Huntington disease (HD), an autosomal dominant, progressive neurodegenerative disorder, is caused by an expanded CAG repeat sequence leading to an increase in the number of glutamine residues in the encoded protein. The normal CAG repeat range is 5-36, whereas 38 or more repeats are found in the diseased state; the severity of disease is roughly proportional to the number of CAG repeats. HD shows anticipation, in which subsequent generations display earlier disease onsets due to intergenerational repeat expansion. For longer repeat lengths, somatic instability of the repeat size has been observed both in human cases at autopsy and in transgenic mouse models containing either a genomic fragment of human HD exon 1 (ref. 9) or an expanded repeat inserted into the endogenous mouse gene Hdh (ref. 10). With increasing repeat number, the protein changes conformation and becomes increasingly prone to aggregation, suggesting important functional correlations between repeat length and pathology. Because dinucleotide repeat instability is known to increase when the mismatch repair enzyme MSH2 is missing, we examined instability of the HD CAG repeat by crossing transgenic mice carrying exon 1 of human HD (ref. 16) with Msh2-/- mice. Our results show that Msh2 is required for somatic instability of the CAG repeat.

This investigation was pursued to test the use of intracellular antibodies (intrabodies) as a means of blocking the pathogenesis of Huntington's disease (HD). HD is characterized by abnormally elongated polyglutamine near the N terminus of the huntingtin protein, which induces pathological protein-protein interactions and aggregate formation by huntingtin or its exon 1-containing fragments. Selection from a large human phage display library yielded a single-chain Fv (sFv) antibody specific for the 17 Nterminal residues of huntingtin, adjacent to the polyglutamine in HD exon 1. This anti-huntingtin sFv intrabody was tested in a cellular model of the disease in which huntingtin exon 1 had been fused to green fluorescent protein (GFP). Expression of expanded repeat HD-polyQ-GFP in transfected cells shows perinuclear aggregation similar to human HD pathology, which worsens with increasing polyglutamine length; the number of aggregates in these transfected cells provided a quantifiable model of HD for this study. Coexpression of anti-huntingtin sFv intrabodies with the abnormal huntingtin-GFP fusion protein dramatically reduced the number of aggregates, compared with controls lacking the intrabody. Anti-huntingtin sFv fused with a nuclear localization signal retargeted huntingtin analogues to cell nuclei, providing further evidence of the anti-huntingtin sFv specificity and of its capacity to redirect the subcellular localization of exon 1. This study suggests that intrabody-mediated modulation of abnormal neuronal proteins may contribute to the treatment of neurodegenerative diseases such as HD, Alzheimer's, Parkinson's, prion disease, and the spinocerebellar ataxias. H untington's disease (HD) is a genetic disorder that derives from expanded CAG repeats in the huntingtin gene (1), which then encodes pathological huntingtin protein with abnormally long polyglutamine sequences (polyQ, or Qn). Huntingtin is expressed ubiquitously by human cells, with high levels in the brain, particularly the cortex and striatum. Murine model studies of HD have shown it occurs through dominant gain of function when a single abnormal allele is present (2-6). Polyglutamine lengths ranging up to 35 residues (Q35) are present in healthy individuals, whereas Q40 or longer are associated with HD pathogenesis (1, 7). Larger polyQ expansions are correlated with earlier onset and more severe symptoms. The polyglutamine length-dependent aggregation of huntingtin has been reported to involve interaction with other proteins as well as selfassociation. Intracellular protein aggregates are found in human HD brains at autopsy and in tissues of mice carrying transgenes with expanded-repeat huntingtin (8-11). Here we report on the use of intracellular antibodies (intrabodies) as a potential therapeutic strategy on the basis of their ability to inhibit aberrant protein aggregate formation in a cellular model for HD. This approach may be applicable to other diseases as well, because HD is a paradigm for several adult-onset neurodegenerative diseases tha...

Hypoxanthine-guanine phosphoribosyltransferase (HPRT) deficiency results in Lesch-Nyhan disease (LND), where affected individuals exhibit a characteristic neurobehavioral disorder that has been linked with dysfunction of dopaminergic pathways of the basal ganglia. Since the functions of HPRT, a housekeeping enzyme responsible for recycling purines, have no direct relationships with the dopaminergic pathways, the mechanisms whereby HPRT deficiency affect them remain unknown. The current studies demonstrate that HPRT deficiency influences early developmental processes controlling the dopaminergic phenotype, using several different cell models for HPRT deficiency. Microarray methods and quantitative PCR were applied to 10 different HPRT-deficient (HPRT(-)) sublines derived from the MN9D cell line. Despite the variation inherent in such mutant sublines, several consistent abnormalities were evident. Most notable were increases in the mRNAs for engrailed 1 and 2, transcription factors known to play a key role in the specification and survival of dopamine neurons. The increases in mRNAs were accompanied by increases in engrailed proteins, and restoration of HPRT reverted engrailed expression towards normal levels, demonstrating a functional relationship between HPRT and engrailed. The functional relevance of the abnormal developmental molecular signature of the HPRT(-) MN9D cells was evident in impoverished neurite outgrowth when the cells were forced to differentiate chemically. To verify that these abnormalities were not idiosyncratic to the MN9D line, HPRT(-) sublines from the SK-N-BE(2) M17 human neuroblastoma line were evaluated and an increased expression of engrailed mRNAs was also seen. Over-expression of engrailed occurred even in primary fibroblasts from patients with LND in a manner that suggested a correlation with disease severity. These results provide novel evidence that HPRT deficiency may affect dopaminergic neurons by influencing early developmental mechanisms.

Mutations in the gene encoding the purine salvage enzyme, hypoxanthine-guanine phosphoribosyltransferase (HPRT) cause Lesch-Nyhan disease, a neurodevelopmental disorder characterized by cognitive, neurological, and behavioral abnormalities. Despite detailed knowledge of the enzyme's function, the key pathophysiological changes that accompany loss of purine recycling are unclear. To facilitate delineating the consequences of HPRT deficiency, four independent HPRT-deficient sublines of the human dopaminergic neuroblastoma, SK-N-BE(2) M17, were isolated by targeted mutagenesis with triple helix-forming oligonucleotides. As a group, these HPRT-deficient cells showed several significant abnormalities: (i) impaired purine recycling with accumulation of hypoxanthine, guanine, and xanthine, (ii) reduced guanylate energy charge and GTP : GDP ratio, but normal adenylate energy charge and no changes in any adenine nucleotide ratios, (iii) increased levels of UTP and NADP + , (iv) reduced DOPA decarboxylase, but normal monoamines, and (v) reduction in cell soma size. These cells combine the analytical power of multiple lines and a human, neuronal origin to provide an important tool to investigate the pathophysiology of HPRT deficiency.

A unique sensitivity to specific biochemical processes is responsible for selective vulnerability of midbrain dopamine neurons in several diseases. Prior studies have shown these neurons are susceptible to energy failure and mitochondrial dysfunction, oxidative stress, and impaired disposal of misfolded proteins. These neurons also are especially vulnerable to the loss of purine recycling. In the brains of humans or mice with inherited defects of the purine recycling enzyme hypoxanthine-guanine phosphoribosyltransferase (HPRT), the most prominent defect is loss of basal ganglia dopamine. To investigate the nature of the relationship between HPRT deficiency and dopamine, the mouse MN9D dopaminergic neuronal cell line was used to prepare 10 sublines lacking HPRT. The mutant sublines grew more slowly than the parent line, but without morphological signs of impaired viability. As a group, the mutant sublines had significantly lower dopamine than the parent line. The loss of dopamine in the mutants did not reflect impaired energy status, as judged by ATP levels or vulnerability to inhibitors of energy production. Indeed, the mutant lines as a group appeared energetically more robust than the parent line. The loss of dopamine also was not accompanied by enhanced susceptibility to oxidative stress or proteasome inhibitors. Instead, the loss of dopamine reflected only one aspect of a broad change in the molecular phenotype of the cells affecting mRNAs encoding tyrosine hydroxylase, the dopamine transporter, the vesicular monoamine transporter, monoamine oxidase B, catechol-O-methyltransferase, and GTP-cyclohydrolase. These changes were selective for the dopamine phenotype, since multiple control mRNAs were normal. These studies suggest purine recycling is an intrinsic metabolic process of particular importance to the molecular phenotype of dopaminergic neurons independent of previously established mechanisms involving energy failure, oxidative stress, or proteasome dysfunction.

Animal models of human disease are important tools for revealing the underlying mechanisms of pathophysiology and developing therapeutic strategies. Several unique mouse calcium channel mutants have been identified with nonepileptic, episodic dyskinetic movements that are phenotypically similar to human paroxysmal dyskinesias. In this report, video demonstrations of these motor attacks are provided for two previously described mouse mutants, tottering and lethargic, as well as a new one, rocker. Semiquantitative comparisons using two different rating scales reveal differences in attack morphology, severity, and duration among the strains. These mice provide three independent models of paroxysmal dyskinesia and support for prior proposals that channelopathies may underlie the human disorders. Keywordstottering; lethargic; rocker; paroxysmal dyskinesia; dystonia; channelopathyThe paroxysmal dyskinesias are a group of rare disorders characterized by intermittent attacks of involuntary abnormal movements.1 -3 These movements are often dystonic or choreiform, but may include a variety of other abnormalities. Unlike epilepsy, they are not associated with abnormal activity on electroencephalograms (EEGs).The paroxysmal dyskinesias are divided currently into three groups, which are based on the triggers of attacks, average attack duration, predominant morphology, and other features. [1][2][3] Paroxysmal nonkinesigenic dyskinesia is precipitated by stress, caffeine, or alcohol. The frequency of attacks ranges from a few per day to a few per year, with a duration of a few hours to several days. In contrast, attacks of paroxysmal kinesigenic dyskinesia are brought on by sudden movement, startle, and even passive manipulation of muscles. They are more frequent but shorter, occur many times per day, and last less than a few minutes each. In paroxysmal exertional dyskinesia, attacks are caused by prolonged exercise. Attacks are of intermediate duration with a frequency tied to the level of physical exertion.Recent studies also have revealed paroxysmal dyskinesias in certain strains of mice. The tottering mouse carries a mutation in the Cacna1a gene, which encodes a calcium channel subunit. 4,5 These mutants exhibit mild ataxia at baseline with intermittent attacks of severely disabling dyskinetic movements. 6,7 The lethargic mouse carries a mutation in the Cacnb4 gene, which encodes a different calcium channel subunit. 8 These mutants exhibit mild ataxia and a hypokinetic syndrome at baseline with intermittent attacks of dyskinetic movements. 9 The purpose of this report is to provide a video demonstration of the motor phenotypes of tottering and lethargic mice. In addition, we describe paroxysmal dyskinesia in a third mutant mouse, rocker, which also carries a mutation in the Cacna1a gene, but different from that in tottering

Immunization against extracellular neurotoxic proteins has shown promise in the treatment of several neurodegenerative disorders. We sought to determine whether immunization against mutant huntingtin, the intracellular protein that causes Huntington's disease (HD), could slow disease progression in the HD mouse model HDR6/2. DNA vaccination was used to present the mutant intracellular antigen to the immune system in a physiological context. Assay of a peripheral biomarker, pancreatic insufficiency, was used as an initial test of efficacy. DNA vaccination with a 5' fragment of the HD cDNA prevented development of the HDR6/2 diabetic phenotype. Insulin staining demonstrated that HDR6/2 diabetes may be caused by a severe pancreatic insulin deficiency. Immunoresponsive HDR6/2 mice showed increased insulin staining more closely resembling wild-type levels. These observations suggest that DNA vaccination against toxic intracellular proteins may be therapeutic.

Lesch-Nyhan disease (LND) is a rare disorder caused by a defect of an enzyme in the purine salvage pathway, hypoxanthine phosphoribosyl transferase (HPRT). It is still unknown how the metabolic defect translates into the complex neuropsychiatric phenotype characterized by self-injurious behavior, dystonia and mental retardation. There are abnormalities in purine and pyrimidine nucleotide content in HPRTdeficient cells. We hypothesized that altered nucleotide concentrations in HPRT deficiency change G-protein-mediated signal transduction. Therefore, our original study aim was to examine the high-affinity GTPase activity of G-proteins in membranes from primary human skin and immortalized mouse skin fibroblasts, rat B103 neuroblastoma cells and mouse Neuro-2a neuroblastoma cells. Unexpectedly, in membranes from human fibroblasts, B103-and Neuro-2a cells, V max of low-affinity nucleoside 5¢-triphosphatase (NTPase) activities was decreased up to 7-fold in HPRT deficiency. In contrast, in membranes from mouse fibroblasts, HPRT deficiency increased NTPase activity up to 4-fold. The various systems analyzed differed from each other in terms of K m values for NTPs, absolute V max values and K i values for nucleoside 5¢- [b,c-imido]triphosphates. Our data show that altered membrane NTPase activity is a biochemical hallmark of HPRT deficiency, but species and cell-type differences have to be considered. Thus, future studies on biochemical changes in LND should be conducted in parallel in several HPRT-deficient systems.