Cellular Senescence Is Induced by the Environmental Neurotoxin Paraquat and Contributes to Neuropathology Linked to Parkinson's Disease.

Exposure to the herbicide paraquat (PQ) is associated with an increased risk of idiopathic Parkinson's disease (PD). Therapies based on PQ's presumed mechanisms of action have not, however, yielded effective disease therapies. Cellular senescence is an anticancer mechanism that arrests proliferation of replication-competent cells and results in a pro-inflammatory senescence-associated secretory phenotype (SASP) capable of damaging neighboring tissues. Here, we demonstrate that senescent cell markers are preferentially present within astrocytes in PD brain tissues. Additionally, PQ was found to induce astrocytic senescence and an SASP in vitro and in vivo, and senescent cell depletion in the latter protects against PQ-induced neuropathology. Our data suggest that exposure to certain environmental toxins promotes accumulation of senescent cells in the aging brain, which can contribute to dopaminergic neurodegeneration. Therapies that target senescent cells may constitute a strategy for treatment of sporadic PD, for which environmental exposure is a major risk factor.

A novel iron (II) preferring dopamine agonist chelator D-607 significantly suppresses α-syn- and MPTP-induced toxicities in vivo.

Here, we report the characterization of a novel hybrid D2/D3 agonist and iron (II) specific chelator, D-607, as a multi-target-directed ligand against Parkinson's disease (PD). In our previously published report, we showed that D-607 is a potent agonist of dopamine (DA) D2/D3 receptors, exhibits efficacy in a reserpinized PD animal model and preferentially chelates to iron (II). As further evidence of its potential as a neuroprotective agent in PD, the present study reveals D-607 to be protective in neuronal PC12 cells against 6-OHDA toxicity. In an in vivo Drosophila melanogaster model expressing a disease-causing variant of α-synuclein (α-Syn) protein in fly eyes, the compound was found to significantly suppress toxicity compared to controls, concomitant with reduced levels of aggregated α-Syn. Furthermore, D-607 was able to rescue DAergic neurons from MPTP toxicity in mice, a well-known PD neurotoxicity model, following both sub-chronic and chronic MPTP administration. Mechanistic studies indicated that possible protection of mitochondria, up-regulation of hypoxia-inducible factor, reduction in formation of α-Syn aggregates and antioxidant activity may underlie the observed neuroprotection effects. These observations strongly suggest that D-607 has potential as a promising multifunctional lead molecule for viable symptomatic and disease-modifying therapy for PD.

Regulation of ATP13A2 via PHD2-HIF1α Signaling Is Critical for Cellular Iron Homeostasis: Implications for Parkinson's Disease.

We previously reported that pharmacological inhibition of a class of enzymes known as prolyl hydroxylase domain proteins (PHDs) has neuroprotective effects in various in vitro and in vivo models of Parkinson's disease (PD). We hypothesized that this was due to inhibition of the PHD2 isoform, preventing it from hydroxylating the transcription factor hypoxia inducible factor 1 α (HIF1α), targeting it for eventual proteasomal degradation. HIF1α itself induces the transcription of various cellular stress genes, including several involved in iron metabolism. Although all three isoforms of PHD are expressed within vulnerable dopaminergic (DAergic) substantia nigra pars compacta neurons, only select downregulation of the PHD2 isoform was found to protect against in vivo neurodegenerative effects associated with the mitochondrial neurotoxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine. These findings were corroborated in induced pluripotent stem cell-derived neurons, providing validation in a pertinent human cell model. PHD2 inhibition was found to result in increased expression of ATP13A2, mutation of which is responsible for a rare juvenile form of PD known as Kufor-Rakeb syndrome. Knockdown of ATP13A2 expression within human DAergic cells was found to abrogate restoration of cellular iron homeostasis and neuronal cell viability elicited by inhibition of PHD2 under conditions of mitochondrial stress, likely via effects on lysosomal iron storage. These data suggest that regulation of ATP13A2 by the PHD2-HIF1α signaling pathway affects cellular iron homeostasis and DAergic neuronal survival. This constitutes a heretofore unrecognized process associated with loss of ATP13A2 function that could have wide-ranging implications for it as a therapeutic target for PD and other related conditions. SIGNIFICANCE STATEMENT: Reductions in PHD2 activity within dopaminergic neurons in vivo and in cultured human induced pluripotent stem cell-derived neurons protects against mitochondrial stress-induced neurotoxicity. Protective effects are dependent on downstream HIF-1α expression. Knockdown of ATP13A2, a gene linked to a rare juvenile form of Parkinson's disease and recently identified as a novel HIF1α target, was found to abrogate maintenance of cellular iron homeostasis and neuronal viability elicited by PHD2 inhibition in vivo and in cultured dopaminergic cells under conditions of mitochondrial stress. Mechanistically, this was due to ATP13A2's role in maintaining lysosomal iron stores. This constitutes a novel mechanism by which alterations in ATP13A2 activity may be driving PD-related neuropathology.

The high-affinity D2/D3 agonist D512 protects PC12 cells from 6-OHDA-induced apoptotic cell death and rescues dopaminergic neurons in the MPTP mouse model of Parkinson's disease.

In this study, in vitro and in vivo experiments were carried out with the high-affinity multifunctional D2/D3 agonist D-512 to explore its potential neuroprotective effects in models of Parkinson's disease and the potential mechanism(s) underlying such properties. Pre-treatment with D-512 in vitro was found to rescue rat adrenal Pheochromocytoma PC12 cells from toxicity induced by 6-hydroxydopamine administration in a dose-dependent manner. Neuroprotection was found to coincide with reductions in intracellular reactive oxygen species, lipid peroxidation, and DNA damage. In vivo, pre-treatment with 0.5 mg/kg D-512 was protective against neurodegenerative phenotypes associated with systemic administration of MPTP, including losses in striatal dopamine, reductions in numbers of DAergic neurons in the substantia nigra (SN), and locomotor dysfunction. These observations strongly suggest that the multifunctional drug D-512 may constitute a novel viable therapy for Parkinson's disease.

Pharmacological prolyl hydroxylase domain inhibition as a therapeutic target for Parkinson's disease.

Previously published data from our laboratory demonstrated that pharmacological inhibition of a family of enzymes known as prolyl hydroxylase domain proteins prevents neurotoxicity associated with the acute 1-methyl-4- phenyl-1,2,3,6-tetrahydropyridine intoxication model of Parkinson's disease in young animals. In this study, we assessed whether prolyl hydroxylase domain inhibition was neuroprotective in an inducible genetic dopaminergic glutathione depletion model previously characterized by our laboratory that more closely recapitulates the age-related and progressive nature of the human disease. Pharmacological prolyl hydroxylase domain inhibition via 3,4-dihydroxybenzoate was found to significantly attenuate hallmark mitochondrial dysfunction and loss of dopaminergic substantia nigral pars compacta neurons associated with this model. These studies further validate the possibility that prolyl hydroxylase domain inhibition may constitute a viable therapy for Parkinson's disease.

Chronic expression of H-ferritin in dopaminergic midbrain neurons results in an age-related expansion of the labile iron pool and subsequent neurodegeneration: implications for Parkinson's disease.

While ferritin elevation within dopaminergic (DA) neurons of the substantia nigra (SN) is protective against neurodegeneration elicited by two toxin models of Parkinson's disease (PD), MPTP and paraquat, in young animals, its prolonged elevation results in a selective age-related neurodegeneration. A similar age-related neurodegeneration has been reported in iron regulatory protein 2-deficient (IRP2 -/-) mice coinciding with increased ferritin levels within degenerating neurons. This has been speculated to be due to subsequent reductions in the labile iron pool (LIP) needed for the synthesis of iron-sulfur-containing enzymes. In order to assess whether LIP reduction is responsible for age-related neurodegeneration in our ferritin transgenics, we examined LIP levels in ferritin-expressing transgenics with increasing age. While LIP levels were reduced within DA SN nerve terminals isolated from young ferritin transgenics compared to wildtype littermate controls, they were found to be increased in older transgenic animals at the age at which selective neurodegeneration is first noted. Furthermore, administration of the bioavailable iron chelator, clioquinol (CQ), to older mice was found to protect against both increased LIP and subsequent dopaminergic neurodegeneration. This suggests that age-related neurodegeneration in these mice is likely due to increased iron availability rather than its reduction. This may have important implications for PD and other related neurodegenerative conditions in which iron and ferritin have been implicated.

Metabolic control analysis in a cellular model of elevated MAO-B: relevance to Parkinson's disease.

We previously demonstrated that spare respiratory capacity of the TCA cycle enzyme alpha-ketoglutarate dehydrogenase (KGDH) was completely abolished upon increasing levels of MAO-B activity in a dopaminergic cell model system (Kumar et al., J Biol Chem 278:46432-46439, 2003). MAO-B mediated increases in H(2)O(2) also appeared to result in direct oxidative inhibition of both mitochondrial complex I and aconitase. In order to elucidate the contribution that each of these components exerts over metabolic respiratory control as well as the impact of MAO-B elevation on their spare respiratory capacities, we performed metabolic respiratory control analysis. In addition to KGDH, we assessed the activities and substrate-mediated respiration of complex I, pyruvate dehydrogenase (PDH), succinate dehydrogenase (SDH), and mitochondrial aconitase in the absence and presence of complex-specific inhibitors in specific and mixed substrate conditions in mitochondria from our MAO-B elevated cells versus controls. Data from this study indicates that Complex I and KGDH are the most sensitive to inhibition by MAO-B mediated H(2)O(2) generation, and could be instrumental in determining the fate of mitochondrial metabolism in this cellular PD model system.

A disruption in iron-sulfur center biogenesis via inhibition of mitochondrial dithiol glutaredoxin 2 may contribute to mitochondrial and cellular iron dysregulation in mammalian glutathione-depleted dopaminergic cells: implications for Parkinson's disease.

Parkinson's disease (PD) is characterized by early glutathione depletion in the substantia nigra (SN). Among its various functions in the cell, glutathione acts as a substrate for the mitochondrial enzyme glutaredoxin 2 (Grx2). Grx2 is involved in glutathionylation of protein cysteine sulfhydryl residues in the mitochondria. Although monothiol glutathione-dependent oxidoreductases (Grxs) have previously been demonstrated to be involved in iron-sulfur (Fe-S) center biogenesis, including that in yeast, here we report data suggesting the involvement of mitochondrial Grx2, a dithiol Grx, in iron-sulfur biogenesis in a mammalian dopaminergic cell line. Given that mitochondrial dysfunction and increased cellular iron levels are two important hallmarks of PD, this suggests a novel potential mechanism by which glutathione depletion may affect these processes in dopaminergic neurons. We report that depletion of glutathione as substrate results in a dose-dependent Grx2 inhibition and decreased iron incorporation into a mitochondrial complex I (CI) and aconitase (m-aconitase). Mitochondrial Grx2 inhibition through siRNA results in a corresponding decrease in CI and m-aconitase activities. It also results in significant increases in iron-regulatory protein (IRP) binding, likely as a consequence of conversion of Fe-S-containing cellular aconitase to its non-Fe-S-containing IRP1 form. This is accompanied by increased transferrin receptor, decreased ferritin, and subsequent increases in mitochondrial iron levels. This suggests that glutathione depletion may affect important pathologic cellular events associated with PD through its effects on Grx2 activity and mitochondrial Fe-S biogenesis.

Inducible alterations of glutathione levels in adult dopaminergic midbrain neurons result in nigrostriatal degeneration.

Parkinson's disease is a neurodegenerative disorder characterized by the preferential loss of midbrain dopaminergic neurons in the substantia nigra (SN). One of the earliest detectable biochemical alterations that occurs in the Parkinsonian brain is a marked reduction in SN levels of total glutathione (glutathione plus glutathione disulfide), occurring before losses in mitochondrial complex I (CI) activity, striatal dopamine levels, or midbrain dopaminergic neurodegeneration associated with the disease. Previous in vitro data from our laboratory has suggested that prolonged depletion of dopaminergic glutathione results in selective impairment of mitochondrial complex I activity through a reversible thiol oxidation event. To address the effects of depletion in dopaminergic glutathione levels in vivo on the nigrostriatal system, we created genetically engineered transgenic mouse lines in which expression of gamma-glutamyl cysteine ligase, the rate-limiting enzyme in de novo glutathione synthesis, can be inducibly downregulated in catecholaminergic neurons, including those of the SN. A novel method for isolation of purified dopaminergic striatal synaptosomes was used to study the impact of dopaminergic glutathione depletion on mitochondrial events demonstrated previously to occur in vitro as a consequence of this alteration. Dopaminergic glutathione depletion was found to result in a selective reversible thiol-oxidation-dependent mitochondrial complex I inhibition, followed by an age-related nigrostriatal neurodegeneration. This suggests that depletion in glutathione within dopaminergic SN neurons has a direct impact on mitochondrial complex I activity via increased nitric oxide-related thiol oxidation and age-related dopaminergic SN cell loss.

Increased murine neonatal iron intake results in Parkinson-like neurodegeneration with age.

Iron elevation is well-documented in the Parkinsonian midbrain but its cause and contribution to subsequent neurodegeneration remain unknown. Mice administered iron at doses equivalent to those found in iron-fortified human infant formula during a developmental period equivalent to the first human year of life display progressive midbrain neurodegeneration and enhanced vulnerability to toxic injury. This may have major implications for the impact of neonatal iron intake as a potential risk factor for later development of Parkinson's disease (PD).

In vitro and in vivo neuroprotection by gamma-glutamylcysteine ethyl ester against MPTP: relevance to the role of glutathione in Parkinson's disease.

Glutathione is an abundant intracellular thiol antioxidant whose levels are reduced both in Parkinson's disease itself and in a widely used animal model of the disorder, systemic MPTP administration. Previous in vitro work from our laboratory has suggested that glutathione depletion may be directly responsible for mitochondrial dysfunction, which ultimately leads to dopaminergic cell death associated with the disease. Here, we demonstrate the ability of gamma-glutamylcysteine ethyl ester, a lipid permeable derivative of the major substrate for scavenger glutathione synthesis, to counteract glutathione loss and neurodegeneration associated with in vitro and in vivo administration of MPTP or its derivatives. This data suggests that prevention of glutathione depletion is a likely therapeutic target for the disease.

Genetic or pharmacological iron chelation prevents MPTP-induced neurotoxicity in vivo: a novel therapy for Parkinson's disease.

Studies on postmortem brains from Parkinson's patients reveal elevated iron in the substantia nigra (SN). Selective cell death in this brain region is associated with oxidative stress, which may be exacerbated by the presence of excess iron. Whether iron plays a causative role in cell death, however, is controversial. Here, we explore the effects of iron chelation via either transgenic expression of the iron binding protein ferritin or oral administration of the bioavailable metal chelator clioquinol (CQ) on susceptibility to the Parkinson's-inducing agent 1-methyl-4-phenyl-1,2,3,6-tetrapyridine (MPTP). Reduction in reactive iron by either genetic or pharmacological means was found to be well tolerated in animals in our studies and to result in protection against the toxin, suggesting that iron chelation may be an effective therapy for prevention and treatment of the disease.

Glutathione, iron and Parkinson's disease.

Parkinson's disease (PD) is a progressive neurodegenerative disease involving neurodegeneration of dopaminergic neurons of the substantia nigra (SN), a part of the midbrain. Oxidative stress has been implicated to play a major role in the neuronal cell death associated with PD. Importantly, there is a drastic depletion in cytoplasmic levels of the thiol tripeptide glutathione within the SN of PD patients. Glutathione (GSH) exhibits several functions in the brain chiefly acting as an antioxidant and a redox regulator. GSH depletion has been shown to affect mitochondrial function probably via selective inhibition of mitochondrial complex I activity. An important biochemical feature of neurodegeneration during PD is the presence of abnormal protein aggregates present as intracytoplasmic inclusions called Lewy bodies. Oxidative damage via GSH depletion might also accelerate the build-up of defective proteins leading to cell death of SN dopaminergic neurons by impairing the ubiquitin-proteasome pathway of protein degradation. Replenishment of normal glutathione levels within the brain may hold an important key to therapeutics for PD. Several reports have suggested that iron accumulation in the SN patients might also contribute to oxidative stress during PD.