Development of an Alpha-synuclein Based Rat Model for Parkinson's Disease via Stereotactic Injection of a Recombinant Adeno-associated Viral Vector

Summary

This manuscript describes how viral vector-mediated local gene delivery provides an attractive way to express transgenes in the central nervous system. The protocol outlines all crucial steps to perform a viral vector injection in the substantia nigra of the rat to develop a viral vector-based animal model for Parkinson's disease.

Abstract

In order to study the molecular pathways of Parkinson's disease (PD) and to develop novel therapeutic strategies, scientific investigators rely on animal models. The identification of PD-associated genes has led to the development of genetic PD models. Most transgenic α-SYN mouse models develop gradual α-SYN pathology but fail to display clear dopaminergic cell loss and dopamine-dependent behavioral deficits. This hurdle was overcome by direct targeting of the substantia nigra with viral vectors overexpressing PD-associated genes. Local gene delivery using viral vectors provides an attractive way to express transgenes in the central nervous system. Specific brain regions can be targeted (e.g. the substantia nigra), expression can be induced in the adult setting and high expression levels can be achieved. Further, different vector systems based on various viruses can be used. The protocol outlines all crucial steps to perform a viral vector injection in the substantia nigra of the rat to develop a viral vector-based alpha-synuclein animal model for Parkinson's disease.

Introduction

To study the pathophysiology of PD and to develop novel therapeutic strategies, there is an urgent need for animal models that closely resemble the neuropathology, physiology and motor symptoms of human PD. The higher the predictive value, the better we can translate new therapies from animal models to patients.

The discovery of alpha-synuclein (α-SYN) as the first PARK gene in 1997 led to the development of the first genetic PD models. Many transgenic mice overexpressing human wild-type (WT) or mutant (A30P, A53T) α-SYN have been generated over the last decade. The levels of α-SYN overexpression have proven to be crucial in ....

Protocol

All animal experiments are carried out in accordance with the European Communities Council Directive of 24 November 1986 (86/609/EEC) and approved by the Bioethical Committee of the University of Leuven (Belgium).

1. Recombinant AAV Production and Purification

Note: rAAV vector production and purification was performed by the Leuven Viral Vector Core (LVVC) as previously described¹⁷.

1. Briefly, transfect subconfluent low (<50) passage adherent HEK 293T cells using a 25kD linear polyethylenimine 150 nM NaCl transfection solution and three different plasmids in a ratio of 1:1:1 in DMEM medium 2% foetal bovine serum. After 24 hr of ....

Results

The overall scheme of the experiment is depicted in Figure 1

rAAV 2/7-mediated overexpression of A53T α-SYN induces dopamine-dependent motor deficits.
To examine whether the level of α-SYN overexpression is sufficient to induce motor impairments in the rats, we subjected the rats to the cylinder test to evaluate spontaneous forelimb use (Figure 3A). From 3 weeks .......

Discussion

There are several critical steps within the protocol. The vector titer as well as the vector purity directly influences the phenotypic outcome of the model. Excessive vector titers or insufficiently purified vector batches may result in non-specific toxicity. Therefore, the use of high quality vector batches and appropriate control vectors is indispensable. Further, the exact positioning of the rat's head in the stereotaxic frame and the accurate determination of the coordinates is essential in targeting the substant.......

Materials

Name	Company	Catalog Number	Comments
Female 8 weeks old Wistar rats	Janvier	/	200-250 g
Ketamine (Nimatek)	Eurovet animal health	804132
Medetomidine (Dormitor)	Orion-Pharma/ Janssen Animal Health	1070-499
Local anesthetic for scalp and ears: Xylocaïne 2% gel	Astrazeneca	0137-547
Terramycine	Pfizer	0132-472
Buprénorphine (Vetergesic)	Ecuphar	2623-627
Jodium 1% isopropanol	VWR	0484-0100
stereotactic head frame	Stoeling	/
Hamilton Syringe (30 gauge -20mm -pst 2)	Hamilton/ Filter Service	7803-07
atipamezole (Antisedan)	Orion-Pharma/Elanco	1300-185
rAAV A53T α-SYN vector	LVVC, KU Leuven	/	https://gbiomed.kuleuven.be/english/research/50000715/laboratory-of-molecular-virology-and-gene-therapy/lvvc/
sodium pentobarbital (Nembutal)	Ceva Santé	0059-444
microtome	Microm	HM650
rabbit polyclonal synuclein Ab	Chemicon	5038	1:5000
rabbit polyclonal TH Ab	Chemicon	152	1:1000
Lutetium oxyorthosilicate detector-based FOCUS 220 tomograph	Siemens/ Concorde Microsystems	/
radioligand: 18F-FECT	In house	/
L-dopa: Prolopa 125	Roche		6mg/kg i.p.
DMEM, Glutamax	Life Technologies	N° 31331-093
Foetal bovine serum	Life Technologies	N° 10270-106
25 kD linear polyethylenimine (PEI)	Polysciences	/
OptiPrep Density Gradient Medium: Iodixanol	Sigma	D1556-250ML
Optimen	Life Technologies	N° 51985-026
Paxinos 1 watston steretactic atlas, fourth Edition	Elsevier	/