Suppression of Kv3.3 channels by antisense oligonucleotides reverses biochemical effects and motor impairment in spinocerebellar ataxia type 13 mice

Abstract

Mutations in KCNC3, the gene that encodes the Kv3.3 voltage dependent potassium channel, cause Spinocerebellar Ataxia type 13 (SCA13), a disease associated with disrupted motor behaviors, progressive cerebellar degeneration, and abnormal auditory processing. The Kv3.3 channel directly binds Hax‐1, a cell survival protein. A disease‐causing mutation, Kv3.3‐G592R, causes overstimulation of Tank Binding Kinase 1 (Tbk1) in the cerebellum, resulting in the degradation of Hax‐1 by promoting its trafficking into multivesicular bodies and then to lysosomes. We have now tested the effects of antisense oligonucleotides (ASOs) directed against the Kv3.3 channel on both wild type mice and those bearing the Kv3.3‐G592R‐encoding mutation. Intracerebroventricular infusion of the Kcnc3‐specific ASO suppressed both mRNA and protein levels of the Kv3.3 channel. In wild‐type animals, this produced no change in levels of activated Tbk1, Hax‐1 or Cd63, a tetraspanin marker for late endosomes/multivesicular bodies. In contrast, in mice homozygous for the Kv3.3‐G592R‐encoding mutation, the same ASO reduced Tbk1 activation and levels of Cd63, while restoring the expression of Hax‐1 in the cerebellum. The motor behavior of the mice was tested using a rotarod assay. Surprisingly, the active ASO had no effects on the motor behavior of wild type mice but restored the behavior of the mutant mice to those of age‐matched wild type animals. Our findings indicate that, in mature intact animals, suppression of Kv3.3 expression can reverse the deleterious effects of a SCA13 mutation while having little effect on wild type animals. Thus, targeting Kv3.3 expression may prove a viable therapeutic approach for SCA13.

Targeting Ion Channels and Purkinje Neuron Intrinsic Membrane Excitability as a Therapeutic Strategy for Cerebellar Ataxia

“…The p.G592R variant of Kv3.3 shows a gain-of-function phenotype. It results in the degradation of a cell survival protein (Hax-1) that binds to Kv3.3 directly [ 63 ]. Other loss-of-function mutations in Kv3.3 that have been identified in human SCA13 patients result in a reduction in Kv3.3 current amplitude and/or Kv3.3 activation voltage [ 64 ].…”

“…Antisense oligonucleotides (ASOs) are small, single-stranded DNA molecules that have the capability to reduce the expression of specific proteins by binding to complementary mRNA transcripts [ 122 ]. The therapeutic effects of ASOs have been shown in several mouse models of cerebellar ataxia, including SCA1, SCA2, SCA3, and SCA13 [ 63 , 123 , 124 , 125 ]. In the Knockin mouse model of SCA1 (Atxn1 154Q/2Q ) that displays ataxia and premature lethality, ASO delivery to the right lateral ventricle improves motor performance and prolongs survival [ 123 ].…”

“…Longitudinal ASO delivery targeting mutant Atxn3 in SCA3 mice restores transcript levels of Kv3.3 and Kv1.6 associated with improved Purkinje neuron firing and motor performance [ 49 ]. In the Kv3.3-G592R mouse model of SCA13, intracerebroventricular injection of ASOs targeting mutant Kcnc3 mRNA restores motor performance to a level comparable with wild-type controls [ 63 ].…”

Timing is everything: structural insights into the disease-linked Kv3 channels controlling fast action-potential firing in the brain

“…ref. 5 ). Likely disease-causing mutations have been found extensively throughout the Kv3 channel protein (i.e., in the N- and C-termini as well as VSD and TM domains); so there is clearly much work to do to understand their actual impact on Kv3 function.…”

“…Validation of specific therapeutic approaches targeting Kv3 channels are already beginning to emerge: e.g., successful attenuation of the symptoms of spinocerebellar ataxia in a mouse model of the Kv3.3-linked human disease ( KCNC3 G592R) using an antisense approach has been demonstrated 5 . Currently there are no approved drugs that specifically target the Kv3 class of ion channel either on the market or in late-stage clinical development.…”