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Classification of the trinucleotide repeat, and resulting disease status, depends on the number of CAG repeats Repeat count Classification Disease status <28 Normal Unaffected 28–35 Intermediate Unaffected 36–40 Reduced Penetrance +/- Affected >40 Full Penetrance
Affected Table 1. Trinucleotide repeats and classification of infection
This neuro-degenerative family includes eight other disorders: dentatorubralpallidoluysian atrophy (DRPLA), spinobulbar muscular atrophy (SBMA) and spinocerebellar ataxia (SCA) types 1–3, 6, 7 and 17, also Machado Joseph Disease. Although all of these disease proteins contain extended polyQ tracts (usually >36 glutamines) and are widely expressed in the body and brain, they cause distinct neuropathology in particular brain regions. Transgenic Mice Model
Human trials in HD are difficult, costly and time-consuming due to the slow disease course, dangerous onset and patient-to-patient variability. Identification of molecular biomarkers associated with disease progression will aid development of effective therapies by allowing further confirmation of animal models and by providing more sensitive measures to show progression of the disease.
In the 12 years since Huntington’s disease mutations were identified, considerable progress has been made with modeling pathogenesis in cell and animal models. Mutant huntingtin accumulates in intraneuronal inclusions. Huntingtin is cleaved to form N-terminal fragments consisting of the first 100–150 residues that contain the expanded polyQ tract and these are believed to be the toxic species found in the aggregates (Aggregation of huntingtin in neuronal intranuclear inclusions and dystrophic neurites in brain - DiFiglia et al., 1997)
The extent to which mouse models expressing N-terminal fragments of mutant huntingtin repeat the human disease phenotype is unclear, although studies on mice have shown pathological and behavioral similarities to HD.
Studies on transgenic mice reported in Neuron, Vol. 23, 181–192, May, 1999 have used mice that replicate the full-length HD gene expressed in the same developmental tissue and cell-specific manner as seen in patients with the disease and cloned using homologous recombination in yeast to introduce expanded CAG repeats (46 and 72) into YACs with the same CAG sizes similar to those seen in adult onset (YAC46 containing 46 CAG repeats) or in juvenile cases (YAC72 containing 72 CAG repeats) HD. The YAC46 and YAC72 mice developed progressive electrophysiological abnormalities that precede nuclear translocation and aggregation of htt. YAC72 mice have behavioural abnormalities, with onset influenced by the level of mutant protein.
A mouse expressing mutant htt with 72 glutamines at higher levels presented with an early onset behavioural phenotype and had intranuclear aggregates and neuro-degeneration specifically in the striatum.
Other YAC72 mice expressing lower levels of mutant protein had progressive electrophysiological abnormalities at 6 and 10 months, followed by selective degeneration of medium spiny neurons in the lateral striatum associated with translocation of N-terminal htt fragments to the nucleus. These mice represented the first animal model expressing full length mutant human htt under the control of its own promoter, providing insight into the sequential molecular and cellular events underlying HD.
Neuronal degeneration can be assessed by light microscopy in 1.5um thick toluidine blue-stained sections from the striatum, the cortex adjacent to the lateral striatum, the hippocampus, and the cerebellum. Degenerating neurones were present in the striatum of YAC 46 and YAC72 mouse at 12 month of age. They were hyperchromatic and shrunken. Unlike striatal neurones, which have normally round nuclei and round cellular outlines, degenerating neurons had oval fusiform perikarya and nuclei. The chromatin within degenerating neurones was also condensed and marginated.
Electron microscopy using the same tissues confirmed the degenerative and abnormal nature of these morphologic changes. The changes varied in severity and included reduced neuronal size, the presence of nuclear and plasma membrane irregularities, increased electron density of the cytoplasm and the nucleus, swelling of some mitochondria, nuclear shrinkage and margination of heterochromatin. These degenerative features were consistent with apoptosis.
Significant degeneration was present in the striatum of the YAC72 mouse at 12 months of age. No degeneration was found in the YAC46 or in the wild type controls used. In the striatum of mouse YAC72 there was extensive neuronal degeneration. Selective neurodegeneration affected specifically the medium spiny neurons of the striatum.
Neurodegeneration can occur in the absence of aggregates. The YAC transgenic mice expressing mutant htt with the 46 CAG repeats did not have any clinical phenotype by detailed behavioural analysis for up to 20months of age. However, the electrophysiological abnormalities were evident much earlier. The severity of abnormalities is more obvious in mice with 72 CAG compared with mice with 46 CAG repeats. Therapeutic silencing of mutant huntingtin with siRNA
Therapies aimed at delaying disease progression have been tested in these mouse models. Beneficial effects have been reported in animals treated with substances that increase transcription of neuroprotective genes (histone and deacetylase), prevent apoptosis (caspase inhibitors), enhance energy metabolism (co-enzyme Q_remacemide and creatine), and inhibit the formation of polyglutamine aggregates (trehalose, Congo red, and cystamine). These approaches target downstream and possibly indirect effects of HD gene expression.
When mutant htt is inducibly expressed, pathological and behavioral features of the disease develop, including a characteristic neuronal inclusions and abnormal motor behavior. Upon repression of transgene expression in affected mice, pathological and behavioral features resolved. Thus, reduction of htt expression by using RNA interference (RNAi) may allow protein clearance mechanisms within neurons to normalize mutant htt-induced changes. Direct inhibition of the mutant htt expression will slow or prevent HD associated symptom.
Small interfering RNA (siRNA), are a class of double-stranded RNA molecules, 20-25 nucleotides long, they play a variety of roles in biology. Most notably, siRNA are involved in the RNA interference (RNAi) pathway, where they interfere with the expression of a specific gene.
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Using gene therapy to switch off genes instead of adding new ones could slow down or prevent the fatal brain disorder. Improving motor and neuropathological abnormalities in Huntington’s disease.
This mechanism involves pieces of double-stranded RNA or siRNAs that trigger the degradation of any other RNA in the cell with a matching sequence. If a siRNA is chosen to match the RNA copied from a particular gene, it will stop production of the protein the gene codes for. In HD the mutations in the huntingtin gene result in defective large protein clumps that gradually kill off part of the brain. The above studies in transgenic mice showed that reducing production of the defective protein can slow down the disease. Discussion
Neurons in the brain of transgenic mice, expressing exon 1 of the human HD gene contained an expanded polyQ, it degenerated within those specific areas of the brain known to be affected in HD. Degenerating neurons of similar ultrastructural appearance can be found in postmortem brain from patients with HD, but have never been reported in other neurodegenerative diseases such as Alzheimer’s disease and Parkinson’s disease. Neuronal death found in the brain in HD is by a process that is morphologically and biochemically distinct from apoptosis, and is associated with the presence of intracellular aggregates of mutant protein. This defines a temporal progression of protein aggregation, inclusion formation, appearance of neurological symptoms, and finally neurodegeneration. Potential treatments for HD directed toward prevention of protein aggregation may thus prove more effective than antiapoptotic therapies. |