Lentiviral Vectors as a Gene Delivery System in the Mouse Midbrain: Cellular and Behavioral Improvements in a 6-OHDA Model of Parkinson's Disease Using GDNF

doi:10.1006/exnr.2000.7409

Experimental Neurology

Volume 164, Issue 1, July 2000, Pages 15-24

https://doi.org/10.1006/exnr.2000.7409 Get rights and content

Abstract

Local delivery of therapeutic molecules represents one of the limiting factors for the treatment of neurodegenerative disorders. In vivo gene transfer using viral vectors constitutes a powerful strategy to overcome this limitation. The aim of the present study was to validate the lentiviral vector as a gene delivery system in the mouse midbrain in the perspective of screening biotherapeutic molecules in mouse models of Parkinson's disease. A preliminary study with a LacZ-encoding vector injected above the substantia nigra of C57BL/6j mice indicated that lentiviral vectors can infect approximately 40,000 cells and diffuse over long distances. Based on these results, glial cell line-derived neurotrophic factor (GDNF) was assessed as a neuroprotective molecule in a 6-hydroxydopamine model of Parkinson's disease. Lentiviral vectors carrying the cDNA for GDNF or mutated GDNF were unilaterally injected above the substantia nigra of C57BL/6j mice. Two weeks later, the animals were lesioned ipsilaterally with 6-hydroxydopamine into the striatum. Apomorphine-induced rotation was significantly decreased in the GDNF-injected group compared to control animals. Moreover, GDNF efficiently protected 69.5% of the tyrosine hydroxylase-positive cells in the substantia nigra against 6-hydroxydopamine-induced toxicity compared to 33.1% with control mutated GDNF. These data indicate that lentiviral vectors constitute a powerful gene delivery system for the screening of therapeutic molecules in mouse models of Parkinson's disease.

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All sections were analyzed in the same experiment to keep staining conditions constant. The number of TH-positive cells was determined by counting every fourth 40-μm section, as previously described.33 The delimitation between the ventral tegmental area and the SN was determined by using the medial terminal nucleus of the accessory optic tract as a landmark.
Glial cell-derived neurotrophic factor (GDNF) has a potent action in promoting the survival of dopamine (DA) neurons. Several studies indicate that increasing GDNF levels may be beneficial for the treatment of Parkinson’s disease (PD) by reducing neurodegeneration of DA neurons. Despite a plethora of preclinical studies showing GDNF efficacy in PD animal models, its application in humans remains questionable for its poor efficacy and side effects due to its uncontrolled, ectopic expression. Here we took advantage of SINEUPs, a new class of antisense long non-coding RNA, that promote translation of partially overlapping sense protein-coding mRNAs with no effects on their mRNA levels. By synthesizing a SINEUP targeting Gdnf mRNA, we were able to increase endogenous GDNF protein levels by about 2-fold. Adeno-associated virus (AAV)9-mediated delivery in the striatum of wild-type (WT) mice led to an increase of endogenous GDNF protein for at least 6 months and the potentiation of the DA system’s functions while showing no side effects. Furthermore, SINEUP-GDNF was able to ameliorate motor deficits and neurodegeneration of DA neurons in a PD neurochemical mouse model. Our data indicate that SINEUP-GDNF could represent a new strategy to increase endogenous GDNF protein levels in a more physiological manner for therapeutic treatments of PD.
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Parkinson's disease is a progressive age-related neurodegenerative disorder and the majority of cases are idiopathic. Current treatments can alleviate symptoms, but disease-modifying therapies are lacking. Gene delivery has been used to develop new therapies for decades, but the success so far has been slim. Genetic material can be introduced into cells via viral vectors or nonviral methods. There are two basic approaches in gene therapy for Parkinson's disease: (1) delivery of new genetic material into the brain parenchyma to support dopaminergic neurons and (2) modifying the genome or introducing transgenes into cells that are then transplanted into the brain. In this chapter we will focus only on the first approach. Adeno-associated virus (AAV) has been the primary vector to transduce neurons in the brain, having a good safety profile and diffusion properties. Moreover, although the transgenes introduced to host cells stay episomal, AAVs can produce long-lasting expression in neurons because they are postmitotic cells. AAVs have been used in the clinic both for increasing dopamine neurotransmitter levels by introducing specific enzymes and for disease-modifying therapies by introducing glial cell line–derived neurotrophic factor or its family member neurturin.
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For example, lentiviruses pseudotyped with a modified envelope displayed anti-GLAST (an astrocyte-specific protein) IgG on their surfaces and preferential astrocyte targeting in vivo (Fassler et al., 2013). Interestingly, due to their ability to infect fully differentiated cells, lentiviruses find widespread usage in the study and development of therapies for neurodegenerative diseases, such as Parkinson’s (Azzouz et al., 2004; Bensadoun et al., 2000; Yin et al., 2017) Alzheimer’s (Li et al., 2017; Parsi et al., 2015; Tan et al., 2018) and Huntington’s disease (Cui et al., 2006; Schwab et al., 2017). Adenoviruses (ADVs) are nonintegrating naked viruses that bear double-stranded DNA.
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Parkinson's disease (PD) is a progressive motor neurodegenerative disorder, characterized by a selective loss of dopaminergic neurons in the substantia nigra. The complexity of disease etiology includes both genetic and environmental factors. No effective drug that can modify disease progression and protect dopamine neurons from degeneration is presently available. Human α-Synuclein A30P (A30P) is a mutant gene identified in early onset PD and showed to result selective dopamine neuron loss in transgenic A30P flies and mice. Paraquat (PQ) is an herbicide and an oxidative stress generator, linked to sporadic PD. We hypothesized that vital PD modifier genes are conserved across species and would show unique transcriptional changes to oxidative stress in animals expressing a PD-associated gene, such as A30P. We also hypothesized that manipulation of PD modifier genes would provide neuroprotection across species. To identify disease modifier genes, we performed two independently-duplicated experiments of microarray, capturing genome-wide transcriptional changes in A30P flies, chronically fed with PQ-contaminated food. We hypothesized that the best time point of identifying a disease modifier gene is at time when flies showed maximal combined toxicity of A30P transgene and PQ treatment during an early stage of disease and that effective disease modifiers gene are those showing transcriptional changes to oxidative stress in A30P expressing and not in wild type animals. Fly Neprilysin3 (Nep3) is one identified gene that is highly conserved. Its mouse and human homolog is endothelin-converting enzyme-1 (Ece1). To investigate the neuroprotective effect of Ece1, we used NS1 cells and mouse midbrain neurons expressing A30P, treated with or without PQ. We found that ECE1 expression protected against A30P toxicity on cell viability, on neurite outgrowth and ameliorated A30P accumulation in vitro. Expression of ECE1 in vivo suppressed dopamine neuron loss and alleviated the corresponding motor deficits in mice with A30P-expression. Our study leverages a new approach to identify disease modifier genes using a stress-enhanced PD animal model.
Neuroprotection and neurorestoration as experimental therapeutics for Parkinson's disease
2017, Experimental Neurology
Disease-modifying treatments remain an unmet medical need in Parkinson's disease (PD). Such treatments can be operationally defined as interventions that slow down the clinical evolution to advanced disease milestones. A treatment may achieve this outcome by either inhibiting primary neurodegenerative events (“neuroprotection”) or boosting compensatory and regenerative mechanisms in the brain (“neurorestoration”). Here we review experimental paradigms that are currently used to assess the neuroprotective and neurorestorative potential of candidate treatments in animal models of PD. We review some key molecular mediators of neuroprotection and neurorestoration in the nigrostriatal dopamine pathway that are likely to exert beneficial effects on multiple neural systems affected in PD. We further review past and current strategies to therapeutically stimulate these mediators, and discuss the preclinical evidence that exercise training can have neuroprotective and neurorestorative effects. A future translational task will be to combine behavioral and pharmacological interventions to exploit endogenous mechanisms of neuroprotection and neurorestoration for therapeutic purposes. This type of approach is likely to provide benefit to many PD patients, despite the clinical, etiological, and genetic heterogeneity of the disease.
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Although it is well established that bulbo-spinal serotonergic projections contribute to pain control mechanisms, whether they exert anti- or pro-nociceptive modulations is still a matter of debate. In order to reappraise the role of 5-HT in descending controls, we used RNA interference to selectively inhibit 5-HT synthesis in B3 neurons and assess resulting changes in nociception.
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Regular ArticleLentiviral Vectors as a Gene Delivery System in the Mouse Midbrain: Cellular and Behavioral Improvements in a 6-OHDA Model of Parkinson's Disease Using GDNF

Abstract

Exp. Neurol.

Neuron

Neurosci. Lett.

Exp. Neurol.

Brain Res.

Exp. Neurol.

Brain Res.

Neuroscience

Neuroscience

Cell

Lancet

Estimation of nuclear population from microtome sections

Anat. Rec.

Mesencephalic dopaminergic neurons protected by GDNF from axotomy-induced degeneration in the adult brain

Nature

Attenuation of 6-OHDA-induced neurotoxicity in glutathione peroxidase transgenic mice

Eur. J. Neurosci.

Intrastriatal injection of an adenoviral vector expressing glial-cell-line-derived neurotrophic factor prevents dopaminergic neuron degeneration and behavioral impairment in a rat model of Parkinson disease

Proc. Natl. Acad. Sci. USA

Highly efficient and sustained gene transfer in adult neurons with a lentivirus vector

J. Virol.

Glial cell line-derived neurotrophic factor supports survival of injured midbrain dopaminergic neurons

J. Comp. Neurol.

Dopaminergic neurons protected from degeneration by GDNF gene therapy

Science

A third-generation lentivirus vector with a conditional packaging system

J. Virol.

Self-inactivating lentiviral vectors with enhanced transgene expression as potential gene transfer system in Parkinson's disease

Hum. Gene Ther.

Regular Article
Lentiviral Vectors as a Gene Delivery System in the Mouse Midbrain: Cellular and Behavioral Improvements in a 6-OHDA Model of Parkinson's Disease Using GDNF