Purkinje cells was the main cause of abnormal cerebellar output, it did not allow the scrutiny of this hypothesis. To address these questions RNA interference using small hairpin RNAs (shRNAs) delivered via adeno-associated viruses (AAV) was used to specifically knockdown α3-containing sodium pumps in different regions of the adult mouse brain. Knockdown of the α3-containing sodium pumps mimicked both the behavioral and electrophysiological changes seen in the pharmacologic model of RDP, recapitulating key aspects of the human disorder. Further, we found that knockdown of the α3 isoform altered the intrinsic pacemaking of Purkinje cells, but not the neurons of the deep cerebellar nuclei. Therefore, acute knockdown of proteins associated with inherited dystonias may be a good strategy for developing phenotypic genetic mouse models where traditional transgenic models have failed to produce symptomatic mice.

Current Opinions and Areas of Consensus on the Role of the Cerebellum in Dystonia

“…It was recently shown that in both genetic and pharmacologic models of rapid-onset dystonia parkinsonism (RDP), abnormal bursting cerebellar output underlies dystonia [43, 47, 66]. Studies have shown that acute knockdown or pharmacologic inhibition of the α3 isoform of the sodium pump, the protein mutated in human RDP, converts the normally regular activity of Purkinje cells to burst firing [43, 66]. This erratic Purkinje cell activity in turn modifies the activity of DCN neurons, resulting in highly irregular cerebellar output.…”

“…Data from animal models also implicates olivocerebellar pathways, particularly Purkinje cells and cerebellar nuclear neurons, in the pathophysiology of dystonia [43, 66, 89]. For instance, morphologically/physiologically defective Purkinje cells or Purkinje cell loss has been described in tottering mice, DYT1 knock-in mice, waddles mice and dt rats [89–92].…”

“…In final analysis, abnormal signaling in cerebellar cortex due to dysfunction of Purkinje cells or their afferents (parallel and/or climbing fibers) will manifest as abnormal firing patterns of cerebellar nuclear neurons [64, 89, 98]. Compatible with this model, abnormalities of cerebellar outflow have been reported in humans and animal models of primary or isolated dystonia [40, 43, 66, 74, 99]. …”

Cerebellar Intermittent Theta-Burst Stimulation and Motor Control Training in Individuals with Cervical Dystonia

“…Another dystonic mouse model developed using conditional genetics to regionally limit cerebellar dysfunction revealed Purkinje cell abnormalities causing dystonia-like postures [10] while progressive loss of Purkinje cell neurons reduced dystonia severity [11]. Further studies using dystonic rodent models localised the mechanism of action to Purkinje cell sodium pump dysfunction, leading to aberrant high frequency firing [12,13]. There is also accumulating evidence of cerebellar involvement in CD in humans.…”

MeCP2 and histone deacetylases 1 and 2 in dorsal striatum collectively suppress repetitive behaviors

“…For HDAC1 staining, the sections were treated in 10 mM citric acid (pH 6) for 15 minutes at 95 °C for antigen retrieval prior to the primary antibody incubation. Immunoreactivity was visualized by secondary antibodies conjugated with either Alexa Fluor 594 (HDAC1, Invitrogen, A11012) 8 or Alexa Flour 488 (HDAC2, HA, GFP, Invitrogen, A11008) 42 . The sections were incubated at a 1:200 dilution at room temperature for 2 hours, counter-stained with 4′,6-diamidino-2-phenylindole (DAPI), and then mounted on superfrost plus slides in Vectashield mounting media (Vector Laboratories).…”