Biomedical subjects

Victor M Miller

Publications and source records attributed to Victor M Miller.

8 recordsLinked to original sources

PubMed2006

A novel route for F-box protein-mediated ubiquitination links CHIP to glycoprotein quality control.

In SCF (Skp1/Cullin/F-box protein) ubiquitin ligases, substrate specificity is conferred by a diverse array of F-box proteins. Only in fully assembled SCF complexes, it is believed, can substrates bound to F-box proteins become ubiquitinated. Here we show that Fbx2, a brain-enriched F-box protein implicated in the ubiquitination of glycoproteins discarded from the endoplasmic reticulum, binds the co-chaperone/ubiquitin ligase CHIP (C terminus of Hsc-70-interacting protein) through a unique N-terminal PEST domain in Fbx2. CHIP facilitates the ubiquitination and degradation of Fbx2-bound glycoproteins, including unassembled NMDA receptor subunits. These findings indicate that CHIP acts with Fbx2 in a novel ubiquitination pathway that links CHIP to glycoprotein quality control in neurons. In addition, they expand the repertoire of pathways by which F-box proteins can regulate ubiquitination and suggest a new role for PEST domains as a protein interaction motif.

Amino Acid Sequence↗

PubMed2005

CHIP suppresses polyglutamine aggregation and toxicity in vitro and in vivo.

Huntington's 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.

Animals↗

PubMed2005

Breaks in coordination: DNA repair in inherited ataxia.

Genetic defects in DNA repair are increasingly recognized as being able to cause degenerative ataxia syndromes. It remains a mystery, however, why disruption of a process fundamental to proliferating cells can be selectively toxic to postmitotic neurons. Recent studies now reveal that an ataxia gene, tyrosyl phosphodiesterase 1 (TDP1), repairs single-stranded DNA breaks in nondividing cells. Here we review the implications of this and other findings for a growing list of hereditary ataxias.

Animals↗

PubMed2005

RNA interference in neuroscience: progress and challenges.

1.RNA interference (RNAi) is a recently discovered biological pathway that mediates post-transcriptional gene silencing. The process of RNAi is orchestrated by an increasingly well-understood cellular machinery. 2. The common entry point for both natural and engineered RNAi are double stranded RNA molecules known as short interfering RNAs (siRNAs), that mediate the sequence-specific identification and degradation of the targeted messenger RNA (mRNA). The study and manipulation of these siRNAs has recently revolutionized biomedical research. 3. In this review, we first provide a brief overview of the process of RNAi, focusing on its potential role in brain function and involvement in neurological disease. We then describe the methods developed to manipulate RNAi in the laboratory and its applications to neuroscience. Finally, we focus on the potential therapeutic application of RNAi to neurological disease.

Animals↗

PubMed2004

Targeting Alzheimer's disease genes with RNA interference: an efficient strategy for silencing mutant alleles.

Tau and amyloid precursor protein (APP) are key proteins in the pathogenesis of sporadic and inherited Alzheimer's disease. Thus, developing ways to inhibit production of these proteins is of great research and therapeutic interest. The selective silencing of mutant alleles, moreover, represents an attractive strategy for treating inherited dementias and other dominantly inherited disorders. Here, using tau and APP as model targets, we describe an efficient method for producing small interfering RNA (siRNA) against essentially any targeted region of a gene. We then use this approach to develop siRNAs that display optimal allele-specific silencing against a well-characterized tau mutation (V337M) and the most widely studied APP mutation (APPsw). The allele-specific RNA duplexes identified by this method then served as templates for constructing short hairpin RNA (shRNA) plasmids that successfully silenced mutant tau or APP alleles. These plasmids should prove useful in experimental and therapeutic studies of Alzheimer's disease. Our results suggest guiding principles for the production of allele-specific siRNA, and the general method described here should facilitate the production of gene-specific siRNAs.

Alleles↗

PubMed2003

Allele-specific silencing of dominant disease genes.

Small interfering RNA (siRNA) holds therapeutic promise for silencing dominantly acting disease genes, particularly if mutant alleles can be targeted selectively. In mammalian cell models we demonstrate that allele-specific silencing of disease genes with siRNA can be achieved by targeting either a linked single-nucleotide polymorphism (SNP) or the disease mutation directly. For a polyglutamine neurodegenerative disorder in which we first determined that selective targeting of the disease-causing CAG repeat is not possible, we took advantage of an associated SNP to generate siRNA that exclusively silenced the mutant Machado-Joseph disease/spinocerebellar ataxia type 3 allele while sparing expression of the WT allele. Allele-specific suppression was accomplished with all three approaches currently used to deliver siRNA: in vitro-synthesized duplexes as well as plasmid and viral expression of short hairpin RNA. We further optimized siRNA to specifically target a missense Tau mutation, V337M, that causes frontotemporal dementia. These studies establish that siRNA can be engineered to silence disease genes differing by a single nucleotide and highlight a key role for SNPs in extending the utility of siRNA in dominantly inherited disorders.

Alleles↗

PubMed2003

Toward therapy for DYT1 dystonia: allele-specific silencing of mutant TorsinA.

A three-nucleotide (GAG) deletion in the TOR1A gene is the most common cause of inherited dystonia, DYT1. Because the mutant protein, TorsinA (TA), is thought to act in a dominant manner to cause disease, inhibiting expression from the mutant gene represents a potentially powerful therapeutic strategy. In an effort to develop therapy for this disease, we tested whether small interfering RNA (siRNA) could selectively silence expression of mutant TA. Exploiting the three-base pair difference between wild-type and mutant alleles, we designed siRNAs to silence expression of mutant, wild-type, or both forms of TA. In transfected cells, siRNA successfully suppressed wild-type or mutant TA in an allele-specific manner: for example, mutant-specific siRNA reduced the levels of mutant TA to less than 1% of controls with minimal effect on wild-type TA expression. In cells expressing both alleles, thus simulating the heterozygous state, siRNA-mediated suppression remained robust and allele specific. Our siRNA studies demonstrate allele-specific targeting of a dominant neurogenetic disease gene and suggest the broad therapeutic potential of siRNA for DYT1 dystonia and other dominantly inherited neurological diseases.

Alleles↗

PubMed2002

Live-cell imaging reveals divergent intracellular dynamics of polyglutamine disease proteins and supports a sequestration model of pathogenesis.

Protein misfolding and aggregation are central features of the polyglutamine neurodegenerative disorders, but the dynamic properties of expanded polyglutamine proteins are poorly understood. Here, we use fluorescence recovery after photobleaching (FRAP) and fluorescence loss in photobleaching (FLIP) with green fluorescent protein fusion proteins to study polyglutamine protein kinetics in living cells. Our results reveal markedly divergent mobility states for an expanded polyglutamine protein, ataxin-3, and establish that nuclear inclusions formed by this protein are aggregates. Additional studies of green fluorescent protein-tagged cAMP response element binding protein coexpressed with either of two mutant polyglutamine proteins, ataxin-3 and huntingtin, support a model of disease in which coaggregation of transcriptional components contributes to pathogenesis. Finally, studies of a third polyglutamine disease protein, ataxin-1, reveal unexpected heterogeneity in the dynamics of inclusions formed by different disease proteins, a finding which may help explain disease-specific elements of pathogenesis in these neurodegenerative disorders.

Animals↗