Review
Metallothionein-mediated neuroprotection in genetically engineered mouse models of Parkinson's disease

https://doi.org/10.1016/j.molbrainres.2004.09.011Get rights and content

Abstract

Parkinson's disease is characterized by a progressive loss of dopaminergic neurons in the substantia nigra zona compacta, and in other sub-cortical nuclei associated with a widespread occurrence of Lewy bodies. The cause of cell death in Parkinson's disease is still poorly understood, but a defect in mitochondrial oxidative phosphorylation and enhanced oxidative and nitrative stresses have been proposed. We have studied controlwt (C57B1/6), metallothionein transgenic (MTtrans), metallothionein double gene knock (MTdko), α-synuclein knock out (α-synko), α-synuclein–metallothionein triple knock out (α-syn–MTtko), weaver mutant (wv/wv) mice, and Ames dwarf mice to examine the role of peroxynitrite in the etiopathogenesis of Parkinson's disease and aging. Although MTdko mice were genetically susceptible to 1, methyl, 4-phenyl, 1,2,3,6-tetrahydropyridine (MPTP) Parkinsonism, they did not exhibit any overt clinical symptoms of neurodegeneration and gross neuropathological changes as observed in wv/wv mice. Progressive neurodegenerative changes were associated with typical Parkinsonism in wv/wv mice. Neurodegenerative changes in wv/wv mice were observed primarily in the striatum, hippocampus and cerebellum. Various hallmarks of apoptosis including caspase-3, TNFα, NFκB, metallothioneins (MT-1, 2) and complex-1 nitration were increased; whereas glutathione, complex-1, ATP, and Ser(40)-phosphorylation of tyrosine hydroxylase, and striatal 18F-DOPA uptake were reduced in wv/wv mice as compared to other experimental genotypes. Striatal neurons of wv/wv mice exhibited age-dependent increase in dense cored intra-neuronal inclusions, cellular aggregation, proto-oncogenes (c-fos, c-jun, caspase-3, and GAPDH) induction, inter-nucleosomal DNA fragmentation, and neuro-apoptosis. MTtrans and α-Synko mice were geneticallyresistant to MPTP-Parkinsonism and Ames dwarf mice possessed significantly higher concentrations of striatal coenzyme Q10 and metallothioneins (MT 1, 2) and lived almost 2.5 times longer as compared to controlwt mice. A potent peroxynitrite ion generator, 3-morpholinosydnonimine (SIN-1)-induced apoptosis was significantly attenuated in MTtrans fetal stem cells. These data are interpreted to suggest that peroxynitrite ions are involved in the etiopathogenesis of Parkinson's disease, and metallothionein-mediated coenzyme Q10 synthesis may provide neuroprotection.

Introduction

Zinc-containing neurons in the brain are a subclass of glutamatergic neurons, which are found predominantly in the telencephalon. These neurons store zinc in their presynaptic terminals and release it by a calcium-dependent mechanism. These “vesicular” pools of zinc are viewed as endogenous modulators of ligand-gated and voltage-gated ion channels.

The term metallothionein (MTs) refers to a low-molecular-weight metal-binding protein (Mr=6000–7000) that has unusual biochemical characteristics, such as a high content of cysteine (25–30%), large proportions of serine and lysine (12–18%), and complete absence of histidine and aromatic amino acids such as tyrosine and phenylalanine. The precise functions of metallothioneins, which may vary in different tissues and organisms, have not been established. However, evidence indicates that the hepatic and renal metallothioneins are involved primarily in metal detoxification [15], whereas the brain metallothionein isoforms I–III participate in the homeostasis of essential trace metals, such as zinc, and in scavenging free radicals caused by oxidative stress. In addition to scavenging free radicals, metallothionein isoforms exert their neuroprotective effects, in part, by enhancing the concentration of ubiquinol from ubiquinone [11], [13], [14], [15].

A mutation, by definition, is a change in gene sequence and may be associated with a specific human disease. Genetically engineered mice are produced by induced mutations, including mice with transgenes, mice with targeted mutations (“knockouts”), and mice with retroviral or chemically induced mutations. Transgenic mice possess a segment of foreign DNA that is inserted in the host cell genome via pronuclear microinjection (non-homologous recombination), via infection with a retroviral vector, or in some situations via homologous recombination. Gene knockout mice are prepared by first introducing gene disruptions, replacements, or duplications into embryonic stem (ES) cells by homologous recombination. Genetically modified ES cells are microinjected in the host embryos at the eight-cell blastocyst stage. The embryos are then introduced in the pseudo-pregnant females that bear chimeric progeny. The chimeric progeny that carry the knockout gene in their germ line are subsequently bred to establish the gene knockout mice. Chemically induced mutations are produced by mutagens, such as ethyl–nitrosourea (ENU), which induces point mutations. ENU mutation is induced by treating male mice with ENU, and then breeding is performed with untreated females. The progeny are screened for phenotypes of interest carrying point mutations by polymerase chain reaction (PCR) analysis of tail DNA. Detailed information and research applications for different genetically engineered mice are now available on the JAX® Mice Database.

Various experimental models have been proposed to explore basic molecular mechanisms of neurodegeneration in Parkinson's disease (PD) [2], [8], [18]; however, an appropriate animal model of PD remains unavailable. Like any other experimental model, genetically engineered animals have limitations. On occasions, they do not breed well, or their growth potential may be impaired due to a compromised immune system. Irrespective of these limitations, genetically engineered animals have provided unique opportunities to understand the disease process and novel therapeutic strategies for neurodegenerative disorders. Genetically manipulated mice have been and are being used to investigate amyotrophic lateral sclerosis [1], [6], Alzheimer's disease [7], motor neuron disease [5], [19], Prion diseases [20], [24], [25], catecholaminergic regulatory systems [3], and Huntington's disease [22], [26], [27].

We have prepared seven different types of genetically engineered mouse models in our laboratories with the primary objectives of exploring the molecular basis of Parkinson's' disease and understanding and preventing neurodegenerative disorders of aging. All the experimental genotypes used in the study were genotyped by tail DNA-PCR analysis. They included controlwt (C57Bl/6J) mice, metallothionein double gene knockout (MTdko) mice (129S7/SvEvBrd-Mt1 tm1Bri Mt2 tm1Bri/J; JAX, stock no. 002211), metallothionein transgenic (MTtrans) mice (Tg(Mt1)174Bri/J; stock no. 002209), α-synuclein knockout mice (α-Synko) mice, α-synuclein–metallothionein triple knockout mice (α-Syn–MTtko), homozygous weaver mutant mice (wv/wv) (B6CBACaAw-J/A-Kcnj6 wv), and Ames dwarf (p/pProp1 df/J; stock no. 001618) mice. Our studies have shown that metallothionein transgenic (MTtrans) and α-Synko mice are highly suitable to explore basic molecular mechanisms of neuroprotection in PD and aging, whereas MTdko and homozygous weaver mutant (wv/wv) mice can be utilized to understand the basic molecular mechanism of progressive dopaminergic neurodegeneration in PD [16]. Furthermore, Ames dwarf mice can be used to explore specifically the molecular mechanisms of aging and longevity [4]. This review forms a brief report from our laboratories dealing with in vivo and in vitro gene manipulation in order to better understand the basic molecular mechanism(s) of dopaminergic neurodegeneration and to establish the neuroprotective potential of metallothionein isoforms I–III [14], [15] in PD and aging.

Section snippets

Metallothionein gene-manipulated mice

In view of the above, we have studied the effects of various neurotoxins causing parkinsonism in some of these genotypes. Our studies have indicated that MTtrans mice are genetically resistant to MPTP, 6-OHDA, salsolinol, and rotenone-induced parkinsonism as compared to MTdko mice, which exhibited an enhanced genetic susceptibility to these agents [10]. In order to understand the basic molecular mechanism of genetic susceptibility of MTdko and resistance of MTtrans mice, we exposed these mice

α-Syn–MTtko mice

In order to further explore the basic molecular mechanisms of α-Syn and MTs in the etiopathogenesis of PD, we cross-bred α-Syn males with MTdko female mice and obtained a rare colony of α-Syn–MTtko mice. We examined the effect of chronic rotenone in these genotypes. Our studies have shown that rotenone (1–200 nM) enhanced NFκβ expression and inhibited complex-1 expression in α-Syn–MTtko mice [9], [29]. Although the litter size, body size, and lifespans of α-Syn–MTtko mice were significantly

MTs inhibit salsolinol- and 1-benzyl-tetrahydroisoquinoline induced neurotoxicity

Salsolinol and 1-benzyl-tetrahydroisoquinoline (1-Bn-TIQ) induced α-Syn expression, and apoptosis was suppressed upon MT-1 gene overexpression [28]. MTtrans fetal stem cells were genetically resistant to peroxynitrite ion-generating compound, 3-morpholinosydnonimine (SIN-1)-induced dopamine oxidation product dihydroxyl phenyl acetaldehyde (DOPAL), and homovalinic acid (HVA)-induced apoptosis.

We have also discovered MT-3 transcripts in human neuroblastoma (SK-N-SH and SH-SY-5Y) cells. MT-3

MTs attenuate DOPAL apoptosis

Dopamine oxidation product DOPAL-induced apoptosis in the human dopaminergic (SK-N-SH) cell line was examined in order to validate the observed neuroprotection. Overnight exposure to DOPAL induced apoptosis that was characterized by spherical appearance, plasma membrane perforations, and nuclear DNA condensation as illustrated in Fig. 1. DOPAL-induced apoptosis was also accentuated in aging mitochondrial genome knockout (RhOmgko) neurons. Transfection of RhOmgko neurons with MT-1sense

MTs inhibit MPP+ apoptosis

In order to determine whether MT overexpression provides dopaminergic neuroprotection, we exposed SK-N-SH neurons to MPP+ overnight in culture and performed single cell gel electrophoresis. MT-1sense oligonucloetide-transfected neurons exhibited green comet tails representing mitochondrial damage, whereas MT-1antisense oligonucleotide-transfected neurons exhibited red comet tails representing nuclear DNA damage, indicating that MT down-regulation may induce severe neurodegeneration in

SIN-1 enhanced MTs and α-Syn expression

A potent peroxynitrite ion generator, SIN-1, induced MT-1 and α-Syn expression in SK-N-SH neurons in a concentration-dependent manner. SIN-1-induced increases in MT-1 and α-Syn expression were inhibited by either selegiline (10 μM) or coenzyme Q10 (10 μM) pretreatment, suggesting the neuroprotective role of MTs in peroxynitrite-induced oxidative- and nitrative stresses [28].

MTs enhance mitochondrial function by augmenting coenzyme Q10 synthesis

Transfection of cultured dopaminergic (SK-N-SH) neurons with antisense oligonucleotides to MT-1 accentuated—whereas transfection with MT-1sense oligonucleotides significantly attenuated—MPP+-, 6-OHDA-, rotenone-, and salsolinol-induced apoptosis [28], [30], [32]. These data further suggest a neuroprotective role of metallothionein isoforms I–III in PD.

MTs stabilize mitochondrial genome

Metallothionein stabilization of the mitochondrial genome was confirmed by preparing a cellular model of aging. The cellular model of aging was prepared by selectively knocking out the mitochondrial genome using 5 μg/l DNA-intercalating agent, ethidium bromide, for 2–3 months in selection medium with appropriate concentrations of glutamine, pyruvate in Dulbecco's modified Eagle's medium (DMEM), 3.7g/l sodium bicarbonate, and 5% fetal bovine serum. A detailed procedure to prepare and identify RhO

Homozygous weaver mutant (wv/wv) mice and PD

A single gene point mutation (G156S) in the G-protein-coupled inward-rectifying potassium channel of wv/wv mice [23] induces loss of the external granular layer neurons in the cerebellum [25], [37] and dopaminergic cells in the striatum [17], [28], [40], [43]. As a consequence of progressive dopaminergic degeneration, wv/wv mice exhibit instability of gait, poor limb coordination, and resting and intention tremors [39], similar to those observed in PD and chronic drug addiction [12], [21].

Neurodegenerative changes in wv/wv mice

By employing digital fluorescence imaging microscopy, we have demonstrated neurodegenerative changes in the striatal and cerebellar regions of wv/wv mice. The neurodegenerative changes were represented by apoptotic cell death, which was observed primarily in the external granular layer of the cerebellar cortex. Some of the cells from the external granular layer exhibited apoptosis during migration in the internal granular layer, whereas the neurons in the striatal region exhibited aggregated

18F-DOPA distribution kinetics and uptake in wv/wv mice

Recently, we have studied in vivo distribution kinetics of 18F-DOPA in wv/wv mice using the high-resolution Concorde microPET imaging system and CTI-RDS-111 cyclotron. We correlated and confirmed microPET findings with conventional neurochemical analysis. The microPET imaging data provided a regression line with a correlation coefficient of 0.89, suggesting that high-resolution microPET imaging can be used to determine the brain regional dopaminergic metabolism in vivo. MicroPET imaging is

Abnormal function of α-Syn in wv/wv mice

We have also demonstrated that SDS-soluble α-Syn is significantly reduced in wv/wv mice, which renders them highly susceptible to dopaminergic neurodegeneration and hence parkinsonism. By using digital immunofluorescence imaging, we discovered α-Syn aggregates in the degenerating striatal dopaminergic neurons of wv/wv mice [13]. However, the basic molecular mechanisms of α-Syn aggregation and progressive loss of dopaminergic neurotransmission in wv/wv remain enigmatic. It seems that SDS-soluble

MTtrans striatal fetal stem cell transplantation

In an in vitro analysis, we confirmed that MTtrans striatal fetal stem cells are genetically resistant to the dopamine oxidation product DOPAL and to 1-methyl-4-phenyl pyridinium ion (MPP+)-induced apoptosis [30]. Since MTtrans striatal fetal stem cells are genetically resistant to environmental and parkinsonian neurotoxins, and wv/wv mice exhibit progressive dopaminergic neurodegeneration in the NS system during aging, we have now proposed to explore the structural and functional integrity of

Neuroprotective role of TRPC1 in PD

Mammalian homologues of the Drosophila canonical transient receptor potential (TRP) protein have been implicated to function as Ca2+ channels [36]. In a recent study, we have explored the molecular mechanism of neuroprotection by TRPC1 in human dopaminergic (SH-SY-5Y) cells. TRPC1 expression was significantly inhibited by exposing the human dopaminergic (SH-SY-5Y) cells to MPP+ or salsolinol. Overexpression of TRPC1 in SH-SY-5Y neurons provided protection against these parkinsonian neurotoxins

Increased lifespan of Ames dwarf mice

We have discovered that Ames dwarf mice survived 2.5 longer than controlwt mice [4]. Studies conducted on long-living Ames mice have shown that these mice express higher levels of metallothionein isoforms and antioxidant enzymes, produce fewer free radicals, and exhibit less DNA and protein oxidative damage when compared to age-matched wild-type mice [45], [46], [47], [48]. In addition, dwarf mice outsurvive controlwt mice when challenged in vivo with the systemic oxidative stressor, paraquat.

Conclusions

Pharmacological interventions involving brain regional induction of MTs, TRPC1, and regulating growth hormone expression might be helpful in the clinical management of PD and other neurodegenerative disorders of unknown etiopathogenesis. Brain regional induction of MTs, in addition to metal detoxification, helps in reactive oxygen species scavenging, coenzyme Q10 synthesis, complex-1 activation, attenuation of α-Syn nitration, and suppression of pro-inflammatory cytokines TNFα and NFκβ involved

Acknowledgments

This research was supported by a grant from the Counter Drug Technology Assessment Center, Office of National Drug Control Policy (no. DATMO5-02C-1252, to M.E.), a grant from NINDS (no. 2R01 NS3456609, to M.E.), and grant R01 AG17059-09 (to M.E.). The excellent secretarial skills of Mrs. Dani Stramer in preparing this manuscript are gratefully acknowledged.

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