Bilirubin, the end product of heme redox metabolism, has cytoprotective properties and is an essential metabolite associated with cardiovascular disease, inflammatory bowel disease, type 2 diabetes, ...and neurodegenerative diseases including Parkinson's disease (PD). PD is characterized by progressive degeneration of nigral dopaminergic neurons and is associated with elevated oxidative stress due to mitochondrial dysfunction. In this study, using a ratiometric bilirubin probe, we revealed that the mitochondrial inhibitor, rotenone, which is widely used to create a PD model, significantly decreased intracellular bilirubin levels in HepG2 cells. Chemical screening showed that BRUP‐1 was a top hit that restored cellular bilirubin levels that were lowered by rotenone. We found that BRUP‐1 up‐regulated the expression level of heme oxygenase‐1 (HO‐1), one of the rate‐limiting enzyme of bilirubin production via nuclear factor erythroid 2‐related factor 2 (Nrf2) activation. In addition, we demonstrated that this Nrf2 activation was due to a direct inhibition of the interaction between Nrf2 and Kelch‐like ECH‐associated protein 1 (Keap1) by BRUP‐1. Both HO‐1 up‐regulation and bilirubin restoration by BRUP‐1 treatment were significantly abrogated by Nrf2 silencing. In neuronal PC12D cells, BRUP‐1 also activated the Nrf2‐HO‐1 axis and increased bilirubin production, resulted in the suppression of neurotoxin‐induced cell death, reactive oxygen species production, and protein aggregation, which are hallmarks of PD. Furthermore, BRUP‐1 showed neuroprotective activity against rotenone‐treated neurons derived from induced pluripotent stem cells. These findings provide a new member of Keap1‐Nrf2 direct inhibitors and suggest that chemical modulation of heme metabolism using BRUP‐1 may be beneficial for PD treatment.
Since bilirubin levels were decreased in patients with Parkinson's disease (PD), we searched for compounds that restore bilirubin levels by using a ratiometric bilirubin probe, Flag‐UnaG‐2A‐mCherry, and identified BRUP‐1. We also found that BRUP‐1 inhibited the binding of Keap1 to Nrf2 leading to the activation of Nrf2‐HO‐1 pathway, thereby increasing bilirubin level. BRUP‐1 showed potent neuroprotective activity through the activation of Nrf2‐HO‐1‐bilirubin pathway in a PD model using neuronal PC12D cells. Neuroprotective activity of BRUP‐1 was also shown in iPSC‐derived neurons, which suggests that chemical modulation of heme metabolism using BRUP‐1 may be beneficial for treatment of PD.
Parkinson’s disease (PD) is characterized as a chronic and progressive neurodegenerative disorder, and the deposition of specific protein aggregates of α-synuclein, termed Lewy bodies, is evident in ...multiple brain regions of PD patients. Although there are several available medications to treat PD symptoms, these medications do not prevent the progression of the disease. Soluble epoxide hydrolase (sEH) plays a key role in inflammation associated with the pathogenesis of PD. Here we found that MPTP (1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine)-induced neurotoxicity in the mouse striatum was attenuated by subsequent repeated administration of TPPU, a potent sEH inhibitor. Furthermore, deletion of the sEH gene protected against MPTP-induced neurotoxicity, while overexpression of sEH in the striatum significantly enhanced MPTP-induced neurotoxicity. Moreover, the expression of the sEH protein in the striatum from MPTP-treated mice or postmortem brain samples from patients with dementia of Lewy bodies (DLB) was significantly higher compared with control groups. Interestingly, there was a positive correlation between sEH expression and phosphorylation of α-synuclein in the striatum. Oxylipin analysis showed decreased levels of 8,9-epoxy-5Z,11Z,14Z-eicosatrienoic acid in the striatum of MPTP-treated mice, suggesting increased activity of sEH in this region. Interestingly, the expression of sEH mRNA in human PARK2 iPSC-derived neurons was higher than that of healthy control. Treatment with TPPU protected against apoptosis in human PARK2 iPSC-derived dopaminergic neurons. These findings suggest that increased activity of sEH in the striatum plays a key role in the pathogenesis of neurodegenerative disorders such as PD and DLB. Therefore, sEH may represent a promising therapeutic target for α-synuclein–related neurodegenerative disorders.
Parkinson's disease (PD) is a common neurodegenerative disorder that results from the loss of dopaminergic neurons. Mutations in coiled-coil-helix-coiled-coil-helix domain containing 2 (CHCHD2) gene ...cause a familial form of PD with α-Synuclein aggregation, and we here identified the pathogenesis of the T61I mutation, the most common disease-causing mutation of CHCHD2. In Neuro2a cells, CHCHD2 is in mitochondria, whereas the T61I mutant (CHCHD2
) is mislocalized in the cytosol. CHCHD2
then recruits casein kinase 1 epsilon/delta (Csnk1e/d), which phosphorylates neurofilament and α-Synuclein, forming cytosolic aggresomes. In vivo, both Chchd2
knock-in and transgenic mice display neurodegenerative phenotypes and aggresomes containing Chchd2
, Csnk1e/d, phospho-α-Synuclein, and phospho-neurofilament in their dopaminergic neurons. Similar aggresomes were observed in a postmortem PD patient brain and dopaminergic neurons generated from patient-derived iPS cells. Importantly, a Csnk1e/d inhibitor substantially suppressed the phosphorylation of neurofilament and α-Synuclein. The Csnk1e/d inhibitor also suppressed the cellular damage in CHCHD2
-expressing Neuro2a cells and dopaminergic neurons generated from patient-derived iPS cells and improved the neurodegenerative phenotypes of Chchd2
mutant mice. These results indicate that Csnk1e/d is involved in the pathogenesis of PD caused by the CHCHD2
mutation.
Increasing evidence shows that metabolic abnormalities in body fluids are distinguishing features of the pathophysiology of Parkinson's disease. However, a non-invasive approach has not been ...established in the earliest or pre-symptomatic phases. Here, we report comprehensive double-cohort analyses of the metabolome using capillary electrophoresis/liquid chromatography mass-spectrometry. The plasma analyses identified 18 Parkinson's disease-specific metabolites and revealed decreased levels of seven long-chain acylcarnitines in two Parkinson's disease cohorts (n = 109, 145) compared with controls (n = 32, 45), respectively. Furthermore, statistically significant decreases in five long-chain acylcarnitines were detected in Hoehn and Yahr stage I. Likewise, decreased levels of acylcarnitine(16:0), a decreased ratio of acylcarnitine(16:0) to fatty acid(16:0), and an increased index of carnitine palmitoyltransferase 1 were identified in Hoehn and Yahr stage I of both cohorts, suggesting of initial β-oxidation suppression. Receiver operating characteristic curves produced using 12-14 long-chain acylcarnitines provided a large area of under the curve, high specificity and moderate sensitivity for diagnosing Parkinson's disease. Our data demonstrate that a primary decrement of mitochondrial β-oxidation and that 12-14 long-chain acylcarnitines decreases would be promising diagnostic biomarkers for Parkinson's disease.
This study aimed to clarify the genomic factors associated with the diagnosis and prognosis of oral squamous cell carcinoma via next‐generation sequencing. We evaluated data from 220 cases of oral ...squamous cell carcinoma. Genomic DNA was eluted using formalin‐fixed, paraffin‐embedded samples, and targeted resequencing of 50 cancer‐related genes was performed. In total, 311 somatic mutations were detected in 220 patients, consisting of 68 synonymous mutations and 243 non‐synonymous mutations. Genes carrying mutations included TP53, CDKN2A, and PIK3CA in 79 (35.9%), 35 (15.9%), and 19 patients (8.6%), respectively. Copy number analysis detected amplification of PIK3CA and AKT1 in 38 (17.3%) and 11 patients (5.0%), respectively. Amplification of receptor tyrosine kinases was found in 37 patients (16.8%). Distant metastasis was noted in nine of 37 patients (24%) with receptor tyrosine kinase amplification, accounting for 43% of the 21 cases of distant metastasis. The cumulative 5‐year survival rate was 64.6% in the receptor tyrosine kinase amplification group vs 85.2% in the no receptor tyrosine kinase amplification group. Moreover, we identified significantly poorer prognosis in the TP53 mutation/receptor tyrosine kinase amplification group, for which the cumulative 5‐year survival rate was 41.6%. In conclusion, the results of this study demonstrated that receptor tyrosine kinase amplification is a prognostic factor for distant metastasis of oral squamous cell carcinoma, indicating the necessity of using next‐generation sequencing in clinical sequencing.
We evaluated data from 220 cases of oral squamous cell carcinoma. Targeted resequencing of 50 cancer‐related genes was performed. Receptor tyrosine kinase amplification is a prognostic factor for distant metastasis of oral squamous cell carcinoma, indicating the necessity of using next‐generation sequencing in clinical sequencing.
Chemical intervention of autophagy has been investigated in clinical trials for various age-related conditions such as sarcopenia and neurodegeneration. However, at present, no autophagy inducer has ...been established as a disease-modifying agent against neurodegenerative diseases.
We screened a library consisting of 796 medicines clinically approved (in Japan) for autophagy enhancers as potential neurodegeneration therapeutics using HeLa cells stably expressing green fluorescent protein-microtubule-associated protein light chain 3 (GFP-LC3) followed by an analysis of the molecular mechanisms using various neuronal models.
The primary screening identified 152 hits in a static cellular state. A widely available Alzheimer's disease drug, memantine, which antagonizes N-Methyl-d-aspartate receptor (NMDAR), was one of the hits. Memantine increased the levels of LC3-II in a dose-dependent and time-dependent manner, and upregulated autophagic flux. In addition, the pharmacological effects of memantine on autophagy were independent of mTORC1 activity and NMDAR activation. Furthermore, a VPS34 inhibitor suppressed the memantine-induced LC3-II upregulation, suggesting that memantine may affect VPS34 complex activity. Notably, intracellular Huntington's disease-specific aggregates of elongated huntingtin, a well-established autophagy substrate, were significantly decreased by memantine. In addition, memantine enhanced elimination of degraded mitochondrial in neurons derived from induced pluripotent stem cells of PARK2 or PARK6 patients, who exhibited defective PINK1/parkin-mediated mitophagy, suggests that memantine accelerated the clearance of damaged mitochondria.
These findings indicate that memantine may be beneficial for the treatment of neurodegeneration characterized by the abnormal accumulation of autophagy or mitophagy substrates.
•From a screen of 796 chemicals, memantine was identified as a novel autophagy inducer.•Memantine, clinically used for dementia, induced neuroprotective macroautophagy.•Memantine enhanced elimination of degraded mitochondrial in neurons derived from PD-iPS cells.
The CMT1A variant accounts for over 60% of cases of Charcot-Marie-Tooth disease (CMT), one of the most common human neuropathies. The cause of CMT1A has been identified as the duplication of PMP22, a ...myelin protein expressed in Schwann cells. Yet, the pathological mechanisms have not been elucidated, and no treatment is currently available. In our study, we established an iPS cell line from a CMT1A patient with PMP22 duplication. The generated iPSCs maintain pluripotency and in vitro differentiation potency.
PARK2 is the most common autosomal recessive form of Parkinson’s disease and is caused by mutations in parkin that result in early-onset loss of dopaminergic neurons in the substantia nigra. In this ...study, we established an induced pluripotent stem cell (iPSC) line from a patient harboring a homozygous exon 3 deletion in PARK2. The established iPSCs showed pluripotency, the capacity to differentiate into the three germ layers, and normal karyotypes.
Patient-specific induced pluripotent stem cells (iPSCs) show promise for use as tools for in vitro modeling of Parkinson's disease. We sought to improve the efficiency of dopaminergic (DA) neuron ...induction from iPSCs by the using surface markers expressed in DA progenitors to increase the significance of the phenotypic analysis. By sorting for a CD184high/CD44- fraction during neural differentiation, we obtained a population of cells that were enriched in DA neuron precursor cells and achieved higher differentiation efficiencies than those obtained through the same protocol without sorting. This high efficiency method of DA neuronal induction enabled reliable detection of reactive oxygen species (ROS) accumulation and vulnerable phenotypes in PARK2 iPSCs-derived DA neurons. We additionally established a quantitative system using the mt-mKeima reporter system to monitor mitophagy in which mitochondria fuse with lysosomes and, by combining this system with the method of DA neuronal induction described above, determined that mitophagy is impaired in PARK2 neurons. These findings suggest that the efficiency of DA neuron induction is important for the precise detection of cellular phenotypes in modeling Parkinson's disease.
•CD184high/CD44- sorting enriches for DA neurons during neural differentiation from hiPSCs.•Highly purified DA neurons from PD-iPSCs stably reproduce PD phenotypes.•The mt-mKeima reporter system allows visualization of impaired mitophagy in PARK2 DA neurons.
PDF
