Parkinson's disease: Mechanisms and models

Biology of the Mouse Histocompatibility-2 Complex

Animal models of Parkinson's disease (PD) have proved highly effective in the discovery of novel treatments for motor symptoms of PD and in the search for clues to the underlying cause of the illness. Models based on specific pathogenic mechanisms may subsequently lead to the development of neuroprotective agents for PD that stop or slow disease progression. The array of available rodent models is large and ranges from acute pharmacological models, such as the reserpine- or haloperidol-treated rats that display one or more parkinsonian signs, to models exhibiting destruction of the dopaminergic nigro-striatal pathway, such as the classical 6-hydroxydopamine (6-OHDA) rat and 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) mouse models. All of these have provided test beds in which new molecules for treating the motor symptoms of PD can be assessed. In addition, the emergence of abnormal involuntary movements (AIMs) with repeated treatment of 6-OHDA-lesioned rats with L-DOPA has allowed for examination of the mechanisms responsible for treatment-related dyskinesia in PD, and the detection of molecules able to prevent or reverse their appearance. Other toxin-based models of nigro-striatal tract degeneration include the systemic administration of the pesticides rotenone and paraquat, but whilst providing clues to disease pathogenesis, these are not so commonly used for drug development. The MPTP-treated primate model of PD, which closely mimics the clinical features of PD and in which all currently used anti-parkinsonian medications have been shown to be effective, is undoubtedly the most clinically-relevant of all available models. The MPTP-treated primate develops clear dyskinesia when repeatedly exposed to L-DOPA, and these parkinsonian animals have shown responses to novel dopaminergic agents that are highly predictive of their effect in man. Whether non-dopaminergic drugs show the same degree of predictability of response is a matter of debate. As our understanding of the pathogenesis of PD has improved, so new rodent models produced by agents mimicking these mechanisms, including proteasome inhibitors such as PSI, lactacystin and epoximycin or inflammogens like lipopolysaccharide (LPS) have been developed. A further generation of models aimed at mimicking the genetic causes of PD has also sprung up. Whilst these newer models have provided further clues to the disease pathology, they have so far been less commonly used for drug development. There is little doubt that the availability of experimental animal models of PD has dramatically altered dopaminergic drug treatment of the illness and the prevention and reversal of drug-related side effects that emerge with disease progression and chronic medication. However, so far, we have made little progress in moving into other pharmacological areas for the treatment of PD, and we have not developed models that reflect the progressive nature of the illness and its complexity in terms of the extent of pathology and biochemical change. Only when this occurs are we likely to make progress in developing agents to stop or slow the disease progression. The overarching question that draws all of these models together in the quest for better drug treatments for PD is how well do they recapitulate the human condition and how predictive are they of successful translation of drugs into the clinic? This article aims to clarify the current position and highlight the strengths and weaknesses of available models.

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This article is part of a themed issue on Translational Neuropharmacology. To view the other articles in this issue visit http://dx.doi.org/10.1111/bph.2011.164.issue-4

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Animal models of Parkinson's disease: a source of novel treatments and clues to the cause of the disease

The T-locus of the mouse

Publisher Summary This chapter discusses the ecdysone receptors and their biological actions. It also summarizes the insect endocrinology and the roles of these steroids in the molting and metamorphosis. Natural hormones that lead to molting and metamorphosis are ecdysones. Molecules whose structures resemble those of the natural hormones are called ecdysteroids. The receptor for ecdysone is a member of the nuclear receptor superfamily that acts as a ligand-dependent transcription factor. The vertebrate steroid hormone receptors act as homodimers whereas the functional ecdysone receptor is always a heterodimer of receptor for ecdysone (EcR) with another member of the nuclear receptor (NR) superfamily, Ultraspiracle, the insect homolog of the vertebrate retinoid X receptor (RXR). Two new technologies promise to transform the environment for investigations of insect hormones, ecdysone receptor, and metamorphosis. The microarrays of expressed sequence tags are constructed (ESTs) and used hybridization to catalog changes in gene expression during metamorphosis. The first technological achievement is reviewed: the complete sequence of the Drosophila genome is obtained and is about to be released. It will be the first complete insect sequence and also the first genomic sequence from an organism that has served as a model for nuclear receptor endocrinology.

Ecdysone receptors and their biological actions.

The human gene that codes for the protein alpha-synuclein has been transferred into the Drosophila melanogaster genome. The transgenic flies recapitulate some of the essential features of Parkinson's disease. These include the degeneration of certain dopaminergic neurons in the brain accompanied by the appearance of age-dependent abnormalities in locomotor activity. In the present study, we tested the locomotor response of these transgenic flies to prototypes of the major classes of drugs currently used to treat this disorder. A time course study was first conducted to determine when impaired locomotor activity appeared relative to normal "wild-type" flies. A climbing or negative geotaxis assay measuring the ability of the organisms to climb up the walls of a plastic vial was used. Based on the results obtained, normal and transgenic flies were treated with each of the drugs in their food for 13 days and then assayed. The activity of transgenic flies treated with L-DOPA was restored to normal. Similarly, the dopamine agonists pergolide, bromocriptine, and 2,3,4,5-tetrahydro-7,8-dihydroxy- 1-phenyl-1H-3-benzazepine (SK&F 38393) were substantially effective. Atropine, the prototypical muscarinic cholinergic receptor antagonist, was also effective but to a lesser extent than the other antiparkinson compounds. p-Chlorophenylalanine, an inhibitor of serotonin synthesis, was without beneficial effect as was alpha-methyl-p-tyrosine, an inhibitor of tyrosine hydroxylase, the rate-limiting step in catecholamine biosynthesis. This behavioral study further demonstrates the utility of this model in studying Parkinson's disease and reinforces the concept that inhibition of the action of alpha-synuclein may be useful in its treatment as may dopamine D(1) receptor agonists.

Effects of pharmacological agents upon a transgenic model of Parkinson's disease in Drosophila melanogaster.

The brain of the adult fruit fly, Drosophila melanogaster, contains tyrosine hydroxylase, the rate-limiting enzyme required for catecholamine biosynthesis, as well as dopa decarboxylase. Catecholamines, principally dopamine, are also present. We have previously shown that pharmacological inhibition of tyrosine hydroxylase with α-methyl-p-tyrosine results in a dose-related inhibition of locomotor activity in adult organisms. Similar results were found with reserpine, a well-known inhibitor of catecholamine uptake into storage granules. The drug-induced inhibition could be prevented in each case by the concomitant administration of l-dopa. The single-copy gene coding for tyrosine hydroxylase in Drosophila is pale (ple). Both null and temperature-sensitive loss of function mutant alleles of ple are recessive embryonic lethals. Heterozygous null mutant flies have normal locomotor activity demonstrating that only a single dose of the wild type form of ple is required to support normal function. Both hemizygous and homozygous temperature-sensitive ple mutants (plets1) also show normal locomotor activity at the permissive temperature for this mutant allele (18°C), which progressively declines as the temperature is increased to its restrictive level (29°C). These abnormal locomotor effects are reversible by l-dopa. Thus the effects on locomotor activity resulting from the pharmacological inhibition of catecholamine synthesis or storage are the same as those resulting from lack of tyrosine hydroxylase expression. These findings indicate that brain catecholamine loss decreases locomotor activity in the fly, as it does in mammals, and demonstrate the ability of functional genomic studies to mimic that of pharmacological inhibition of enzyme function or other similar processes.

Effects of tyrosine hydroxylase mutants on locomotor activity in Drosophila: a study in functional genomics.

In Drosophila melanogaster embryos cuticle formation occurs between 12 and 16 hours of development at 25°C. The formation of the cuticulin and the protein epicuticular layers is simultaneous in the hypoderm, the tracheoblasts, and the fore- and hindgut cells. The cuticulin forms as a dual lamina, aggregating from granules secreted by the hypodermal cells. This is followed by the formation of a granular protein epicuticle and finally by the secretion of a mixed fibrous and granular endocuticle.



All secretory cells are relatively simple in their ultrastructure. The secretory process is a membrane phenomenon, occurring at the tips of hypodermal microvillae on cells at the surface of the embryo and on those hypodermal cells lining the lumen of the fore- and hindgut. It also occurs along the entire surface of the tracheoblast lumen as well as on the outer surface of those cells which form exoskeletal chitinous setae. The process involves a specialization of the plasma membrane with the formation of secretory granules intracellularly beneath the membrane and the extrusion of these granules through the membrane to the outside where final cuticle formation occurs.

Cuticle formation in the embryo of Drosophila melanogaster

A statistical study of embryos obtained from both spontaneously ovulated and superovulated +/t12 females, mated inter se, shows that the range of the lethal phenocritical period of the t12 allele in a homozygous condition is from the 8–12 cell stage to the early blastocyst stage. The majority of t12 homozygotes are developmentally arrested as late morula and the nuclei of these embryos contain lipid droplets and fibrillo-granular bodies. These same inclusions are found in other t12 embryos which are developmentally arrested either earlier or later than the late morula stage and distinguish 30–40% of the embryos (presumably t12 homozygotes) from their litter-mates at the 2- and 4-cell stages. Ultrastructural-cytochemical studies of the fibrillo-granular bodies show that the fibrillar areas are sensitive to pepsin and the granules to ribonuclease and are thus structurally and chemically similar to definitive nucleoli. Binucleate cells are also present in a high frequency of t12 homozygous embryos. This condition is considered an additional phenotypic expression of the genotype.



Prior to developmental arrest, the nuclear and cytoplasmic organelles of t12 embryos do not differ from those of similarly staged litter-mates or control +/+ embryos. Homozygous mutant embryos examined shortly following developmental arrest contain cells ranging from structurally normal to degenerative. Asynchronous cell death is common to all t12 homozygous embryos. A chronological description of degenerative cellular changes is presented.

Ralph Hillman

Papers

Effects of pharmacological agents upon a transgenic model of Parkinson's disease in Drosophila melanogaster.

Effects of tyrosine hydroxylase mutants on locomotor activity in Drosophila: a study in functional genomics.

Cuticle formation in the embryo of Drosophila melanogaster

Ultrastructural studies of cleavage stage t12/t12 mouse embryos.

Reproduction and development in Drosophila are dependent upon catecholamines