Edgardo Rodríguez-Lebrón

CHIP Suppresses Polyglutamine Aggregation and Toxicity In Vitro and In Vivo

Huntingtons disease (HD) and other polyglutamine (polyQ) neurodegenerative diseases are characterized by neuronal accumulation of the disease protein, suggesting that the cellular ability to handle abnormal proteins is compromised. As both a cochaperone and ubiquitin ligase, the C-terminal Hsp70 (heat shock protein 70)-interacting protein (CHIP) links the two major arms of protein quality control, molecular chaperones, and the ubiquitin-proteasome system. Here, we demonstrate that CHIP suppresses polyQ aggregation and toxicity in transfected cell lines, primary neurons, and a novel zebrafish model of disease. Suppression by CHIP requires its cochaperone function, suggesting that CHIP acts to facilitate the solubility of mutant polyQ proteins through its interactions with chaperones. Conversely, HD transgenic mice that are haploinsufficient for CHIP display a markedly accelerated disease phenotype. We conclude that CHIP is a critical mediator of the neuronal response to misfolded polyQ protein and represents a potential therapeutic target in this important class of neurodegenerative diseases.

Allele-specific RNA interference for neurological disease

Suppressing the expression of toxic genes through RNAi holds great promise for the treatment of human disease. Allele-specific approaches have now been used to silence dominant toxic genes implicated in several neurological disorders. Here, we review strategies used to achieve allele-specific silencing in light of recent developments in the field of RNAi biology. In particular, new insights into siRNA and miRNA processing may be used to improve efficiency and specificity of RNAi therapy. We further discuss steps that can be taken to maximize the therapeutic benefits of this powerful technology.

RNAi medicine for the brain: progresses and challenges

RNAi interference (RNAi) is a powerful gene silencing technology that has immense potential for treating a vast array of human ailments, for which suppressing disease-associated genes may provide clinical benefit. Here, we review the development of RNAi as a therapeutic modality for neurodegenerative diseases affecting the central nervous system (CNS). We overview promising preclinical data for the application of RNAi in the CNS and discuss key challenges (e.g. delivery and specificity) that remain as these approaches transition to the clinic.

Allele-specific RNAi Mitigates Phenotypic Progression in a Transgenic Model of Alzheimer's Disease

Despite recent advances suggesting new therapeutic targets, Alzheimers disease (AD) remains incurable. Aberrant production and accumulation of the Abeta peptide resulting from altered processing of the amyloid precursor protein (APP) is central to the pathogenesis of disease, particularly in dominantly inherited forms of AD. Thus, modulating the production of APP is a potential route to effective AD therapy. Here, we describe the successful use of an allele-specific RNA interference (RNAi) approach targeting the Swedish variant of APP (APPsw) in a transgenic mouse model of AD. Using recombinant adeno-associated virus (rAAV), we delivered an anti-APPsw short-hairpin RNA (shRNA) to the hippocampus of AD transgenic mice (APP/PS1). In short- and long-term transduction experiments, reduced levels of APPsw transprotein were observed throughout targeted regions of the hippocampus while levels of wild-type murine APP remained unaltered. Moreover, intracellular production of transfer RNA (tRNA)-valine promoter-driven shRNAs did not lead to detectable neuronal toxicity. Finally, long-term bilateral hippocampal expression of anti-APPsw shRNA mitigated abnormal behaviors in this mouse model of AD. The difference in phenotype progression was associated with reduced levels of soluble Abeta but not with a reduced number of amyloid plaques. Our results support the development of allele-specific RNAi strategies to treat familial AD and other dominantly inherited neurodegenerative diseases.

Toward RNAi therapy for the polyglutamine disease Machado-Joseph disease

Machado-Joseph disease (MJD) is a dominantly inherited ataxia caused by a polyglutamine-coding expansion in the ATXN3 gene. Suppressing expression of the toxic gene product represents a promising approach to therapy for MJD and other polyglutamine diseases. We performed an extended therapeutic trial of RNA interference (RNAi) targeting ATXN3 in a mouse model expressing the full human disease gene and recapitulating key disease features. Adeno-associated virus (AAV) encoding a microRNA (miRNA)-like molecule, miRATXN3, was delivered bilaterally into the cerebellum of 6- to 8-week-old MJD mice, which were then followed up to end-stage disease to assess the safety and efficacy of anti-ATXN3 RNAi. Despite effective, lifelong suppression of ATXN3 in the cerebellum and the apparent safety of miRATXN3, motor impairment was not ameliorated in treated MJD mice and survival was not prolonged. These results with an otherwise effective RNAi agent suggest that targeting a large extent of the cerebellum alone may not be sufficient for effective human therapy. Artificial miRNAs or other nucleotide-based suppression strategies targeting ATXN3 more widely in the brain should be considered in future preclinical tests.Machado-Joseph disease (MJD) is a dominantly inherited ataxia caused by a polyglutamine-coding expansion in the ATXN3 gene. Suppressing expression of the toxic gene product represents a promising approach to therapy for MJD and other polyglutamine diseases. We performed an extended therapeutic trial of RNA interference (RNAi) targeting ATXN3 in a mouse model expressing the full human disease gene and recapitulating key disease features. Adeno-associated virus (AAV) encoding a microRNA (miRNA)-like molecule, miRATXN3, was delivered bilaterally into the cerebellum of 6- to 8-week-old MJD mice, which were then followed up to end-stage disease to assess the safety and efficacy of anti-ATXN3 RNAi. Despite effective, lifelong suppression of ATXN3 in the cerebellum and the apparent safety of miRATXN3, motor impairment was not ameliorated in treated MJD mice and survival was not prolonged. These results with an otherwise effective RNAi agent suggest that targeting a large extent of the cerebellum alone may not be sufficient for effective human therapy. Artificial miRNAs or other nucleotide-based suppression strategies targeting ATXN3 more widely in the brain should be considered in future preclinical tests.

Altered Purkinje cell miRNA expression and SCA1 pathogenesis

Spinocerebellar ataxia type 1 (SCA1) is a dominantly inherited neurodegenerative disorder caused by polyglutamine repeat expansions in Ataxin-1. Recent evidence supports a role for microRNAs (miRNAs) deregulation in SCA1 pathogenesis. However, the extent to which miRNAs may modulate the onset, progression or severity of SCA1 remains largely unknown. In this study, we used a mouse model of SCA1 to determine if miRNAs are misregulated in pre- and post-symptomatic SCA1 cerebellum. We found a significant alteration in the steady-state levels of numerous miRNAs prior to and following phenotypic onset. In addition, we provide evidence that increased miR-150 levels in SCA1 Purkinje neurons may modulate disease pathogenesis by targeting the expression of Rgs8 and Vegfa.

Unexpected off-targeting effects of anti-huntingtin ribozymes and siRNA in vivo

Gene transfer strategies to reduce levels of mutant huntingtin (mHtt) mRNA and protein by targeting human Htt have shown therapeutic promise in vivo. Previously, we have reported that a specific, adeno-associated viral vector (rAAV)-delivered short-hairpin RNA (siHUNT-2) targeting human Htt mRNA unexpectedly decreased levels of striatal-specific transcripts in both wild-type and R6/1 transgenic HD mice. The goal of this study was to determine whether the siHUNT-2-mediated effect was due to adverse effects of RNA interference (RNAi) expression in the brain. To this end, we designed two catalytically active hammerhead ribozymes directed against the same region of human Htt mRNA targeted by siHUNT-2 and delivered them to wild-type and R6/1 transgenic HD mice. After 10 weeks of continuous expression, these ribozymes, like siHUNT-2, negatively impacted the expression of a subset of genes in the striatum. This effect was independent of rAAV transduction and specific to the targeting of a unique sequence in human Htt mRNA. After consideration of the known potential RNAi-specific toxic mechanisms, only cleavage of an unintended RNA target can account for the data reported herein. Thus, long-term rAAV-mediated RNAi in the brain does not, in and of itself, negatively affect striatal gene expression. These findings have important implications in the development of therapeutic RNAi for the treatment of neurological disease.

Sustained striatal ciliary neurotrophic factor expression negatively affects behavior and gene expression in normal and R6/1 mice

Huntingtons disease (HD) is a neurodegenerative disorder caused by an elongation of CAG repeats in the HD gene, which encodes a mutant copy of huntingtin with an expanded polyglutatmine repeat. Individuals who are affected by the disease suffer from motor, cognitive, and emotional impairments. Levels of certain striatal‐enriched mRNAs decrease in both HD patients and transgenic HD mice prior to the development of motor symptoms and neuronal cell death. Ciliary neurotrophic factor (CNTF) has been shown to protect neurons against chemically induced toxic insults in vitro and in vivo. To test the hypothesis that CNTF might protect neurons from the negative effects of the mutant huntingtin protein in vivo, CNTF was continuously expressed following transduction of the striatum by recombinant adeno‐associated viral vectors (rAAV2). Wild‐type and R6/1 HD transgenic (R6/1) mice that received bilateral or unilateral intrastriatal injections of rAAV2‐CNTF experienced weight loss. The CNTF‐treated R6/1 HD transgenic mice experienced motor impairments at an earlier age than expected compared with age‐matched control R6/1 HD transgenic animals. CNTF also caused abnormal behavior in WT mice. In addition to behavioral impairments, in situ hybridization showed that, in both WT and R6/1 mice, CNTF expression caused a significant decrease in the levels of striatal‐enriched transcripts. Overall, continuous expression of striatal CNTF at the dose mediated by the expression cassette used in this study was detrimental to HD and wild‐type mice.