Parkinson's disease (PD) is most commonly a sporadic illness, and is characterized by degeneration of substantia nigra dopamine (DA) neurons and abnormal cytoplasmic aggregates of ␣-synuclein. Rarely, PD may be caused by missense mutations in ␣-synuclein. MPTP, a neurotoxin that inhibits mitochondrial complex I, is a prototype for an environmental cause of PD because it produces a pattern of DA neurodegeneration that closely resembles the neuropathology of PD. Here we show that ␣-synuclein null mice display striking resistance to MPTP-induced degeneration of DA neurons and DA release, and this resistance appears to result from an inability of the toxin to inhibit complex I. Contrary to predictions from in vitro data, this resistance is not due to abnormalities of the DA transporter, which appears to function normally in ␣-synuclein null mice. Our results suggest that some genetic and environmental factors that increase susceptibility to PD may interact with a common molecular pathway, and represent the first demonstration that normal ␣-synuclein function may be important to DA neuron viability.T he concept of genetic predisposition to disease suggests that one's genes influence susceptibility to environmental insult. However, the relationship between genetic and environmental factors is poorly understood; most models of disease focus on single genes or toxins. A major challenge of postgenomic biology will be to link the molecular pathways modified by diseaseassociated alleles to the environmental factors implicated in disease susceptibility.There is increasing evidence for genetic susceptibility to Parkinson's disease (PD) (1-3). Additionally, dysfunction of a common molecular pathway has been implicated in the familial and sporadic forms of PD. Mutations in the gene that encodes ␣-synuclein cause a rare form of dominantly inherited PD, and ␣-synuclein is an abundant protein in Lewy bodies, the proteinaceous neuronal inclusions that are the pathological hallmark of sporadic PD (4-6). The ␣-synuclein pathway is also implicated in an autosomal recessive form of PD caused by mutations in the gene encoding parkin (7,8). Epidemiological and twin studies suggest that environmental factors alter susceptibility to PD (9). The fact that exposure of humans to the environmental toxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) causes a syndrome that mimics the core neurological symptoms and relatively selective dopamine (DA) neuron degeneration of PD lends support to this concept (10, 11).We asked whether a model neurotoxin for an environmental cause of PD might act on a molecular pathway implicated in genetic and sporadic forms of the disease by generating ␣-synuclein null mice, and testing whether they display altered sensitivity to MPTP-induced degeneration of substantia nigra (SN) DA neurons. MethodsAnimal Generation. A 5.7-kb EcoRV mouse ␣-synuclein fragment (Fig. 1A) was used to generate the targeting construct. A DNA fragment containing, in order, LoxP-phosphoglycerate kinaseNeomycin-transcription blocking '...

mTOR is a regulator of cell growth and survival, protein synthesis-dependent synaptic plasticity, and autophagic degradation of cellular components. When triggered by mTOR inactivation, macroautophagy degrades long-lived proteins and organelles via sequestration into autophagic vacuoles. mTOR further regulates synaptic plasticity, and neurodegeneration occurs when macroautophagy is deficient. Nevertheless, whether macroautophagy is a modulator of presynaptic function was previously unknown. We find that the mTOR inhibitor, rapamycin, induces formation of autophagic vacuoles in prejunctional dopaminergic axons with associated decreased axonal profile volumes, synaptic vesicle numbers, and evoked dopamine release. Evoked dopamine secretion was enhanced and recovery accelerated in transgenic mice in which macroautophagy deficiency was restricted to dopaminergic neurons; rapamycin failed to decrease evoked dopamine release in the striatum of these mice. Macroautophagy that follows mTOR inhibition in presynaptic terminals, therefore, rapidly alters presynaptic structure and neurotransmission.

There is increasing evidence that neuron death in neurodegenerative diseases, such as Parkinson's disease, is due to the activation of programmed cell death. However, the upstream mediators of cell death remain largely unknown. One approach to the identification of upstream mediators is to perform gene expression analysis in disease models. Such analyses, performed in tissue culture models induced by neurotoxins, have identified up-regulation of CHOP/ GADD153, a transcription factor implicated in apoptosis due to endoplasmic reticulum stress or oxidative injury. To evaluate the disease-related significance of these findings, we have examined the expression of CHOP/GADD153 in neurotoxin models of parkinsonism in living animals. Nuclear expression of CHOP protein is observed in developmental and adult models of dopamine neuron death induced by intrastriatal injection of 6-hydroxydopamine (6OHDA) and in models induced by 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP). CHOP is a mediator of neuron death in the adult 60HDA model because a null mutation results in a reduction in apoptosis. In the chronic MPTP model, however, while CHOP is robustly expressed, the null mutation does not protect from the loss of neurons. We conclude that the role of CHOP depends on the nature of the toxic stimulus. For 6OHDA, an oxidative metabolite of dopamine, it is a mediator of apoptotic death.

Despite promising preclinical studies, neurotrophic factors have not yet achieved an established role in the treatment of human neurodegenerative diseases. One impediment has been the difficulty in providing these macromolecules in sufficient quantity and duration at affected sites. An alternative approach is to directly activate, by viral vector transduction, intracellular signaling pathways that mediate neurotrophic effects. We have evaluated this approach in dopamine neurons of the substantia nigra, neurons affected in Parkinson's disease, by adeno-associated virus 1 transduction with a gene encoding a myristoylated, constitutively active form of the oncoprotein Akt/PKB. Adeno-associated virus Myr-Akt has pronounced trophic effects on dopamine neurons of adult and aged mice, including increases in neuron size, phenotypic markers, and sprouting. Transduction confers almost complete protection against apoptotic cell death in a highly destructive neurotoxin model. Activation of intracellular neurotrophic signaling pathways by vector transfer is a feasible approach to neuroprotection and restorative treatment of neurodegenerative disease.apoptosis ͉ dopamine ͉ neurotrophic ͉ programmed cell death ͉ substantia nigra

There are currently no therapies that provide either protection or restoration of neuronal function for adult-onset neurodegenerative diseases such as Parkinson's disease (PD). Many clinical efforts to provide such benefits by infusion of neurotrophic factors have failed, in spite of robust effects in preclinical assessments. One important reason for these failures is the difficulty, due to diffusion limits, of providing these protein molecules in sufficient amounts to the intended cellular targets in the central nervous system. This challenge suggests an alternative approach, that of viral vector transduction to directly activate the intracellular signaling pathways that mediate neurotrophic effects. To this end we have investigated the ability of a constitutively active form of the GTPase Rheb, an important activator of mammalian target of rapamycin (mTor) signaling, to mediate neurotrophic effects in dopamine neurons of the substantia nigra (SN), a population of neurons affected in PD. We find that constitutively active hRheb(S16H) induces many neurotrophic effects in mice, including abilities to both preserve and restore the nigrostriatal dopaminergic axonal projections in a highly destructive neurotoxin model. We conclude that direct viral vector transduction of vulnerable neuronal populations to activate intracellular neurotrophic signaling pathways offers promise for the treatment of neurodegenerative disease.

Objective-A prevailing concept in neuroscience has been that the adult mammalian central nervous system is incapable of restorative axon regeneration. Recent evidence, however, has suggested that reactivation of intrinsic cellular programs regulated by Akt/mTor signaling may restore this ability.Methods-To assess this possibility in the brain, we have examined the ability of adenoassociated virus mediated transduction of dopaminergic neurons of the substantia nigra with constitutively active forms of the kinase Akt and the GTPase Rheb to induce re-growth of axons after they have been destroyed by neurotoxin lesion.Results-Both constitutively active myristoylated Akt and hRheb(S16H) induce regrowth of axons from dopaminergic neurons to their target, the striatum. Histological analysis demonstrates that these new axons achieve morphologically accurate reinnervation. In addition, functional reintegration into target circuitry is achieved, as indicated by partial behavioral recovery.Interpretation-We conclude that regrowth of axons within the adult nigro-striatal projection, a system that is prominently affected in Parkinson's disease, can be achieved by activation of Akt/ mTor signaling in surviving endogenous mesencephalic dopaminergic neurons by viral vector transduction.

Axon degeneration is a hallmark of neurodegenerative diseases, including Alzheimer's disease and Parkinson's disease. Such degeneration is not a passive event but rather an active process mediated by mechanisms that are distinct from the canonical pathways of programmed cell death that mediate destruction of the cell soma. Little is known of the diverse mechanisms involved, particularly those of retrograde axon degeneration. We have previously observed in living animal models of degeneration in the nigrostriatal projection that a constitutively active form of the kinase, myristoylated Akt (Myr-Akt), demonstrates an ability to suppress programmed cell death and preserve the soma of dopamine neurons. Here, we show in both neurotoxin and physical injury (axotomy) models that Myr-Akt is also able to preserve dopaminergic axons due to suppression of acute retrograde axon degeneration. This cellular phenotype is associated with increased mammalian target of rapamycin (mTor) activity and can be recapitulated by a constitutively active form of the small GTPase Rheb, an upstream activator of mTor. Axon degeneration in these models is accompanied by the occurrence of macroautophagy, which is suppressed by Myr-Akt. Conditional deletion of the essential autophagy mediator Atg7 in adult mice also achieves striking axon protection in these acute models of retrograde degeneration. The protection afforded by both Myr-Akt and Atg7 deletion is robust and lasting, because it is still observed as protection of both axons and dopaminergic striatal innervation weeks after injury. We conclude that acute retrograde axon degeneration is regulated by Akt/Rheb/mTor signaling pathways.

The transduction of dopaminergic (DA) neurons with human ras homolog enriched in brain, which has a S16H mutation [hRheb(S16H)] protects the nigrostriatal DA projection in the 6-hydroxydopamine (6-OHDA)-treated animal model of Parkinson's disease (PD). However, it is still unclear whether the expression of active hRheb induces the production of neurotrophic factors such as glial cell line-derived neurotrophic factor (GDNF) and brain-derived neurotrophic factor (BDNF), which are involved in neuroprotection, in mature neurons. Here, we show that transduction of nigral DA neurons with hRheb(S16H) significantly increases the levels of phospho-cyclic adenosine monophosphate (cAMP) response element-binding protein (p-CREB), GDNF, and BDNF in neurons, which are attenuated by rapamycin, a specific inhibitor of mammalian target of rapamycin complex 1 (mTORC1). Moreover, treatment with specific neutralizing antibodies for GDNF and BDNF reduced the protective effects of hRheb(S16H) against 1-methyl-4-phenylpyridinium (MPP(+))-induced neurotoxicity. These results show that activation of hRheb/mTORC1 signaling pathway could impart to DA neurons the important ability to continuously produce GDNF and BDNF as therapeutic agents against PD.