Using CRISPR/Cas9 to reduce neuronal damage in a Huntington's disease mouse model by Ellen Wu

Original Article

ellen_wu29 — 2018-11-26 21:07:10 UTC

Yang, S., Chang, R., Yang, H., Zhao, T., Hong, Y., Kong, H. E., . . . Li, X. (2017). CRISPR/Cas9-mediated gene editing ameliorates neurotoxicity in mouse model of Huntington’s disease. Journal of Clinical Investigation,127(7), 2719-2724. doi:10.1172/jci92087

ellen_wu29 — 2018-12-04 18:12:58 UTC

Construction of separate AAV vectors containing either HTT-gRNA or Cas9. Both driven by CMV promoter.
Stereotaxic injection of vectors into the striatum of 9-month-old homozygous HD140Q-KI mice. One side of the brain injected with Cas9 vector only, the contralateral side injected with both vectors.
Left Panels Immunostaining shows high mHTT expression (GFP) when only the Cas9 vector is delivered. Absence of HTT-gRNA (RFP) confirms the lack of gene editing.
Right Panels When both Cas9 and gRNA vectors are delivered, mHTT expression level is significantly reduced. This suggests that CRISPR/Cas9 gene editing effectively reduces mHTT level in homozygous HD140Q-KI mouse.

ellen_wu29 — 2018-12-04 18:53:00 UTC

Figure 4. Western Blot Analysis of WT and CRISPR/Cas9 edited homozygous HD mouse

ellen_wu29 — 2018-12-04 19:25:55 UTC

Figure 7. Grip strength test

ellen_wu29 — 2018-12-04 19:55:53 UTC

Figure 8 Body weight

Rotarod

ellen_wu29 — 2018-12-04 19:59:10 UTC

Balance beam

ellen_wu29 — 2018-12-04 20:01:48 UTC

See Beam-balance Testing section

ellen_wu29 — 2018-12-04 20:03:52 UTC

Figure 5. Rotarod Test. Red represents Treated HD, blue represents control HD, and black represents WT.

ellen_wu29 — 2018-12-04 20:10:15 UTC

Figure 6. Balance beam test.

ellen_wu29 — 2018-12-04 20:19:19 UTC

Figure 3. Immunofluorescence of CRISPR/Cas9 edited HD mouse striatum

ellen_wu29 — 2018-12-04 20:27:58 UTC

Injection of Cas9 vector and either HTT-gRNA or Control-gRNA into homozygous HD mouse brains.
Western blot analysis on the cortex, striatum, and hippocampus of WT, HTT-gRNA, and Control-gRNA
Within all three brain regions, mHTT and GFAP (indicate reactive astrocytes) protein levels are reduced in HTT-gRNA compared to Control-gRNA, suggesting that HTT-guided CRISPR/Cas9 editing ameliorates neurotoxicity.
This reduction is protein-specific, since the levels of NeuN (mature neuron), p62 (autophagy), Caspase3 (apoptosis), and Vinculin (loading control) are comparable across all three groups.

ellen_wu29 — 2018-12-04 20:46:56 UTC

Together, these behaviour tests demonstrate that CRISPR/Cas9 brings an improvement in motor functions to HD mouse, at a level that is comparable with WT.

ellen_wu29 — 2018-12-04 21:39:48 UTC

In summary, CRISPR/Cas9 ameliorates neurotoxicity associated with Huntington's disease by reducing mHTT aggregates and reactive astrocytes.

ellen_wu29 — 2018-12-04 21:42:18 UTC

ellen_wu29 — 2018-12-04 21:57:28 UTC

Figure 2. Research Design

CRISPR/Cas9 reduces mHTT expression in homozygous HD140Q-KI mouse

ellen_wu29 — 2018-12-04 22:06:14 UTC

CRISPR/Cas9 reduces mHTT and GFAP levels in homozygous HD mouse

ellen_wu29 — 2018-12-04 22:06:36 UTC

CRISPR/Cas9 improves Rotarod performance in heterzygous HD mouse

ellen_wu29 — 2018-12-04 22:09:22 UTC

ellen_wu29 — 2018-12-04 22:10:21 UTC

Heterozygous HD140Q-KI mouse used to access motor function
Treated HD Group AAV-U6-gRNA-CMV-RFP and AAV-pMECP2-SpCas9 vectors injected into both sides of the striatum of heterozygous HD mouse
Control HD Group Same criteria as treated HD, except that control gRNA is used instead
WT control
n=12 for each group.
2-way ANOVA with Bonferroni's test, comparing Treated HD and Control HD
*p<0.05, **p<0.012, ***p<0.001
WT and Treated HDs are able to stay on the Rotarod longer than Control HDs, suggesting that gene editing leads to better motor performance in heterozygous HDs.

CRISPR/Cas9 improves Balance beam performance in heterozygous HD mouse

ellen_wu29 — 2018-12-04 22:15:23 UTC

ellen_wu29 — 2018-12-04 22:17:30 UTC

Treated HDs and WTs take significantly less time to cross the balance beam compared to Control HDs, suggesting an improvement in motor function brought by CRISPR/Cas9 gene editing in heterozygous HD mouse.

CRISPR/Cas9 improves grip strength in heterozygous HD mouse

ellen_wu29 — 2018-12-04 22:19:32 UTC

ellen_wu29 — 2018-12-04 22:19:56 UTC

Treated HD and WT controls can have significantly higher grip strength, measured in grams of force, compared to Control HDs.

Body weight unaffected by HD phenotype or CRISPR/Cas9 treatment

ellen_wu29 — 2018-12-04 22:21:40 UTC

ellen_wu29 — 2018-12-04 22:21:48 UTC

No significant difference in body weight between Treated HD, Control HD, and WT.

Future Directions

2018-12-06 05:10:19 UTC

A study showed that cognitive performance declined in pre-motormanifest HD gene expansion carriers and became worse to late stage HD. (Baake, Reijintjes, Dumas& Tompson, 2017) Based on the critical analysis, an experiment testing the safety of depletion of HTT non-allele specifically should be designed. 50 mice should be injected with either AAV-HTT-gRNA/AAV-MECP2-Cas9 or AAV-control-gRNA/AAV-MECP2-Cas9, and their cognitive functions need to be monitored for 3 months. The two groups of treated knock in mice and WT controls should be tested for their ability to discriminate two complex visual stimuli.

If the treated KI mice did not show significant reduction in cognitive function compared to the wild type control, depletion of HTT in a non-allele specific way is really a safe therapeutic approach. It is not safe if otherwise and is this case, other mechanism like epigenetics might also contributed in the development of Huntington disease.(Lee J, Hwang YJ, Kim KY, Kowall NW& Ryu H, 2013)

Critical Analysis

2018-12-06 05:12:47 UTC

The hypothesis was depletion of HTT using CRISPR/Cas9 in a non–allele-specific way could efficiently and permanently eliminate neuronal toxicity in adult brain.

Overall, the observations were valid and agreed with hypothesis. However, there was data that did not fit with the argument being ignored in this study in Figure 3 (D). The decreased GFAP levels by HTT-gRNA compared with control-gRNA were not explained in the study. Other studies raised the possibility that the loss of uptake activity and the recovery of GLT1 protein could be different in the GFAP-negative astrocyte population of the striatum. (Vagner, Dvorzhak, Wójtowicz, Harms& Grantyn, 2016)

The control procedure was assessing the effects of CRISPR/Cas9-mediated neuronal mHTT depletion in both homozygous and heterozygous knock in mice. Two kinds of combination were injected in two sides of the striatum as control subjects. These control procedures were necessary for comparative purposes to eliminate the effect of confounding variables.

Critical analysis

2018-12-07 07:02:28 UTC

One of the finding was that CRISPR/Cas9-mediated gene inactivation could reverse the neuropathology and behavioural phenotypes. This finding of the study seemed over interpreted because the motor function improvement assessed by rotarod test did not seem significantly different from the control subject.

The statistical support and analysis were not used sufficiently in Figure 2(C) and the possible explanation was that the sample size was only 24 mice and not enough data was collected in this study.

The implication of this study was CRISPR/Cas9 could more efficiently deplete the expression of mHTT than previous therapeutic approaches. Other study showed results agreeing with the conclusion of this study. (Kolli, Lu, Rossignol& Dunbar, 2017)

In order to test whether depletion of HTT could be a safe therapy, the authors should include cognitive testing in addition to the motor function testing because there was a study saying that mice models exhibited cognitive inflexibility and psychomotor slowing, which were similar to cognitive symptoms described in HD patients. (Farrar et al., 2014)

Huntington's Disease

jeremyho98 — 2018-12-07 08:40:51 UTC

Huntington’s Disease (HD) is a progressive neurodegenerative disease, characterized by loss of striatal tissue (1). There is currently no cure for HD (1).
Significant physical, cognitive and psychiatric symptoms, such as: jerky involuntary movements, impaired walking and coordination, difficulty producing speech and swallowing; difficulty focusing and organizing thoughts; irritability and depression(1).
Huntingtin (HTT) gene is located on chromosome 4, and contains 67 exons. Exon 1 has a CAG repeat domain, which typically contains 1-35 repeats of CAG. CAG encodes glutamine, so it is referred to as the Polyglutamine or PolyQ chain.
HTT is necessary for neural development, and has over 200 identified binding partners(2). It is involved in many processes such as neurogenesis, signal transduction, and stress response (3).

Mutation

jeremyho98 — 2018-12-07 08:41:49 UTC

Mutations that cause increase in number of CAG repeats beyond 35 repeat threshold cause HD. The greater then number of repeats, the earlier the onset of HD (4).
Mutant HTT (mHTT) contains an elongated PolyQ chain. Elongated PolyQ chain is targeted by caspases for degradation, forming fragments that aggregate (5).

Mechanism

jeremyho98 — 2018-12-07 08:43:37 UTC

Nature of aggregates is controversial, thought to be origin of neurotoxicity.
Aggregates may be coping mechanism to sequester mutant fragments and protect the neuron (6). However, they also attract and sequester chaperone proteins, inhibiting them from preventing protein misfolding (7).
mHTT increases sensitivity and permeability of NMDA receptor subunits, leading to increased glutamate signalling and enhanced excitotoxic cell death in striatum (8).
mHTT is also associated with repression of dopamine (DA) transporters and receptors, decreasing dopaminergic inputs from substantia nigra to striatum (9), and loss of dopaminergic neurons in both structures (10).
Lower levels of DA thought to contribute to physical deficits in HD.
Decreased DA levels also activate pro-apoptotic c-Jun kinase pathway in the presence of mHTT (11)

Videos

ellen_wu29 — 2018-12-07 16:22:03 UTC

Nature Video (2015, April 23). Retrieved December 07, 2018, from https://www.youtube.com/watch?v=JL9Y3P870jU.

Engineers, M. (2018, June 02). Retrieved December 07, 2018, from https://www.youtube.com/watch?v=T38fDS2i13k

Hausser, N., Johnson, K., Parsley, M. A., Guptarak, J., Spratt, H., & Sell, S. L. (2018, January 30). Retrieved December 07, 2018, from https://www.jove.com/video/56044/detecting-behavioral-deficits-in-rats-after-traumatic-brain-injury

Reference for critique and future directions

2018-12-09 02:58:41 UTC

1. Baake, V., Reijntjes, Robert H. A. M., Dumas, E. M., Thompson, J. C., & Roos, R. A. C. (2017). Cognitive decline in huntington's disease expansion gene carriers. Cortex, 95, 51-62.
2. Farrar AM et al. (2014). Cognitive deficits in transgenic and knock-in HTT mice parallel those in huntington's disease. J Huntingtons Dis., 3(2), 145-58.
3. Lee J, Hwang YJ, Kim KY, Kowall NW& Ryu H. (2013). Epigenetic mechanisms of neurodegeneration in huntington’s disease. Neurotherapeutics., 10(4), 664–676.
4. Kolli, N., Lu, M., Maiti, P., Rossignol, J., & Dunbar, G. L. (2017). CRISPR-Cas9 mediated gene-silencing of the mutant huntingtin gene in an in vitro model of huntington’s disease. Int. J. Mol. Sci., 18(4), 754.
5. Vagner, T., Dvorzhak, A., Wójtowicz, A. M., Harms, C., & Grantyn, R. (2016). Systemic application of AAV vectors targeting GFAP-expressing astrocytes in Z-Q175-KI huntington's disease mice. Mol Cell Neurosci., 77:76-86.

Figure 1: CAG repeat domain and PolyQ chain of Huntingtin

jeremyho98 — 2018-12-09 22:27:59 UTC

CRISPR

jeremyho98 — 2018-12-09 22:36:08 UTC

Cas9 endonuclease causes double stranded DNA breaks, and can introduce indel mutations.
Uses complementary guide RNA (gRNA) to target Cas9 to sequence of interest.
Possibility to use Cas9 to target expanded CAG repeat domain in mHTT for deletion, as a permanent cure for Huntington's Disease.

Reference for Background Image

jeremyho98 — 2018-12-09 23:15:37 UTC

Q. (2016). DNA String Biology[Painting]. Retrieved November 10, 2018, from https://pixabay.com/en/dna-string-biology-3d-1811955/

Reference for Background

jeremyho98 — 2018-12-10 11:48:29 UTC

1.https://www.huntingtonsociety.ca/learn-about-hd/what-is-huntingtons/

2. Kaltenbach, L., et al. (2007). Huntingtin interacting proteins are genetic modifiers of neurodegeneration. PLoS Gen. 3(5):e82.

3) MacDonald, M. 2003. Huntingtin: alive and well and working in middle management. Sci STKE. 207:pe48.
4) Duyao, M., Ambrose, C., Myers, R., Novelletto, A., Persichetti, F., Frotnali, M., Folstein, S., Ross, C., Franz, M., Abbott, M., et al. (1993). Trinucleotide repeat length instability and age of onset in Huntington's disease. Nat Genet. 4(4):387-392.

5) Kim, Y.J., Yi, Y., Sapp, E., Wang, Y., Cuiffo, B., Kegel, K.B., Qin, Z.H., Aronin, N. & DiFiglia, M. (2001) Caspase 3‐cleaved N‐terminal fragments of wild‐type and mutant huntingtin are present in normal and Huntington’s disease brains, associate with membranes, and undergo calpain‐dependent proteolysis. Proc Natl Acad Sci USA. 98:2784–12789.
6) Gunawardena, S. & Goldstein, L.S. (2005) Polyglutamine diseases and transport problems: deadly traffic jams on neuronal highways. Arch. Neurol., 62, 46–51.

7) Ho, L., Carmichael, J., Swart, J., Wyttenbach, A., Rankin, J. & Rubinsztein, D. (2001) The molecular biology of Huntington’s disease. Psychol. Med., 31, 3–14.

8) Zeron, M., Hansson, O., Chen, N., Wellington, C., Leavitt, B., Brundin, P., Hayden, M. & Raymond, L. (2002). Increased sensitivity to N-methyl-D-aspartate receptor-mediated excitotoxicity in a mouse model of Huntington's disease. Neuron. 33(6):849-860.

9) Augood, S., Faull, R. & Emson, P. (1997). Dopamine D1 and D2 receptor gene expression in the striatum in Huntington’s disease. Ann Neurol. 42(2):215-221.

10)Huot, P., Lévesque, M. & Parent, A. (2007) The fate of striatal dopaminergic neurons in Parkinson’s disease and Huntington’s chorea. Brain. 130:222–232.

11) Charvin, D., Vanhoutte, P., Pages, C., Borrelli, E. & Caboche, J. (2005). Proc Natl Acad Sci USA. 102(34) :122-23.

Conclusion

jason_ning9 — 2018-12-10 12:11:02 UTC

1. Deletion of the polyQ domain in mHTT with CRISPR/Cas 9 gene editing technique effectively reduces nuclear accumulation and aggregation of mHTT in Homozygous HD140Q-KI mice.

2. Attenuation of GFAP proteins in association with mHTT gene editing resulted in decreased nuclear accumulated of mHTT and reactive astrocytes in homozygous mouse.

3. Data illustrates a strong inverse relationship between percentage of mHTT and performance on motor tests in heterozygous mice, demonstrating the potential effectiveness of CRISPR/Cas 9 gene editing as a tool to treat Huntington’s disease.

Significance

jason_ning9 — 2018-12-10 12:11:09 UTC

Although the research paper by Su Yang and her team were not the first to publish a paper regarding the use of CRISPR/Cas 9 gene editing technique to treat Huntington’s disease, they are the first research group to demonstrate phenotypic improvement of motor functions after treatment using various motor tests.

An earlier research paper by Jun wan Shin and his team in 2016 indicated use of CRISPR/Cas 9 gene editing technique to completely prevent generation of mHTT nuclear aggregation (1). However, this paper only focused mostly on using the gene editing technique to effectively reduce mHTT expression. In early 2017, Alex Monteys and his team published an article using CRISPR/Cas 9 gene editing technique to see if reduction of mHTT is viable in humans as are in mice (2). Unfortunately, they only examined the effect of mHTT reduction at a genomic level and failed to test for phenotypic changes. Su Yang and her team discovered that mHTT gene inactivation could reverse neuropathology and behavioural phenotypes in mice that has already developed.

Despite the fact that the researchers did not test if there were deleterious effects due to gene editing, their results already demonstrate the potential use of CRISPR/Cas 9 to treat neurodegenerative diseases caused by a toxic gain of function in mutant genes.

Reference for Discussion

jason_ning9 — 2018-12-10 15:17:11 UTC

1. Shin, Jun Wan, et al. “Permanent Inactivation of Huntington's Disease Mutation by Personalized Allele-Specific CRISPR/Cas9.” Human Molecular Genetics, 2016, doi:10.1093/hmg/ddw286.

2. Monteys, Alex Mas, et al. “CRISPR/Cas9 Editing of the Mutant Huntingtin Allele In Vitro and In Vivo.” Molecular Therapy, vol. 25, no. 1, Jan. 2017, pp. 12–23., doi:10.1016/j.ymthe.2016.11.010.