J Korean Neurosurg Soc.  2020 Sep;63(5):579-589. 10.3340/jkns.2019.0182.

An Optimization of AAV-82Q-Delivered Rat Model of Huntington’s Disease

• So KH
• Choi JH
• Islam J
• KC E
• Moon HC
• Won SY
• Kim HK
• Kim S
• Hyun SH
• Park YS

• KMID: 2506021
• DOI: http://doi.org/10.3340/jkns.2019.0182

Abstract

Objective
: No optimum genetic rat Huntington model both neuropathological using an adeno-associated virus (AAV-2) vector vector has been reported to date. We investigated whether direct infection of an AAV2 encoding a fragment of mutant huntingtin (AV2-82Q) into the rat striatum was useful for optimizing the Huntington rat model.
Methods
: We prepared ten unilateral models by injecting AAV2-82Q into the right striatum, as well as ten bilateral models. In each group, five rats were assigned to either the 2×10¹² genome copies (GC)/mL of AAV2-82Q (×1, low dose) or 2×10¹³ GC/mL of AAV2-82Q (×10, high dose) injection model. Ten unilateral and ten bilateral models injected with AAV-empty were also prepared as control groups. We performed cylinder and stepping tests 2, 4, 6, and 8 weeks after injection, tested EM48 positive mutant huntingtin aggregates.
Results
: The high dose of unilateral and bilateral AAV2-82Q model showed a greater decrease in performance on the stepping and cylinder tests. We also observed more prominent EM48-positive mutant huntingtin aggregates in the medium spiny neurons of the high dose of AAV2-82Q injected group.
Conclusion
: Based on the results from the present study, high dose of AAV2-82Q is the optimum titer for establishing a Huntington rat model. Delivery of high dose of human AAV2-82Q resulted in the manifestation of Huntington behaviors and optimum expression of the huntingtin protein in vivo.

Keyword

Huntington disease; Adeno-associated virus vector; Huntingtin protein; Neurodegenerative diseases; Gene delivery

Figure

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  Fig. 1. Schematic of the study protocol. AAV2 : adeno-associated viral vector, CPU : caudate putamen, MRI : magnetic resonance imaging.

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  Fig. 2. The number of steps in the stepping test recorded at 2, 4, 6, and 8 weeks after the AAV2 injection. A : The use of the left paw by the ×1 and ×10 subgroups of the unilateral group as significantly decreased compared with the control subgroup after two weeks. Additionally, the use of the left paw was significantly decreased over time in both the ×1 and ×10 subgroups. At week 8, a significant difference was observed between the ×1 and ×10 subgroups. B : No differences were observed among the three subgroups. C and D : The use of both paws was significantly decreased in the ×1 and ×10 subgroups of the bilateral group compared to the control subgroup after 4 weeks. At week 8, a significant difference was observed between the ×1 and ×10 subgroups. Additionally, the use of both paws was significantly decreased over time in both the ×1 and ×10 subgroups. *p<0.001. † p<0.01. ‡ p<0.05.

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  Fig. 3. The asymmetry scores for the cylinder test at 2, 4, 6, and 8 weeks after the AAV2 injection. A : The asymmetry scores of the ×1 and ×10 subgroups of the unilateral group were significantly decreased compared with the control subgroup at 6 and 8 weeks. B : The asymmetry scores did not differ among the three subgroups of the bilateral group. *p<0.001.

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  Fig. 4. Immunohistochemical staining with the EM48 antibody showed aggregation of the hTT protein in the striatum of the hD rats. A and B : Formation of mutant hTT detected by the EM48 antibody at 11 weeks after AAV2 injection into the contralateral and ipsilateral striatum of the hD rats. Arrows indicate intranuclear aggregates, and arrowheads indicate smaller neuropil aggregates outside the nucleus. C and D : The number of EM48 aggregated cells counted was significantly different between the hD ×1 and hD ×10 subgroups in unilateral ipsilateral striatum (C) and bilateral striata (D) (one-way ANOVA with Turkey’s post-hoc test). *p<0.05. hTT : huntingtin gene, hD : huntington’s disease, AAV2 : adeno-associated viral vector, ANOVA : analysis of variance.

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  Fig. 5. The huntingtin gene (hTT) protein expression was detected by an hTT-specific antibody (mAB2166) in the striatum of AAV-82Q-infected rat brain. The mAb2166 positive cells (cells with brown color) were found with higher expression in the AAV2-82Q ×10 infected subgroup (D and h) than in the x1 subgroup (C and G). The mAb2166 was scarcely positive in the control subgroups (A, B, E, and F). Scale bar, A-D : 50 μm; E-h : 20 μm. Brian tissue was embedded in paraffin, and hematoxyin was used. A-D are 10× image and E-h are 20× image. hD : huntington’s disease, AAV2 : adeno-associated virus.

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  Fig. 6. The histological characters of the AAV-82Q huntingtin infected rat brain. Brain sections showed the activation of microglia and astrocytes in AAV-82Q rats. higher expression of an oligodendrocyte marker (Olig2) and an apoptosis marker (Caspase3) were observed in the AAV2-82Q ×10 group compared to control groups and the ×1 group. The neuronal cell marker (NeuN) was not different between control and injected groups. Scale bar : 50 μm. hD : huntington’s disease, AAV2 : adeno-associated virus.

Reference

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