Nikolai Kholodilov

CHOP/GADD153 is a mediator of apoptotic death in substantia nigra dopamine neurons in an in vivo neurotoxin model of parkinsonism.

There is increasing evidence that neuron death in neurodegenerative diseases, such as Parkinsons 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.

Oncoprotein Akt/PKB induces trophic effects in murine models of Parkinson's disease

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 Parkinsons 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.

Increased Expression of Rat Synuclein in the Substantia Nigra Pars Compacta Identified by mRNA Differential Display in a Model of Developmental Target Injury

Abstract : Human α‐synuclein was identified on the basis of proteolytic fragments derived from senile plaques of Alzheimers disease, and it is the locus of mutations in some familial forms of Parkinsons disease. Its normal function and whether it may play a direct role in neural degeneration remain unknown. To explore cellular responses to neural degeneration in the dopamine neurons of the substantia nigra, we have developed a rodent model of apoptotic death induced by developmental injury to their target, the striatum. We find by mRNA differential display that synuclein is up‐regulated in this model, and thus it provides an opportunity to examine directly whether synuclein plays a role in the death of these neurons or, alternatively, in compensatory responses. Up‐regulation of mRNA is associated with an increase in the number of neuronal profiles immunostained for synuclein protein. At a cellular level, synuclein is almost exclusively expressed in normal neurons, rather than apoptotic profiles. Synuclein is up‐regulated throughout normal postnatal development of substantia nigra neurons, but it is not further up‐regulated during periods of natural cell death. We conclude that up‐regulation of synuclein in the target injury model is unlikely to mediate apoptotic death and propose that it may be due to a compensatory response in neurons destined to survive.

Akt Suppresses Retrograde Degeneration of Dopaminergic Axons by Inhibition of Macroautophagy

Axon degeneration is a hallmark of neurodegenerative diseases, including Alzheimers disease and Parkinsons 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.

Activation of Caspase-3 in Developmental Models of Programmed Cell Death in Neurons of the Substantia Nigra

Abstract: Programmed cell death has been proposed to play a role in the death of neurons in acute and chronic degenerative neurologic disease. There is now evidence that the caspases, a family of cysteine proteases, mediate programmed cell death in various cells. In neurons, caspase‐3 (CPP32/Yama/apopain), in particular, has been proposed to play a role. We examined the expression of caspase‐3 in three models of programmed cell death affecting neurons of the substantia nigra in the rat: natural developmental neuron death and induced developmental death following either striatal target injury with quinolinic acid or dopamine terminal lesion with intrastriatal injection of 6‐hydroxydopamine. Using an antibody to the large (p17) subunit of activated caspase‐3, we have found that activated enzyme is expressed in apoptotic profiles in all models. Increased p17 immunostaining correlated with increased enzyme activity. The sub‐cellular distribution of activated caspase‐3 differed among the models: In natural cell death and the target injury model, it was strictly nuclear, whereas in the toxin model, it was also cytoplasmic. We conclude that p17 immunostaining is a useful marker for programmed cell death in neurons of the substantia nigra.

Dopaminergic pathway reconstruction by Akt/Rheb-induced axon regeneration.

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 protein kinase B (Akt)/mammalian target of rapamycin (mTor) signaling may restore this ability.

Regulation of the development of mesencephalic dopaminergic systems by the selective expression of glial cell line-derived neurotrophic factor in their targets

Glial cell line-derived neurotrophic factor (GDNF) has been shown to protect and restore dopamine (DA) neurons in injury models and is being evaluated for the treatment of Parkinsons disease. Nevertheless, little is known of its physiological role. We have shown that GDNF suppresses apoptosis in DA neurons of the substantia nigra (SN) postnatally both in vitro and during their first phase of natural cell death in vivo. Furthermore, intrastriatal injection of neutralizing antibodies augments cell death, suggesting that endogenous GDNF plays a role as a target-derived factor. Such a role would predict that overexpression of GDNF in striatum would increase the surviving number of SN DA neurons. To test this hypothesis, we used the tetracycline-dependent transcription activator (tTA)/tTA-responsive promoter system to create mice that overexpress GDNF selectively in the striatum, cortex, and hippocampus. These mice demonstrate an increased number of SN DA neurons after the first phase of natural cell death. However, this increase does not persist into adulthood. As adults, these mice also do not have increased dopaminergic innervation of the striatum. They do, however, demonstrate increased numbers of ventral tegmental area (VTA) neurons and increased innervation of the cortex. This morphologic phenotype is associated with an increased locomotor response to amphetamine. We conclude that striatal GDNF is necessary and sufficient to regulate the number of SN DA neurons surviving the first phase of natural cell death, but it is not sufficient to increase their final adult number. GDNF in VTA targets, however, is sufficient to regulate the adult number of DA neurons.

Synuclein-1 is selectively up-regulated in response to nerve growth factor treatment in PC12 cells

Mutations in the α‐synuclein gene have recently been identified in families with inherited Parkinsons disease and the protein product of this gene is a component of Lewy bodies, indicating that α‐synuclein is involved in Parkinsons disease pathogenesis. A role for normal α‐synuclein in synaptic function, apoptosis or plasticity responses has been suggested. We show here that in rat pheochromocytoma PC12 cells synuclein‐1, the rat homolog of human α‐synuclein, is highly and selectively up‐regulated at the mRNA and protein levels after 7 days of nerve growth factor treatment. Synuclein‐1 expression appears neither sufficient nor necessary for the neuritic sprouting that occurs within 1–2 days of nerve growth factor treatment. Rather, it likely represents a component of a late neuronal maturational response. Synuclein‐1 redistributes diffusely within the cell soma and the neuritic processes in nerve growth factor‐treated PC12 cells. Cultured neonatal rat sympathetic neurones express high levels of synuclein‐1, with a diffuse intracellular distribution, similar to neuronal PC12 cells. These results suggest that levels of synuclein‐1 may be regulated by neurotrophic factors in the nervous system and reinforce a role for α‐synuclein in plasticity‐maturational responses. In contrast, there is no correlation between synuclein expression and apoptotic death following trophic deprivation.

AAV Transduction of Dopamine Neurons With Constitutively Active Rheb Protects From Neurodegeneration and Mediates Axon Regrowth

There are currently no therapies that provide either protection or restoration of neuronal function for adult-onset neurodegenerative diseases such as Parkinsons 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.

JNK2 and JNK3 combined are essential for apoptosis in dopamine neurons of the substantia nigra, but are not required for axon degeneration.

Activation of c‐jun N‐terminal kinase (JNK) by the mitogen‐activated protein kinase cascade has been shown to play an important role in the death of dopamine neurons of the substantia nigra, one of the principal neuronal populations affected in Parkinson’s disease. However, it has remained unknown whether the JNK2 and JNK3 isoforms, either singly or in combination, are essential for apoptotic death, and, if so, the mechanisms involved. In addition, it has been unclear whether they play a role in axonal degeneration of these neurons in disease models. To address these issues we have examined the effect of single and double jnk2 and jnk3 null mutations on apoptosis in a highly destructive neurotoxin model, that induced by intrastriatal 6‐hydroxydopamine. We find that homozygous jnk2/3 double null mutations result in a complete abrogation of apoptosis and a prolonged survival of the entire population of dopamine neurons. In spite of this complete protection at the cell soma level, there was no protection of axons. These studies provide a striking demonstration of the distinctiveness of the mechanisms that mediate cell soma and axon degeneration, and they illustrate the need to identify and target pathways of axon degeneration in the development of neuroprotective therapeutics.