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

Leucine-rich repeat kinase 2 (LRRK2) is the causative molecule of the autosomal dominant hereditary form of Parkinson's 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-3ß (GSK-3ß) 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.

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 Parkinson's 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 Snap25(S187A/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 Ser(129)-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 Snap25(S187A/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.

Loss of DARPP-32 and calbindin in multiple system atrophy.

We evaluated the immunohistochemical intensities of α-synuclein, phosphorylated α-synuclein (p-syn), dopamine- and cAMP-regulated phosphoprotein of 32 kDa (DARPP-32), calbindin-D 28k, calpain-cleaved carboxy-terminal 150-kDa spectrin fragment, and tyrosine hydroxylase in multiple system atrophy (MSA). The caudate head, anterior putamen, posterior putamen, substantia nigra, pontine nucleus, and cerebellar cortex from six MSA brains, six age-matched disease control brains (amyotrophic lateral sclerosis), and five control brains were processed for immunostaining by standard methods. Immunostaining for α-synuclein, p-syn, or both was increased in all areas examined in oligodendrocytes in MSA. Immunostaining for DARPP-32 and calbindin-D 28k was most prominently decreased in the posterior putamen, where neuronal loss was most prominent. Immunostaining for DARPP-32 and calbindin-D 28k was also diminished in the anterior putamen and caudate head, where neuronal loss was less prominent or absent. Calbindin immunostaining was also decreased in the dorsal tier of the substantia nigra and cerebellar cortex. Loss of immunostaining for DARPP-32 and calbindin-D 28k compared with that of neurons indicates calcium toxicity and disturbance of the phosphorylated state of proteins as relatively early events in the pathogenesis of MSA.

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.

α-Synuclein (α-syn) is a key protein in Parkinson's disease (PD), and its abnormal accumulation is implicated only not in the loss of dopaminergic neurons in the substantia nigra but also in impairment of olfactory bulb (OB) in PD. Olfactory dysfunction could arise from these OB changes as an early symptom in PD. We reported previously the impairment of neuronal stem cell (NSC) proliferation in the subventricular zone, which is upstream of OB in PD models. Reduction of NSC generation could potentially lead to olfactory dysfunction, which is commonly associated with and precedes the motor symptoms by several years in PD. Here, we investigated neurosphere formation in vitro and migration of NSCs in vivo after transduction of α-syn-encoding retroviral vector to characterize the function of α-syn in NSC. Over-expression of α-syn caused less effective formation of neurospheres and induced morphological changes. Fluorescence-activated cell sorting showed diminished NSC cell cycle progression induced by over-expression of α-syn. Intriguingly, suppression of NSC migration along the rostral migratory stream was observed when the α-syn-encoding vector was directly injected into the subventricular zone of mice in vivo. These results indicate that α-syn affects the generation of NSC and suggest that this protein could serve as a tool for the design of potentially useful therapy for PD patients.

Effects of UCH-L1 on alpha-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-L1(Ile93Met)). 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 alpha-synuclein (alphaSyn). Immunohistochemistry of midbrain sections of a patient with sporadic PD showed alphaSyn- 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 alphaSyn for 4 weeks significantly enhanced the loss of nigral DA cell bodies in UCH-L1(Ile93Met)-Tg mice, but had weak effects in age-matched UCH-L1(wild-type)-Tg mice and non-Tg littermates. In contrast, the extent of alphaSyn-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-L1(Ile93Met) mutant, and suggest that regulation of UCH-L1 in nigral DA cells could be a future target for treatment of PD.

The isolation of neural stem cells from the olfactory bulb of Parkinson's disease model.

Hyposmia is one of the characteristic symptoms of PD. We isolated the neurosphere forming cells (NSFCs) from the olfactory bulb (OB) after dopaminergic neuronal loss induced by 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), which is a model of Parkinson's disease. We used BrdU to label dividing cells and isolated NSFCs from the OB of adult mice with or without MPTP to confirm the function of OB in PD models. Seven days after MPTP treatment, BrdU-positive cells were significantly increased in the OB, especially in the glomerular layer (GL) and the subependymal zone (SEZ). The number of neurospheres derived from the adult OB was not decreased in groups receiving MPTP, instead, it was significantly increased at 21 days post-injection and only returned to control levels 40 days after MPTP administration. We also evaluated the differentiation of NSFCs into neural subtypes and found that these NSFCs could be well infected with retrovirus. Adult neurogenesis may be enhanced as a repair system in the tyrosine hydroxylase (TH) positive cells of the OB after MPTP administration. The isolation of neural stem cells from the OB after MPTP administration has helped to establish the cellular basis of neurogenesis and supports a role for the transplant-mediated treatment of 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 Parkinson's 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.

Gene transfer targeting mouse vestibule using adenovirus and adeno-associated virus vectors.

HYPOTHESIS: The present study assessed how to inject a gene into the mouse vestibule and which is the optimum gene to the mouse vestibule adenovirus (AdV) vector or adeno-associated virus (AAV) vector. BACKGROUND: Loss of vestibular hair cell is seen in various balance disorder diseases. There have been some reports concerning gene delivery to the mouse vestibule in recent years. To effectively induce transgene expression at the vestibule, we assessed the efficiency of inoculating the mouse inner ear using various methods. METHODS: We employed an AdV- and AAV-carrying green fluorescent protein using a semicircular canal approach (via a canalostomy) and round window approach. RESULTS: AAV injection via canalostomy induced gene expression at the hair cells, supporting cells, and fibrocytes at the vestibular organs without auditory or balance dysfunction, suggesting it was the most suitable transfection method. This method is thus considered to be a promising strategy to prevent balance dysfunction. CONCLUSION: AAV injection via canalostomy to the vestibule is the noninvasive and highly efficient transfection method, and this study may have the potential to repair balance disorders in human in the future.

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

BACKGROUND: Parkinson'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. RESULTS: Here, 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. CONCLUSIONS: Thus, 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.

Adeno-associated viral vector-mediated gene transduction in mesencephalic slice culture.

Adeno-associated viral (AAV) vector is a non-pathogenic vehicle that is suitable for the delivery of foreign genes into non-dividing neuronal cells. This vector has been utilized for in vivo neurological research and in clinical trials of gene therapy for neurodegenerative disorders. Viral vector-mediated gene delivery has the limitation that progressive changes in cellular phenotype cannot be monitored in living animals. To visualize living neurons transduced with foreign genes in vitro, we used cultured mesencephalic tissue harboring living dopaminergic (DA) neurons and examined cellular tropism of serotype-1 and serotype-2 AAV vectors in a culture system. The viability of DA neurons was evaluated using transgenic mice carrying enhanced green fluorescent protein under the control of the rat tyrosine hydroxylase (TH) promoter, which enables the visualization of living DA cells in the substantia nigra. Apoptosis of a subset of neuronal cells was noted within one day of culture. After 7 days, the serotype-1 AAV vector had successfully delivered the foreign gene into neurons and astrocytes, and serotype-2 AAV vector was able to transduce TH-positive DA neurons efficiently. Our method should be useful for in vitro investigations of pathological changes in DA neurons following transduction with foreign genes.

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 the neuroprotective actions of parkin in vivo. Here, we investigated mechanisms of neuroprotection of overexpressed parkin in a modified long-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 α-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.

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

Parkinson's 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.