The development of transgenic animal models is dependent on identifying the potential role of genes of interest to the etiology of PD. A transgenic mouse is an animal in which a specific gene of interest has been altered through one of several techniques, including (i) the excision of the host gene (knock-out), (ii) the introduction of a mutant gene (knock-in), and (iii) the alteration of gene expression (knock-down, null, or over-expression). In PD, one source of transgenic targeting is derived from genes identified through epidemiological and linkage analysis studies. Alpha-synuclein and parkin are examples of genes that have been identified through linkage analysis in familial forms of parkinsonism. Once the transgene has been constructed, the degree of its expression and its impact on the phenotype of the animal depends on many factors, including the selection of sequence (mutant vs. wild-type), site of genomic integration, number of copies recombined, selection of transcription promoter, and upstream controlling elements (enhancers). Other important factors may include the background strain and age of the animal. These different features may account for some of the biochemical and pathological variations observed among transgenic mouse lines.
Rare cases of autosomal dominant familial forms of PD (the Contursi, German, and Iowa kindreds) have been linked to A30P and A53T substitution mutations in the gene encoding alpha-synuclein or triplicate of the gene (141-143). The normal function of alpha-synuclein is unknown, but its localization and developmental expression suggests a role in neuroplasticity, neurotransmission, and vesicular function (144-146). The disruption of normal neuronal function may lead to the loss of synaptic maintenance and subsequent degeneration. It is interesting that mice with knockout of alpha-synuclein are viable suggesting that a "gain-in-function" phenotype or other protein-protein interactions may contribute to neurodegeneration. Although no mutant forms of alpha-synuclein have been identified in idiopathic PD, its localization to Lewy bodies (including PD and related disorders) has suggested a patho-physiological link between alpha-synuclein aggregation and neurodegenerative disease (147,148). An interesting caveat is that the mutant allele of alpha-synuclein in the Contursi kindred is identical to the wild-type mouse suggesting that protein expression and/or protein-protein interactions, leading to a yet unidentified gain of function may be more important than loss of function due to missense mutation. Since the identification of alpha-synuclein in familial PD, many groups have developed transgenic mouse models (149-163).
A review of the transgene construction parameters (species and/or mutant forms), promoter selection (neuron or glia specific), and gene and protein expression patterns or levels demonstrates a high degree of variability in the resulting trans-genic strains. Some transgenic mouse lines show neurochemical or pathological changes in dopaminergic neurons (including inclusions, decreased striatal dopamine, and loss of striatal tyrosine hydroxylase immunoreactivity) and behavioral deficits (rotarod and attenuation of dopamine-dependent locomotor response to amphetamine), whereas other lines show no deficits. No group has reported the specific loss of substantia nigra dopaminergic neurons despite inclusion pathology or cell death in other areas of the brain. This range of results with different alpha-synuclein constructs from different laboratories underscores the important link between protein expression (mutant vs. wild-type alleles) and pathological and behavioral outcome. Important applications of alpha-synuclein transgenic mice are occurring at the level of understanding the role of this protein in basal ganglia function. For example, the response of alpha-synuclein expression to neurotoxic injury as well as interactions with other proteins, including parkin, will provide valuable insights into mechanisms important to neurodegeneration (164). Some groups report evidence of neuronal dysfunction (either physiological or motor behavioral changes) without cell death. This suggests that cell death may in fact be a component of the late phase in the progression of basal ganglia degeneration while neuronal dysfunction may occur at the level of the synapse and connectivity.
An autosomal recessive form of juvenile parkinsonism (AR-JP) led to the identification of a gene on chromosome 6q27 called parkin (165,166). Mutations in parkin may account for the majority of autosomal recessive familial cases of PD. Parkin protein has a large N-terminal ubiquitin-like domain and C-terminal cysteine ring structure and is expressed in the brain (167-169). Recent biochemical studies indicate that parkin protein may play a critical role in mediating interactions with a number of different proteins involved in the proteasome-mediated degradation pathway, including alpha-synuclein (164,170). Null mutations in mice appear normal with respect to motor behavior with no evidence of cell loss; however, striatal dopamine levels are elevated with enhanced synaptic excitability in striatal neurons (171). Mutations of the parkin gene have been introduced into transgenic mice. At present, there is very little known about pathological or behavioral alterations due to mutations in parkin protein. However, parkin transgenic models enable investigation of the ubiquitin-mediated protein degradation pathways and its relationship to neurodegenerative disease.
Mutations in the DJ-1 gene are associated with rare forms of autosomal recessive early-onset PD (172). Mice with knock-out of DJ-1 appear hypoactive, show no apparent loss of midbrain dopaminergic neurons, but do display altered electrophysiological properties in striatal neurons that can be rescued by targeting dopamine receptor D1 (173). Analysis of the structure, function, and pattern of expression of DJ-1 in mice, Drosophila, and in cell culture indicate that DJ-1 protein interacts with mitochondria playing a role in oxidative stress and apoptosis (174,175). DJ-1 has been shown to interact with a number of other proteins implicated in familial parkinsonism, including alpha-synuclein, thus regulating its function and potential toxicity (176).
UCH-L1 is a member of the family of de-ubiquitinating enzymes responsible for mediating monomeric subunits from poly-ubiquitin chains (177). Mutations in UCH-L1 result in impaired clearance of ubiquitin and ubiquitinated proteins, therefore leading to elevated cell toxicity, protein accumulation, and cell death. At present, transgenic mice with altered UCH-L1 expression have been examined in the context of spermatogenesis and not extensively on alterations in basal ganglia function (178,179). Some studies have reported increased synuclein accumulation and neu-ropathology in UCH-L1 transgenic mice supporting interactions between synuclein and the UPS (180).
The leucine-rich repeat kinase 2 gene (LRRK2), also called Dardarin, encodes a large polymer of 2527 amino acids protein with multiple structural motifs (181). Mutations in this gene have been identified in familial forms of PD that result in autosomal dominant late-onset PD (182-184). The precise function of LRRK2 is currently unknown, but recent studies have suggested that this cytoplasmic protein can associate with other PD proteins, including parkin, and possesses kinase activity (185,186). Ongoing structural/functional analysis of this large complex protein will reveal more precisely its role in neurodegenerative disorders and will guide the development of transgenic animals for study.
A locus for a rare familial form of PD maps to chromosome 1p36 and is termed PINK1 (phosphatase and tensin homolog induced kinase-1) (187). The function of PINK1 is thought to be in the protection of mitochondria from oxidative stress (188,189). In addition, PINK1 may serve to control interactions with regulatory factors involved in apoptosis (189). Transgenic mice affecting PINK1 have not yet been reported.
Nurr1 is a transcription factor that is highly expressed in early development, and disruption of this gene results in the failure to develop nigrostriatal dopaminergic neurons in postnatal life (190,191). Nurr1 expression decreases with age and in patients with PD, suggesting that this protein may play a role in maintaining dopamine cell function and integrity (192). Transgenic mice disrupting Nurr1 expression show lack of development of nigrostriatal dopaminergic neurons based on tyrosine hydroxylase immunoreactivity (193-197). Although the time course of midbrain dopaminergic cell death in PD is unclear, the Nurr1 transgenic strains may provide insight into understanding dopamine cell development, potential susceptibility to PD in the context of dopamine dysfunction, and elucidation of the role of Nurrl may act to guide stem cells as a therapeutic replacement for lost neurons.
The function of the basal ganglia is dependent on a wide range of proteins involved in dopamine biosynthesis, metabolism, uptake, and neurotransmission. To elucidate the role of numerous proteins in basal ganglia development, function, dysfunction, and their potential role in PD and its treatments, a wide spectrum of transgenic animals have been developed. These include transgenic mice targeting tyrosine hydrox-ylase, DAT, monoamine oxidase A and B, catechol-O-methyl-transferase (COMT), dopamine receptors, and vesicular monoamine transporter 2 (vmat-2). These mice are instrumental in elucidating the regulation of dopamine neurotransmission and its link to motor behavior. In addition, genes and proteins involved in other features of basal ganglia function or susceptibility to toxicity, but not directly involved in dopamine neurotransmission, have also been developed, including those for neu-rotrophic factors [such as brain-derived neurotrophic factor (BDNF), glial-derived neurotrophic factor (GDNF), and their receptors]; immune response components (IL-6, TNF-alpha); and other neurotransmitter systems, including those for glutamate, adenosine, and acetylcholine. It is important to recognize the importance of these various genetic models and their potential impact in understanding normal and diseased basal ganglia function and identifying new therapeutic treatments.
It should be noted that with the advent of new vector technologies based on infectious viruses, including lentivirus and adenovirus, genes of interest are introduced directly into the brain using stereotaxic targeting. This allows genes to be introduced to adult animals avoiding the potential confounder in transgenic lines where some gene manipulations are embryonic lethal, fail to thrive postnatal, or other systems may compensate in development for a specific gene deficiency. For example, induction of neurodegeneration can be achieved by direct targeting of alpha-synuclein or tau into the midbrain dopaminergic neurons (198-201). Genes beneficial to neuron protection and repair can also be delivered directly to their site of action in the brain. These include those genes encoding neurotrophic factors like GDNF or dopamine biosynthesis (202-206). Targeting to specific regions to regulate basal ganglia function such as inhibition of the subthalamic nucleus has been reported with some success (207). Studies are still underway to evaluate different parameters of delivery, stability, toxicity, and long-term efficacy, as well as evaluation in nonhuman primate models prior to clinical applications. A number of clinical trials using viral vectors are currently in early phase studies.
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