Inclusion formation in Huntington's disease R6/2 mouse muscle cultures

Huntington's disease (HD) is an autosomal dominant disorder caused by an expansion in the number of glutamine repeats in the N-terminal region of the huntingtin protein. Nuclear and cytoplasmic aggregates of the N-terminal portion of huntingtin have been found in the brains of HD patients and the brains and non-neuronal tissues of the R6/2 HD transgenic mouse. We have cultured myoblasts and myotubes from transgenic R6/2 mice and littermate controls to investigate the formation of these inclusions in post mitotic cells. Huntingtin immunoreactivity was intense in differentiating, desmin positive myoblasts and myotubes from both control and R6/2 mice suggesting that it may play a role in myotube differentiation. Following differentiation huntingtin and ubiquitin positive aggregates were observed in R6/2 but not control cultures. After 3 weeks in differentiation medium cytoplasmic huntingtin and ubiquitin immunoreactive aggregates were observed in non-myotube cells, while nuclear huntingtin aggregates were seen in a proportion of myotubes after 6 weeks. Growth in the absence of serum resulted in a marked increase in the number of R6/2 myotubes containing nuclear inclusions after 6 weeks demonstrating that environmental factors influenced huntingtin aggregate formation in these cells. Consequently, cultured myotubes from R6/2 mice may be a useful post mitotic cell culture model to study both the biochemical consequences of huntingtin aggregates and the factors that may influence aggregate formation.     

L-Glutamate toxicity in Huntington's disease fibroblasts

Brain degeneration in Huntington's Disease is thought to occur primarily in the regions of high L-glutamate concentrations. Huntington's Disease fibroblast cultures have a sensitivity to these high concentrations of L-glutamate. These cells show degeneration and loss of viability, within 12 hrs, following treatment with 30 mM L-glutamate. This effect appears to be specific for L-glutamate, can be prevented by glutamine, and is not observed in matched control cultures. The observed glutamate sensitivity may serve as a biochemical genetic marker and the excessive effect of glutamate on neuronal membranes could initiate the characteristic neuronal degeneration observed in Huntington's Disease brains.     

CGS21680 attenuates symptoms of Huntington's disease in a transgenic mouse model

Huntington's disease (HD) is an autosomal dominant neurodegenerative disease caused by a CAG trinucleotide expansion in exon 1 of the Huntingtin (Htt) gene. We show herein that in an HD transgenic mouse model (R6/2), daily administration of CGS21680 (CGS), an A(2A) adenosine receptor (A(2A)-R)-selective agonist, delayed the progressive deterioration of motor performance and prevented a reduction in brain weight. 3D-microMRI analysis revealed that CGS reversed the enlarged ventricle-to-brain ratio of R6/2 mice, with particular improvements in the left and right ventricles. (1)H-MRS showed that CGS significantly reduced the increased choline levels in the striatum. Immunohistochemical analyses further demonstrated that CGS reduced the size of ubiquitin-positive neuronal intranuclear inclusions (NIIs) in the striatum of R6/2 mice and ameliorated mutant Htt aggregation in a striatal progenitor cell line overexpressing mutant Htt with expanded polyQ. Moreover, chronic CGS treatment normalized the elevated blood glucose levels and reduced the overactivation of a major metabolic sensor [5'AMP-activated protein kinase (AMPK)] in the striatum of R6/2 mice. Since AMPK is a master switch for energy metabolism, modulation of energy dysfunction caused by the mutant Htt might contribute to the beneficial effects of CGS. Collectively, CGS is a potential drug candidate for the treatment of HD.     

Galectin-1 expression in innervated and denervated skeletal muscle

Galectin-1 is a soluble carbohydrate-binding protein with a particularly high expression in skeletal muscle. Galectin-1 has been implicated in skeletal muscle development and in adult muscle regeneration, but also in the degeneration of neuronal processes and/or in peripheral nerve regeneration. Exogenously supplied oxidized galectin-1, which lacks carbohydrate-binding properties, has been shown to promote neurite outgrowth after sciatic nerve sectioning. In this study, we compared the expression of galectin-1 mRNA and immunoreactivity in innervated and denervated mouse and rat hind-limb and hemidiaphragm muscles. The results show that galectin-1 mRNA expression and immunoreactivity are up-regulated following denervation. The galectin-1 mRNA is expressed in the extrasynaptic and perisynaptic regions of the muscle, and its immunoreactivity can be detected in both regions by Western blot analysis. The results are compatible with a role for galectin-1 in facilitating reinnervation of denervated skeletal muscle.     

Semaphorin 6C expression in innervated and denervated skeletal muscle

Semaphorins are secreted or transmembrane proteins important for axonal guidance and for the structuring of neuronal systems. Semaphorin 6C, a transmembrane Semaphorin, has growth cone collapsing activity and is expressed in adult skeletal muscle. In the present study the expression of Semaphorin 6C mRNA and immunoreactivity has been compared in innervated and denervated mouse hind-limb and hemidiaphragm muscles. Microscopic localization of immunoreactivity was studied in innervated and denervated rat skeletal muscle. The results show that Semaphorin 6C mRNA expression and immunoreactivity on Western blots are down-regulated following denervation. The mRNA of Semaphorin 6C as well as immunoreactivity determined by Western blots are expressed in extrasynaptic as well as perisynaptic regions of muscle. Immunohistochemical studies, however, show Semaphorin 6C-like immunoreactivity to be concentrated at neuromuscular junctions. The results suggest a role for Semaphorin 6C in neuromuscular communication.     

Neurodegenerative processes in Huntington's disease

Huntington''s disease (HD) is a complex and severe disorder characterized by the gradual and the progressive loss of neurons, predominantly in the striatum, which leads to the typical motor and cognitive impairments associated with this pathology. HD is caused by a highly polymorphic CAG trinucleotide repeat expansion in the exon-1 of the gene encoding for huntingtin protein. Since the first discovery of the huntingtin gene, investigations with a consistent number of in-vitro and in-vivo models have provided insights into the toxic events related to the expression of the mutant protein. In this review, we will summarize the progress made in characterizing the signaling pathways that contribute to neuronal degeneration in HD. We will highlight the age-dependent loss of proteostasis that is primarily responsible for the formation of aggregates observed in HD patients. The most promising molecular targets for the development of pharmacological interventions will also be discussed.     

BDNF prevents NMDA‐induced toxicity in models of Huntington's disease: the effects are genotype specific and adenosine A2A receptor is involved

NMDA receptor‐mediated excitotoxicity is thought to play a pivotal role in the pathogenesis of Huntington's disease (HD). The neurotrophin brain‐derived neurotrophic factor (BDNF), which is also highly involved in HD and whose effects are modulated by adenosine A_2ARs, influences the activity and expression of striatal NMDA receptors. In electrophysiology experiments, we investigated the role of BDNF toward NMDA‐induced effects in HD models, and the possible involvement of A_2ARs. In corticostriatal slices from wild‐type mice and age‐matched symptomatic R6/2 mice (a model of HD), NMDA application (75 μM) induced a transient or a permanent (i.e., toxic) reduction of field potential amplitude, respectively. BDNF (10 ng/mL) potentiated NMDA effects in wild‐type, while it protected from NMDA toxicity in R6/2 mice. Both effects of BDNF were prevented by A_2AR blockade. The protective effect of BDNF against NMDA‐induced toxicity was reproduced in a cellular model of HD. These findings may have very important implications for the neuroprotective potential of BDNF and A_2AR ligands in HD.     

Defining the role of the Bcl-2 family proteins in Huntington's disease

B-cell lymphoma 2 (Bcl-2) family proteins regulate survival, mitochondria morphology dynamics and metabolism in many cell types including neurons. Huntington''s disease (HD) is a neurodegenerative disorder caused by an expanded CAG repeat tract in the IT15 gene that encodes for the protein huntingtin (htt). In vitro and in vivo models of HD and HD patients'' tissues show abnormal mitochondrial function and increased cell death rates associated with alterations in Bcl-2 family protein expression and localization. This review aims to draw together the information related to Bcl-2 family protein alterations in HD to decipher their potential role in mutated htt-related cell death and mitochondrial dysfunction.     

Iron dysregulation in Huntington's disease

Huntington's disease (HD) is one of many neurodegenerative diseases with reported alterations in brain iron homeostasis that may contribute to neuropathogenesis. Iron accumulation in the specific brain areas of neurodegeneration in HD has been proposed based on observations in post‐mortem tissue and magnetic resonance imaging studies. Altered magnetic resonance imaging signal within specific brain regions undergoing neurodegeneration has been consistently reported and interpreted as altered levels of brain iron. Biochemical studies using various techniques to measure iron species in human samples, mouse tissue, or in vitro has generated equivocal data to support such an association. Whether elevated brain iron occurs in HD, plays a significant contributing role in HD pathogenesis, or is a secondary effect remains currently unclear.

     

Study on the variation in X-ray sensitivity of human diploid skin fibroblasts. Normal radiosensitivity of cells derived from patients with Huntington's disease

9 cell strains derived from patients with Huntington's disease and 9 from age- and sex-matched controls were investigated for X-ray sensitivity. No differences in radiosensitivity were observed for the two groups. The two groups taken together reveal a dependence of radiosensitivity on intrinsic cloning efficiency which in turn correlates with donor age. A difference in radiosensitivity between males and females is also indicated although at the borderline of significance. As a parameter for radiosensitivity the dose needed to obtain 0.1% survival appears superior to the Do.     

Creatine therapy provides neuroprotection after onset of clinical symptoms in Huntington's disease transgenic mice

While there have been enormous strides in the understanding of Huntington's disease (HD) pathogenesis, treatment to slow or prevent disease progression remains elusive. We previously reported that dietary creatine supplementation significantly improves the clinical and neuropathological phenotype in transgenic HD mice lines starting at weaning, before clinical symptoms appear. We now report that creatine administration started after onset of clinical symptoms significantly extends survival in the R6/2 transgenic mouse model of HD. Creatine treatment started at 6, 8, and 10 weeks of age, analogous to early, middle, and late stages of human HD, significantly extended survival at both the 6- and 8-week starting points. Significantly improved motor performance was present in both the 6- and 8-week treatment paradigms, while reduced body weight loss was only observed in creatine-supplemented R6/2 mice started at 6 weeks. Neuropathological sequelae of gross brain and neuronal atrophy and huntingtin aggregates were delayed in creatine-treated R6/2 mice started at 6 weeks. We show significantly reduced brain levels of both creatine and ATP in R6/2 mice, consistent with a bioenergetic defect. Oral creatine supplementation significantly increased brain concentrations of creatine and ATP to wild-type control levels, exerting a neuroprotective effect. These findings have important therapeutic implications, suggesting that creatine therapy initiated after diagnosis may provide significant clinical benefits to HD patients.     

Responses of Huntington's disease and ataxia telangiectasia lymphoblastoid cells to bleomycin

Ionizing radiation sensitive, mutant human lymphoblastoid cell lines derived from patients with Huntington's disease (HD), or ataxia telangiectasia (AT) both showed cross sensitivity to bleomycin, as assayed by reduced cell viability and increased frequency of chromosome aberrations compared to normal controls. In contrast to AT cells which failed to show inhibition of DNA synthesis after exposure to ionizing radiation, or bleomycin treatment, the sensitive cells from HD patients had depressed rates of DNA synthesis after damage with these agents, similar to that seen in normal cells. In terms of progression through the cell cycle bleomycin damaged AT cells moved from G1 into S and from S to G2 + M at almost the same rate as untreated cells. Bleomycin treated HD cells showed a large proportion of cells blocked in G1, cells were slowed down in S, the rate of entry to G2 + M was reduced and only 5% of cycling cells reached G2. Progress through the cell cycle in normal cells exposed to bleomycin showed a partial block in G1 and the rate of entry to G2 + M was reduced. These differences in response of normal, AT and HD cells to ionizing radiation and bleomycin treatment indicates that the defect underlying the sensitivity is different in HD cells from that in AT cells.     

Alterations in striatal synaptic transmission are consistent across genetic mouse models of Huntington's disease

Since the identification of the gene responsible for HD (Huntington''s disease), many genetic mouse models have been generated. Each employs a unique approach for delivery of the mutated gene and has a different CAG repeat length and background strain. The resultant diversity in the genetic context and phenotypes of these models has led to extensive debate regarding the relevance of each model to the human disorder. Here, we compare and contrast the striatal synaptic phenotypes of two models of HD, namely the YAC128 mouse, which carries the full-length huntingtin gene on a yeast artificial chromosome, and the CAG140 KI (knock-in) mouse, which carries a human/mouse chimaeric gene that is expressed in the context of the mouse genome, with our previously published data obtained from the R6/2 mouse, which is transgenic for exon 1 mutant huntingtin. We show that striatal MSNs (medium-sized spiny neurons) in YAC128 and CAG140 KI mice have similar electrophysiological phenotypes to that of the R6/2 mouse. These include a progressive increase in membrane input resistance, a reduction in membrane capacitance, a lower frequency of spontaneous excitatory postsynaptic currents and a greater frequency of spontaneous inhibitory postsynaptic currents in a subpopulation of striatal neurons. Thus, despite differences in the context of the inserted gene between these three models of HD, the primary electrophysiological changes observed in striatal MSNs are consistent. The outcomes suggest that the changes are due to the expression of mutant huntingtin and such alterations can be extended to the human condition.     

Shaping the role of mitochondria in the pathogenesis of Huntington's disease

Intense research on the pathogenesis of Huntington's disease (HD), a genetic neurodegenerative disease caused by a polyglutamine expansion in the Huntingtin (Htt) protein, revealed multiple potential mechanisms, among which mitochondrial alterations had emerged as key determinants of the natural history of the disease. Pharmacological and genetic animal models of mitochondrial dysfunction in the striatum, which is mostly affected in HD corroborated a key role for these organelles in the pathogenesis of the disease. Here, we will give an account of the recent evidence indicating that the mitochondria-shaping machinery is altered in HD models and patients. Since its correction can counteract HD mitochondrial dysfunction and cellular damage, drugs impacting on mitochondrial shape are emerging as a new possibility of treatment for this devastating condition.     

Conditioned medium from amniotic cells protects striatal degeneration and ameliorates motor deficits in the R6/2 mouse model of Huntington's disease

Inflammation significantly impacts the progression of Huntington's disease (HD) and the mutant HTT protein determines a pro‐inflammatory activation of microglia. Mesenchymal stem/stromal cells (MSC) from the amniotic membrane (hAMSC), and their conditioned medium (CM‐hAMSC), have been shown to possess protective effects in vitro and in vivo in animal models of immune‐based disorders and of traumatic brain injury, which have been shown to be mediated by their immunomodulatory properties. In this study, in the R6/2 mouse model for HD we demonstrate that mice treated with CM‐hAMSC display less severe signs of neurological dysfunction than saline‐treated ones. CM‐hAMSC treatment significantly delayed the development of the hind paw clasping response during tail suspension, reduced deficits in rotarod performance, and decreased locomotor activity in an open field test. The effects of CM‐hAMSC on neurological function were reflected in a significant amelioration in brain pathology, including reduction in striatal atrophy and the formation of striatal neuronal intranuclear inclusions. In addition, while no significant increase was found in the expression of BDNF levels after CM‐hAMSC treatment, a significant decrease of microglia activation and inducible nitric oxide synthase levels were observed. These results support the concept that CM‐hAMSC could act by modulating inflammatory cells, and more specifically microglia.