Experimental animal models of a-synucleinopathies are being produced by a number of laboratories (Table 2). They are essential for studying disease pathogenesis and for identifying ways to interfere with the disease process. Several transgenic mouse lines that express wild-type or mutant human
Animal Models of a-Synucleinopathies
Toxin Model Genetic Models
Rotenone Worm Fly Mouse Rat Marmoset
Nerve cell loss
Filamentous + - + + u u a-Synuclein inclusions
The rotenone model is in the rat. The genetic models in worm, fly, and mouse are based on the transgenic expression of human a-synuclein (wild-type and mutant), whereas the rat and marmoset models are based on viral vector-mediated transfer of human a-synuclein (wild-type and mutant). u = unknown.
a-synuclein in nerve cells have been described. One study has reported on the effects of a-synuclein overexpression in glial cells. In all published studies, mice developed numerous a-synuclein-immu-noreactive cell bodies and processes.
The first study to be published described the expression of wild-type human a-synuclein driven by the human platelet-derived growth factor-p promoter (117). The mice developed cytoplasmic and nuclear intraneuronal inclusions in neocortex, hippocampus, olfactory bulb, and substantia nigra. These inclusions were a-synuclein-immunoreactive, with some being ubiquitin-positive as well. By electron microscopy, they consisted of amorphous, nonfilamentous material. The mice showed a reduction in dopaminergic nerve terminals in the striatum and signs of impaired motor function, but they failed to exhibit nerve cell loss in the substantia nigra. Two studies have reported the expression of wild-type and A53T or A30P mutant human a-synuclein driven by the murine Thy-1 promoter. In one study (118), mice expressing wild-type or A53T mutant a-synuclein developed an early-onset motor impairment that was associated with axonal degeneration in the ventral roots and signs of muscle atrophy. Some a-synuclein inclusions were argyrophilic and ubiquitin-immunoreactive, but they lacked the filaments characteristic of the human diseases. In the second study (119,120), mice expressing A30P mutant a-synuclein developed a neurodegenerative phenotype consisting of the accumulation of protease-resistant human a-synuclein phosphorylated at S129. Occasional inclusions were also ubiquitin-positive. By electron microscopy, filamentous structures were observed, although they were not shown to be made of a-synuclein. The transgenic mice showed a progressive deterioration of motor function. It remains to be seen whether the accumulation of mutant a-synuclein was accompanied by nerve cell loss.
Three studies have described the expression of wild-type and A53T or A30P mutant human a-synuclein under the control of the murine prion protein promoter (121-123). A severe movement disorder was observed that was accompanied by the accumulation of a-synuclein in nerve cells and their processes. One study documented the presence of abundant a-synuclein filaments in brain and spinal cord of mice transgenic for A53T a-synuclein (121). The formation of filamentous inclusions closely correlated with the appearance of clinical symptoms, suggesting a possible cause-and-effect relationship. A minority of inclusions was ubiquitin-immunoreactive. Signs of Wallerian degeneration were much in evidence in ventral roots, but nerve cell numbers in the ventral horn of the spinal cord were unchanged. A major difference with PD was the absence of significant pathology in dopam-
inergic nerve cells of the substantia nigra. In mice, these neurons appear to be relatively resistant to the effects of a-synuclein expression. This is further supported by reports showing that the expression of wild-type and mutant human a-synuclein under the control of the tyrosine hydroxylase promoter did not lead to the formation of inclusions or neurodegeneration (124,125).
Mouse lines transgenic for wild-type human a-synuclein under the control of a proteolipid protein promoter were generated to give high levels of expression in oligodendroglia (126). Expression of human a-synuclein phosphorylated at S129 was obtained, but there was no sign of argyrophilic glial cytoplasmic inclusions or abnormal filaments. Behavioral changes were also not observed.
One of the first reports describing the overexpression of a-synuclein made use of Drosophila melanogaster, an organism without synucleins (127). Expression of wild-type and A30P or A53T mutant human a-synuclein in nerve cells of D. melanogaster resulted in the formation of filamentous Lewy body-like inclusions and an age-dependent loss of some dopaminergic nerve cells (Table 2). The inclusions were a-synuclein- and ubiquitin-immunoreactive. An age-dependent locomotor defect was observed that could be reversed by the administration of L-DOPA or several dopamine agonists, underscoring the validity of this model for PD (128). Overexpression of wild-type and A53T mutant human a-synuclein in nerve cells of Caenorhabditis elegans resulted in a loss of dopaminergic nerve cells and motor deficits, in the apparent absence of filamentous a-synuclein inclusions (Table 2) (129).
Disease models in D. melanogaster and C. elegans offer some advantages over mouse models, in particular with regard to the speed and relative ease with which genetic modifiers of disease pheno-type can be discovered and pharmacological modifiers can be screened. Coexpression of human heat-shock protein 70 alleviated the toxicity of a-synuclein in transgenic flies (130). Conversely, a reduction in the fly chaperone system exacerbated nerve cell loss. Increasing chaperone activity through the administration of geldanamycin delayed neurodegeneration in transgenic flies (131). It thus appears that chaperones can modulate the neurotoxicity resulting from the overexpression of human a-synuclein.
Viral vector-mediated gene transfer differs from standard transgenic approaches by being targeted to a defined region of the central nervous system and by being inducible at any point during the life of the animal. Recombinant adeno-associated virus and recombinant lentivirus vector systems have been used to express wild-type and mutant a-synuclein in the substantia nigra of rat and marmoset (Table 2).
In the rat, expression of a-synuclein was maximal 2-3 wk after virus injection (132-134). At 8-10 wk, numerous swollen, dystrophic axons and dendrites were observed in conjunction with a-synuclein inclusions. Nerve cell bodies contained Lewy body-like inclusions and substantial nerve cell loss (3080%) was present in the substantia nigra. The inclusions were a-synuclein-positive and ubiquitin-negative, but it remains to be determined whether they were also filamentous. Behavior-ally, about a quarter of animals were impaired in spontaneous and drug-induced motor behaviors. Similar findings were reported for wild-type and A30P or A53T mutant human a-synuclein. One study has reported that degeneration of transduced nigral cells was seen upon expression of human, but not rat, a-synuclein (134). In the marmoset, dopaminergic nerve cells of the substantia nigra were transduced with high efficiency and human a-synuclein was expressed (135). Similar to the rat, a-synuclein-positive inclusions were observed, together with a loss of 40-75% of tyrosine hydroxylase-positive neurons. Overexpression of disease-causing gene products by using recombinant viral vectors constitutes a promising way forward for modeling human neurodegenerative diseases in a number of species, including primates.
A model of a-synuclein pathology has been developed in the rat by using the chronic administration of the pesticide rotenone, a high-affinity inhibitor of complex I, one of the five enzyme complexes of the inner mitochondrial membrane involved in oxidative phosphorylation (136,137). The rats developed a progressive degeneration of nigrostriatal neurons and Lewy body-like inclusions that were immunoreactive for a-synuclein and ubiquitin (Table 2). Behaviorally, they showed bradykinesia, postural instability, and some evidence of resting tremor. Using this regime of rotenone administration, the inhibition of complex I was only partial, indicating that a bioenergetic defect with ATP (adenosine 5'-triphosphate) depletion was probably not involved. Instead, oxidative damage might have contributed to this condition, as partial inhibition of complex I by rotenone is known to stimulate the production of reactive oxygen species. It would therefore seem that oxidative stress can lead to the assembly of a-synuclein into filaments. Although it remains to be seen how robust a model rotenone administration is, it appears clear that it can lead to the degeneration of nigrostriatal dopaminergic nerve cells in association with a-synuclein-positive inclusions. This has so far not been achieved following the administration of either 6-hydroxydopamine or MPTP, the two most widely used toxin models of PD. It has been reported that the systemic administration of rotenone leads to the specific degeneration of dopaminergic nerve cells in the substantia nigra. However, a subsequent study has described additional nerve cell loss in the striatum (138), raising the question of how valid a model rotenone intoxication is for PD.
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