Nonallele-specific Silencing of Mutant and Wild-type Huntingtin Demonstrates Therapeutic Efficacy in Huntington’s Disease Mice

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Human Genetics and Genomics- Nonallele-specific Silencing of Mutant and Wild-type Huntingtin Demonstrates Therapeutic Efficacy in Huntington’s Disease Mice

Huntington`s Disease (HD) is an autosomal dominant neurodegenerative disease affecting 4-to-7 per 100,000 individuals. It is classed as a trinucleotide repeat disorder (Marcy et al, 1993) due to the fact that it results from an expanded CAG repeat which leads to a polyglutamine strand of variable length at the N-terminus (Walker, 2007). The normal number of CAG repeats is 10-35 times, however, in HD this repeat will occur 36 times or more and as CAG codes for glutamine, HD is classified as a polyglutamine disease. The huntingtin gene which regulates brain-derived neurotrophic factor (BDNF) that promotes the survival of neurons in the brain is affected (Zuccato et al, 2010). In Huntington`s Disease, mutated protein aggregates within the neuronal cells of the caudate and putamen of the basal ganglia causing neuronal cell death. The pathology of HD will show severe atrophy of the caudate nucleus and putamen. Cell death might be related to excitotoxicity which is the excessive signaling of these neurons, leading to high intracellular calcium (Raymond, 2003). The symptoms involve the central nervous system which will have an effect on movement (chorea and athetosis), cognitive disturbance, and mood. The loss of small neurons will also cause the expansion of the lateral ventricles and this can lead to psychological problems such as dementia, personality changes and depression.

Brain regions affected by HD have decreased GABA and acetylcholine but increased dopamine levels which helps explain why neuroleptics, which are dopamine receptor antagonists and tetrabenazine which depletes dopamine are used to treat chorea in people with HD but have no effect on overall survival. The average age of onset is around 40 years old (Vonsattel et al, 1985) but could be earlier depending on the number of repeats. Death happens within 10-20 years of diagnosis often by aspirational pneumonia on account of dis-coordinated swallowing or suicide.

The idea of using mice to silence mutant htt and wild type htt through RNA interference has shown therapeutic benefit in HD mice, mainly in silencing the wild type htt. Mouse HD genes are 81% in similarity to that of the human HD gene when expressed at DNA level (Liou, 2010). Knock in mice models have more precise portrayal of the disease`s genotype as scientists are able to replace specific gene regions of the HD gene with human copies. In 1996 the first transgenic mouse models (R6/1 and R6/2) were developed (Mangiarini et al., 1996) by inserting a part of the htt gene from a juvenile HD patient into the mouse genome. The purpose of these mice was to be able to study and understand repeat expansion which leads to Huntington`s Disease. However these models lacked the full length of mutant htt (mainly the R6/2) and the rapid progression of disease was extreme. They also had excessive weight loss and early death at usually 12-14 weeks (Crook and Housman, 2015). Due to this, other mouse models were developed such as the N171-82Q-17. These mice express the human htt cDNA which encodes glutamine and the first 171 amino acids bearing 82 CAG repeats (Schilling et al., 1999).

The aim of this study was to investigate whether therapeutic benefit is attainable via nonallelle-specific silencing of mutant and wild type htt in HD-N171-82Q mice (Boudreau et al., 2009). Previous studies of utilising RNAi induced by short hairpin RNAs (shRNAs) to reduce expression of mutant htt have shown that there is a possibility of improving abnormalities relating to HD disease in a mouse model (S.Q Harper et al, 2005). Pfister et al (2009) also suggested that targeting SNPs to degrade mutant htt while presenting the wild type htt is a promising approach. However, the number of SNP candidates is very limited in comparison to that of nonallele-specific strategy as there are countless sequences that could be targeted (Valerie et al., 2014). Although this study aims to find therapeutic benefit through nonallele-specific silencing of both the mutant and wild type huntingtin gene which codes for the huntingtin protein, it has been noted that the effects of nonallelle-specific silencing of the mutant htt still remains unknown. Allele specific targeting of mutant htt may be ideal considering that the wild type htt plays an important role in the development of neuronal nerve cells and other cellular processes (Zuccato et al., 2003).

During the study the mutant mice were injected with formulation buffer (FB), AAV1-hrGFP or AAV1-mi2.4 and the wild type mice were injected with only the formulation buffer into the striatum. Adeno-associated 2/1 viral vectors were used (AAV1-hrGFP, AAV1-mi2.4, AAV1-sh2.4 and AAV1-sh2.8). Using AAV is beneficial because they integrate into the active gene in the mice. Also, there is a lack of pathogenicity. ShRNA targets exons in HD mRNA and shRNA 2.4 and shRNA 2.8 have been known to reduce the expression of endogenous htt protein in mice C2C12 cells (McBride et al., 2008). Although this study aims to find the benefit of nonallele-specific silencing of the mutant and wild type htt, the use of AAV1 delivered shRNA in order to decrease striatal expression of the mutant htt allele has shown to improve motor and neuropathological defects in HD mouse models (Harper et al., 2005), for that reason, this suggests that it might be more beneficial for the suppression to be allele specific because reduction of wild type htt might not be well tolerated in the brain.

Immunohistochemical analysis (IHC) was used to analyse striatal neurons after they had been stained using goat anti-rabbit IgG secondary antibody. IHC is a procedure that is used for protein expression analysis. During this study, IHC was used to assess neurotoxicity by looking at DARPP-32 and Iba1. The researchers found that the mice that had been treated with AAV1-sh2.4 showed a loss of DARPP-32 which plays an important role in the regulation of dopamine-ion channels. There was also an increase in the activation of microglia which responds to neuronal damage. Mice that were injected with AAV1-mi2.4 and the buffer solution had an opposite reaction. In Huntington`s Disease the striatum is usually the most severely affected region, therefore the analysis of the striatal neurons helped to conclude that neuronal loss contributes to motor impairment but not chorea (Guo et al., 2012) as the mutant mice injected with the AAV1-mi2.4 managed to perform better in the rotarod test. The benefits of using immunohistochemical analysis are that it is relatively inexpensive, the results can be generated quite quickly as it is a rapid automated system and there is good cell morphology preservation as the aim of this technique is to use the least amount of antibody in order to cause the least damage to cells. The limitations are that it is hard to quantitate, success depends on the antibody that is used and the interpretation of the results could be subjective.

Another method that was used to analyse microglial activation is real time PCR (QPCR). This was used for analysis of CD11b mRNA and the results showed that there was a fivefold increase in sh2.4 HD-N171-82Q mice. An increased expression of CD11b represents neurodegenerative inflammation (Roy et al., 2006). These results show that mi2.4 has an improved safety in comparison to sh2.4 in HD-N171-82Q mice. Although this can be used in order to develop a much safer vector, the use of artificial miRNAs in mouse models with neurodegenerative disorders for therapeutic efficacy has not yet been published. QPCR is highly sensitive so it allows the detection of low copy targets and it has a wide dynamic range as 1-1011 of gene copies can be detected within a single run. It is also highly quantitative and accurate which means that the absolute copy number and gene expression can be measured accurately in comparison to traditional PCR. Moreover, the closed tube format it uses eliminates risk of cross contamination which is why results are measured accurately. It is also fast as no agarose gel is needed and safe because ethidium bromide is not used to stain DNA gels. The disadvantages of QPCR are that it is quite costly and might be complex so setting up requires high technical skill.

Northern blot analysis was also used to assess htt-specific antisense RNA levels in AAV1-RNAi treated striata after the striata was separated from tissue punches and then stained with ethidium bromide. Antisense RNA binds to mRNA to prevent its translation and if there is any neuronal damage, there will be an accumulation of it. Benefits of using northern blot analysis are that it has a high specificity so false positive results are reduced, it is less time consuming and requires no protection against radiation and the membranes can be stored and re-probed for years later after blotting. However, it has low sensitivity in comparison to other methods such as real time PCR.

The capacity of mi2.4 to effectively silence both mutant and wild-type mouse htt alleles was assessed by using western blot analysis. This method detects specific proteins in a sample and during this study it was used to assess the human protein, myc, and mouse htt (mHtt) proteins. In order to be able to assess this, myc-tagged human HD-N171-82Q htt fragment cells were overexpressed in C2C12, also, RNAi (mi2.4) expression plasmids were co-transfected and a reduction in the human mutant and endogenous wild-type htt produced by C2C12 cells was recorded. Previous studies have shown that in separate experimental systems, mi2.4 is able to silence both the mutant and wild type htt, the results from this western blot have highlighted that it is possible to simultaneously silence both the alleles in HD-N171-82Q mice models. Advantages of using western blot analysis are that it has a high sensitivity and specificity as it can detect as little as ng of protein in a sample and will only detect the protein of interest. However, it is time consuming and has a high demand in terms of experience.

Another method used in this study was microarray analyses to look for transcriptional changes between AAV1-hrGFP treated group and AAV1-RNAi groups. They therefore evaluated the transcriptional profile changes resulting from RNAi-mediated silencing of striatal htt, with the purposes of wanting to gain additional insight into the functions of wild-type htt and to further evaluate the safety of suppressing wild-type htt in adult mouse striatum. This procedure revealed htt as the most downregulated gene meaning that htt-related cellular pathways are disrupted after specific RNAi treatment. The benefits of using microarray is that it makes it possible to examine the expression of thousands of genes simultaneously, however, hybridisation is potentially non-specific. The method relies on existing knowledge about the genome sequence and may be expensive as it has a need for highly trained personnel. In order to be able to perform the microarray, a Tukey-Kramer test was completed first in order to cut down the number of genes that were expressed from 761 to 473. This test is used to compare the mean values of the experimental group (RNAi) against the control group (hrGFP). A p value of 0.05 was considered to be significant and a low p value means a higher chance of the hypothesis being true. The hypothesis of the study being that nonallelle-specific silencing of mutant and wild-type htt provides therapeutic benefit in HD mice. This test reduces the chances of type 1 errors being made, however, it is not designed to text complex comparisons.

The HD-N171-82Q mice were also subjected to a rotarod performance test which evaluates motor coordination and balance. Mice were tested at 10, 14 and 18 weeks and killed at 20 weeks for histological analyses. Mutant mice injected with FB, AAV1-hrGFP or AAV1-mi2.4 performed better than the wild type injected with FB. However, this test has no negative impact on motor behaviour and animals that have poor coordination will fall off at the start making it partially subjective.

Summary of results

When the mice were killed at 20 weeks of age in order to perform histological analyses and QPCR analyses in order to evaluate long-term suppression of mutant human and wild-type mouse htt transcripts, the researchers found that HD-N171-82Q mice treated with AAV1-mi2.4 showed significant reductions (~75%) in human and mouse htt mRNA levels, relative to control-treated mice (P < 0.01). These results, together with data from the rotarod analyses demonstrate that nonallele-specific silencing of mutant and wild-type htt provides therapeutic benefit in HD-N171-82Q mice.

The methods employed in this study were appropriate and helped to answer the research question, however some of them, such as the use of vectors would not have been strong enough to answer the question without relating to findings of previous studies as the results from that showed that it might not be safe to use shRNA compared to mi2.4.

Impact of findings

There are no current treatments for neurodegeneration but only medication to manage the symptoms. The findings of this study have an impact as they show that it is achievable to be able to reduce mutant genes in genetic disorders and hopefully lead to the treatment of them. The researchers found that the mice were able to tolerate a reduction in both the mutant and wild type htt. The mammalian brain is able to tolerate an up to 75% reduction in wild type htt mRNA in the striatum for several months. This research can be useful towards the development of treatment for other trinucleotide disorders such as myotonic dystrophy and spinocerebellar ataxia type 7. Recently, there have been studies into the nonallele-specific silencing of ataxin 7. There is evidence that nonallele specific silencing of ataxin-7 by RNAi is well tolerated in a SCA7 mouse model (Ramachandran et al., 2014).

Some stem cell research on HD mouse models has also shown that it might be possible to take stem cells from an individual with Huntington’s disease, correct the HD mutation and implant it back into the person`s brain. An alternative to this would be to mobilise the neural stem cells that are already in the brain and revive with a specific growth factor delivered to the brain.

References

Becanovic, K., Pouladi, M., Lim, R., Kuhn, A., Pavlidis, P., Luthi-Carter, R., Hayden, M. and Leavitt, B. (2010). Transcriptional changes in Huntington disease identified using genome-wide expression profiling and cross-platform analysis. Human Molecular Genetics, 19(8), pp.1438-1452.
Bilsen, P., Jaspers, L., Lombardi, M., Odekerken, J., Burright, E. and Kaemmerer, W. (2008). Identification and Allele-Specific Silencing of the Mutant Huntingtin Allele in Huntington’s Disease Patient-Derived Fibroblasts. Human Gene Therapy, 19(7), pp.710-718.
Boudreau, R., McBride, J., Martins, I., Shen, S., Xing, Y., Carter, B. and Davidson, B. (2009). Nonallele-specific Silencing of Mutant and Wild-type Huntingtin Demonstrates Therapeutic Efficacy in Huntington’s Disease Mice. Molecular Therapy, 17(6), pp.1053-1063.
Cattaneo, E. (2003). Dysfunction of Wild-Type Huntingtin in Huntington disease. Physiology, 18(1), pp.34-37.
Crook, Z. and Housman, D. (2011). Huntington’s Disease: Can Mice Lead the Way to Treatment?. Neuron, 69(5), p.1038.
Drouet, V., Ruiz, M., Zala, D., Feyeux, M., Auregan, G., Cambon, K., Troquier, L., Carpentier, J., Aubert, S., Merienne, N., Bourgois-Rocha, F., Hassig, R., Rey, M., Dufour, N., Saudou, F., Perrier, A., Hantraye, P. and Déglon, N. (2014). Allele-Specific Silencing of Mutant Huntingtin in Rodent Brain and Human Stem Cells. PLoS ONE, 9(6), p.e99341.
Guo, Z., Rudow, G., Pletnikova, O., Codispoti, K., Orr, B., Crain, B., Duan, W., Margolis, R., Rosenblatt, A., Ross, C. and Troncoso, J. (2012). Striatal neuronal loss correlates with clinical motor impairment in Huntington’s disease. Movement Disorders, 27(11), pp.1379-1386.
Harper, S., Staber, P., He, X., Eliason, S., Martins, I., Mao, Q., Yang, L., Kotin, R., Paulson, H. and Davidson, B. (2005). RNA interference improves motor and neuropathological abnormalities in a Huntington’s disease mouse model. Proceedings of the National Academy of Sciences, 102(16), pp.5820-5825.
Li, X., Chang, R., Liu, X. and Li, S. (2015). Transgenic animal models for study of the pathogenesis of Huntington’s disease and therapy. Drug Design, Development and Therapy, p.2179.
Mangiarini, L., Sathasivam, K., Seller, M., Cozens, B., Harper, A., Hetherington, C., Lawton, M., Trottier, Y., Lehrach, H., Davies, S. and Bates, G. (1996). Exon 1 of the HD Gene with an Expanded CAG Repeat Is Sufficient to Cause a Progressive Neurological Phenotype in Transgenic Mice. Cell, 87(3), pp.493-506.
McBride, J., Boudreau, R., Harper, S., Staber, P., Monteys, A., Martins, I., Gilmore, B., Burstein, H., Peluso, R., Polisky, B., Carter, B. and Davidson, B. (2008). Artificial miRNAs mitigate shRNA-mediated toxicity in the brain: Implications for the therapeutic development of RNAi. Proceedings of the National Academy of Sciences, 105(15), pp.5868-5873.
Ramachandran, P., Boudreau, R., Schaefer, K., La Spada, A. and Davidson, B. (2014). Nonallele Specific Silencing of Ataxin-7 Improves Disease Phenotypes in a Mouse Model of SCA7. Molecular Therapy, 22(9), pp.1635-1642.
Raymond, L. (2003). Excitotoxicity in Huntington disease. Clinical Neuroscience Research, 3(3), pp.121-128.
Roy, A., Fung, Y., Liu, X. and Pahan, K. (2006). Up-regulation of Microglial CD11b Expression by Nitric Oxide. Journal of Biological Chemistry, 281(21), pp.14971-14980.
Swanson, P. (2015). Immunohistochemistry as a Surrogate for Molecular Testing. Applied Immunohistochemistry & Molecular Morphology, 23(2), pp.81-96.
onsatell, J., Myers, H., Stevens, T., Ferrante, R., Bird, E. and Richardson, E. (1985). Neuropathological classification of Huntington’s disease. pp.559-77.
Wallace, L., Moreo, A., Clark, K. and Harper, S. (2013). Dose-dependent Toxicity of Humanized Renilla reniformis GFP (hrGFP) Limits Its Utility as a Reporter Gene in Mouse Muscle. Molecular Therapy – Nucleic Acids, 2, p.e86.

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Nonallele-specific Silencing of Mutant and Wild-type Huntingtin Demonstrates Therapeutic Efficacy in Huntington’s Disease Mice

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