Therapeutic Effects of Selenium on Alpha-Synuclein Accumulation in Substantia Nigra Pars Compacta in a Rat Model of Parkinson's Disease: Behavioral and Biochemical Outcomes.

Parkinson's disease (PD) is the second most common progressive neurodegenerative disorder characterized by the accumulation of accumulated alpha-synuclein (α-Syn) in substantia nigra. Research has shown that selenium (Se) can protect neural cells through the actions of selenoproteins, including selenoprotein P (SelP) and selenoprotein S (SelS), which participate in endoplasmic reticulum-associated protein degradation (ERAD). In this study, we investigated the potential protective role of Se in a pre-clinical PD rat model.We aimed to evaluate the therapeutic effects of Se administration in the 6-hydroxydopamine (6-OHDA) induced unilateral rat PD model. Male Wistar rats were utilised for unilateral PD animal model which were subjected to stereotaxic surgery and injected with 20 µg 6-OHDA/5 µl 0.2% ascorbate saline. After confirming the model, the rats were intraperitoneally injected with 0.1, 0.2, and 0.3 mg/kg of sodium selenite for 7 days. We then performed behavioral tests, including apomorphine-induced rotation, hanging, and rotarod tests. Following sacrifice, we analysed the substantia nigra area of the brain and serum for protein quantification, element analysis, and gene expression analysis.Our results indicate that the administration of 0.3 mg/kg of Se improved the motor deficiency in hanging, rotarod, and apomorphine-induced rotational tests. While there was no significant improvement in the expression of α-Syn, Se increased the expression of selenoproteins. Additionally, levels of selenoproteins, Se, and α-Syn both brain and serum were re-established by the treatment, suggesting the role of Se on the α-Syn accumulation. Furthermore, Se improved PD-induced biochemical deficits by increasing the levels of SelS and SelP (p<0.005).In conclusion, our findings suggest that Se may have a protective role in PD. 0.3 mg/kg dosage of Se increased the expression of selenoproteins, reduced the accumulation of α-Syn in the brain, and improved PD-induced motor deficits. These results suggest that Se may be a potential therapeutic option for PD treatment.

Chronic high-sugar diet in adulthood protects Caenorhabditis elegans from 6-OHDA-induced dopaminergic neurodegeneration.

BACKGROUND: Diets high in saturated fat and sugar, termed "Western diets," have been associated with several negative health outcomes, including increased risk for neurodegenerative disease. Parkinson's disease (PD) is the second most prevalent neurodegenerative disease and is characterized by the progressive death of dopaminergic neurons in the brain. We build upon previous work characterizing the impact of high-sugar diets in Caenorhabditis elegans to mechanistically evaluate the relationship between high-sugar diets and dopaminergic neurodegeneration. RESULTS: Adult high-glucose and high-fructose diets, or exposure from day 1 to 5 of adulthood, led to increased lipid content, shorter lifespan, and decreased reproduction. However, in contrast to previous reports, we found that adult chronic high-glucose and high-fructose diets did not induce dopaminergic neurodegeneration alone and were protective from 6-hydroxydopamine (6-OHDA) induced degeneration. Neither sugar altered baseline electron transport chain function and both increased vulnerability to organism-wide ATP depletion when the electron transport chain was inhibited, arguing against energetic rescue as a basis for neuroprotection. The induction of oxidative stress by 6-OHDA is hypothesized to contribute to its pathology, and high-sugar diets prevented this increase in the soma of the dopaminergic neurons. However, we did not find increased expression of antioxidant enzymes or glutathione levels. Instead, we found evidence suggesting downregulation of the dopamine reuptake transporter dat-1 that could result in decreased 6-OHDA uptake. CONCLUSIONS: Our work uncovers a neuroprotective role for high-sugar diets, despite concomitant decreases in lifespan and reproduction. Our results support the broader finding that ATP depletion alone is insufficient to induce dopaminergic neurodegeneration, whereas increased neuronal oxidative stress may drive degeneration. Finally, our work highlights the importance of evaluating lifestyle by toxicant interactions.

Co-transplantation of autologous Treg cells in a cell therapy for Parkinson's disease.

The specific loss of midbrain dopamine neurons (mDANs) causes major motor dysfunction in Parkinson's disease, which makes cell replacement a promising therapeutic approach1-4. However, poor survival of grafted mDANs remains an obstacle to successful clinical outcomes5-8. Here we show that the surgical procedure itself (referred to here as 'needle trauma') triggers a profound host response that is characterized by acute neuroinflammation, robust infiltration of peripheral immune cells and brain cell death. When midbrain dopamine (mDA) cells derived from human induced pluripotent stem (iPS) cells were transplanted into the rodent striatum, less than 10% of implanted tyrosine hydroxylase (TH)+ mDANs survived at two weeks after transplantation. By contrast, TH- grafted cells mostly survived. Notably, transplantation of autologous regulatory T (Treg) cells greatly modified the response to needle trauma, suppressing acute neuroinflammation and immune cell infiltration. Furthermore, intra-striatal co-transplantation of Treg cells and human-iPS-cell-derived mDA cells significantly protected grafted mDANs from needle-trauma-associated death and improved therapeutic outcomes in rodent models of Parkinson's disease with 6-hydroxydopamine lesions. Co-transplantation with Treg cells also suppressed the undesirable proliferation of TH- grafted cells, resulting in more compact grafts with a higher proportion and higher absolute numbers of TH+ neurons. Together, these data emphasize the importance of the initial inflammatory response to surgical injury in the differential survival of cellular components of the graft, and suggest that co-transplanting autologous Treg cells effectively reduces the needle-trauma-induced death of mDANs, providing a potential strategy to achieve better clinical outcomes for cell therapy in Parkinson's disease.

Menstrual blood-derived endometrial stem cells alleviate neuroinflammation by modulating M1/M2 polarization in cell and rat Parkinson's disease models.

BACKGROUND: Neuroinflammation is closely related to the development of Parkinson's disease (PD). Because of the extensive sources, non-invasive and periodical collection method, human menstrual blood-derived endometrial stem cells (MenSCs) have been explored as a promising tool for treatment of PD. This study aimed to investigate if MenSCs could inhibit neuroinflammation in PD rats by regulating M1/M2 polarization and to excavate the underlying mechanisms. METHODS: MenSCs were co-cultured with 6-OHDA-exposed microglia cell lines. Then the morphology of microglia cells and the level of inflammatory factors were assessed by immunofluorescence and qRT-PCR. After MenSCs were transplanted into the brain of PD rats, animal motor function, the expression of tyrosine hydroxylase, and the level of inflammatory factors in the cerebrospinal fluid (CSF) and serum were detected to evaluate the therapeutic potential of MenSCs. Meanwhile, the expression of M1/M2 phenotype related genes was detected by qRT-PCR. One protein array kit containing 1000 kinds of factors was used to detect the protein components in the conditioned medium of MenSCs. Finally, bioinformatic analysis was performed to analyze the function of factors secreted by MenSCs and the signal pathways involved in. RESULTS: MenSCs could suppress 6-OHDA-induced microglia cell activation and significantly decrease inflammation in vitro. After transplantation into the brain of PD rats, MenSCs improved animal motor function, which was indicated by the increased movement distance, ambulatory episodes, exercise time on the rotarod, and less contralateral rotation. Additionally, MenSCs reduced the loss of dopaminergic neurons and down-regulated the level of pro-inflammatory factors in the CSF and serum. Moreover, q-PCR and WB results showed the transplantation of MenSCs significantly down-regulated the expression of M1 phenotype cell markers and meanwhile up-regulated the expression of M2 phenotype cell markers in the brain of PD rats. 176 biological processes including inflammatory response, negative regulation of apoptotic process, and microglial cell activation were enriched by GO-BP analysis. 58 signal pathways including PI3K/Akt and MAPK were enriched by KEGG analysis. CONCLUSIONS: In conclusion, our results provide preliminary evidence for the anti-inflammation capacity of MenSCs by regulating M1/M2 polarization. We firstly demonstrated the biological process of factors secreted by MenSCs and the signal pathways involved in using protein array and bioinformatic analysis.

Potential Therapeutic Effects of Policosanol from Insect Wax on Caenorhabditis elegans Models of Parkinson's Disease.

Parkinson's disease (PD) is the second most common neurodegenerative disease worldwide. The standard treatments for PD focus on symptom relief rather than attempting to address the underlying degenerative processes completely. This study aimed to evaluate the potential therapeutic effects of policosanol derived from insect wax (PIW) by investigating improvements in disease symptoms represented in Caenorhabditis elegans models of PD. For our assessments, we used the following three models: NL5901, which is a transgenic model for α-synuclein aggregation; wild-type N2 induced with 6-hydroxydopamine (6-OHDA); and 6-OHDA-induced BZ555 as a model for loss of dopaminergic neurons (DNs). Specifically, we examined the effects of PIW treatment on α-synuclein aggregation, the loss of DNs, lipid abundance, and the lifespan of treated organisms. Further, we examined treatment-related changes in the levels of reactive oxygen species (ROS), malondialdehyde (MDA), adenosine triphosphate (ATP), glutathione S-transferase (GST), and superoxide dismutase (SOD), as well as the mRNA production profiles of relevant genes. A 10 µg/mL dose of PIW reduced the aggregation of α-synuclein in NL5901 and suppressed the loss of DNs in 6-OHDA-induced BZ555. Overall, PIW treatment decreased ROS and MDA levels, restored lipid abundance, and prolonged the lifespans of worms in all the three models, which may be associated with changes in the expression profiles of genes related to cell survival and oxidative stress response pathways. Our findings show that PIW alleviated the symptoms of PD in these models, possibly by regulating the stress responses initiated by injuries such as α-synuclein aggregation or 6-OHDA treatment.

Transplantation of ERK gene-modified bone marrow mesenchymal stem cells ameliorates cognitive deficits in a 6-hydroxydopamine rat model of Parkinson's disease.

The study aimed to investigate bone marrow mesenchymal stem cells (BMSCs) and extracellular signal-regulated kinase (ERK) gene-modified BMSCs (ERK-BMSCs) transplantation in ameliorating cognitive deficits in Parkinson's disease (PD). The PD rat model was built by 6-hydroxydopamine (6-OHDA) injection into the right striatum for 8 weeks, then successful PD rats were randomly divided into three groups and respectively transplanted in the same position of striatum as modeling with PBS, BMSCs and ERK-BMSCs for another 8 weeks. The 6-OHDA-induced PD rat model was successfully established, as demonstrated by reduced active avoidance response (AAR) times, percentage of time exploring in the light area (Ltime%) and platform quadrant time (PQT), as well as p-ERK expression. Compared with PBS rats, both BMSCs and ERK-BMSCs transplantation significantly reduced the left turn number, while increased AAR, Ltime%, PQT and p-ERK expression, suggesting improved cognitive abilities through restoring p-ERK expression. In addition, ERK-BMSCs injection exhibited higher therapeutic efficacy against cognitive deficits compared with BMSCs injection. These results demonstrated that BMSCs transplantation ameliorated cognitive deficits, and ERK-BMSCs exerted synergistic effects, which may prove beneficial against cognitive impairments in PD.

Citronellol Prevents 6-OHDA-Induced Oxidative Stress, Mitochondrial Dysfunction, and Apoptosis in Parkinson Disease Model of SH-SY5Y Cells via Modulating ROS-NO, MAPK/ERK, and PI3K/Akt Signaling Pathways.

Parkinson disease is a neurodegenerative disorder distinguished by dopaminergic shortage in the striatum and the accumulation of α-synuclein neuronal aggregates in the brains of patients. Since, there is no accurate treatment available for Parkinson disease, researches are designed to alleviate the pathognomonic symptoms such as neuroinflammation, oxidative stress, mitochondrial dysfunction, and apoptosis. Accordingly, a number of compounds have been reported to inhibit these pathognomonic symptoms. In this study, we have assessed the neuroprotective potential of citronellol against 6-OHDA-induced neurotoxicity in SH-SY5Y cells. The results found that citronellol treatment effectively hindered the cell death caused by 6-OHDA and thereby maintaining the cell viability in SH-SY5Y cells at 50 µg/mL concentration. As expected, the citronellol treatment significantly reduced the 6-OHDA-induced secretion of inflammatory factors (IL-1ß, IL-6, and TNF-α), which was obtained through ELISA technique. Similarly, citronellol hindered the 6-OHDA-induced oxidative stress by lowering the intracellular ROS and NO level and MDA leakage along with increased expression of SOD level in SH-SY5Y cells. The JC-1 staining showed that 6-OHDA increased the number of green fluorescent dots with ruptured mitochondrial membrane potential, while citronellol increased the amount of red fluorescent, showing the rescue potential against the 6-OHDA-induced mitochondrial dysfunction. Furthermore, citronellol hampered the 6-OHDA-induced apoptosis via the suppression of Bcl-2/Bax pathway. The western blotting results hypothesized that citronellol rescued SH-SY5Y cells from 6-OHDA-induced neurotoxicity via modulating ROS-NO, MAPK/ERK, and PI3K/Akt signaling pathways. However, further clinical trials are required to verify the anti-Parkinson efficacy.

L-Ornithine L-Aspartate Restores Mitochondrial Function and Modulates Intracellular Calcium Homeostasis in Parkinson's Disease Models.

The altered crosstalk between mitochondrial dysfunction, intracellular Ca2+ homeostasis, and oxidative stress has a central role in the dopaminergic neurodegeneration. In the present study, we investigated the hypothesis that pharmacological strategies able to improve mitochondrial functions might prevent neuronal dysfunction in in vitro models of Parkinson's disease. To this aim, the attention was focused on the amino acid ornithine due to its ability to cross the blood-brain barrier, to selectively reach and penetrate the mitochondria through the ornithine transporter 1, and to control mitochondrial function. To pursue this issue, experiments were performed in human neuroblastoma cells SH-SY5Y treated with rotenone and 6-hydroxydopamine to investigate the pharmacological profile of the compound L-Ornithine-L-Aspartate (LOLA) as a new potential therapeutic strategy to prevent dopaminergic neurons' death. In these models, confocal microscopy experiments with fluorescent dyes measuring mitochondrial calcium content, mitochondrial membrane potential, and mitochondrial ROS production, demonstrated that LOLA improved mitochondrial functions. Moreover, by increasing NCXs expression and activity, LOLA also reduced cytosolic [Ca2+] thanks to its ability to modulate NO production. Collectively, these results indicate that LOLA, by interfering with those mitochondrial mechanisms related to ROS and RNS production, promotes mitochondrial functional recovery, thus confirming the tight relationship existing between cytosolic ionic homeostasis and cellular metabolism depending on the type of insult applied.

Agathobaculum butyriciproducens Shows Neuroprotective Effects in a 6-OHDA-Induced Mouse Model of Parkinson's Disease.

Parkinson's disease (PD) is the second-most prevalent neurodegenerative disease and is characterized by dopaminergic neuronal death in the midbrain. Recently, the association between alterations in PD pathology and the gut microbiota has been explored. Microbiota-targeted interventions have been suggested as a novel therapeutic approach for PD. Agathobaculum butyriciproducens SR79T (SR79) is an anaerobic bacterium. Previously, we showed that SR79 treatment induced cognitive improvement and reduced Alzheimer's disease pathologies in a mouse model. In this study, we hypothesized that SR79 treatment may have beneficial effects on PD pathology. To investigate the therapeutic effects of SR79 on PD, 6-hydroxydopamine (6-OHDA)-induced mouse models were used. D-Amphetamine sulfate (d-AMPH)-induced behavioral rotations and dopaminergic cell death were analyzed in unilateral 6-OHDA-lesioned mice. Treatment with SR79 significantly decreased ipsilateral rotations induced by d-AMPH. Moreover, SR79 treatment markedly activated the AKT/GSK3ß signaling pathway in the striatum. In addition, SR79 treatment affected the Nrf2/ARE signaling pathway and its downstream target genes in the striatum of 6-OHDA-lesioned mice. Our findings suggest a protective role of SR79 in 6-OHDA-induced toxicity by regulating the AKT/Nrf2/ARE signaling pathway and astrocyte activation. Thus, SR79 may be a potential microbe-based intervention and therapeutic strategy for PD.

Human gingival mesenchymal stem cells improve movement disorders and tyrosine hydroxylase neuronal damage in Parkinson disease rats.

BACKGROUND AIMS: Gingival mesenchymal stem cells (GMSCs) demonstrate high proliferation, trilineage differentiation and immunomodulatory properties. Parkinson disease (PD) is the second most common type of neurodegenerative disease. This study aimed to explore the effect and mechanism of GMSC-based therapy in 6-hydroxydopamine-induced PD rats. METHODS: RNA sequencing and quantitative proteomics technology was used to validate the neuroprotective role of GMSCs therapeutic in 6-Hydroxydopamine -induced PD model in vitro and in vivo. Western blotting, immunofluorescence and real-time quantitative PCR verified the molecular mechanism of GMSCs treatment. RESULTS: Intravenous injection of GMSCs improved rotation and forelimb misalignment behavior, enhanced the anti-apoptotic B-cell lymphoma 2/B-cell lymphoma 2-associated X axis, protected tyrosine hydroxylase neurons, decreased the activation of astrocytes and reduced the astrocyte marker glial fibrillary acidic protein and microglia marker ionized calcium-binding adaptor molecule 1 in the substantia nigra and striatum of PD rats. The authors found that GMSCs upregulated nerve regeneration-related molecules and inhibited metabolic disorders and the activation of signal transducer and activator of transcription 3. GMSCs showed a strong ability to protect neurons and reduce mitochondrial membrane potential damage and reactive oxygen species accumulation. The safety of GMSC transplantation was confirmed by the lack of tumor formation following subcutaneous transplantation into nude mice for up to 8 weeks. CONCLUSIONS: The authors' research helps to explain the mechanism of GMSC-based therapeutic strategies and promote potential clinical application in Parkinson disease.

[Inhibition connexin 43 by mimetic peptide Gap27 mediates protective effects on 6-hydroxydopamine induced Parkinson's disease mouse model].

OBJECTIVE: To explore whether the using of mimetic peptide Gap27, a selective inhibitor of connexin 43 (Cx43), could block the death of dopamine neurons and influence the expression of Cx43 in 6-hydroxydopamine (6-OHDA)-induced Parkinson's disease mouse models. METHODS: Eighteen C57BL/6 mice were randomly divided into control group, 6-OHDA group and 6-OHDA+Gap27 group, with 6 mice in each group. Bilateral substantia nigra stereotactic injection was performed. The control group was injected with ascorbate solution, 6-OHDA group was injected with 6-OHDA solution, and 6-OHDA+Gap27 group was injected with 6-OHDA and Gap27 mixed solution. Immuno-histochemical staining was used to detect the number of dopamine neurons, quantitative real-time polymerase chain reaction (qRT-PCR) was used to detect the expression of Cx43 messenger ribonucleic acid (mRNA), immuno-fluorescence staining was used to detect the distribution of Cx43 protein, the contents of Cx43 protein and Cx43 phosphorylation at serine 368 (Cx43-ps368) in mouse midbrain were detected by Western blot. RESULTS: After injection of 6-OHDA, numerous dopamine neurons in substantia nigra died as Cx43 content increased, Cx43-ps368 content decreased. Mixing Gap27 while injecting 6-OHDA could reduce the number of death dopamine neurons and weaken the changes of Cx43 and Cx43-ps368 content caused by 6-OHDA. The number of tyrosine hydroxylase (TH) immunoreactive positive neurons in 6-OHDA group decreased to 27.7% ± 0.02% of the control group (P < 0.01); The number of TH immunoreactive positive neurons in 6-OHDA+Gap27 group was (1.64±0.16) times higher than that in 6-OHDA group (P < 0.05); The content of total Cx43 protein in 6-OHDA group was (1.44±0.07) times higher than that in 6-OHDA+Gap27 group (P < 0.05) while (1.68±0.07) times higher than that in control group (P < 0.01). In 6-OHDA group, the content of Cx43-ps368 protein and its proportion in total Cx43 protein were significantly lower than that in 6-OHDA+Gap27 group (P < 0.05). CONCLUSION: In 6-OHDA mouse models, mimetic peptide Gap27 played a protective role in reducing the damage to substantia nigra dopamine neurons, which was induced by 6-OHDA. The overexpression of Cx43 protein might have neurotoxicity to dopamine neuron. Meanwhile, decreasing Cx43 protein level and keeping Cx43-ps368 protein level may be the protective mechanisms of Gap27.

Potential anti-Parkinsonian's effect of S-(+)-linalool from Cinnamomum osmophloeum ct. linalool leaves are associated with mitochondrial regulation via gas-1, nuo-1, and mev-1 in Caenorhabditis elegans.

Parkinson's disease (PD) is one of the prevalent neurodegenerative diseases, and developing new treatments from natural products is of particular interest. Essential oils from Cinnamomum osmophloeum ct. linalool leaves contain high levels (~95%) of S-(+)-linalool. The neuroprotective effects of linalool have been previously described, yet the underlying molecular mechanisms remain largely unknown. This study aimed to investigate the potential anti-Parkinsonian's effect of S-(+)-linalool on mitochondrial regulation and decipher the underlying molecular mechanisms in Caenorhabditis elegans PD model. Essential oils at 20 mg/L and 20 mg/L S-(+)-linalool each significantly attenuated the damaging effects of 6-hydroxydopamine (6-OHDA) on dopaminergic (DA) neurons and decreased the mitochondrial unfolded protein response (UPRmt ) to antimycin. RNAi knockdown of mitochondrial complex I (gas-1, nuo-1), and complex II (mev-1) genes prevented the improvement of mitochondrial activity by S-(+)-linalool. The protective effects of S-(+)-linalool on 6-OHDA-induced behavior changes were absent in a DA-specific strain of C. elegans produced by gas-1, nuo-1, and mev-1 RNAi knockdown. These results suggest the potential anti-Parkinsonian's effect of S-(+)-linalool is associated with mitochondrial activity and regulated by gas-1, nuo-1, and mev-1 in C. elegans. Our findings suggest that S-(+)-linalool might be a promising candidate for therapeutic application to inhibit the progression of PD.

Repressing PTBP1 fails to convert reactive astrocytes to dopaminergic neurons in a 6-hydroxydopamine mouse model of Parkinson's disease.

Lineage reprogramming of resident glial cells to dopaminergic neurons (DAns) is an attractive prospect of the cell-replacement therapy for Parkinson's disease (PD). However, it is unclear whether repressing polypyrimidine tract binding protein 1 (PTBP1) could efficiently convert astrocyte to DAns in the substantia nigra and striatum. Although reporter-positive DAns were observed in both groups after delivering the adeno-associated virus (AAV) expressing a reporter with shRNA or CRISPR-CasRx to repress astroglial PTBP1, the possibility of AAV leaking into endogenous DAns could not be excluded without using a reliable lineage-tracing method. By adopting stringent lineage-tracing strategy, two other studies show that either knockdown or genetic deletion of quiescent astroglial PTBP1 fails to obtain induced DAns under physiological condition. However, the role of reactive astrocytes might be underestimated because upon brain injury, reactive astrocyte can acquire certain stem cell hallmarks that may facilitate the lineage conversion process. Therefore, whether reactive astrocytes could be genuinely converted to DAns after PTBP1 repression in a PD model needs further validation. In this study, we used Aldh1l1-CreERT2-mediated specific astrocyte-lineage-tracing method to investigate whether reactive astrocytes could be converted to DAns in a 6-hydroxydopamine (6-OHDA) mouse model of PD. However, we found that no astrocyte-originated DAn was generated after effective and persistent knockdown of astroglial PTBP1 either in the substantia nigra or in striatum, while AAV 'leakage' to nearby neurons was easily observed. Our results confirm that repressing PTBP1 does not convert astrocytes to DAns, regardless of physiological or PD-related pathological conditions.

NOX2-derived hydrogen peroxide impedes the AMPK/Akt-mTOR signaling pathway contributing to cell death in neuronal cells.

Oxidative stress is closely related to the pathogenesis of Parkinson's disease (PD), a typical neurodegenerative disease. NADPH oxidase 2 (NOX2) is involved in hydrogen peroxide (H2O2) generation. Recently, we have reported that treatment with H2O2 and PD toxins, including 6-hydroxydopamine (6-OHDA), 1-Methyl-4-phenylpyridin-1-ium (MPP+) and rotenone, induces neuronal apoptosis by inhibiting the mTOR pathway. Here, we show that treatment with 6-OHDA, MPP+ or rotenone induced H2O2 generation by upregulating the levels of NOX2 and its regulatory proteins (p22phox, p40phox, p47phox, p67phox, and Rac1), leading to apoptotic cell death in PC12 cells and primary neurons. Inhibition of NOX2 with apocynin or diphenyleneiodonium, or knockdown of NOX2 powerfully attenuated PD toxins-evoked NOX2 and H2O2, thereby hindering activation of AMPK, inhibition of Akt/mTOR, and induction of apoptosis in neuronal cells. Pretreatment with catalase, a H2O2-scavenging enzyme, blocked the effects of PD toxins on NOX2-dependent H2O2 production, AMPK/Akt/mTOR signaling and apoptosis in the cells. Similar effects were also seen in the cells pretreated with Mito-TEMPO, a mitochondria-selective superoxide scavenger, implying a mitochondrial H2O2-dependent mechanism involved. Further research revealed that ectopic expression of constitutively active Akt or dominant negative AMPKα, or inhibition of AMPK with compound C suppressed PD toxins-induced expression of NOX2 and its regulatory proteins, as well as consequential H2O2 production and apoptosis in the cells. Taken together, these results indicate that certain PD toxins can impede the AMPK/Akt-mTOR signaling pathway leading to neuronal apoptosis by eliciting NOX2-derived H2O2 production. Our findings suggest that neuronal loss in PD may be prevented by regulating the NOX2, AMPK/Akt-mTOR signaling and/or applying antioxidants to ameliorate oxidative stress.

6-Hydroxydopamine Induces Abnormal Iron Sequestration in BV2 Microglia by Activating Iron Regulatory Protein 1 and Inhibiting Hepcidin Release.

Disrupted iron homeostasis in the substantia nigra pars compacta (SNpc) is an important pathological mechanism in Parkinson's disease (PD). It is unclear what role microglia play in iron metabolism and selective iron deposition in the SNpc of PD brain. In this study, we observed that 6-hydroxydopamine (6-OHDA) induced the expression of divalent metal transporter-1 (DMT1) and iron influx in BV2 microglia cells, which might be associated with the upregulation of iron regulatory protein 1 (IRP1) expression. Moreover, we found that 6-OHDA had no significant effect on the expression of ferroportin 1 (FPN1) and iron efflux in BV2 microglial cells, which might be the combined action of IRP1 upregulation and reduced hepcidin levels. Furthermore, 6-OHDA treatment activated BV2 microglia and enhanced the release of pro-inflammatory cytokines. Interestingly, iron overloading suppressed IRP1 expression, thus downregulating DMT1 and upregulating FPN1 levels in these microglial cells. On the contrary, iron deficiency activated IRP1, leading to increased expression of DMT1 and decreased expression of FPN1-which indicates that activated IRP1 induces iron overloading in 6-OHDA-treated microglia, but not iron overloading modulates the expression of IRP1. Taken together, our data suggest that 6-OHDA can regulate the expression of DMT1 and FPN1 by activating IRP1 and inhibiting hepcidin release, thus leading to abnormal iron sequestration in microglia. In addition, 6-OHDA can activate microglia, which leads to increased release of pro-inflammatory factors that can further induce genome damage in dopaminergic neurons.

Cryopreservation of Induced Pluripotent Stem Cell-Derived Dopaminergic Neurospheres for Clinical Application.

BACKGROUND: Pluripotent stem cell (PSC)-derived dopaminergic (DA) neurons are an expected source of cell therapy for Parkinson's disease. The transplantation of cell aggregates or neurospheres, instead of a single cell suspension has several advantages, such as keeping the 3D structure of the donor cells and ease of handling. For this PSC-based therapy to become a widely available treatment, cryopreservation of the final product is critical in the manufacturing process. However, cryopreserving cell aggregates is more complicated than cryopreserving single cell suspensions. Previous studies showed poor survival of the DA neurons after the transplantation of cryopreserved fetal ventral-mesencephalic tissues. OBJECTIVE: To achieve the cryopreservation of induced pluripotent stem cell (iPSC)-derived DA neurospheres toward clinical application. METHODS: We cryopreserved iPSC-derived DA neurospheres in various clinically applicable cryopreservation media and freezing protocols and assessed viability and neurite extension. We evaluated the population and neuronal function of cryopreserved cells by the selected method in vitro. We also injected the cells into 6-hydroxydopamine (6-OHDA) lesioned rats, and assessed their survival, maturation and function in vivo. RESULTS: The iPSC-derived DA neurospheres cryopreserved by Proton Freezer in the cryopreservation medium Bambanker hRM (BBK) showed favorable viability after thawing and had equivalent expression of DA-specific markers, dopamine secretion, and electrophysiological activity as fresh spheres. When transplanted into 6-OHDA-lesioned rats, the cryopreserved cells survived and differentiated into mature DA neurons, resulting in improved abnormal rotational behavior. CONCLUSION: These results show that the combination of BBK and Proton Freezer is suitable for the cryopreservation of iPSC-derived DA neurospheres.

Towards chronic deep brain stimulation in freely moving hemiparkinsonian rats: applicability and functionality of a fully implantable stimulation system.

Objective.This study aimed at investigating a novel fully implantable deep brain stimulation (DBS) system and its ability to modulate brain metabolism and behavior through subthalamic nucleus (STN) stimulation in a hemiparkinsonian rat model.Approach.Twelve male rats were unilaterally lesioned with 6-hydroxydopamine in the medial forebrain bundle and received a fully implantable DBS system aiming at the ipsilesional STN. Each rat underwent three cylinder tests to analyze front paw use: a PRE test before any surgical intervention, an OFF test after surgery but before stimulation onset and an ON test under DBS. To visualize brain glucose metabolism in the awake animal, two [18F]FDG scans were conducted in the OFF and ON condition. At least 4 weeks after surgery, an [18F]FDOPA scan was used to check for dopaminergic integrity.Main results.In general, STN DBS increased [18F]FDG uptake ipsilesionally and decreased it contralesionally. More specifically, bilateral orbitofrontal cortex, ipsilateral caudate putamen, sensorimotor cortex and nucleus accumbens showed significantly higher tracer uptake in ON compared to OFF condition. Contralateral cingulate and secondary motor cortex, caudate putamen, amygdala, hippocampus, retrosplenial granular cortex, superior colliculus, and parts of the cerebellum exhibited significantly higher [18F]FDG uptake in the OFF condition. On the behavioral level, stimulation was able improve use of the contralesional affected front paw suggesting an effective stimulation produced by the implanted system.Significance.The fully implantable stimulation system developed by us and presented here offers the output of arbitrary user-defined waveforms, patterns and stimulation settings and allows tracer accumulation in freely moving animals. It is therefore a suitable device for implementing behavioral PET studies. It contributes immensely to the possibilities to characterize and unveil the effects and mechanisms of DBS offering valuable clues for future improvements of this therapy.

Zinc oxide nanoparticles modulate the gene expression of ZnT1 and ZIP8 to manipulate zinc homeostasis and stress-induced cytotoxicity in human neuroblastoma SH-SY5Y cells.

Zinc ions (Zn2+) are important messenger molecules involved in various physiological functions. To maintain the homeostasis of cytosolic Zn2+ concentration ([Zn2+]c), Zrt/Irt-related proteins (ZIPs) and Zn2+ transporters (ZnTs) are the two families of proteins responsible for decreasing and increasing the [Zn2+]c, respectively, by fluxing Zn2+ across the membranes of the cell and intracellular compartments in opposite directions. Most studies focus on the cytotoxicity incurred by a high concentration of [Zn2+]c and less investigate the [Zn2+]c at physiological levels. Zinc oxide-nanoparticle (ZnO-NP) is blood brain barrier-permeable and elevates the [Zn2+]c to different levels according to the concentrations of ZnO-NP applied. In this study, we mildly elevated the [Zn2+]c by ZnO-NP at concentrations below 1 µg/ml, which had little cytotoxicity, in cultured human neuroblastoma SH-SY5Y cells and characterized the importance of Zn2+ transporters in 6-hydroxy dopamine (6-OHDA)-induced cell death. The results show that ZnO-NP at low concentrations elevated the [Zn2+]c transiently in 6 hr, then declined gradually to a basal level in 24 hr. Knocking down the expression levels of ZnT1 (located mostly at the plasma membrane) and ZIP8 (present in endosomes and lysosomes) increased and decreased the ZnO-NP-induced elevation of [Zn2+]c, respectively. ZnO-NP treatment reduced the basal levels of reactive oxygen species and Bax/Bcl-2 mRNA ratios; in addition, ZnO-NP decreased the 6-OHDA-induced ROS production, p53 expression, and cell death. These results show that ZnO-NP-induced mild elevation in [Zn2+]c activates beneficial effects in reducing the 6-OHDA-induced cytotoxic effects. Therefore, brain-delivery of ZnO-NP can be regarded as a potential therapy for neurodegenerative diseases.

Early Expression of Neuronal Dopaminergic Markers in a Parkinson's Disease Model in Rats Implanted with Enteric Stem Cells (ENSCs).

BACKGROUND: Parkinson's Disease (PD) is a common neurodegenerative disorder affecting the dopaminergic (DAergic) system. Replacement therapy is a promising alternative aimed at reconstructing the cytoarchitecture of affected brain regions in PD. Experimental approaches, such as the replacement of DAergic neurons with cells obtained from the Enteric Nervous System (ENS) has yet to be explored. OBJECTIVE: To establish and characterize a cell replacement strategy with ENS Cells (ENSCs) in a PD model in rats. METHODS: Since ENSCs can develop mature DAergic phenotypes, here we cultured undifferentiated cells from the myenteric plexus of newborn rats, establishing that they exhibit multipotential characteristics. These cells were characterized and further implanted in the Substantia nigra pars compacta (SNpc) of adult rats previously lesioned by a retrograde degenerative model produced by intrastriatal injection of 6-Hydroxydopamine (6-OHDA). DAergic markers were assessed in implants to validate their viability and possible differentiation once implanted. RESULTS: Cell cultures were viable, exhibited stem cell features and remained partially undifferentiated until the time of implant. The retrograde lesion induced by 6-OHDA produced DAergic denervation, reducing the number of fibers and cells in the SNpc. Implantation of ENSCs in the SNpc of 6-OHDAlesioned rats was tracked after 5 and 10 days post-implant. During that time, the implant increased selective neuronal and DAergic markers, Including Microtubule-Associated Protein 2 (MAP-2), Dopamine Transporter (DAT), and Tyrosine Hydroxylase (TH). CONCLUSION: Our novel results suggest that ENSCs possess a differentiating, proliferative and restorative potential that may offer therapeutic modalities to attenuate neurodegenerative events with the inherent demise of DAergic neurons.

Protective effects of valproic acid on 6-hydroxydopamine-induced neuroinjury.

Oxidative stress may play critically important roles in the etiology of Parkinson's disease (PD). 6-Hydroxydopamine (6-OHDA) is a physiological neurotoxin reported to induce oxidative-induced apoptosis of dopaminergic neurons in PD mice models. Valproic acid (VPA), a clinical mood stabilizer, is a HDAC inhibitor with neuroprotective capacities. In the study, we aim at examining the feasibility of VPA as a protector for dopaminergic neurons against damage from 6-OHDA, and the intracellular mechanisms. The 6-OHDA-induced neurotoxicity to the human dopaminergic cell line SH-SY5Y was applied for examining VPA protective effects. Pretreatment with VPA was able to improve cell viability and reduce 6-OHDA-induced reactive oxygen species. Furthermore, a significant suppression of apoptotic caspases including cleaved caspase-3, caspase-7, and caspase-9 was observed. The results also revealed VPA decreased the 6-OHDA-induced Bax/Bcl2 ratio, as measured at protein level. These novel findings indicate that VPA may be capable of protecting the SH-SY5Y dopaminergic neuronal cells from 6-OHDA-induced toxicity via the deceasing of apoptotic caspases (cleaved caspase-3, caspase-7, and caspase-9) and reducing of the Bax/Bcl2 ratio. Very possibly, VPA could serve as not only a mood stabilizer but also a potential antidote for PD prevention.