TH Protein Research Articles

A pilot study on the mechanism of Guasha in treating Parkinson's disease based on molecular level and ultra-trace proteomics analysis

Guasha is a widely applied non-pharmacological therapy of traditional Chinese medicine (TCM), which has been proved to be effective in the treatment of Parkinson's disease (PD). The objective of this study was to explore the mechanisms of Guasha therapy in the pathological processes including neuroinflammation in PD. After 3 Guasha courses, a reduction in α-synuclein aggregation and alleviation of microglia activation were observed in the substantia nigra pars compacta (SNpc). Furthermore, levels of TNF-α, IL-1β and IFN-γ in the SNpc decreased, while plasma TGF-β, IL-4 and IL-10 increased. Moreover, both TH protein and mRNA levels in the SNpc, as well as dopamine levels in the striatum, exhibited an increase. The proteomics analysis results based on plasma-derived exosomes revealed a total of 943 differentially expressed proteins identified. Compared to the model group, the Guasha group screened for 82 differential proteins, with 30 upregulated and 52 downregulated. The improvement of pathological changes in the PD model mice treated with Guasha primarily involves biological processes such as oxidative stress, immune response and inflammation, and cellular structure regulation. It involves signaling pathways related to aldosterone synthesis and secretion, adrenergic signaling in myocardial cells, COVID-19-related inflammatory signaling, and neurotrophic signaling. Collectively, the mechanisms of Guasha for treating PD might be closely related to inhibiting microglial cell activation-mediated neuroinflammation, regulating oxidative stress, cellular structure, aldosterone synthesis and secretion-mediated electrolyte balance, as well as noradrenergic signaling-mediated neuroprotection. These findings provided new insight for Guasha in treating PD and would potentially enhance therapeutic interventions.

α-synuclein regulates Cyclin D1 to promote abnormal initiation of the cell cycle and induce apoptosis in dopamine neurons

The etiology of Parkinson's disease (PD) is characterized by the death of dopamine neurons in the substantia nigra pars compacta, while misfolding and abnormal aggregation of α-synuclein (α-syn) are core pathological features. Previous studies have suggested that damage to dopamine neurons may be related to cell cycle dysregulation, but the specific mechanisms remain unclear. In this study, a PD mouse model was induced by stereotactic injection of α-syn into the nucleus, and treated with the cell cycle inhibitor, roscovitine (Rosc). The results demonstrated that Rosc improved behavioral disorders caused by α-syn, increased TH protein expression, inhibited α-syn and p-α-syn protein expression, and reduced the expression levels of G1/S phase cell cycle genes Cyclin D1, Cyclin E, CDK2, CDK4, E2F and pRB. Additionally, Rosc decreased Bax and Caspase-3 expression caused by α-syn, while increasing Bcl-2 protein expression. Meanwhile, we observed that α-syn can influence neuronal cell autophagy by decreasing the expression level of Beclin 1 and increasing the expression level of P62. However, Rosc can improve this phenomenon. In a cell model induced by α-syn in dopamine neuron injury cells, knockdown of Cyclin D1 led to similar results as those observed in animal experiments: Knocking down Cyclin D1 improved the abnormal initiation of the cell cycle caused by α-syn and regulated cellular autophagy, resulting in a reduction of apoptosis in dopamine neurons. In summary, exogenous α-syn can lead to the accumulation of α-syn and phosphorylated α-syn in dopamine neurons, increase key factors of the G1/S phase cell cycle such as Cyclin D1, and regulate downstream related indicators, causing the cell cycle to restart and leading to apoptosis of dopamine neurons. This exacerbates PD symptoms. However, knockdown of Cyclin D1 inhibits the progression of the cell cycle and can reverse this situation. These findings suggest that a Cyclin D inhibitor may be a novel therapeutic target for treating PD.

Electroacupuncture pretreatment mediates sympathetic nerves to alleviate myocardial ischemia–reperfusion injury via CRH neurons in the paraventricular nucleus of the hypothalamus

BackgroundMyocardial ischemia–reperfusion can further exacerbate myocardial injury and increase the risk of death. Our previous research found that the paraventricular nucleus (PVN) of the hypothalamus plays a crucial role in the improvement of myocardial ischemia–reperfusion injury (MIRI) by electroacupuncture (EA) pretreatment, but its mechanism of action is still unclear. CRH neurons exhibit periodic concentrated expression in PVN, but further research is needed to determine whether they are involved in the improvement of MIRI by EA pretreatment. Meanwhile, numerous studies have shown that changes in sympathetic nervous system innervation and activity are associated with many heart diseases. This study aims to investigate whether EA pretreatment improves MIRI through sympathetic nervous system mediated by PVNCRH neurons.MethodsIntegrated use of fiber-optic recording, chemical genetics and other methods to detect relevant indicators: ECG signals were acquired through Powerlab standard II leads, and LabChart 8 calculated heart rate, ST-segment offset, and heart rate variability (HRV); Left ventricular ejection fraction (LVEF), left ventricular short-axis shortening (LVFS), left ventricular end-systolic internal diameter (LVIDs) and interventricular septal thickness (IVSs) were measured by echocardiography; Myocardial infarct area (IA) and area at risk (AAR) were calculated by Evans-TTC staining. Pathological changes in cardiomyocytes were observed by HE staining; Changes in PVNCRH neuronal activity were recorded by fiber-optic photometry; Sympathetic nerve discharges were recorded for in vivo electrophysiology; NE and TH protein expression was assayed by Western blot.ResultsOur data indicated that EA pretreatment can effectively alleviate MIRI. Meanwhile, we found that in the MIRI model, the number and activity of CRH neurons co labeled with c-Fos in the PVN area of the rat brain increased, and the frequency of sympathetic nerve discharge increased. EA pretreatment could reverse this change. In addition, the results of chemical genetics indicated that inhibiting PVNCRH neurons has a similar protective effect on MIRI as EA pretreatment, and the activation of PVNCRH neurons can counteract this protective effect.ConclusionEA pretreatment can inhibit PVNCRH neurons and improve MIRI by inhibiting sympathetic nerve, which offers fresh perspectives on the application of acupuncture in the management of cardiovascular disease.

Resveratrol protects dopaminergic neurons in a mouse model of Parkinson's disease by regulating the gut-brain axis via inhibiting the TLR4 signaling pathway

To investigate the protective effect of resveratrol on intestinal barrier in 1-methyl-4-phenyl-1, 2, 3, 6-tetrahydropyridine (MPTP)-induced Parkinson's disease (PD) mouse models and its mechanism for regulating TLR4/MyD88/NF-κB signaling to protect dopaminergic neurons. Fifty-two C57BL/6J mice were randomized into control group (n= 12), MPTP group (n=14), MPTP + resveratrol (30 mg/kg) group (n=13), and MPTP + resveratrol (90 mg/kg) group (n=13), and mouse models were established by intraperitoneal MPTP (30 mg/kg) injection for 7 days in the latter 3 groups. Behavioral tests were conducted to evaluate the effect of resveratrol on motor symptoms of the mice. Western blotting was used to detect the expression of TH, α-syn, ZO-1, Claudin-1, TLR4, MyD88, and NF-κB in the brain tissues of the mice. Immunohistochemistry, immunofluorescence, ELISA and transmission electron microscopy were used to verify the effect of resveratrol for suppressing inflammation and protecting the intestinal barrier. Compared with those in the normal control group, the mice in MPTP group showed significant changes in motor function, number of dopaminergic neurons, neuroinflammation, levels of LPS and LBP, and expressions of tight junction proteins in the intestinal barrier. Resveratrol treatment significantly improved motor function of the PD mice (P < 0.01), increased the number of neurons and TH protein expression (P < 0.05), down-regulated the expressions of GFAP, Iba-1, and TLR4, lowered fecal and plasma levels of LPS and LBP (P < 0.05), restored the expression levels of ZO-1 and Claudin-1 (P < 0.01), and down-regulated the expressions of TLR4, MyD88, and NF-κB in the colon tissue (P < 0.05). The mice with resveratrol treatment at 30 mg/kg showed normal morphology of the tight junction complex with neatly and tightly arranged intestinal villi. Resveratrol repairs the intestinal barrier by inhibiting TLR4/MyD88/NF-κB signaling pathway-mediated inflammatory response, thereby improving motor function and neuropathy in mouse models of MPTP-induced PD.

Secretory Clusterin Inhibits Dopamine Neuron Apoptosis in MPTP Mice by Preserving Autophagy Activity

Secretory clusterin (sCLU) plays an important role in the research progress of nervous system diseases. However, the physiological function of sCLU in Parkinson's disease (PD) are unclear. The purpose of this study was to examine the effects of sCLU-mediated autophagy on cell survival and apoptosis inhibition in a 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced mouse model of PD. We found that MPTP administration induced prolonged pole-climbing time, shortened traction time and rotarod time, significantly decreased TH protein expression in the SN tissue of mice. In contrast, sCLU -treated mice took less time to climb the pole and had an extended traction time and rotating rod time. Meanwhile, sCLU intervention induced increased expression of the TH protein in the SN of mice. These results indicated that sCLU intervention could reduce the loss of dopamine neurons in the SN area and alleviate dyskinesia in mice. Furthermore, MPTP led to suppressed viability, enhanced apoptosis, an increased Bax/Bcl-2 ratio, and cleaved caspase-3 in the SN of mice, and these effects were abrogated by sCLU intervention. In addition, MPTP increased the levels of P62 protein, decreased Beclin1 protein, decreased the ratio of LC3B-II/LC3B-I, and decreased the numbers of autophagosomes and autophagolysosomes in the SN tissues of mice. These effects were also abrogated by sCLU intervention. Activation of PI3K/AKT/mTOR signaling with MPTP inhibited autophagy in the SN of MPTP mice; however, sCLU treatment activated autophagy in MPTP-induced PD mice by inhibiting PI3K/AKT/mTOR signaling. These data indicated that sCLU treatment had a neuroprotective effect in an MPTP-induced model of PD.

Cotransplantation of NSCs and ethyl stearate promotes synaptic plasticity in PD rats by Drd1/ERK/AP-1 signaling pathway

Ethnopharmacological relevanceTraditional Chinese medicine views kidney shortage as a significant contributor to the aetiology of Parkinson's disease (PD), a neurodegenerative condition that is closely linked to aging. In clinical, patients with Parkinson's disease are often treated with Testudinis Carapax et Plastrum (Plastrum Testudinis, PT), a traditional Chinese medication that tonifies the kidney. Previous research has demonstrated that ethyl stearate (PubChem CID: 8122), an active component of Plastrum Testudinis Extracted with ethyl acetate (PTE), may encourage neural stem cells (NSCs) development into dopaminergic (DAergic) neurons. However, the effectiveness and mechanism of cotransplantation of ethyl stearate and NSCs in treating PD model rats still require further investigation. Aim of the studyPD is a neurodegenerative condition marked by the loss and degradation of dopaminergic neurons in the substantia nigra of the midbrain. Synaptic damage is also a critical pathology in PD. Because of their self-renewal, minimal immunogenicity, and capacity to differentiate into dopaminergic (DAergic) neurons, NSCs are a prospective treatment option for Parkinson's disease cell transplantation therapy. However, encouraging transplanted NSCs to differentiate into dopaminergic neurons and enhancing synaptic plasticity in vivo remains a significant challenge in improving the efficacy of NSCs transplantation for PD. This investigation seeks to examine the efficacy of cotransplantation of NSCs and ethyl stearate in PD model rats and its mechanism related to synaptic plasticity. Materials and methodsOn 6-hydroxydopamine-induced PD model rats, we performed NSCs transplantation therapy and cotransplantation therapy involving ethyl stearate and NSCs. Rotating behavior induced by apomorphine (APO) and pole climbing tests were used to evaluate behavioral changes. Using a variety of methods, including Western blotting (WB), immunofluorescence analysis, enzyme-linked immunosorbent assay, and quantitative real-time polymerase chain reaction (qRT-PCR), we examined the function and potential molecular mechanisms of ethyl stearate in combined NSCs transplantation therapy. ResultsIn the rat PD model, cotransplantation of ethyl stearate with NSCs dramatically reduced motor dysfunction, restored TH protein levels, and boosted dopamine levels in the striatum, according to our findings. Furthermore, the expression levels of SYN1 and PSD95, markers of synaptic plasticity, and BDNF, closely related to synaptic plasticity, were significantly increased. Cotransplantation with ethyl stearate and NSCs also increased the expression levels of Dopamine Receptor D1 (Drd1), an important receptor in the dopamine neural circuit, accompanied by an increase in MMP9 levels, ERK1/2 phosphorylation levels, and c-fos protein levels. ConclusionsAccording to the results of our investigation, cotransplantation of ethyl stearate and NSCs significantly improves the condition of PD model rats. We found that cotransplantation of ethyl stearate and NSCs may promote the expression of MMP9 by regulating the Drd1-ERK-AP-1 pathway, thus improving synaptic plasticity after NSCs transplantation. These findings provide new experimental support for the treatment of PD with the kidney tonifying Chinese medicine Plastrum Testudinis and suggest a potential therapeutic strategy for PD based on cotransplantation therapy.

Dihydromyricetin alleviates pyroptosis and necroptosis in mice with MPTP-induced chronic Parkinson's disease by inducing autophagy

To investigate the effect of 8-week dihydromyricetin (DHM) treatment on motor ability of mice with MPTP/probenecid-induced Parkinson's disease (PD) and explore the molecular mechanism. Sixty C57BL/6 mice were randomized into the control group, PD model group, PD+DHM group and PD+NEC-1 group (n=15). In the latter 3 groups, the mice were treated with 25 mg·kg-1·d-1 MPTP and 250 mg·kg-1·d-1 probenecid twice a week for 5 weeks to establish PD models; DHM (100 mg·kg-1·d-1) was administered 5 times a week via gavage for 8 weeks and NEC-1 (6.25 mg·kg-1·d-1, twice a week) via intraperitoneal injection for 5 weeks. The changes in motor function of the mice were assessed, and the expressions of TH, GFAP and Iba-1 in the substantia nigra were detected with immunofluorescence assay; serum levels of IL-1β and LDH were detected using ELISA. The mRNA expressions of TNF-α and IL-6 were determined with RT-PCR, and the expressions of TH and proteins associated with pyroptosis, neuroinflammation, necroptosis and autophagy in the striatum were detected using Western blotting. MPP +-activated Bv-2 cells were treated with different concentrations of DHM or 3-MA, and the expressions of proteins associated with autophagy and NLRP3 were detected using Western blotting; PI staining was used to detect cell necroptosis. The PD mouse models showed significantly reduced TH-positive cells and TH protein expression (P < 0.001). DHM obviously ameliorated motor deficits and TH loss in PD mice, increased TH expression (P=0.0023), decreased α-syn levels (P < 0.001), lowered the protein expressions of GFAP (P=0.045) and Iba-1 (P < 0.001) and the mRNA and protein levels of TNF-α (P=0.0015) and IL-6 (P < 0.001), and increased IL-4 level (P < 0.001). The 8-week DHM treatment significantly suppressed pyroptosis and necroptosis and activated autophagy in the striatum of the PD mice. In MPP +-induced Bv-2 cells, DHM treatment effectively reversed autophagy impairment and inhibited NLRP3 and TNF-α, IL-6 and IL-1β release, and the anti--inflammatory effects of DHM was obviously blunted by 3-MA. DHM can improve motor function of PD mice probably by activating autophagy to inhibit pyroptosis and necroptosis and reduce neuroinflammation.

Qiji Shujiang granules alleviates dopaminergic neuronal injury of parkinson's disease by inhibiting NLRP3/Caspase-1 pathway mediated pyroptosis

BackgroundThe Qiji Shujiang granule (QJG) is a traditional Chinese drug widely used in treating PD patients. However, the potential mechanism of QJG in PD therapy is still unclear. PurposeThis study aims to examine the neuroprotective effects of QJG and the specific mechanism by which QJG alleviates MPTP/Probenecid-induced pyroptosis and offers an alternative for PD treatment. Study Design and MethodsWe first employed network pharmacology along with molecular docking to identify potential molecular targets and pathways. Subsequently, we validated our findings of RNA-sequencing (RNA-seq) analysis and experiments in vivo and vitro. Lentiviral systems and inhibitors were used for experiments. ResultsThe protein-protein interactions (PPI) core genes network consists of NLRP3, CASP1 (caspase-1), TP53, and MAPK8. Pathway enrichment analysis revealed that inflammatory responses related to pyroptosis were significantly enriched. The molecular docking findings showed the highest degree of centrality regarding the top three bioactive compounds following the online database. RNA-seq analysis identified that NLRP3 inflammasome was significantly downregulated in the QJG group while it was significantly upregulated in the model group. Our findings revealed that QJG dose-dependently increased the total traveled distances, enhanced the dopaminergic neurons, and accelerated the restoration of the TH protein level, showing a good antioxidant capacity through increasing the SOD levels and decreasing MDA levels. QJG significantly reduced the expression levels of NLRP3, GSDMD-N, IL-1β, and caspase-1 in striatum tissue. Furthermore, the group treated with OE-NLRP3 decreased cell viability, increased ROS and MDA levels, and promoted NLRP3, GSDMD-N, and caspase-1, in addition to IL-1β expression levels. Furthermore, OE-NLRP3+QJG treatment significantly reversed the effect. In vivo experiments, QJG dose-dependently alleviated motor impairment by increasing the total traveled distances, rescued dopaminergic neurons, inhibited oxidative stress through increasing the SOD levels and decreasing MDA levels and suppressed NLRP3-mediated pyroptosis by reducing the expression levels of NLRP3, GSDMD-N, IL-1β, and caspase-1 in MPTP induced PD Mice. Moreover, in vitro experiments, the OE-NLRP3 treated group decreased cell viability, increased ROS and MDA levels, and promoted NLRP3, GSDMD-N, caspase-1, in addition to IL-1β expression levels. Furthermore, OE-NLRP3+QJG treatment significantly reversed the effect. ConclusionsThis study provides pharmacological support for the use of QJG in the treatment of PD. Herein, we concluded that QJG induced the alleviation of pyroptosis by inhibiting the NLRP3/caspase-1 pathway to exert a neuroprotective effect.

Dyrk1a Phosphorylation of α-Synuclein Mediating Apoptosis of Dopaminergic Neurons in Parkinson's Disease.

To investigate the role of aberrant Dyrk1a expression in phosphorylation modification at the α-synuclein serine 129 (Ser129) site to analyze its molecular mechanism in mediating apoptosis of PD. The protein level of P-α-synuclein (Ser129), α-synuclein, Bcl-2, Bax, active caspase 3, GSK3β, PI3K, AKT, and cyclinD1 were detected. The mRNA transcript levels of Dyrk1a and DAT and protein levels of IL-1β, IL-6, COX-2, and TNF-α were detected. P-α-synuclein (Ser129), α-synuclein, Bax, active caspase 3, GSK3β, and cyclinD1 expressions were decreased in Dyrk1a-AAV-ShRNA (P < 0.05), and Bcl-2, AKT, and PI3K expressions were increased (P < 0.05). Increased TH protein expression was shown in Dyrk1a-AAV-ShRNA (P < 0.05). Dyrk1a mRNA was decreased in the Dyrk1a-AAV-ShRNA group (P < 0.05), and DAT mRNA was increased (P < 0.05). IL-1β, IL-6, COX-2, and TNF-α protein levels were decreased in Dyrk1al-AAV-Sh-RNA (P < 0.05). Transcriptome sequencing showed that Fam220a, which was expected to activate STAT family protein binding activity and participate in the negative regulation of transcription through RNA polymerase II and protein dephosphorylation showed differentially upregulated expression. The untargeted metabolome showed that the major compounds in the Dyrk1a-AAV-ShRNA group were hormones and transmission mediators and the most metabolism-related pathways. Fam220a showed differentially upregulated expression, and differentially expressed genes were enriched for the neuroactive ligand-receptor interaction, vascular smooth muscle contraction, and melanogenesis-related pathways. Abnormal Dyrk1a expression can affect α-synuclein phosphorylation modifications, and dyrk1a knockdown activates the PI3K/AKT pathway and reduces dopaminergic neuron apoptosis. It provides a theoretical basis for the group to further investigate the molecular mechanism.

The Mitochondrial-Derived Peptide (MOTS-c) Interacted with Nrf2 to Defend the Antioxidant System to Protect Dopaminergic Neurons Against Rotenone Exposure.

MOTS-c is a 16-amino acid mitochondrial-derived peptide reported to be involved in regulating energy metabolism. However, few studies have reported the role of MOTS-c on neuron degeneration. In this study, it was aimed to explore the action of MOTS-c in rotenone-induced dopaminergic neurotoxicity. In an in vitro study, it was observed that rotenone could influence the expression and localization of MOTS-c significantly in PC12 cells, with more MOTS-c translocating into the nucleus from mitochondria. Further study showed that the translocation of MOTS-c from the mitochondria into the nucleus could directly interact with Nrf2 to regulate HO-1 and NQO1 expression in PC12 cells exposed to rotenone, which had been suggested to be involved in the antioxidant defense system. In vivo and in vitro experiments demonstrated that exogenous MOTS-c pretreatment could protect PC12 cells and rats from mitochondrial dysfunction and oxidative stress induced by rotenone. Moreover, MOTS-c pretreatment significantly decreased the loss of TH, PSD95, and SYP protein expression in the striatum of rats exposed to rotenone. In addition, MOTS-c pretreatment could clearly alleviate the downregulated expression of Nrf2, HO-1, and NQO1, as well as the upregulated Keap1 protein expression in the striatum of rotenone-treated rats. Taken together, these findings suggested that MOTS-c could directly interact with Nrf2 to activate the Nrf2/HO-1/NQO1 signal pathway to defend the antioxidant system to prevent dopaminergic neurons from rotenone-induced oxidative stress and neurotoxicity in vitro and in vivo.

Fluorescence microscopy-based sensitive method to quantify dopaminergic neurodegeneration in a Drosophila model of Parkinson's disease.

Death of dopaminergic (DAergic) neurons in the substantia nigra pars compacta of the human brain is the characteristic pathological feature of Parkinson's disease (PD). On exposure to neurotoxicants, Drosophila too exhibits mobility defects and diminished levels of brain dopamine. In the fly model of sporadic PD, our laboratory has demonstrated that there is no loss of DAergic neuronal number, however, a significant reduction in fluorescence intensity (FI) of secondary antibodies that target the primary antibody-anti-tyrosine hydroxylase (TH). Here, we present a sensitive, economical, and repeatable assay to characterize neurodegeneration based on the quantification of FI of the secondary antibody. As the intensity of fluorescence correlates with the amount of TH synthesis, its reduction under PD conditions denotes the depletion in the TH synthesis, suggesting DAergic neuronal dysfunction. Reduction in TH protein synthesis is further confirmed through Bio-Rad Stain-Free Western Blotting. Quantification of brain DA and its metabolites (DOPAC and HVA) using HPLC-ECD further demonstrated the depleted DA level and altered DA metabolism as evident from enhanced DA turnover rate. Together all these PD marker studies suggest that FI quantification is a refined and sensitive method to understand the early stages of DAergic neurodegeneration. FI quantification is performed using ZEN 2012 SP2, a licensed software from Carl Zeiss, Germany. This method will be of good use to biologists, as it with few modifications, can also be implemented to characterize the extent of degeneration of different cell types. Unlike the expensive and cumbersome confocal microscopy, the present method using fluorescence microscopy will be a feasible option for fund-constrained neurobiology laboratories in developing countries.

Trimethylamine N-Oxide Exacerbates Neuroinflammation and Motor Dysfunction in an Acute MPTP Mice Model of Parkinson's Disease.

Observational studies have shown abnormal changes in trimethylamine N-oxide (TMAO) levels in the peripheral circulatory system of Parkinson's disease (PD) patients. TMAO is a gut microbiota metabolite that can cross the blood-brain barrier and is strongly related to neuroinflammation. Neuroinflammation is one of the pathological drivers of PD. Herein, we investigated the effect of TMAO on 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced PD model mice. TMAO pretreatment was given by adding 1.5% (w/v) TMAO to the drinking water of the mice for 21 days; then, the mice were administered MPTP (20 mg/kg, i.p.) four times a day to construct an acute PD model. Their serum TMAO concentrations, motor function, dopaminergic network integrity, and neuroinflammation were then assayed. The results showed that TMAO partly aggravated the motor dysfunction of the PD mice. Although TMAO had no effect on the dopaminergic neurons, TH protein content, and striatal DA level in the PD mice, it significantly reduced the striatal 5-HT levels and aggravated the metabolism of DA and 5-HT. Meanwhile, TMAO significantly activated glial cells in the striatum and the hippocampi of the PD mice and promoted the release of inflammatory cytokines in the hippocampus. In summary, higher-circulating TMAO had adverse effects on the motor capacity, striatum neurotransmitters, and striatal and hippocampal neuroinflammation in PD mice.

Programmed Death-1 Deficiency Aggravates Motor Dysfunction in MPTP Model of Parkinson's Disease by Inducing Microglial Activation and Neuroinflammation in Mice.

Abundant reactive gliosis and neuroinflammation are typical pathogenetic hallmarks of brains in Parkinson's disease (PD) patients, but regulation mechanisms are poorly understood. We are interested in role of programmed death-1 (PD-1) in glial reaction, neuroinflammation and neuronal injury in PD pathogenesis. Using PD mouse model and PD-1 knockout (KO) mice, we designed wild-type-control (WT-CON), WT-1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (WT-MPTP), PD-1-KO-control (KO-CON) and PD-1-KO-MPTP (KO-MPTP), and observed motor dysfunction of animal, morphological distribution of PD-1-positive cells, dopaminergic neuronal injury, glial activation and generation of inflammatory cytokines in midbrains by motor behavior detection, immunohistochemistry and western blot. WT-MPTP mouse model exhibited decrease of PD-1/Iba1-positive microglial cells in the substantia nigra compared with WT-CON mice. By comparison of four groups, PD-1 deficiency showed exacerbation in motor dysfunction of animals, decreased expression of TH protein and TH-positive neuronal protrusions. PD-1 deficiency enhanced microglial activation, production of proinflammatory cytokines like inducible nitric oxide synthase, tumor necrosis factor-α, interleukin-1β and interleukin-6, and expression and phosphorylation of AKT and ERK1/2 in the substantia nigra of MPTP model. We concluded that PD-1 deficiency could aggravate motor dysfunction of MPTP mouse model by inducing microglial activation and neuroinflammation in midbrains, suggesting that PD-1 signaling abnormality might be possibly involved in PD pathogenesis.

Effects of Psychological Stress on Central Dopaminergic Neurons

Objective — To evaluate the effects of psychological stress on central dopaminergic neurons. Methods — A test group of 90 rats was subjected to psychological stress for 30 minutes a day for 14 days. The rats were divided into three groups, and treated with tyrosine 250mg/kg, 500mg/kg, 1000mg/kg per day, 30 rats as psychological stress group and 30 rats as control group. After stress, TH and fos protein immunohistochemical staining were carried out on the relevant brain tissue sections of VTA (lateral tegmental area), Nac (nucleus accumbens) and mPFC (prefrontal cortex), and the results of TH immunohistochemical staining, Fos protein immunohistochemical staining and Fos/TH double staining were analyzed. Results — In VTA, the number of TH positive neurons in stress group was lower than that in control group (P<0.05), and the number of TH positive neurons in 500mg/kg and 1000mg/kg tyrosine groups was higher than that in stress group (P<0.05). The expression of Fos protein in VTA, mPFC and Nac of control group was lower than that of stress group, and the expression of Fos protein in VTA, mPFC and Nac of 500mg/kg and 1000mg/kg tyrosine groups was lower than that of stress group (P<0.05). Conclusion — Repeated psychological stress can damage central dopaminergic neurons, decrease the number of TH positive neurons and increase the expression of Fos protein. Tyrosine, the precursor of dopamine, can effectively control the damage.

Naringenin alleviates paraquat-induced dopaminergic neuronal loss in SH-SY5Y cells and a rat model of Parkinson's disease

Parkinson's disease (PD), a common neurodegenerative disease is characterized by the progressive loss of dopaminergic neurons in the substantia nigra. The cause of dopaminergic loss in PD remains unknown for a long time, however, recent reports suggest oxidative stress plays a key role in the pathogenesis of PD. Paraquat (PQ), a widely used herbicide is an oxidative stress inducer that has been implicated as a potential risk factor for the development of PD. Flavonoids are naturally occurring polyphenolic compounds that display a variety of therapeutic properties against oxidative stress. Naringenin (NAR), a natural flavonoid, exhibits neuroprotection against PD-related pathology. However, studies on its neuroprotective role and the underlying mechanisms are scarce, therefore the present study explored the potential neuroprotective role of NAR in PQ-induced parkinsonism in SH-SY5Y cells and rat model. The effect of NAR on PQ-induced cellular toxicity was determined by measuring cell viability, oxidative stress, ATP levels and the same effect was determined by assessing behavioral, biochemical, immunohistochemical, qRT-PCR and Western blot in rat model. NAR treatment in SH-SY5Y cells resulted in increased cell viability, reduced oxidative stress, elevated mitochondrial membrane potential, and higher cellular ATP levels. In rats, NAR treatment resulted in significant neuroprotection against PQ-induced behavioral deficits, oxidative stress, mitochondrial dysfunction, and astrocytosis. NAR treatment significantly modulated PQ-induced mRNA expressions of DRD2, DAT, LRRK2, SNCA, β-catenin, caspase-3, BDNF genes. NAR treatment increased TH protein expression and modulated its immunoreactivity in rat striatum. Also, GFAP decreased in response to NAR treatment. So, in the present study, NAR exhibits neuroprotection against PQ-induced neurotoxicity and neurodegeneration indicating its novel therapeutic potential against PD.

Inhalation of particulate and emissions from 3 dimensional (3D) printers: Effects on the olfactory bulb (OB) and hypothalamic‐pituitary‐endocrine axes

There is increasing concern regarding the potential health effects associated with exposure to plastic nanoparticles and the chemicals released when plastic is heated or dissolved in water. Although exposure to plastic particles and their composite chemicals can be generated by a number of different processes, 3D printers used in manufacturing, schools and homes, are sources of fumes and particulate that humans are exposed to. This study examined whether inhalation of particulate and fumes generated during 3D printing with polycarbonate (PC) affects the OB and neuroendocrine system, and if there is a dose-response relationship between exposure levels and measures of cell function. Methods Male rats (n= 6/group, 200 – 250 g at arrival) were exposed to filtered air, or black PC particulate and fumes generated by 3 desktop 3D-printers (exposure was 4 h/day). The 4h average particulate concentration delivered to the animals breathing space was 500 µg/m3. Animals were exposed 4 days/week until the specified number of days of exposure (1, 4, 8, 15 or 30) had been reached. The morning after the last exposure, animals were euthanized, blood was collected and circulating hormone levels were measured in serum samples using ELISAs. The OB and brain were also collected, frozen, sectioned and stored at -80°C until processed for immunohistochemistry. Results In the OB, exposure to 3D-emissions resulted in reductions in tyrosine hydroxylase (TH; rate limiting enzyme in dopamine synthesis) and glial derived neurotrophic factor immunolabeling. There were also reductions in myelin basic protein and cAMP nucleotide phosphatase staining, suggesting that there was myelin damage and dysfunction in the OB. In the hypothalamus, the major effects of exposure to 3D-emissions were reductions in immunostaining for TH and glial fibrillary acidic protein, a marker of astrocyte activation. Changes in immunostaining in the OB suggest there is cellular dysfunction and potentially an alteration in the transmission of olfactory information to the brain. Changes in activity and morphology of astrocytes in the hypothalamus have been shown to affect the release of hormones, including those regulating reproductive and metabolic processes; In the current study, exposure to 3D-emissions also resulted in changes in circulating concentrations of follicle stimulating hormone, and thyroid stimulating hormone. Conclusions Inhalation of PC particulate and/or fumes may act through olfactory pathways and affect neuroendocrine function. Although is not clear if these changes are the result of particulate matter interfering with various cellular and physiological processes, or due to endocrine disrupting bisphenols that are present in polycarbonate plastics, the results of this study are consistent with other experiments showing that exposure to PC particulate and the composite chemicals interfere with neuroendocrine processes that regulate reproduction and metabolism.

PGC-1α-siRNA suppresses inflammation in substantia nigra of PD mice by inhibiting microglia

Background and purpose: Parkinson’s disease is a common degenerative disease of the central nervous system with complex pathogenesis. More and more studies have found that inflammatory response promotes the occurrence and development of the disease, in which the activation of microglia plays an important role. PGC-1α (peroxisome proliferator activated receptor-γ coactivator-1α) is the main factor in mitochondrial biogenetic, and is closely related to the inflammatory response. Our immunofluorescence test results showed that PGC-1α and microglia (Iba1) have double-labeled phenomenon. The expression of microglia in the MPTP (1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine) group increased, and PGC-1α/Iba1 double label increased. To test whether lowering the expression of PGC-1α can reduce the activation of microglia and protect the substantia nigra dopaminergic neurons, we constructed PGC-1α interference lentivirus. Methods: Immunofluorescence, western blot, and ELISA were used to detect microglial phenotype. Results: The results showed that PGC-1α interfering with lentivirus can transfect microglial cells in substantia nigra, and the PGC-1α protein level decreased in substantia nigra accordingly; TH protein expression had no statistical difference compared with MPTP group; PGC-1α interfering lentivirus reduced microglia number and activation, and at the same time the expression of iNOS and Arg1 significantly reduced compared with MPTP group. The IL-6 expression in blood detected using ELISA was significantly reduced compared with MPTP group. Conclusion: PGC-1α downregulation inhibited microglia activity, and both M1 and M2 microglial activities are reduced.

Tcf4 Is Involved in Subset Specification of Mesodiencephalic Dopaminergic Neurons

During development, mesodiencephalic dopaminergic (mdDA) neurons form into different molecular subsets. Knowledge of which factors contribute to the specification of these subsets is currently insufficient. In this study, we examined the role of Tcf4, a member of the E-box protein family, in mdDA neuronal development and subset specification. We show that Tcf4 is expressed throughout development, but is no longer detected in adult midbrain. Deletion of Tcf4 results in an initial increase in TH-expressing neurons at E11.5, but this normalizes at later embryonic stages. However, the caudal subset marker Nxph3 and rostral subset marker Ahd2 are affected at E14.5, indicating that Tcf4 is involved in correct differentiation of mdDA neuronal subsets. At P0, expression of these markers partially recovers, whereas expression of Th transcript and TH protein appears to be affected in lateral parts of the mdDA neuronal population. The initial increase in TH-expressing cells and delay in subset specification could be due to the increase in expression of the bHLH factor Ascl1, known for its role in mdDA neuronal differentiation, upon loss of Tcf4. Taken together, our data identified a minor role for Tcf4 in mdDA neuronal development and subset specification.

Transcription Factors Phox2a/2b Upregulate Expression of Noradrenergic and Dopaminergic Phenotypes in Aged Rat Brains.

The present study investigated the effects of forced overexpression of Phox2a/2b, two transcription factors, in the locus coeruleus (LC) of aged rats on noradrenergic and dopaminergic phenotypes in brains. Results showed that a significant increase in Phox2a/2b mRNA levels in the LC region was paralleled by marked enhancement in expression of DBH and TH per se. Furthermore, similar increases in TH protein levels were observed in the substantial nigra and striatum, as well as in the hippocampus and frontal cortex. Overexpression of Phox2 genes also significantly increased BrdU-positive cells in the hippocampal dentate gyrus and NE levels in the striatum. Moreover, this manipulation significantly improved the cognition behavior. The in vitro experiments revealed that norepinephrine treatments may increase the transcription of TH gene through the epigenetic action on the TH promoter. The results indicate that Phox2 genes may play an important role in improving the function of the noradrenergic and dopaminergic neurons in aged animals, and regulation of Phox2 gene expression may have therapeutic utility in aging or disorders involving degeneration of noradrenergic neurons.

Evidence that Sympathetic Activity is Increased Selectively to the Heart in a Mouse Model of Hypertrophic Cardiomyopathy

Hypertrophic cardiomyopathy (HCM), the most common genetic heart disease, is characterized by cardiac hypertrophy, fibrosis, and increased risk of arrhythmias and sudden death, even in the absence of heart failure. We have studied young (&lt;20 wks) transgenic mice with cardiac‐specific expression of mutated alpha‐tropomyosin (Glu180Gly), an established model of HCM, to investigate autonomic mechanisms. In early experiments, abnormal heart rate and blood pressure responses to β‐adrenergic receptor blockade and ganglionic blockade indicated high cardiac accompanied by low vasomotor sympathetic tone in HCM vs. wild‐type (WT) mice. The aim of this study was to further characterize regional sympathetic activity in HCM vs. WT littermates by measuring expression/activity of tyrosine hydroxylase (TH, rate‐limiting enzyme for norepinephrine (NE) synthesis) and the norepinephrine transporter (NET) in sympathetic ganglia (stellate, superior cervical, celiac/mesenteric), and expression of β1 receptors in left ventricle (LV). Results (below) are expressed relative to WT (n=4 male mice each group). TH protein expression (Western blot) (standardized to neuronal content with PGP9.5) was significantly higher in stellate (cardiac) ganglia in HCM mice (HCM 162±12 vs. WT 100±7; p&lt;0.05), but not significantly different in superior cervical (HCM 82±4 vs. WT 100±14; p&gt;0.05) or celiac‐mesenteric (HCM 95±7 vs. WT 100±4; p&gt;0.05) ganglia. TH/PGP9.5 protein expression was also higher in the LV of HCM (135±15) vs. WT (100±1) mice (p&lt;0.05). LV β1 receptor protein expression was significantly lower in HCM (60±1) vs. WT (100±4) mice (p&lt;0.05), consistent with increased local NE levels. NET protein (immunofluorescence) was lower in stellate ganglia from HCM mice vs. WT (n=3 mice per group, 3 sections/mouse, p&lt;0.05). NET activity was assessed by measuring the rate of uptake of a fluorescent NET substrate (Molecular Devices) into individual sympathetic neurons isolated from stellate and superior cervical ganglia. NET activity measured in stellate neurons was significantly lower in HCM (4.5±0.3 a.u.*min−1, n=115 neurons, from 4 mice) vs. WT (5.3±0.4 a.u.*min−1, n=98 neurons, from 4 mice). NET activity in superior cervical ganglia did not differ between genotypes. In conclusion, increased TH expression (enhanced ‐NE synthesis) and decreased NET expression/activity (reduced NE reuptake) in stellate ganglia are consistent with increased cardiac sympathetic activity in HCM, whereas the lack of differences between HCM and WT in other sympathetic ganglia are consistent with normal sympathetic activity to non‐cardiac target organs in HCM.Support or Funding InformationNIH F32HL140880