Brain-specific knockdown of miR-29 results in neuronal cell death and ataxia in mice

Abstract

Several microRNAs have been implicated in neurogenesis, neuronal differentiation, neurodevelopment, and memory. Development of miRNA-based therapeutics, however, needs tools for effective miRNA modulation, tissue-specific delivery, and in vivo evidence of functional effects following the knockdown of miRNA. Expression of miR-29a is reduced in patients and animal models of several neurodegenerative disorders, including Alzheimer's disease, Huntington's disease, and spinocerebellar ataxias. The temporal expression pattern of miR-29b during development also correlates with its protective role in neuronal survival. Here, we report the cellular and behavioral effect of in vivo, brain-specific knockdown of miR-29. We delivered specific anti-miRNAs to the mouse brain using a neurotropic peptide, thus overcoming the blood-brain-barrier and restricting the effect of knockdown to the neuronal cells. Large regions of the hippocampus and cerebellum showed massive cell death, reiterating the role of miR-29 in neuronal survival. The mice showed characteristic features of ataxia, including reduced step length. However, the apoptotic targets of miR-29, such as Puma, Bim, Bak, or Bace1, failed to show expected levels of up-regulation in mice, following knockdown of miR-29. In contrast, another miR-29 target, voltagedependent anion channel1 (VDAC1), was found to be induced several fold in the hippocampus, cerebellum, and cortex of mice following miRNA knockdown. Partial restoration of apoptosis was achieved by down-regulation of VDAC1 in miR-29 knockdown cells. Our study suggests that regulation of VDAC1 expression by miR-29 is an important determinant of neuronal cell survival in the brain. Loss of miR-29 results in dysregulation of VDAC1, neuronal cell death, and an ataxic phenotype.

VDAC as a Potential Target in Huntingtons Disease Therapy: The State of the Art

“…Thus, VDAC may play a central role in mitochondria dysfunction detected in HD ( Figure 1) and its attenuation and consequently in HD treatment. However, searching of PubMed provides only two review papers pointing at putative contribution of VDAC to neurodegeneration, including HD [27,46] and one experimental paper concerning the role of VDAC expression regulation in neuronal cell survival in the brain and the significance of the mechanism to neurodegenerative disease including HD [108]. Accordingly, our preliminary results suggest interaction of human VDAC isoforms with both Htt and mHtt (unpublished results).…”

“…It has been demonstrated that the known neuroprotective activity of asiatic acid (a triterpenoid) may result from its free radical scavenging activity as well as from regulation of VDAC expression at the level of transcription and translation [107]. Moreover, VDAC is also included in development of microRNA-based therapeutics for several neurodegenerative disorders [108]. …”

A Simple Alternative to Stereotactic Injection for Brain Specific Knockdown of miRNA

“…This method, originally developed for knockdown of a neurotropic virus, is amenable to modular changes in the three domains that produced cell type specific knockdown of miRNA, namely, the cell penetrating peptide, the neurotropic peptide and the anti-sense molecule. For instance, we found that using a different miR-29 specific antagomir leads to knockdown but fails to produce the behavioural effects 9 . Besides cell penetrating peptides, non-viral methods of gene delivery to neuronal cells include nano-particles, lipids and cationic polymers.…”

“…Such a peptide derived from the Rabies Virus Glycoprotein (RVG), was previously used to deliver siRNA against Japanese Encephalitis Virus in mice 8 . We found that using the peptide for antagomir delivery, miRNAs can be effectively knocked down in the mouse brain 9 .…”

“…To standardize the protocol any fluorescently labeled oligonucleotide (FLO) can be used in place of the antagomir 9 . The peptides should be of high purity (HPLC grade, 98% purity).Sequences and charge of peptide and antagomir are given in Table 1. 1.…”