Huntington’s Disease (HD) is a fatal autosomal dominant neurodegenerative disease that is caused by a mutation in the Huntington gene, or HTT gene.  The HTT gene encodes for the protein huntingtin. Wild type HTT gene contains a CAG trinucleotide of 10-35 repeats on exon 1 of chromosome 4. In mutated HTT, the CAG segment can be repeated from 36 to 120 times. The increased size of the CAG repeat encodes for an elongated form of the HTT protein. Once the elongated protein is produced, it is then cut into smaller toxic fragments. These fragments will bind to one another, build in neurons and form aggregates. This inhibits neurons from fully functioning and affects neurons specifically located in the basal ganglia, the area of the brain responsible for motor coordination. 

One vital aspect of Huntington’s Disease research is the use of transgenic mouse models. The R6/2 and YAC128 strain are excellent murine models for HD because it displays a neurological phenotype that mimics many symptoms of HD in addition to intranuclear inclusions. However, an immune deficient mouse model to study human based cell therapies is needed. Previous studies have shown that MSC’s (mesenchymal stem cells) could only be tolerated in vivo for up to 7 days without an immunosuppression regime. Here we generated and characterized an immune deficient and xeno-tolerant mouse model of HD by cross-breeding the NSG mice, a well-known immune deficient mouse strain, with R6/2 mice via blastocyst injection. Likewise, we assessed the function of the human immune system in the pathogenesis of HD via humanizing the novel R6/2-NSG strain. It is hypothesized that the increased levels of activated microglia, that responds to the mutated HTT protein aggregates, can contribute to the progression of HD.  The generation of this novel strain of mice will help progress the field towards eliminating the need for immunosuppression in cellular therapy, and by elucidating the role of neuroinflammation in the pathogenesis of Huntington’s disease in a murine model. 

The experimental design of this project consists of isolation of Hematopoietic stem cell from cord blood, humanization of R6/2/NSG mice, and conducting behavior assays and testing their and motor function skills via rotarod performance test, clasping, open field for a year. After behavioral data is collected, mice will be euthanized by CO2 asphyxiation and perfused with saline followed by fixative. Brains will be collected after perfusion, processed for histology and immunohistochemistry. Mutant Htt inclusions, human microglia, activated microglia, striatal volume, in conjunction with total brain atrophy, lateral ventricle size, striosome size, cell loss, and cell types will be evaluated using staining and IHC.

The main goal of our experimentation was to determine what the role of the immune Huntington Disease in R6/2 NSG. In a previous study done by the Nolta lab, YAC128/NSGs were humanized and the behavioral phenotype was characterized along with striatal volume analysis.

In addition, immunohistochemistry was conducted on YAC128/NSG mice to assess human microglia. This protocol is currently being optimized to yield better results.  Moreover, results from a preliminary volumetric showed that when compared YAC128/NSG non-humanized mice and parental strain, humanized mice had higher brain atrophy. This demonstrates that the immune system is implicated in progression of Huntington’s Disease. This project was designed to be a blinded study, therefore most of the analysis would be done after all data have been collected.