Keiko Imamura

D‐β‐hydroxybutyrate protects dopaminergic SH‐SY5Y cells in a rotenone model of Parkinson's disease

It has been postulated that the pathogenesis of Parkinsons disease (PD) is associated with mitochondrial dysfunction. Rotenone, an inhibitor of mitochondrial complex I, provides models of PD both in vivo and in vitro. We investigated the neuroprotective effect of D‐β‐hydroxybutyrate (bHB), a ketone body, against rotenone toxicity by using SH‐SY5Y dopaminergic neuroblastoma cells. SH‐SY5Y cells, differentiated by all‐trans‐retinoic acid, were exposed to rotenone at concentrations ranging from 0 to 1,000 nM. We evaluated cellular oxidation reduction by the alamarBlue assay, viability by lactate dehydrogenase (LDH) assay, and survival/death ratio by live/dead assays. Exposure to rotenone for 48 hr oxidized cells and decreased their viability and survival rate in a concentration‐dependent manner. Pretreatment of cells with 8 mM bHB provided significant protection to SH‐SY5Y cells. Whereas rotenone caused the loss of mitochondrial membrane potential, released cytochrome c into the cytosol, and reduced cytochrome c content in mitochondria, addition of bHB blocked this toxic effect. bHB also attenuated the rotenone‐induced activation of caspase‐9 and caspase‐3. Administration of 0–10 mM 3‐nitropropionic acid, a complex II inhibitor, also decreased the reducing power of SH‐SY5Y cells measured by alamarBlue assay. Pretreatment with 8 mM bHB attenuated the decrease of alamarBlue fluorescence. These data demonstrated that bHB had a neuroprotective effect that supported the mitochondrial respiration system by reversing the inhibition of complex I or II. Ketone bodies, the alternative energy source in the mammalian brain, appear to have therapeutic potential in PD.

Increased Plasma Matrix Metalloproteinase‐9 Levels in Migraineurs

Background and Objective.— Cortical spreading depression and neurogenic inflammation have been hypothesized to be key steps in the development of migraine headache. Recent studies have highlighted matrix metalloproteinase‐9 (MMP‐9) in cortical spreading depression, neurogenic inflammation, and cerebral ischemia. To seek their possible association, we investigated plasma MMP‐9 levels in migraineurs during headache‐free periods.

Executive dysfunction in non-demented Parkinson's disease patients with hallucinations.

Objective –  We investigated executive function in Parkinson’s disease (PD) patients, and focused on executive dysfunction in PD with hallucinations, but without dementia.

Prevalence of dementia in the rural island town of Ama-cho, Japan.

Background: With the striking increase in the number of elderly people in Japan, dementia has not only become a medical but also a social issue. Methods: We studied the prevalence of dementing disorders in a rural island town of Japan (Ama-cho), using a door-to-door 2-phase design. Results: Of the 120 persons screened as having cognitive impairment, 104 people were diagnosed as having dementia. The prevalence (cases/100 persons aged 65 years and older) was 11.0 for all types of dementia, 7.0 for Alzheimer’s disease, 1.7 for vascular dementia, 0.53 for dementia with Lewy bodies, 0.74 for Parkinson’s disease dementia, 0.21 for progressive supranuclear palsy, 0.11 for frontotemporal lobar degeneration and 0.74 for other dementia. The overall prevalence was higher in women for Alzheimer’s disease and Parkinson’s disease dementia, and in men, for vascular dementia and dementia with Lewy bodies. Conclusion: We confirmed the overall prevalence of dementia in the elderly population aged 65 years and older to be 11.0. This finding is higher compared with previous reports in Japan.

The Src/c-Abl pathway is a potential therapeutic target in amyotrophic lateral sclerosis

Analysis of ALS patient iPSC-derived motor neurons indicates that Src/c-Abl inhibitors may have potential for treating ALS. A stepping stone to ALS drug discovery ALS is a heterogeneous motor neuron disease for which there is no treatment and for which a common therapeutic target has yet to be identified. In a new study, Imamura et al. developed a drug screen using motor neurons generated from ALS patient induced pluripotent stem cells (iPSCs). They screened existing drugs and showed that inhibitors of Src/c-Abl kinases promoted autophagy and rescued ALS motor neurons from degeneration. One of the drugs was effective for promoting survival of motor neurons derived from ALS patients with different genetic mutations. The Src/c-Abl pathway may be a potential therapeutic target for developing new drugs to treat ALS. Amyotrophic lateral sclerosis (ALS), a fatal disease causing progressive loss of motor neurons, still has no effective treatment. We developed a phenotypic screen to repurpose existing drugs using ALS motor neuron survival as readout. Motor neurons were generated from induced pluripotent stem cells (iPSCs) derived from an ALS patient with a mutation in superoxide dismutase 1 (SOD1). Results of the screen showed that more than half of the hits targeted the Src/c-Abl signaling pathway. Src/c-Abl inhibitors increased survival of ALS iPSC-derived motor neurons in vitro. Knockdown of Src or c-Abl with small interfering RNAs (siRNAs) also rescued ALS motor neuron degeneration. One of the hits, bosutinib, boosted autophagy, reduced the amount of misfolded mutant SOD1 protein, and attenuated altered expression of mitochondrial genes. Bosutinib also increased survival in vitro of ALS iPSC-derived motor neurons from patients with sporadic ALS or other forms of familial ALS caused by mutations in TAR DNA binding protein (TDP-43) or repeat expansions in C9orf72. Furthermore, bosutinib treatment modestly extended survival of a mouse model of ALS with an SOD1 mutation, suggesting that Src/c-Abl may be a potentially useful target for developing new drugs to treat ALS.

Pramipexole has astrocyte-mediated neuroprotective effects against lactacystin toxicity

Pramipexole, a dopamine D2/D3 receptor agonist used in the treatment of Parkinsons disease, has been reported to have neuroprotective potential. We investigated the effect of pramipexole against cell death induced by a proteasome inhibitor, lactacystin, using primary mecencephalic neuronal cultures and SH-SY5Y cells. In E14 rat primary mesencephalic cultures, the number of surviving tyrosine hydroxylase (TH)-positive neurons and microtubule associated protein 2 (MAP2)-positive neurons was decreased by exposure to 1-5 microM lactacystin in a dose-dependent manner. Pretreatment with 100 microM pramipexole rescued TH-positive neurons and MAP2-positive neurons from the toxicity of lactacystin. The protective effect of pramipexole was not selective for TH-positive dopaminergic neurons. However, the treatment with 100 microM pramipexole did not protect SH-SY5Y cells against lactacystin-induced cell toxicity and proteasome dysfunction. We hypothesized that the protective effect of pramipexole against the lactacystin-toxicity was not direct but a secondary effect mediated by astrocytes. Therefore, we investigated the efficacy of conditioned medium collected from mecencephalic astrocytes treated with pramipexole. The conditioned medium increased the viability of SH-SY5Y cells against the toxicity of lactacystin. Pramipexole increased the levels of brain derived neurotrophic factor (BDNF) in the conditioned medium of astrocyte cultures. These protective effects were not significantly inhibited by dopamine D2 or D3 receptor antagonists. We demonstrated that pramipexole had the protective effect against lactacystin toxicity, mediated by a neurotrophic effect of astrocyte-produced factors including BDNF.

Serum heart-fatty acid binding protein levels in patients with Lewy body disease

OBJECTIVE In order to examine a possible role of serum heart-fatty acid binding protein (H-FABP) in patients with Lewy body disease, we measured serum levels of H-FABP in patients with dementia with Lewy bodies (DLB), Parkinsons disease (PD), and Alzheimers disease (AD). METHODS Serum levels of H-FABP were measured using a solid-phase enzyme-linked immunoassay. Iodine-123 metaiodobenzylguanidine ((123)I-MIBG) cardiac scintigraphy was performed on each patient and the heart to mediastinum (H/M) ratio was calculated. Receiver operating characteristic (ROC) analysis was used to calculate the optimal cutoff values of the H-FABP between DLB and AD patients. Independent predictive variables for serum H-FABP levels were analyzed using multivariate regression analysis. RESULTS Serum levels of H-FABP were significantly higher in DLB patients and PD patients than in AD patients. H/M ratios of the DLB and PD patients were significantly lower than those of AD patients. The diagnostic value of the serum H-FABP levels between AD and DLB patients was inferior to that of the delayed H/M ratio of (123)I-MIBG cardiac scintigraphy. Multivariate regression analysis revealed that the delayed H/M ratio predicted serum H-FABP levels in the PD patients. CONCLUSIONS Examination of serum H-FABP levels did not allow discrimination between DLB and AD patients. Cardiac sympathetic nerve dysfunction may be associated with elevation of serum H-FABP in Lewy body disease patients.

Calcium dysregulation contributes to neurodegeneration in FTLD patient iPSC-derived neurons.

Mutations in the gene MAPT encoding tau, a microtubules-associated protein, cause a subtype of familial neurodegenerative disorder, known as frontotemporal lobar degeneration tauopathy (FTLD-Tau), which presents with dementia and is characterized by atrophy in the frontal and temporal lobes of the brain. Although induced pluripotent stem cell (iPSC) technology has facilitated the investigation of phenotypes of FTLD-Tau patient neuronal cells in vitro, it remains unclear how FTLD-Tau patient neurons degenerate. Here, we established neuronal models of FTLD-Tau by Neurogenin2-induced direct neuronal differentiation from FTLD-Tau patient iPSCs. We found that FTLD-Tau neurons, either with an intronic MAPT mutation or with an exonic mutation, developed accumulation and extracellular release of misfolded tau followed by neuronal death, which we confirmed by correction of the intronic mutation with CRISPR/Cas9. FTLD-Tau neurons showed dysregulation of the augmentation of Ca2+ transients evoked by electrical stimulation. Chemogenetic or pharmacological control of neuronal activity-relevant Ca2+ influx by the introduction of designer receptors exclusively activated by designer drugs (DREADDs) or by the treatment with glutamate receptor blockers attenuated misfolded tau accumulation and neuronal death. These data suggest that neuronal activity may regulate neurodegeneration in tauopathy. This FTLD-Tau model provides mechanistic insights into tauopathy pathogenesis and potential avenues for treatments.

Endogenous catecholamine enhances the dysfunction of unfolded protein response and α-synuclein oligomerization in PC12 cells overexpressing human α-synuclein

Parkinsons disease (PD) is a neurodegenerative disorder characterized by the selective loss of dopaminergic neurons and the presence of Lewy bodies. alpha-Synuclein is a major component of Lewy bodies. Recently, many studies have focused on the interaction between alpha-synuclein and catecholamine in the pathogenesis of PD. However, no detailed relationship between cathecholamine and alpha-synuclein cytotoxicity has been elucidated. Therefore, this study established PC12 cell lines which overexpress human alpha-synuclein in a tetracycline-inducible manner. The overexpression of human alpha-synuclein increased the number of apoptotic cells in a long-term culture. Moreover, human alpha-synuclein expressing PC12 cells demonstrated an increased vulnerability to several stressors in a short culture period. Thapsigargin increased the SDS soluble oligomers of alpha-synuclein associated with catecholamine-quinone. The unfolded protein response (UPR) study showed that thapsigargin increased eIF2alpha phosphorylation and nuclear GADD153/CHOP induction under alpha-synuclein overexpressed conditions. The activities of the ATF6alpha and IRE1alpha pathways decreased. These findings suggest that an overexpression of alpha-synuclein partly inactivates the UPR. alpha-Methyltyrosine inhibited the dysfunction of the UPR caused by an overexpression of human alpha-synuclein. Therefore, these findings suggest that the coexistence of human alpha-synuclein with catecholamine enhances the endoplasmic reticulum stress-related toxicity in PD pathogenesis.

Homocysteine is toxic for dopaminergic neurons in primary mesencephalic culture.

Hyperhomocysteinemia associated with L-3,4-dihydroxyphenylalanine (L-dopa) treatment has been observed in patients with Parkinsons disease. We investigated the toxicity of homocysteine (Hcy) on E14-rat-primary mesencephalic culture. Exposure to 0–5 mM Hcy decreased number of tyrosine hydroxylase (TH)-positive dopaminergic neurons and microtubule associated protein 2 (MAP2)-positive neurons in a dose-dependent manner. TH-positive neurons had vulnerability to the insult of Hcy compared with the other MAP2-positive neurons. In dopaminergic neurons, 5 μM reserpine enhanced the Hcy toxicity, whereas 50 μM &agr;-methyltyrosine attenuated the toxic effect, showing that the intracellular dopamine increased the cytotoxicity of Hcy. Hcy enhanced the toxicity of 1-methyl-4-phenylpyridinium (MPP+) for dopaminergic neurons. It was suggested that the Hcy toxicity was associated with the oxidative stress. Hcy is toxic for dopaminergic neurons, and hyperhomocysteinemia may modify the clinical course of Parkinsons disease.