Downregulation of Protease Cathepsin D and Upregulation of Pathologic α-Synuclein Mediate Paucity of DNAJC6-Induced Degeneration of Dopaminergic Neurons.

A homozygous mutation of the DNAJC6 gene causes autosomal recessive familial type 19 of Parkinson's disease (PARK19). To test the hypothesis that PARK19 DNAJC6 mutations induce the neurodegeneration of dopaminergic cells by reducing the protein expression of functional DNAJC6 and causing DNAJC6 paucity, an in vitro PARK19 model was constructed by using shRNA-mediated gene silencing of endogenous DANJC6 in differentiated human SH-SY5Y dopaminergic neurons. shRNA targeting DNAJC6 induced the neurodegeneration of dopaminergic cells. DNAJC6 paucity reduced the level of cytosolic clathrin heavy chain and the number of lysosomes in dopaminergic neurons. A DNAJC6 paucity-induced reduction in the lysosomal number downregulated the protein level of lysosomal protease cathepsin D and impaired macroautophagy, resulting in the upregulation of pathologic α-synuclein or phospho-α-synucleinSer129 in the endoplasmic reticulum (ER) and mitochondria. The expression of α-synuclein shRNA or cathepsin D blocked the DNAJC6 deficiency-evoked degeneration of dopaminergic cells. An increase in ER α-synuclein or phospho-α-synucleinSer129 caused by DNAJC6 paucity activated ER stress, the unfolded protein response and ER stress-triggered apoptotic signaling. The lack of DNAJC6-induced upregulation of mitochondrial α-synuclein depolarized the mitochondrial membrane potential and elevated the mitochondrial level of superoxide. The DNAJC6 paucity-evoked ER stress-related apoptotic cascade, mitochondrial malfunction and oxidative stress induced the degeneration of dopaminergic neurons via activating mitochondrial pro-apoptotic signaling. In contrast with the neuroprotective function of WT DNAJC6, the PARK19 DNAJC6 mutants (Q789X or R927G) failed to attenuate the tunicamycin- or rotenone-induced upregulation of pathologic α-synuclein and stimulation of apoptotic signaling. Our data suggest that PARK19 mutation-induced DNAJC6 paucity causes the degeneration of dopaminergic neurons via downregulating protease cathepsin D and upregulating neurotoxic α-synuclein. Our results also indicate that PARK19 mutation (Q789X or R927G) impairs the DNAJC6-mediated neuroprotective function.

HCH6-1, an antagonist of formyl peptide receptor-1, exerts anti-neuroinflammatory and neuroprotective effects in cellular and animal models of Parkinson's disease.

Microglial activation-induced neuroinflammation contributes to onset and progression of sporadic and hereditary Parkinson's disease (PD). Activated microglia secrete pro-inflammatory and neurotoxic IL-1ß, IL-6 and TNF-α, which subsequently promote neurodegeneration. Formyl peptide receptor-1 (FPR1) of CNS microglia functions as pattern recognition receptor and is activated by N-formylated peptides, leading to microglial activation, induction of inflammatory responses and resulting neurotoxicity. In this study, it was hypothesized that FPR1 activation of microglia causes loss of dopaminergic neurons by activating inflammasome and upregulating IL-1ß, IL-6 or TNF-α and that FPR1 antagonist HCH6-1 exerts neuroprotective effect on dopaminergic neurons. FPR1 agonist fMLF induced activation of microglia cells by causing activation of NLRP3 inflammasome and upregulation and secretion of IL-1ß, IL-6 or TNF-α. Conditioned medium (CM) of fMLF-treated microglia cells, which contains neurotoxic IL-1ß, IL-6 and TNF-α, caused apoptotic death of differentiated SH-SY5Y dopaminergic neurons by inducing mitochondrial oxidative stress and activating pro-apoptotic signaling. FPR1 antagonist HCH6-1 prevented fMLF-induced activation of inflammasome and upregulation of pro-inflammatory cytokines in microglia cells. HCH6-1 co-treatment reversed CM of fMLF-treated microglia-induced apoptotic death of dopaminergic neurons. FPR1 antagonist HCH6-1 inhibited rotenone-induced upregulation of microglial marker Iba-1 protein level, cell death of dopaminergic neurons and motor impairment in zebrafish. HCH6-1 ameliorated rotenone-induced microglial activation, upregulation of FPR1 mRNA, activation of NLRP3 inflammasome, cell death of SN dopaminergic neurons and PD motor deficit in mice. Our results suggest that FPR1 antagonist HCH6-1 possesses anti-neuroinflammatory and neuroprotective effects on dopaminergic neurons by inhibiting microglial activation and upregulation of inflammasome activity and pro-inflammatory cytokines.

TOMM40 Genetic Variants Cause Neuroinflammation in Alzheimer's Disease.

Translocase of outer mitochondrial membrane 40 (TOMM40) is located in the outer membrane of mitochondria. TOMM40 is essential for protein import into mitochondria. TOMM40 genetic variants are believed to increase the risk of Alzheimer's disease (AD) in different populations. In this study, three exonic variants (rs772262361, rs157581, and rs11556505) and three intronic variants (rs157582, rs184017, and rs2075650) of the TOMM40 gene were identified from Taiwanese AD patients using next-generation sequencing. Associations between the three TOMM40 exonic variants and AD susceptibility were further evaluated in another AD cohort. Our results showed that rs157581 (c.339T > C, p.Phe113Leu, F113L) and rs11556505 (c.393C > T, p.Phe131Leu, F131L) were associated with an increased risk of AD. We further utilized cell models to examine the role of TOMM40 variation in mitochondrial dysfunction that causes microglial activation and neuroinflammation. When expressed in BV2 microglial cells, the AD-associated mutant (F113L) or (F131L) TOMM40 induced mitochondrial dysfunction and oxidative stress-induced activation of microglia and NLRP3 inflammasome. Pro-inflammatory TNF-α, IL-1ß, and IL-6 released by mutant (F113L) or (F131L) TOMM40-activated BV2 microglial cells caused cell death of hippocampal neurons. Taiwanese AD patients carrying TOMM40 missense (F113L) or (F131L) variants displayed an increased plasma level of inflammatory cytokines IL-6, IL-18, IL-33, and COX-2. Our results provide evidence that TOMM40 exonic variants, including rs157581 (F113L) and rs11556505 (F131L), increase the AD risk of the Taiwanese population. Further studies suggest that AD-associated mutant (F113L) or (F131L) TOMM40 cause the neurotoxicity of hippocampal neurons by inducing the activation of microglia and NLRP3 inflammasome and the release of pro-inflammatory cytokines.

Deficiency of RAB39B Activates ER Stress-Induced Pro-apoptotic Pathway and Causes Mitochondrial Dysfunction and Oxidative Stress in Dopaminergic Neurons by Impairing Autophagy and Upregulating α-Synuclein.

Deletion and missense or nonsense mutation of RAB39B gene cause familial Parkinson's disease (PD). We hypothesized that deletion and mutation of RAB39B gene induce degeneration of dopaminergic neurons by decreasing protein level of functional RAB39B and causing RAB39B deficiency. Cellular model of deletion or mutation of RAB39B gene-induced PD was prepared by knocking down endogenous RAB39B in human SH-SY5Y dopaminergic cells. Transfection of shRNA-induced 90% reduction in RAB39B level significantly decreased viability of SH-SY5Y dopaminergic neurons. Deficiency of RAB39B caused impairment of macroautophagy/autophagy, which led to increased protein levels of α-synuclein and phospho-α-synucleinSer129 within endoplasmic reticulum (ER) and mitochondria. RAB39B deficiency-induced increase of ER α-synuclein and phospho-α-synucleinSer129 caused activation of ER stress, unfolded protein response, and ER stress-induced pro-apoptotic cascade. Deficiency of RAB39B-induced increase of mitochondrial α-synuclein decreased mitochondrial membrane potential and increased mitochondrial superoxide. RAB39B deficiency-induced activation of ER stress pro-apoptotic pathway, mitochondrial dysfunction, and oxidative stress caused apoptotic death of SH-SY5Y dopaminergic cells by activating mitochondrial apoptotic cascade. In contrast to neuroprotective effect of wild-type RAB39B, PD mutant (T168K), (W186X), or (G192R) RAB39B did not prevent tunicamycin- or rotenone-induced increase of neurotoxic α-synuclein and activation of pro-apoptotic pathway. Our results suggest that RAB39B is required for survival and macroautophagy function of dopaminergic neurons and that deletion or PD mutation of RAB39B gene-induced RAB39B deficiency induces apoptotic death of dopaminergic neurons via impairing autophagy function and upregulating α-synuclein.

Functional variant rs17525453 within RAB35 gene promoter is possibly associated with increased risk of Parkinson's disease in Taiwanese population.

Our previous study suggests that upregulated RAB35 is implicated in etiology of Parkinson's disease (PD). We hypothesized that upregulated RAB35 results from single nucleotide polymorphisms (SNPs) in RAB35 gene promoter. We identified SNPs within RAB35 gene promoter by analyzing DNA samples of discovery cohort and validation cohort. SNP rs17525453 within RAB35 gene promoter (T>C at position of -66) was significantly associated with idiopathic PD patients. Compared to normal controls, sporadic PD patients had higher C allele frequency. CC and CT genotype significantly increased risk of PD compared with TT genotype. SNP rs17525453 within RAB35 gene promoter leads to formation of transcription factor TFII-I binding site. Results of EMSA and supershift assay indicated that TFII-I binds to rs17525453 sequence of RAB35 gene promoter. Luciferase reporter assays showed that rs17525453 variant of RAB35 gene promoter possesses an augmented transcriptional activity. Our results suggest that functional variant rs17525453 within RAB35 gene promoter is likely to enhance transcriptional activity and upregulate RAB35 protein, which could lead to increased risk of PD in Taiwanese population.

(D620N) VPS35 causes the impairment of Wnt/ß-catenin signaling cascade and mitochondrial dysfunction in a PARK17 knockin mouse model.

Patients with familial type 17 of Parkinson's disease (PARK17) manifest autosomal dominant pattern and late-onset parkinsonian syndromes. Heterozygous (D620N) mutation of vacuolar protein sorting 35 (VPS35) is genetic cause of PARK17. We prepared heterozygous VPS35D620N/+ knockin mouse, which is an ideal animal model of (D620N) VPS35-induced autosomal dominant PARK17. Late-onset loss of substantia nigra pars compacta (SNpc) dopaminergic (DAergic) neurons and motor deficits of Parkinson's disease were found in 16-month-old VPS35D620N/+ mice. Normal function of VPS35-containing retromer is needed for activity of Wnt/ß-catenin cascade, which participates in protection and survival of SNpc DAergic neurons. It was hypothesized that (D620N) VPS35 mutation causes the malfunction of VPS35 and resulting impaired activity of Wnt/ß-catenin pathway. Protein levels of Wnt1 and nuclear ß-catenin were reduced in SN of 16-month-old VPS35D620N/+ knockin mice. Downregulated protein expression of survivin, which is a target gene of nuclear ß-catenin, and upregulated protein levels of active caspase-8 and active caspase-9 were observed in SN of VPS35D620N/+ mice at age of 16 months. VPS35 is involved in controlling morphology and function of mitochondria. Impaired function of VPS35 caused by (D620N) mutation could lead to abnormal morphology and malfunction of mitochondria. A significant decrease in mitochondrial size and resulting mitochondrial fragmentation was found in tyrosine hydroxylase-positive and neuromelanin-positive SNpc DAergic neurons of 16-month-old VPS35D620N/+ mice. Mitochondrial complex I activity or complex IV activity was reduced in SN of 16-month-old VPS35D620N/+ mice. Increased level of mitochondrial ROS and oxidative stress were found in SN of 16-month-old VPS35D620N/+ mice. Levels of cytosolic cytochrome c and active caspase-3 were increased in SN of VPS35D620N/+ mice aged 16 months. Our results suggest that PARK17 mutant (D620N) VPS35 impairs activity of Wnt/ß-catenin signaling pathway and causes abnormal morphology and dysfunction of mitochondria, which could lead to neurodegeneration of SNpc DAergic cells.

Assembly and Detachment of Hyaluronic Acid on a Protein-Conjugated Gold Nanoparticle.

The assembly-disassembly of hyaluronic acid (HA) with a bovine serum albumin-conjugated gold nanoparticle (BSA-AuNP) was demonstrated using a gas-phase electrophoresis approach, electrospray-differential mobility analysis (ES-DMA). Physical sizes, number and mass concentrations, and degrees of aggregation of HA, BSA, and AuNP were successfully quantified using ES-DMA hyphenated with inductively coupled plasma mass spectrometry. Attenuated total reflection Fourier transform infrared (ATR-FTIR) spectroscopy was employed complementarily for an orthogonal characterization of the assembly of HA with BSA-AuNP and the subsequent HA detachment. The results show that the surface packing density of HA on BSA-AuNP was proportional to the concentration of HA (CHA) when CHA ≤ 5 × 10-3 µmol/L, and the equilibrium binding constant of HA on BSA-AuNP was identified as ≈ 4 × 105 L/mol at pH 3. The pH-sensitive and enzyme-induced detachments of HA from BSA-AuNP were both successfully characterized using ES-DMA and ATR-FTIR. In the absence of enzymatic catalysis, the rate constant of HA detachment (k) was shown to increase by at least 3.7 times on adjusting the environmental acidity from pH 3 to pH 7. A significant enzyme-induced HA detachment was identified at pH 7, showing a remarkable increase of k by at least two times in the presence of an enzyme. This work provides a proof of concept for assembly of HA-based hybrid colloidal nanomaterials through the tuning of surface chemistry in the aqueous phase with the ability of in situ quantitative characterization, which has shown promise for the development of a variety of HA-derivative biomedical applications (e.g., drug delivery).

Alda-1, an activator of ALDH2, ameliorates Achilles tendinopathy in cellular and mouse models.

Achilles tendinopathy has a high re-injury rate and poor prognosis. Development of effective therapy for Achilles tendinopathy is important. Excessive accumulation of ROS and resulting oxidative stress are believed to cause tendinopathy. Overproduction of hydrogen peroxide (H2O2), the most common ROS, could lead to the tendinopathy by causing oxidative damage, activation of endoplasmic reticulum (ER) stress and apoptotic death of tenocytes. Activation of mitochondrial aldehyde dehydrogenase 2 (ALDH2) is expected to alleviate oxidative stress and ER stress. Alda-1 is a selective and potent activator of ALDH2. In this study, we examined the cytoprotective benefit of Alda-1, an activator of ALDH2, on H2O2-induced Achilles tendinopathy in cellular and mouse models. We prepared cellular and mouse models of Achilles tendinopathy by treating cultured Achilles tenocytes and Achilles tendons with oxidative stressor H2O2. Subsequently, we studied the protective benefit of Alda-1 on H2O2-induced Achilles tendinopathy. Alda-1 pretreatment attenuated H2O2-induced cell death of cultured Achilles tenocytes. Treatment of Alda-1 prevented H2O2-induced oxidative stress and depolarization of mitochondrial membrane potential in tenocytes. Application of Alda-1 attenuated H2O2-triggered mitochondria- and ER stress-mediated apoptotic cascades in cultured tenocytes. Alda-1 treatment ameliorated the severity of H2O2-induced Achilles tendinopathy in vivo by preventing H2O2-induced pathological histological features of Achilles tendons, apoptotic death of Achilles tenocytes and upregulated expression of inflammatory cytokines IL-1ß and TNF-α. Our results provide the evidence that ALDH2 activator Alda-1 ameliorates H2O2-induced Achilles tendinopathy. Alda-1 could be used for preventing and treating Achilles tendinopathy.

Characterization of Microbial Dynamics and Volatile Metabolome Changes During Fermentation of Chambourcin Hybrid Grapes From Two Pennsylvania Regions.

Microbial diversity present on grapes in wineries, and throughout fermentation has been associated with important metabolites for final wine quality. Although microbiome-metabolome associations have been well characterized and could be used as indicators of wine quality, the impact of regionality on the microbiome and metabolome is not well known. Additionally, studies between microbiome and metabolome have been conducted on single species grape such as Vitis vinifera instead of other species and interspecific hybrids. Although the Pennsylvania wine industry is relatively young compared to California, the industry has been experiencing rapid growth over the past decade and is expected to continue to grow in the future. Pennsylvania's climate of cold winters and high levels of rainfall throughout the growing season favors cultivation of interspecific hybrid grapes such as Vitis ssp. Chambourcin, one of the most commonly grown hybrid varieties in the state. Chambourcin is a prime candidate for studying the impact of regionality on microbiome-metabolome interactions as interspecific hybrid varieties could shape the future of winemaking. Here, we identify for the first time the regional distribution of microbial communities and their interactions with volatile metabolome during fermentation (0-20 days) by integrating high throughput Illumina sequencing (16S and ITS) and headspace-solid phase microextraction-gas chromatography-mass spectrometry. Analyzing 88 samples from nine wineries in the Central and East Pennsylvania regions, we observed high microbial diversity during early stages of fermentation (1-4 days) where non-Saccharomyces yeasts such as Starmerella and Aureobasidium and non-Oenococcus bacteria, Sphingomonas, likely contribute to microbial terroir to the resulting wines. Furthermore, key differentiators between two regions in Pennsylvania, as identified by LEfSe analysis, include the fungal genera Cladosporium and Kazachstania and the bacterial genera Lactococcus and Microbacterium. Moreover, 29 volatile fermentation metabolites were discriminated significantly (variable importance in projection > 1) between the two regions as shown by Partial Least Squares-Discriminant Analysis. Finally, Spearman's correlation identified regional differences of microbial-metabolite associations throughout fermentation that could be used for targeted microbiome manipulation to improve wine quality and preserve regionality. In summary, these results demonstrate the microbial signatures during fermentation and differential microorganisms and metabolites further support impact of regionality on Chambourcin wines in Pennsylvania.

Combined Assessment of Serum Alpha-Synuclein and Rab35 is a Better Biomarker for Parkinson's Disease.

BACKGROUND AND PURPOSE: It is essential to develop a reliable predictive serum biomarker for Parkinson's disease (PD). The accumulation of alpha-synuclein (αSyn) and up-regulated expression of Rab35 participate in the etiology of PD. The purpose of this investigation was to determine whether the combined assessment of serum αSyn and Rab35 is a useful predictive biomarker for PD. METHODS: Serum levels of αSyn or Rab35 were determined in serum samples from 59 sporadic PD patients, 19 progressive supranuclear palsy (PSP) patients, 20 multiple system atrophy (MSA) patients, and 60 normal controls (NC). Receiver operating characteristics (ROC) curves were calculated to determine the diagnostic accuracy of αSyn or/and Rab35 in discriminating PD patients from NC or atypical parkinsonian patients. RESULTS: The levels of αSyn and Rab35 were increased in PD patients. The serum level of Rab35 was positively correlated with that of αSyn in PD patients. Compared to analyzing αSyn or Rab35 alone, the combined analysis of αSyn and Rab35 produced a larger area under the ROC curve and performed better in discriminating PD patients from NC, MSA patients, or PSP patients. When age was dichotomized at 55, 60, 65, or 70 years, the combined assessment of αSyn and Rab35 for classifying PD was better in the group below the cutoff age than in the group above the cutoff age. CONCLUSIONS: Combined assessment of serum αSyn and Rab35 is a better biomarker for discriminating PD patients from NC or atypical parkinsonian patients, and is a useful predictive biomarker for younger sporadic PD patients.

Upregulated Expression of MicroRNA-204-5p Leads to the Death of Dopaminergic Cells by Targeting DYRK1A-Mediated Apoptotic Signaling Cascade.

MicroRNAs (miRs) downregulate or upregulate the mRNA level by binding to the 3'-untranslated region (3'UTR) of target gene. Dysregulated miR levels can be used as biomarkers of Parkinson's disease (PD) and could participate in the etiology of PD. In the present study, 45 brain-enriched miRs were evaluated in serum samples from 50 normal subjects and 50 sporadic PD patients. The level of miR-204-5p was upregulated in serum samples from PD patients. An upregulated level of miR-204-5p was also observed in the serum and substantia nigra (SN) of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) mouse model of PD. Expression of miR-204-5p increased the level of α-synuclein (α-Syn), phosphorylated (phospho)-α-Syn, tau, or phospho-tau protein and resulted in the activation of endoplasmic reticulum (ER) stress in SH-SY5Y dopaminergic cells. Expression of miR-204-5p caused autophagy impairment and activation of c-Jun N-terminal kinase (JNK)-mediated apoptotic cascade in SH-SY5Y dopaminergic cells. Our study using the bioinformatic method and dual-luciferase reporter analysis suggests that miR-204-5p positively regulates mRNA expression of dual-specificity tyrosine phosphorylation regulated kinase 1A (DYRK1A) by directly interacting with 3'UTR of DYRK1A. The mRNA and protein levels of DYRK1A were increased in SH-SY5Y dopaminergic cells expressing miR-204-5p and SN of MPTP-induced PD mouse model. Knockdown of DYRK1A expression or treatment of the DYRK1A inhibitor harmine attenuated miR-204-5p-induced increase in protein expression of phospho-α-Syn or phospho-tau, ER stress, autophagy impairment, and activation of JNK-mediated apoptotic pathway in SH-SY5Y dopaminergic cells or primary cultured dopaminergic neurons. Our results suggest that upregulated expression of miR-204-5p leads to the death of dopaminergic cells by targeting DYRK1A-mediated ER stress and apoptotic signaling cascade.

Generation of induced pluripotent stem cells from a young-onset Parkinson's disease patient carrying the compound heterozygous PLA2G6 p.D331Y/p.M358IfsX mutations.

Mutations in PLA2G6 gene cause PLA2G6-associated neurodegeneration, including recessive familial type 14 of Parkinson's disease (PARK14). Previously, we identified PARK14 patients with compound heterozygous c.991G > T/c.1077G > A (p.D331Y/p.M358IfsX) mutations. The c.1077G > A mutation led to a four base-pairs deletion and frameshift mutation (p.M358IfsX) of PLA2G6 mRNA. We established induced pluripotent stem cells (iPSCs) from peripheral blood mononuclear cells of a female patient with compound heterozygous c.991G > T/c.1077G > A (p.D331Y/ p.M358IfsX) mutations by using Sendai-virus delivery system. The iPSCs exhibited pluripotency and in vivo differentiation potential. The iPSCs can be used for studying the molecular pathogenic mechanism of PARK14.

A facile quantification of hyaluronic acid and its crosslinking using gas-phase electrophoresis.

We report a facile, high-resolution approach to quantitatively characterize hyaluronic acid (HA) and study its crosslinking reaction using electrospray-differential mobility analysis (ES-DMA). Mobility size distributions, number concentrations, molecular mass distributions, and polydispersity index of HAs were obtained successfully via a rapid analysis by ES-DMA (< 30 min). The limit of detection, the limit of quantification, and the precision of the mobility size measurement achieve 2.5 nm, 4.0 nm, and 0.3 nm, respectively. Size exclusion chromatography (SEC) was employed as an orthogonal approach, showing that the averaged molecular mass and polydispersity index of HA measured by ES-DMA were close to the results of SEC on a semi-quantitative basis. The 1,4-butanediol diglycidyl ether (BDDE)-induced crosslinking of HA was also able to be successfully characterized through a time-dependent study using ES-DMA, which has shown the promise of direct analysis of solution-based reactions. Both the extent and the rate of HA crosslinking (induced by BDDE) were proportional to reaction temperature and concentration ratio of HA to BDDE. The activation energy of the reaction-limited BDDE-induced crosslinking of HA was found to be ≈ 21 kJ/mol. The prototype study demonstrates ES-DMA as a new method for a rapid quantitative characterization of HA and its derivative product and providing a capability of real-time monitoring of the HA crosslinking during formulation process. Graphical abstract.

PARK14 (D331Y) PLA2G6 Causes Early-Onset Degeneration of Substantia Nigra Dopaminergic Neurons by Inducing Mitochondrial Dysfunction, ER Stress, Mitophagy Impairment and Transcriptional Dysregulation in a Knockin Mouse Model.

PARK14 patients with homozygous (D331Y) PLA2G6 mutation display motor deficits of pure early-onset Parkinson's disease (PD). The aim of this study is to investigate the pathogenic mechanism of mutant (D331Y) PLA2G6-induced PD. We generated knockin (KI) mouse model of PARK14 harboring homozygous (D331Y) PLA2G6 mutation. Then, we investigated neuropathological and neurological phenotypes of PLA2G6D331Y/D331Y KI mice and molecular pathogenic mechanisms of (D331Y) PLA2G6-induced degeneration of substantia nigra (SN) dopaminergic neurons. Six-or nine-month-old PLA2G6D331Y/D331Y KI mice displayed early-onset cell death of SNpc dopaminergic neurons. Lewy body pathology was found in the SN of PLA2G6D331Y/D331Y mice. Six-or nine-month-old PLA2G6D331Y/D331Y KI mice exhibited early-onset parkinsonism phenotypes. Disrupted cristae of mitochondria were found in SNpc dopaminergic neurons of PLA2G6D331Y/D331Y mice. PLA2G6D331Y/D331Y mice displayed mitochondrial dysfunction and upregulated ROS production, which may lead to activation of apoptotic cascade. Upregulated protein levels of Grp78, IRE1, PERK, and CHOP, which are involved in activation of ER stress, were found in the SN of PLA2G6D331Y/D331Y mice. Protein expression of mitophagic proteins, including parkin and BNIP3, was downregulated in the SN of PLA2G6D331Y/D331Y mice, suggesting that (D331Y) PLA2G6 mutation causes mitophagy dysfunction. In the SN of PLA2G6D331Y/D331Y mice, mRNA levels of eight genes that are involved in neuroprotection/neurogenesis were decreased, while mRNA levels of two genes that promote apoptotic death were increased. Our results suggest that PARK14 (D331Y) PLA2G6 mutation causes degeneration of SNpc dopaminergic neurons by causing mitochondrial dysfunction, elevated ER stress, mitophagy impairment, and transcriptional abnormality.

PARK14 PLA2G6 mutants are defective in preventing rotenone-induced mitochondrial dysfunction, ROS generation and activation of mitochondrial apoptotic pathway.

Mutations in the gene encoding Ca2+-independent phospholipase A2 group 6 (PLA2G6) cause the recessive familial type 14 of Parkinson's disease (PARK14). Mitochondrial dysfunction is involved in the pathogenesis of Parkinson's disease (PD). PLA2G6 is believed to be required for maintaining mitochondrial function. In the present study, rotenone-induced cellular model of PD was used to investigate possible molecular pathogenic mechanism of PARK14 mutant PLA2G6-induced PD. Overexpression of wild-type (WT) PLA2G6 ameliorated rotenone-induced apoptotic death of SH-SY5Y dopaminergic cells. PARK14 mutant (D331Y), (G517C), (T572I), (R632W), (N659S) or (R741Q) PLA2G6 failed to prevent rotenone-induced activation of mitochondrial apoptotic pathway and exert a neuroprotective effect. WT PLA2G6, but not PARK14 mutant PLA2G6, prevented rotenone-induced mitophagy impairment. In contrast to WT PLA2G6, PARK14 mutant PLA2G6 was ineffective in attenuating rotenone-induced decrease in mitochondrial membrane potential and increase in the level of mitochondrial superoxide. WT PLA2G6, but not PARK14 PLA2G6 mutants, restored enzyme activity of mitochondrial complex I and cellular ATP content in rotenone-treated SH-SY5Y dopaminergic cells. In contrast to WT PLA2G6, PARK14 mutant PLA2G6 failed to prevent rotenone-induced mitochondrial lipid peroxidation and cytochrome c release. These results suggest that PARK14 PLA2G6 mutants lose their ability to maintain mitochondrial function and are defective inpreventing mitochondrial dysfunction, ROS production and activation of mitochondrial apoptotic pathway in rotenone-induced cellular model of PD.

Increased Rab35 expression is a potential biomarker and implicated in the pathogenesis of Parkinson's disease.

Parkinson's disease (PD) is the second common neurodegenerative disease. Identification of biomarkers for early diagnosis and prediction of disease progression is important. The present comparative proteomic study of serum samples using two-dimensional fluorescence differential gel electrophoresis followed by ELISA confirmation demonstrated that protein expression of Rab35 was increased in PD patients compared with matched control subjects and other parkinsonian disorders, progressive supranuclear palsy (PSP) and multiple system atrophy (MSA). The serum level of Rab35 was significantly correlated with the age at onset of PD. The median age of onset in patients with higher Rab35 serum level was 5 years younger than those with lower Rab35 serum level. There was a positive correlation between the Rab35 level and disease duration of PD. Moreover, the protein expression of Rab35 was increased in the substantia nigra but not in the striatum of mouse models of PD, including MPTP-treated mice, rotenone-treated mice, (R1441C) LRRK2 or (G2019S) LRRK2 transgenic mice. Furthermore, overexpression of Rab35 increased the aggregation and secretion of mutant A53T α-synuclein in dopaminergic SH-SY5Y cells. Co-expression of Rab35 with wild-type or A53T α-synuclein in SH-SY5Y cells deteriorated cell death. Our results suggest that Rab35 is potentially useful in the differential diagnosis of parkinsonian disorders and is implicated in the pathogenesis of PD.

(R1441C) LRRK2 induces the degeneration of SN dopaminergic neurons and alters the expression of genes regulating neuronal survival in a transgenic mouse model.

Mutation of leucine-rich repeat kinase 2 (LRRK2) is the most common genetic cause of both familial and sporadic Parkinson's disease (PD) cases. Several mutations in LRRK2 gene were reported in PD patients. R1441 is the second most frequent site of LRRK2 mutation. We generated (R1441C) LRRK2 transgenic mice that displayed motor deficits at the age of 16 months. Compared with wild-type mice, 16-month-old (R1441C) LRRK2 mice exhibited a significant reduction in the number of substantia nigra (SN) dopaminergic neurons. To elucidate molecular pathogenic pathways involved in (R1441C) LRRK2-induced death of SN dopaminergic neurons, we performed microarray analysis to visualize altered mRNA expressions in the SN of (R1441C) LRRK2 mouse. In the SN of (R1441C) LRRK2 transgenic mouse, the mRNA expression of three genes that promote cell death was upregulated, while the mRNA expression of seven genes that contribute to neurogenesis/neuroprotection was significantly downregulated. Our results suggest that altered expression of these genes involved in regulating neuronal survival may contribute to the pathogenesis of (R1441C) LRRK2-induced PD.

T1-11 and JMF1907 ameliorate polyglutamine-expanded ataxin-3-induced neurodegeneration, transcriptional dysregulation and ataxic symptom in the SCA3 transgenic mouse.

More studies are required to develop therapeutic agents for treating spinocerebellar ataxia type 3 (SCA3), which is caused by mutant polyglutamine-expanded ataxin-3 and is the most prevalent subtype of spinocerebellar ataxias. T1-11 [N6-(4-Hydroxybenzyl) adenosine], isolated from a Chinese medicinal herb Gastordia elata, is an adenosine A2A receptor agonist. SCA3 and Huntington's disease (HD) belong to a family of polyglutamine neurodegenerative diseases. T1-11 exerted a therapeutic effect on HD transgenic mouse by decreasing protein level of polyglutamine-expanded huntingtin in the striatum. In the present study, we test the possibility that T1-11 or JMF1907 [N6-(3-Indolylethyl) adenosine], a synthetic analog of T1-11, alleviates pontine neuronal death, cerebellar transcriptional downregulation and ataxic symptom in the SCA3 transgenic mouse expressing HA-tagged polyglutamine-expanded ataxin-3-Q79 (ataxin-3-Q79HA). Daily oral administration of T1-11 or JMF1907 prevented neuronal death of pontine nuclei in the SCA3 mouse with a dose-dependent manner. Oral application of T1-11 or JMF1907 reversed mutant ataxin-3-Q79-induced cerebellar transcriptional repression in the SCA3 transgenic mouse. T1-11 or JMF1907 ameliorated the symptom of motor incoordination displayed by SCA3 mouse. Oral administration of T1-11 or JMF1907 significantly decreased protein level of ataxin-3-Q79HA in the pontine nuclei or cerebellum of SCA3 mouse. T1-11 or JMF1907 significantly augmented the chymotrypsin-like activity of proteasome in the pontine nuclei or cerebellum of SCA3 mouse. Our results suggests that T1-11 and JMF1907 alleviate pontine neuronal death, cerebellar transcriptional downregulation and ataxic symptom of SCA3 transgenic mouse by augmenting the proteasome activity and reducing the protein level of polyglutamine-expanded ataxin-3-Q79 in the pontine nuclei and cerebellum.

Neuroprotective effects of aldehyde dehydrogenase 2 activation in rotenone-induced cellular and animal models of parkinsonism.

Many studies have shown that mitochondrial aldehyde dehydrogenase 2 (ALDH2) functions as a cellular protector against oxidative stress by detoxification of cytotoxic aldehydes. Within dopaminergic neurons, dopamine is metabolized by monoamine oxidase to yield 3,4-dihydroxyphenylacetaldehyde (DOPAL) then converts to a less toxic acid product by ALDH. The highly toxic and reactive DOPAL has been hypothesized to contribute to the selective neurodegeneration in Parkinson's disease (PD). In this study, we investigated the neuroprotective mechanism and therapeutic effect of ALDH2 in rotenone models for parkinsonism. Overexpression of wild-type ALDH2 gene, but not the enzymatically deficient mutant ALDH2*2 (E504K), reduced rotenone-induced cell death. Application of a potent activator of ALDH2, Alda-1, was effective in protecting against rotenone-induced apoptotic cell death in both SH-SY5Y cells and primary cultured substantia nigra (SN) dopaminergic neurons. In addition, intraperitoneal administration of Alda-1 significantly reduced rotenone- or MPTP-induced death of SN tyrosine hydroxylase (TH)-positive dopaminergic neurons. The attenuation of rotenone-induced apoptosis by Alda-1 resulted from decreasing ROS accumulation, reversal of mitochondrial membrane potential depolarization, and inhibition of activation of proteins related to mitochondrial apoptotic pathway. The present study demonstrates that ALDH2 plays a crucial role in maintaining normal mitochondrial function to protect against neurotoxicity and that Alda-1 is effective in ameliorating mitochondrial dysfunction and inhibiting mitochondria-mediated apoptotic pathway. These results indicate that ALDH2 activation could be a neuroprotective therapy for PD.

Transglutaminase 2 expression is increased as a function of malignancy grade and negatively regulates cell growth in meningioma.

Most meningiomas are benign, but some clinical-aggressive tumors exhibit brain invasion and cannot be resected without significant complications. To identify molecular markers for these clinically-aggressive meningiomas, we performed microarray analyses on 24 primary cultures from 21 meningiomas and 3 arachnoid membranes. Using this approach, increased transglutaminase 2 (TGM2) expression was observed, which was subsequently validated in an independent set of 82 meningiomas by immunohistochemistry. Importantly, the TGM2 expression level was associated with increasing WHO malignancy grade as well as meningioma recurrence. Inhibition of TGM2 function by siRNA or cystamine induced meningioma cell death, which was associated with reduced AKT phosphorylation and caspase-3 activation. Collectively, these findings suggest that TGM2 expression increases as a function of malignancy grade and tumor recurrence and that inhibition of TGM2 reduces meningioma cell growth.