The Parkinson's-Reversing Breakthrough

Treatment Options for Parkinson Disease

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The inadvertent self-administration of MPTP by heroin addicts in the 1980s induced an acute form of parkinsonism whose clinical and biochemical features were indistinguishable from idiopathic PD (35,36). Like PD, this MPTP cohort demonstrated an excellent response to levodopa and dopamine agonist treatment but developed motor complications within weeks. The rapidity with which these motor complications appeared presumably reflected the severity of substantia nigra neuronal degeneration induced by MPTP. Given the similarities between the human model of MPTP-induced parkinsonism and PD, it became evident that MPTP could be used to develop animal models of PD.

The subsequent administration of MPTP to a number of different animals has demonstrated a wide variety of sensitivity to the toxic effects of MPTP. These differences were shown to be species, strain, and age dependent. For example, the nonhuman primate is the most sensitive to the toxic effects of MPTP. The mouse, cat, dog, and guinea pig are less sensitive and the rat is the least sensitive. Even within species there are strain differences. For example, the C57BL/6 mouse is the most sensitive of all mouse strains tested while strains such as CD-1 appear almost resistant (37,38). Some differences among strains may also depend on the supplier, as seen with variability in the Swiss Webster strain (39). In addition to strain, animal sensitivity to the neurotoxicant effects of MPTP may be influenced by the animal's age with older mice, for example, being more sensitive (40,41). Studies suggest that age-dependent differences may be due to differences in MPTP metabolism (42). To bypass potential confounders involved in MPTP delivery to the brain and its conversion to the 1-methyl-4-pyridinium (MPP+) toxin form, some investigators have utilized stereotaxic delivery via cannulae into the striatum (43). Similar to 6-OHDA lesioning, this approach still has technical issues regarding targeting and diffusion, and has yet to be tested in a wide range of species.

The mechanism of MPTP toxicity has been thoroughly investigated. The meperidine analog MPTP is converted to MPP+ by monoamine oxidase B. MPP+ acts as a substrate of the DAT, leading to the inhibition of mitochondrial complex I, the depletion of adenosine triphosphate (ATP), and cell death of dopaminergic neurons. MPTP administration to mice and nonhuman primates selectively destroys dopaminergic neurons of the SNpc, the same neurons affected in PD (44). Similar to PD, other catecholaminergic neurons, such as those in the VTA and locus coeruleus, may be affected to a lesser degree. In addition, dopamine depletion occurs in both the putamen and caudate nucleus. The preferential lesioning of either the putamen or caudate nucleus may depend on animal species and regimen of MPTP administration (45-47). Unlike PD, Lewy bodies have not been reported; however, eosinophilic inclusions (reminiscent of Lewy bodies) have been described in aged nonhuman primates (48). The time course of MPTP-induced neurodegeneration is rapid, and therefore represents a major difference with idiopathic PD, which is a chronic progressive disease. Interestingly, data from humans exposed to MPTP indicate that the toxic effects of MPTP may be more protracted than initially believed (49). Details of MPTP toxicity, safety, and utility have been described in reviews (50,51).

The MPTP-Lesioned Mouse Model

The administration of MPTP to mice results in behavioral alterations that may resemble human parkinsonism. For example, hypokinesia, bradykinesia, and akinesia can be observed through various behavioral analyses, including open field activity monitoring, swim test, pole test, grip coordination, and rotarod. Whole body tremor and postural abnormalities have also been reported, but primarily in the acute phase (52). Cognitive changes have been reported with respect to spatial learning (53). In general, these behavioral alterations tend to be highly variable with some mice showing severe deficits while others show little or no behavioral change [for review, see Ref. (52)]. This behavioral variability may be due to a number of factors, including the degree of lesioning, mouse strain, time course after lesioning, and the reliability and validity of the behavioral analysis (54-58).

The MPTP-lesioned mouse model has proven valuable to investigate potential mechanisms of neurotoxic-induced dopaminergic cell death. For example, mechanisms under investigation have included mitochondrial dysfunction, energy (ATP) depletion, free-radical production, apoptosis, and glutamate excitotoxicity (51). In addition to its utility in studying acute cell death, the MPTP-lesioned model also provides an opportunity to study injury-induced neuroplasticity. The MPTP-lesioned mouse displays the return of striatal dopamine several weeks to months after lesion-ing (45,47,59,60). The molecular mechanism of this neuroplasticity of the injured basal ganglia is an area of investigation in our laboratory and in others, and appears to encompass both neurochemical and morphological components. In addition, it has been shown that this plasticity may be facilitated through activity-dependent processes using treadmill training (61,62).

MPTP-Lesioned Nonhuman Primate

Administration of MPTP to nonhuman primates results in parkinsonian symptoms, including bradykinesia, postural instability, and rigidity. In some species, resting or action/postural tremor has been observed (63). Similar to PD, the MPTP-lesioned nonhuman primate responds to traditional anti-parkinsonian therapies, such as lev-odopa and dopamine agonists. Following the administration of MPTP, the nonhuman primate progresses through acute (hours), sub-acute (days), and chronic (weeks) behavioral phases of toxicity that are due to the peripheral and central effects of MPTP. The acute phase is characterized by sedation and a hyper-adrenergic state, the sub-acute phase by the development of varying degrees of parkinsonian features, and the chronic phase by initial recovery (by some, but not all animals) followed by the stabilization of motor deficits (64). In general, the behavioral response to MPTP-lesioning may vary at both the inter- and intra-species level. Variability may be due to age and species phylogeny. For example, older animals and Old World monkeys (such as rhesus, Macaca mulatta or African Green, Cercopithecus aethiops) tend to be more sensitive than young and New World monkeys (such as the squirrel monkey, Saimiri sciureus or marmoset, Callithrix jacchus) (65-67).

Behavioral recovery after MPTP-induced parkinsonism has been reported in most species of nonhuman primate. The degree and time course of behavioral recovery is dependent on age, species, and mode of MPTP administration (64). In general, the more severely affected animal is less likely to recover (63). Study of the molecular mechanisms underlying behavioral recovery of the nonhuman primate has identified that the mechanisms underlying recovery may include alterations in dopamine biosynthesis (increased tyrosine hydroxylase protein and mRNA expression) and turnover; down-regulation of DAT; increased dopamine metabolism; sprouting and branching of tyrosine hydroxylase fibers; alterations of other neurotransmitter systems, including glutamate and serotonin; and alterations of signal transduction pathways in both the direct (D1) and indirect (D2) pathways (68,69).

The administration of MPTP through a number of different dosing regimens has led to the development of several distinct models of parkinsonism in the nonhuman primate. Each model is characterized by unique behavioral and neurochem-ical parameters. As a result, numerous studies addressing a variety of hypotheses have been conducted. These studies consist of new pharmacological treatments, transplantation, mechanisms of motor complications, deep brain stimulation, behavioral recovery, cognitive impairment, and the development of novel neuro-protective and restorative therapies. For example, in some models, there is profound striatal dopamine depletion and denervation with little or no dopaminergic axons or terminals remaining. This model provides an optimal setting to test fetal tissue grafting since the presence of any tyrosine hydroxylase positive axons or sprouting cells would be due to transplanted tissue survival. Other models have less extensive dopamine depletion and only partial denervation, with a modest to moderate degree of dopaminergic axons and terminals remaining. This partially denervated model best resembles mild to moderately affected PD patients. Therefore, sufficient dopaminer-gic neurons and axons as well as compensatory mechanisms are likely to be present. The effects of growth factors (inducing sprouting) or neuroprotective factors (promoting cell survival) are best evaluated in this situation. The following section reviews the most commonly used MPTP-lesioned nonhuman primate models.

In the systemic lesioned model, MPTP may be administered via intra-muscular, intra-venous, intra-peritoneal, or subcutaneous injection (70-73). This leads to bilateral depletion of striatal dopamine and nigrostriatal cell death. A feature of this model is that the degree of lesioning can be titrated, resulting in a range (mild to severe) of parkinsonian symptoms. The presence of clinical asymmetry is common, with one side more severely affected. Levodopa administration leads to the reversal of all behavioral signs of parkinsonism in a dose-dependent fashion. After several days to weeks of levodopa administration, animals develop reproducible motor complications, both wearing-off and dyskinesia. Animal behavior in this model and others may be assessed using cage-side or video-based observation, automated activity measurements in the cage through infrared-based motion detectors or accelerometers, and examination of hand-reaching movement tasks. The principal advantage of this model is that the behavioral syndrome closely resembles the clinical features of idiopathic PD. The systemic model has partial dopaminergic denervation bilaterally and probably best represents the degree of loss seen in all stages of PD, including end stage disease where some dopaminergic neurons are still present. This model is well suited for therapeutics that interact with remaining dopamin-ergic neurons, including growth factors, neuroprotective agents, and dopamine modulation. The easily reproducible dyskinesia in this model allows for extensive investigation of its underlying mechanism and treatment. Disadvantages of this model include spontaneous recovery in mildly affected animals. Alternatively, bilaterally severely affected animals may require extensive veterinary care and dopamine supplementation.

Administration of MPTP via unilateral intracarotid infusion has been used to induce a hemiparkinsonian state in the primate, called the hemiparkinsonian model

(74). The rapid metabolism of MPTP to MPP+ in the brain may account for the localized toxicity to the hemisphere ipsilateral to the infusion. Motor impairments appear primarily on the contralateral side. Hemi-neglect, manifested by a delayed motor reaction time, also develops on the contralateral side. In addition, spontaneous ipsilateral rotation may develop. Levodopa administration reverses the parkinsonian symptoms and induces contralateral rotation. Substantia nigra neurodegeneration and striatal dopamine depletion (greater than 99%) on the ipsilateral side to the injection is more extensive than seen in the systemic model. The degree of unilateral lesioning in this model is dose dependent.

Major advantages of the hemi-lesioned model include the ability for animals to feed and maintain themselves without supportive care, the availability of the unaffected limb on the ipsilateral side to serve as a control, and the utility of the dopamine-induced rotation for pharmacological testing. In addition, due to the absence of dopaminergic innervation in the striatum, the hemi-lesioned model is well suited for examining neuronal sprouting of transplanted tissue. A disadvantage of this model is that only a subset of parkinsonian features are evident and are restricted to one side of the body, a situation not seen in idiopathic PD.

The bilateral intracarotid model employs an intracarotid injection of MPTP followed several months later by another intracarotid injection on the opposite side

(75). This model combines the less debilitating features of the carotid model as well as creating bilateral clinical features, a situation more closely resembling idiopathic PD. The advantage of this model is its prolonged stability and limited inter-animal variability. Similar to the hemi-lesioned model, where there is extensive striatal dopamine depletion and denervation, the bilateral intracarotid model is well suited for evaluation of transplanted tissue. However, levodopa administration may result in only partial improvement of parkinsonian motor features and food retrieval tasks. This can be a disadvantage since high doses of test drug may be needed to demonstrate efficacy, increasing the risk for medication-related adverse effects.

A novel approach to MPTP lesioning is the administration of MPTP via intracarotid infusion, followed by a systemic injection. This over-lesioned model is characterized by severe dopamine depletion ipsilateral to the MPTP-carotid infusion and a partial depletion on the contralateral side due to the systemic MPTP injection. Consequently, animals are still able to maintain themselves due to a relatively intact side. The behavioral deficits consist of asymmetric parkinsonian features. The more severely affected side is contralateral to the intracarotid injection (76). Levodopa produces a dose-dependent improvement in behavioral features; however, the complications of levodopa therapy, such as dyskinesia, have not been as consistently observed. This model combines some of the advantages of both the systemic and intracarotid MPTP models, including stability. This model is suitable for both transplant studies, utilizing the more depleted side, and neuro-regeneration with growth factors, utilizing the partially depleted side where dopaminergic neurons still remain.

Finally, the chronic low dose model consists of intravenous injections of a low dose of MPTP administration over a 5- to 13-month period (77). This model is characterized by cognitive deficits consistent with frontal lobe dysfunction reminiscent of PD or normal-aged monkeys. These animals have impaired attention and short-term memory processes and perform poorly in tasks of delayed response or delayed alternation. Since gross parkinsonian motor symptoms are essentially absent at least in early stages, this model is well adapted for studying cognitive deficits analogous to those that accompany idiopathic PD.

The MPTP-lesioned nonhuman primate has provided a valuable tool for investigating potential mechanisms underlying motor complications related to long-term levodopa use in human idiopathic PD. The MPTP-lesioned nonhuman primate has been shown to demonstrate both wearing-off and dyskinesia. Although the etiology of dyskinesia is unknown, electrophysiological, neurochemical, molecular, and neu-roimaging studies in the nonhuman primate models suggest that the pulsatile delivery of levodopa may lead to changes in the neuronal firing rate and pattern of the globus pallidus and subthalamic nucleus; enhancement of D1- and/or D2-receptor mediated signal transduction pathways; super-sensitivity of the D2 receptor; alterations in the phosphorylation state and subcellular localization of glutamate (NMDA subtype) receptors; modifications in the functional links between dopamine receptor subtypes (D1 and D2, and D1 and D3); changes in glutamate receptors (AMPA and NMDA receptor subtypes); and enhancement of opiod-peptide mediated neurotransmission (78-82).

While the presence of a nigral lesion has long been considered an important prerequisite for the development of dyskinesia in the MPTP model, recent studies demonstrate that even normal nonhuman primates when given sufficiently large doses of levodopa over two to eight weeks may develop peak-dose dyskinesia (83). The high levels of plasma levodopa in this dosing regimen may serve to exhaust the buffering capacity within the striatum of the normal animal, and therefore lead to pulsatile delivery of levodopa and priming of postsynaptic dopaminergic sites for dyskinesia.

In addition to its central effects, the administration of MPTP may lead to systemic effects that may prove detrimental to any animal during the induction of a parkinsonian state. For example, the peripheral conversion of MPTP to MPP+ in the liver could lead to toxic injury of the liver and heart. To address these potential peripheral effects of MPTP, squirrel monkeys were administered MPTP (a series of six subcutaneous injections of 2 mg/kg, free-base, two weeks apart) and were given a comprehensive exam 1, 4, and 10 days after each injection. This exam included measurements of body weight, core body temperature, heart rate, blood pressure, liver and kidney function, and white blood cell count. Biochemical markers of hepa-tocellular toxicity were evident within days of MPTP lesioning and persisted for several weeks after the last injection. In addition, animals had significant hypothermia within 48 hours after lesioning that persisted for up to 10 days after the last MPTP injection (Petzinger et al. in preparation). The pathophysiology of these effects may be directly related to MPTP itself and/or its metabolites. The systemic effects of MPTP on animal models should be taken into consideration during the design of any pharmacological study.

MPTP-Lesioning in Other Species

While mice and nonhuman primates continue to remain the primary species in the majority of studies with MPTP, researchers have reported the effects of MPTP in a wide range of other species. These include the leech (Hirudo medicinakis), planarian flatworm (Dugesia japonica), rainbow trout (Oncorhynchus mykiss), goldfish (Carassius auratus), zebra fish (Brachydanio rerio), frog (Rana pipiens and Rana clamitans), salamander (Taricha torosa), snake (Elaphe obsolete and Nerodia fasciata), lizard (Anolis car-olinensis), chicken (Gallus gallus), rat (Rattus rattus and Rattus norvegicus), guinea pig (Cavia porcellus), rabbit (Oryctolagus cuniculus), dog (Canis familiaris), and pig (Suss-crofa domestica). Some of these species may have some advantages, such as the zebra fish, where powerful genetic tools involved in large-scale screening can be applied (84). Despite the novel application of MPTP to these species, there are limitations that restrict their popularity, including animal availability, biosafety exposure and disposal, genetic background, and standardization of lesioning regimen and its efficacy.

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