Tomoko Nihira

Mitochondrial dysfunction associated with increased oxidative stress and α-synuclein accumulation in PARK2 iPSC-derived neurons and postmortem brain tissue

BackgroundParkinson’s disease (PD) is a neurodegenerative disease characterized by selective degeneration of dopaminergic neurons in the substantia nigra (SN). The familial form of PD, PARK2, is caused by mutations in the parkin gene. parkin-knockout mouse models show some abnormalities, but they do not fully recapitulate the pathophysiology of human PARK2.ResultsHere, we generated induced pluripotent stem cells (iPSCs) from two PARK2 patients. PARK2 iPSC-derived neurons showed increased oxidative stress and enhanced activity of the nuclear factor erythroid 2-related factor 2 (Nrf2) pathway. iPSC-derived neurons, but not fibroblasts or iPSCs, exhibited abnormal mitochondrial morphology and impaired mitochondrial homeostasis. Although PARK2 patients rarely exhibit Lewy body (LB) formation with an accumulation of α-synuclein, α-synuclein accumulation was observed in the postmortem brain of one of the donor patients. This accumulation was also seen in the iPSC-derived neurons in the same patient.ConclusionsThus, pathogenic changes in the brain of a PARK2 patient were recapitulated using iPSC technology. These novel findings reveal mechanistic insights into the onset of PARK2 and identify novel targets for drug screening and potential modified therapies for PD.

Neuronal specificity of α-synuclein toxicity and effect of parkin co-expression in primates

Abstract Recombinant adeno-associated viral (rAAV) vector-mediated overexpression of α-synuclein (αSyn) protein has been shown to cause neurodegeneration of the nigrostriatal dopaminergic pathway in rodents and primates. Using serotype-2 rAAV vectors, we recently reported the protective effect of Parkin on αSyn-induced nigral dopaminergic neurodegeneration in a rat model. Here we investigated the neuronal specificity of αSyn toxicity and the effect of Parkin co-expression in a primate model. We used another serotype (type-1) of AAV vector that was confirmed to deliver genes of interest anterogradely and retrogradely to neurons in rats. The serotype-1 rAAV (rAAV1) carrying α Syn cDNA (rAAV1-αSyn), and a cocktail of rAAV1-αSyn and rAAV1 carrying parkin cDNA (rAAV1-parkin) were unilaterally injected into the striatum of macaque monkeys, resulting in protein expression in striatonigral GABAergic and nigrostriatal dopaminergic neurons. Injection of rAAV1-αSyn alone decreased tyrosine hydroxylase immunoreactivity in the striatum compared with the contralateral side injected with a cocktail of rAAV1-αSyn and rAAV1-parkin. Immunostaining of striatonigral GABAergic neurons was similar on both sides. Overexpression of Parkin in GABAergic neurons was associated with less accumulation of αSyn protein and/or phosphorylation at Ser129 residue. Our results suggest that the toxicity of accumulated αSyn is not induced in non-dopaminergic neurons and that the αSyn-ablating effect of Parkin is exerted in virtually all neurons in primates.

Effects of UCH-L1 on α-synuclein over-expression mouse model of Parkinson’s disease

The rare inherited form of Parkinson’s disease (PD), PARK5, is caused by a missense mutation in ubiquitin carboxy‐terminal hydrolase‐L1 (UCH‐L1) gene, resulting in Ile93Met substitution in its gene product (UCH‐L1Ile93Met). PARK5 is inherited in an autosomal‐dominant mode, but whether the Ile93Met mutation gives rise to a gain‐of‐toxic‐function or loss‐of‐function of UCH‐L1 protein remains controversial. Here, we investigated the selective vulnerabilities of dopaminergic (DA) neurons in UCH‐L1‐transgenic (Tg) and spontaneous UCH‐L1‐null gracile axonal dystrophy mice to an important PD‐causing insult, abnormal accumulation of α‐synuclein (αSyn). Immunohistochemistry of midbrain sections of a patient with sporadic PD showed αSyn‐ and UCH‐L1‐double‐positive Lewy bodies in nigral DA neurons, suggesting physical and/or functional interaction between the two proteins in human PD brain. Recombinant adeno‐associated viral vector‐mediated over‐expression of αSyn for 4 weeks significantly enhanced the loss of nigral DA cell bodies in UCH‐L1Ile93Met‐Tg mice, but had weak effects in age‐matched UCH‐L1wild‐type‐Tg mice and non‐Tg littermates. In contrast, the extent of αSyn‐induced DA cell loss in gracile axonal dystrophy mice was not significantly different from wild‐type littermates at 13‐weeks post‐injection. Our results support the hypothesis that PARK5 is caused by a gain‐of‐toxic‐function of UCH‐L1Ile93Met mutant, and suggest that regulation of UCH‐L1 in nigral DA cells could be a future target for treatment of PD.

Correlation between levels of pigment epithelium-derived factor and vascular endothelial growth factor in the striatum of patients with Parkinson's disease

Parkinsons disease (PD) is caused by progressive degeneration of nigrostriatal dopaminergic neurons and can potentially be treated by intrastriatal delivery of neurotrophic factors. Pigment epithelium-derived factor (PEDF), which exhibits protective effects on various neuronal populations, is up-/down-regulated in the cerebrospinal fluid in some neurodegenerative conditions. Here we investigated the level of PEDF protein in the striatum and immunoreactivity for PEDF in the substantia nigra (SN) of patients with PD to assess its role in the pathophysiology of PD. We also studied changes in PEDF expression in the striatum of the 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) mouse model of PD. We found a transient and rapid up-regulation of PEDF transcripts and a marked increase in immunoreactivity for PEDF protein in response to MPTP administration in mice. However, there were no significant changes in striatal levels of PEDF and immunoreactivity for PEDF in the SN of PD patients compared with age-matched non-PD patients. Intriguingly, the striatal levels of PEDF and vascular endothelial growth factor (VEGF), which has opposite functions to PEDF in terms of angiogenesis and vascular permeability, correlated positively in PD patients. Our results suggest up-regulation of PEDF in response to acute insult to the dopaminergic pathway, but such response might be disturbed in patients with advanced PD. The correlation between PEDF and VEGF striatal levels in PD patients suggests that concerted neurotrophic functions of these factors or structural changes in blood vessel walls play an important role in the pathophysiology of PD.

Parkin-Mediated Protection of Dopaminergic Neurons in a Chronic MPTP-Minipump Mouse Model of Parkinson Disease

Loss-of-function mutations in the ubiquitin ligase parkin are the major cause of recessively inherited early-onset Parkinson disease (PD). Impairment of parkin activity caused by nitrosative or dopamine-related modifications may also be responsible for the loss of dopaminergic (DA) neurons in sporadic PD. Previous studies have shown that viral vector-mediated delivery of parkin prevented DA neurodegeneration in several animal models, but little is known about theneuroprotective actions of parkin in vivo. Here, we investigated mechanisms of neuroprotection of overexpressed parkin in a modifiedlong-term mouse model of PD using osmotic minipump administration of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP). Recombinant adeno-associated viral vector-mediated intranigral delivery of parkin prevented motor deficits and DA cell loss in the mice. Ser129-phosphorylated &agr;-synuclein-immunoreactive cells were increased in the substantia nigra of parkin-treated mice. Moreover, delivery of parkin alleviated the MPTP-induced decrease of the active phosphorylated form of Akt. On the other hand, upregulation of p53 and mitochondrial alterations induced by chronic MPTP administration were barely suppressed by parkin. These results suggest that the neuroprotective actions of parkin may be impaired in severe PD.

Effect of melatonin on α-synuclein self-assembly and cytotoxicity

α-Synuclein (αS) assembly has been implicated as a critical step in the development of Lewy body diseases such as Parkinsons disease and dementia with Lewy bodies. Melatonin (Mel), a secretory product of the pineal gland, is known to have beneficial effects such as an antioxidant function and neuroprotection. To elucidate whether Mel has an antiassembly effect, here we used circular dichroism spectroscopy, photoinduced crosslinking of unmodified proteins, thioflavin S fluorescence, size exclusion chromatography, electron microscopy and atomic force microscopy to examine the effects of Mel on the αS assembly. We also examined the effects of Mel on αS-induced cytotoxicity by assaying 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide metabolism in αS-treated, primary neuronal cells. Initial studies revealed that Mel blocked αS fibril formation as well as destabilizing preformed αS fibrils. Subsequent evaluation of the assembly-stage specificity of the effect showed that Mel was able to inhibit protofibril formation, oligomerization, and secondary structure transitions. Importantly, Mel decreased αS-induced cytotoxicity. These data suggest a mechanism of action for Mel, inhibition of assembly of toxic polymers and protection of neurons from their effect.

Accumulation of α-Synuclein Triggered by Presynaptic Dysfunction

Pathological examination of dementia with Lewy bodies patients identified the presence of abnormal α-synuclein (αSyn) aggregates in the presynaptic terminals. αSyn is involved in the regulation of soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) complex. Importantly, αSyn-transgenic mouse and postmortem examination of patients with Parkinsons disease have demonstrated the abnormal distribution of SNARE protein in presynaptic terminals. In this study, we investigated the effects of SNARE dysfunction on endogenous αSyn using Snap25S187A/S187A mutant mice. These mice have homozygous knock-in gene encoding unphosphorylatable S187A-substituted synaptosomal-associated protein of 25 kDa (SNAP-25). The mice displayed a significant age-dependent change in the distribution of αSyn and its Ser129-phosphorylated form in abnormally hypertrophied glutamatergic nerve terminals in the striatum. Electron-microscopic analysis revealed the abnormally condensed synaptic vesicles with concomitant mislocalization of αSyn protein to the periactive zone in the glutamatergic nerve terminals. However, the Snap25S187A/S187A mutant mouse harbored no abnormalities in the nigrostriatal dopaminergic neurons. Our present results suggest that SNARE dysfunction is the initial trigger of mislocalization and accumulation of αSyn, and probably is an important pathomechanism of α-synucleinopathies.

I2020T mutant LRRK2 iPSC-derived neurons in the Sagamihara family exhibit increased Tau phosphorylation through the AKT/GSK-3β signaling pathway

Leucine-rich repeat kinase 2 (LRRK2) is the causative molecule of the autosomal dominant hereditary form of Parkinsons disease (PD), PARK8, which was originally defined in a study of a Japanese family (the Sagamihara family) harboring the I2020T mutation in the kinase domain. Although a number of reported studies have focused on cell death mediated by mutant LRRK2, details of the pathogenetic effect of LRRK2 still remain to be elucidated. In the present study, to elucidate the mechanism of neurodegeneration in PD caused by LRRK2, we generated induced pluripotent stem cells (iPSC) derived from fibroblasts of PD patients with I2020T LRRK2 in the Sagamihara family. We found that I2020T mutant LRRK2 iPSC-derived neurons released less dopamine than control-iPSC-derived neurons. Furthermore, we demonstrated that patient iPSC-derived neurons had a lower phospho-AKT level than control-iPSC-derived neurons, and that the former showed an increased incidence of apoptosis relative to the controls. Interestingly, patient iPSC-derived neurons exhibited activation of glycogen synthase kinase-3β (GSK-3β) and high Tau phosphorylation. In addition, the postmortem brain of the patient from whom the iPSC had been established exhibited deposition of neurofibrillary tangles as well as increased Tau phosphorylation in neurons. These results suggest that I2020T LRRK2-iPSC could be a promising new tool for reproducing the pathology of PD in the brain caused by the I2020T mutation, and applicable as a model in studies of targeted therapeutics.

Compensation of Depleted Neuronal Subsets by New Neurons in a Local Area of the Adult Olfactory Bulb

In the olfactory bulb (OB), loss of preexisting granule cells (GCs) and incorporation of adult-born new GCs continues throughout life. GCs consist of distinct subsets. Here, we examined whether the loss and incorporation of GC subsets are coordinated in the OB. We classified GCs into mGluR2-expressing and -negative subsets and selectively ablated mGluR2-expressing GCs in a local area of the OB with immunotoxin-mediated cell ablation method. The density of mGluR2-expressing GCs showed considerable recovery within several weeks after the ablation. During recovery, an mGluR2-expressing new GC subset was preferentially incorporated over an mGluR2-negative new GC subset in the area of ablation, whereas the preferential incorporation was not observed in the intact area. The area-specific preferential incorporation of mGluR2-expressing new GCs occurred for BrdU analog- and retrovirus-labeled adult-born cells as well as for neonate-derived transplanted cells. The mGluR2-expressing new GCs in the ablated area were synaptically incorporated into the local bulbar circuit. The spine size of mGluR2-expressing new GCs in the ablated area was larger than that of those in the intact area. In contrast, mGluR2-negative new GCs did not show ablated area-specific spine enlargement. These results indicate that local OB areas have a mechanism to coordinate the loss and incorporation of GC subsets by compensatory incorporation of new GC subsets, which involves subset-specific cellular incorporation and subset-specific regulation of spine size.

Ectopic expression of α-synuclein affects the migration of neural stem cells in mouse subventricular zone

J. Neurochem. (2010) 115, 854–863.