Loss Of TH-positive Neurons Research Articles

The Mst1/2-BNIP3 axis is required for mitophagy induction and neuronal viability under mitochondrial stress

Mitophagy induction upon mitochondrial stress is critical for maintaining mitochondrial homeostasis and cellular function. Here, we found that Mst1/2 (Stk3/4), key regulators of the Hippo pathway, are required for the induction of mitophagy under various mitochondrial stress conditions. Knockdown of Mst1/2 or pharmacological inhibition by XMU-MP-1 treatment led to impaired mitophagy induction upon CCCP and DFP treatment. Mechanistically, Mst1/2 induces mitophagy independently of the PINK1-Parkin pathway and the canonical Hippo pathway. Moreover, our results suggest the essential involvement of BNIP3 in Mst1/2-mediated mitophagy induction upon mitochondrial stress. Notably, Mst1/2 knockdown diminishes mitophagy induction, exacerbates mitochondrial dysfunction, and reduces cellular survival upon neurotoxic stress in both SH-SY5Y cells and Drosophila models. Conversely, Mst1 and Mst2 expression enhances mitophagy induction and cell survival. In addition, AAV-mediated Mst1 expression reduced the loss of TH-positive neurons, ameliorated behavioral deficits, and improved mitochondrial function in an MPTP-induced Parkinson’s disease mouse model. Our findings reveal the Mst1/2-BNIP3 regulatory axis as a novel mediator of mitophagy induction under conditions of mitochondrial stress and suggest that Mst1/2 play a pivotal role in maintaining mitochondrial function and neuronal viability in response to neurotoxic treatment.

J24335 exerts neuroprotective effects against 6-hydroxydopamine-induced lesions in PC12 cells and mice

Parkinson's disease is the second most prevalent age-related neurodegenerative disease and disrupts the lives of people aged >60 years. Meanwhile, single-target drugs becoming inapplicable as PD pathogenesis diversifies. Mitochondrial dysfunction and neurotoxicity have been shown to be relevant to the pathogenesis of PD. The novel synthetic compound J24335 (11-Hydroxy-1-(8-methoxy-5-(trifluoromethyl)quinolin-2-yl)undecan-1-one oxime), which has been researched similarly to J2326, has the potential to be a multi-targeted drug and alleviate these lesions. Therefore, we investigated the mechanism of action and potential neuroprotective function of J24335 against 6-OHDA-induced neurotoxicity in mice, and in PC12 cell models. The key target of action of J24335 was also screened. MTT assay, LDH assay, flow cytometry, RT-PCR, LC–MS, OCR and ECAR detection, and Western Blot analysis were performed to characterize the neuroprotective effects of J24335 on PC12 cells and its potential mechanism. Behavioral tests and immunohistochemistry were used to evaluate behavioral changes and brain lesions in mice. Moreover, bioinformatics was employed to assess the drug-likeness of J24335 and screen its potential targets. J24335 attenuated the degradation of mitochondrial membrane potential and enhanced glucose metabolism and mitochondrial biosynthesis to ameliorate 6-OHDA-induced mitochondrial dysfunction. Animal behavioral tests demonstrated that J24335 markedly improved motor function and loss of TH-positive neurons and dopaminergic nerve fibers, and contributed to an increase in the levels of dopamine and its metabolites in brain tissue. The activation of both the CREB/PGC-1α/NRF-1/TFAM and PKA/Akt/GSK-3β pathways was a major contributor to the neuroprotective effects of J24335. Furthermore, bioinformatics predictions revealed that J24335 is a low toxicity and highly BBB permeable compound targeting 8 key genes (SRC, EGFR, ERBB2, SYK, MAPK14, LYN, NTRK1 and PTPN1). Molecular docking suggested a strong and stable binding between J24335 and the 8 core targets. Taken together, our results indicated that J24335, as a multi-targeted neuroprotective agent with promising therapeutic potential for PD, could protect against 6-OHDA-induced neurotoxicity via two potential pathways in mice and PC12 cells.

Sodium butyrate ameliorates gut dysfunction and motor deficits in a mouse model of Parkinson's disease by regulating gut microbiota.

A growing body of evidence showed that gut microbiota dysbiosis might be associated with the pathogenesis of Parkinson's disease (PD). Microbiota-targeted interventions could play a protective role in PD by regulating the gut microbiota-gut-brain axis. Sodium butyrate (NaB) could improve gut microbiota dysbiosis in PD and other neuropsychiatric disorders. However, the potential mechanism associated with the complex interaction between NaB and gut microbiota-gut-brain communication in PD needs further investigation. C57BL/6 mice were subjected to a rotenone-induced PD model and were treated intragastrically with NaB for 4 weeks. The gut function and motor function were evaluated. The α-synuclein expression in colon and substantia nigra were detected by western blotting. Tyrosine hydroxylase (TH)-positive neurons in substantia nigra were measured by immunofluorescence. Moreover, gut microbiota composition was analyzed by 16S rRNA sequencing. Fecal short chain fatty acids (SCFAs) levels were determined by liquid chromatography tandem mass spectrometry (LC-MS). The levels of glucagon like peptide-1 (GLP-1) in tissues and serum were evaluated using enzyme-linked immunosorbent assay (ELISA). NaB ameliorated gut dysfunction and motor deficits in rotenone-induced mice. Meanwhile, NaB protected against rotenone-induced α-synuclein expression in colon and substantia nigra, and prevented the loss of TH-positive neurons. In addition, NaB could remodel gut microbiota composition, and regulate gut SCFAs metabolism, and restore GLP-1 levels in colon, serum, and substantia nigra in PD mice. NaB could ameliorate gut dysfunction and motor deficits in rotenone-induced PD mice, and the mechanism might be associated with the regulation of gut microbiota dysbiosis.

Neuroprotective effects of human umbilical cord mesenchymal stromal cells in PD mice via centrally and peripherally suppressing NLRP3 inflammasome-mediated inflammatory responses

Mesenchymal stromal cells (MSCs) exhibit beneficial anti-inflammatory effects against Parkinson’s disease (PD) via immunomodulatory actions. However, the underlying molecular mechanism remains unclear. This study aimed to investigate the potential neuroprotective effects of MSCs and the possible mechanisms involved by infusing human umbilical cord MSCs (hMSCs) in 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-treated mature male C57BL/6 mice. Subsequently, the striatal content of dopamine (DA) and its metabolites, tyrosine hydroxylase (TH)-positive neurons and activated microglia, circulating inflammatory cytokines, gene expression of cytokines, and NOD-like receptor protein 3 (NLRP3) inflammasome molecules were measured using high-performance liquid chromatography, flow cytometry, immunohistochemistry, immunofluorescent staining, and real-time polymerase chain reaction assays, respectively. Infused hMSCs markedly ameliorated the reduction in striatal DA and loss of TH-positive neurons in the substantia nigra of PD mice. The MPTP-induced activation of microglia and increase in tumor necrosis factor-α and interleukin-1β mRNA expression in the striatum were also attenuated by hMSCs. Furthermore, hMSCs completely reversed the elevated pro-inflammatory cytokine levels in the serum and mRNA expression of cytokines in the peripheral organs of PD mice. Moreover, hMSCs significantly inhibited the expression of caspase-1 and NLRP3 of the NLRP3 inflammasome in both the central and peripheral organs at mRNA level. These data suggest that hMSCs protect dopaminergic neurons in PD mice by suppressing both the central and peripheral inflammatory responses, probably by inhibiting the NLRP3 inflammasome. However, the animals in our study only received several MSC infusions, and the effects of infused MSCs over extended periods on the NLRP3 inflammasome need to be investigated in future studies.

Kaemperfol alleviates pyroptosis and microglia-mediated neuroinflammation in Parkinson's disease via inhibiting p38MAPK/NF-κB signaling pathway

The study aims to investigate whether kaemperfol (KAE) inhibits microglia pyroptosis and subsequent neuroinflammatory response to exert neuroprotective effects, along with the underlying mechanisms. The results showed KAE could ameliorate the behavioral deficits of Parkinson's disease (PD) rats, inhibit the activation of microglia and astrocytes, reduce the loss of TH-positive neurons, down-regulate levels of pyroptosis-related NOD-like receptor family pyrin domain containing 3 (NLRP3), GasderminD-N Term (GSDMD-NT), caspase1, apoptosis-associated speck-like protein containing a caspase-recruitment domain (ASC), interleukin (IL)-1β, and IL-18, and decrease the levels of inflammatory molecules (inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2)) and p38 mitogen-activated protein kinase/nuclear factor-kappaB (p38MAPK/NF-κB) signaling pathway molecules (p38MAPK, p-p38MAPK, NF-κB, and p-NF-κB) in the substantia nigra of PD rats. Further in vitro study indicated that KAE reversed the activation of BV2 cells and down-regulated the expressions of pyrolytic proteins, inflammatory mediators and key molecules in p38MAPK/NF-κB signaling pathway. Collectively, KAE inhibits the microglia pyroptosis and subsequent neuroinflammatory response to exert neuroprotective effects on 6-hydroxydopamine (6-OHDA)-induced PD rats and lipopolysaccharide (LPS)-induced BV2 inflammatory cells through inhibiting p38MAPK/NF-κB signaling pathway.

Neuroprotective Effect of Intrastriatal Caffeic Acid Phenethyl Ester Treatment in 6-OH Dopamine Model of Parkinson's Disease in Rats.

Parkinson's disease (PD) is the second most common neurodegenerative disorder, and the main cause of PD is still not known. Until now, no cure for Parkinson's disease is yet in sight. Caffeic acid phenethyl ester (CAPE) is a polyphenolic component of the propolis, which can be derived from honeybee hive propolis. We aimed to determine the effect of intrastriatal CAPE administration as a neuroprotective agent on 6-hydroxydopamine (6-OHDA)-induced PD model. Adult male Wistar rats weighing 280–320 g were used. The PD model was induced with unilateral intrastriatal 6-OHDA injection. Treatment groups received 20 μmol/5 μL/4 day and 80 μmol/5 μL/4 day CAPE 24 h after 6-OHDA injection. Eight days after 6-OHDA application, behavioral studies (adhesive tape removal test, open-field test, cylinder test, and apomorphine-induced asymmetric rotational behavior) were performed once more to compare the effects of CAPE on behavior tests. Striatal histological verifications, immunohistochemistry, and stereological quantitation were performed. Our results for the first time showed that, besides improving the motor performance, CAPE treatment also prevents 6-OHDA-induced loss of TH-positive neurons. From our results, CAPE may be a promising clinical agent in the treatment of PD.

Depression Induced by Chronic Unpredictable Mild Stress Increases Susceptibility to Parkinson's Disease in Mice via Neuroinflammation Mediated by P2X7 Receptor.

The relationship between depression and Parkinson's disease (PD) is complicated and still not fully understood. We investigated whether depression increased the susceptibility to PD and whether this resulted from neuroinflammation mediated by purinergic ligand-gated ion channel 7 receptor (P2X7R) of microglia in mice. Depression was induced by a 14-day chronic unpredictable mild stress (CUMS), and PD was induced by 1-day acute injection of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP). Before MPTP administration, some mice were given brilliant blue G (BBG), a P2X7R inhibitor. Changes in depression and motor function were assessed by sucrose preference, tail suspension, open field, and rotating rod tests. Differences in P2X7R, caspase-1, NLRP3 inflammasome, interleukin (IL)-1β, tyrosine hydroxylase (TH), and microglial activation among experimental groups were detected by immunofluorescence, immunohistochemistry, western blotting, and ELISA. CUMS-induced depression-like behavior, and MPTP induced PD in mice. CUMS mice had no motor dysfunction, but the dyskinesia and loss of TH-positive neurons in the substantia nigra after MPTP treatment were more serious than with MPTP treatment alone. With behavioral changes, neuroinflammatory markers, such as caspase-1, NLRP3 and IL-1β increased, and microglia were activated as well as expression of P2X7R increased. Additionally, BBG partly reversed the above abnormalities. Summarily, we suggest that CUMS aggravates dyskinesia and death of dopaminergic neurons in an MPTP-PD model via promoting activation of microglia and neuroinflammation, which may be mediated by P2X7R. Inhibition of P2X7R could be a new control strategy for PD associated with depression.

Eupatilin prevents behavioral deficits and dopaminergic neuron degeneration in a Parkinson's disease mouse model

AimsNeuroinflammation and apoptosis play a crucial role in Parkinson's disease (PD) pathogenesis. Eupatilin is a lipophilic flavonoid isolated from Artemisia species and exerts anti-apoptotic and anti-inflammatory activities. In this study, we investigated the effects of Eupatilin on a mouse model of PD induced by 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP). Main methodsThe rotarod test and traction test were constructed to examine the motor function. Immunofluorescent staining was performed to detect the expression of TH, Iba-1 and GFAP. Apoptosis was examined by the TUNEL assay. Real-time PCR was used to determine the mRNA expression and Western blot and ELISA were used to determine the protein expression. Key findingsEupatilin improved behavioral impairment caused by MPTP. A loss of TH positive neurons was observed in the substantia nigra pars compacta of MPTP-lesioned brain, while it was rescued by Eupatilin. Moreover, MPTP administration increased the cell number of microglia and astrocytes and the expression of inflammatory factors TNF-α, IL-1β, and IL-6. Whereas Eupatilin suppressed the activation of neuroinflammation. Eupatilin also decreased cell apoptosis enhanced by MPTP/MPP+ exposure in vivo and in vitro. We further revealed that Eupatilin abolished MPTP-induced downregulation of IκBα expression and accumulation of p65 in the nuclear compartment. Besides, MPTP administration led to dephosphorylation of Akt and GSK-3β, but it was restored by Eupatilin. SignificanceWe demonstrate that Eupatilin alleviates behavioral impairment and dopaminergic neuron loss induced by MPTP through inhibition of neuroinflammation and apoptosis. Our research provides more evidence for Eupatilin as a potential preventative drug for PD.

Quantitative whole-brain 3D imaging of tyrosine hydroxylase-labeled neuron architecture in the mouse MPTP model of Parkinson's disease.

ABSTRACTParkinson's disease (PD) is a basal ganglia movement disorder characterized by progressive degeneration of the nigrostriatal dopaminergic system. Immunohistochemical methods have been widely used for characterization of dopaminergic neuronal injury in animal models of PD, including the MPTP (1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine) mouse model. However, conventional immunohistochemical techniques applied to tissue sections have inherent limitations with respect to loss of 3D resolution, yielding insufficient information on the architecture of the dopaminergic system. To provide a more comprehensive and non-biased map of MPTP-induced changes in central dopaminergic pathways, we used iDISCO immunolabeling, light-sheet fluorescence microscopy (LSFM) and deep-learning computational methods for whole-brain three-dimensional visualization and automated quantitation of tyrosine hydroxylase (TH)-positive neurons in the adult mouse brain. Mice terminated 7 days after acute MPTP administration demonstrated widespread alterations in TH expression. Compared to vehicle controls, MPTP-dosed mice showed a significant loss of TH-positive neurons in the substantia nigra pars compacta and ventral tegmental area. Also, MPTP dosing reduced overall TH signal intensity in basal ganglia nuclei, i.e. the substantia nigra, caudate-putamen, globus pallidus and subthalamic nucleus. In contrast, increased TH signal intensity was predominantly observed in limbic regions, including several subdivisions of the amygdala and hypothalamus. In conclusion, mouse whole-brain 3D imaging is ideal for unbiased automated counting and densitometric analysis of TH-positive cells. The LSFM–deep learning pipeline tracked brain-wide changes in catecholaminergic pathways in the MPTP mouse model of PD, and may be applied for preclinical characterization of compounds targeting dopaminergic neurotransmission.

Treatment Of Magnesium-L-Threonate Elevates The Magnesium Level In The Cerebrospinal Fluid And Attenuates Motor Deficits And Dopamine Neuron Loss In A Mouse Model Of Parkinson's disease.

PurposeEpidemiology research has demonstrated that magnesium (Mg) deficiency is associated with a high incidence of Parkinson’s disease (PD). It is known that the systemic administration of MgSO4 is not able to elevate the Mg concentration in cerebrospinal fluid (CSF). This study aims to verify the protective effect of magnesium-L-threonate (MgT) in 1-methyl-4-phenyl-1, 2, 3, 6-tetrahydropyridine (MPTP) mouse model.MethodsC57BL/6J mice were orally administered MgT or MgSO4 for 4 weeks, and received MPTP in the third week. After analysis of open-field and rotarod tests on the last day, tyrosine hydroxylase (TH) immunopositive cells and protein levels were quantified in the substantia nigra pars compacta (SNpc) and striatum. The expression of inducible nitric oxide synthase (iNOS) level was evaluated. Mg concentration in serum and CSF was measured after oral administration of MgSO4 or MgT in normal mice. Mg concentration in the CSF was increased in the mice treated with MgT but not MgSO4.ResultsThe total distance and mean speed in open-field tests, and the time spent on rotarod in the MgT group were increased, compared with MPTP group. The MgT treatment but not MgSO4 dose-dependently attenuated the loss of TH-positive neurons, and the reduction of the TH expression in the SNpc. The MgT treatment also inhibited the expression of iNOS as measured by immunohistochemistry and Western blots. Double-immunofluorescence staining of TH and iNOS showed iNOS-positive cells were collocalized for TH-positive cells.ConclusionThe treatment with MgT is associated with an increase of Mg in the CSF. MgT, rather than MgSO4, can significantly attenuate MPTP-induced motor deficits and dopamine (DA) neuron loss.

Pilose Antler Extracts (PAEs) Protect against Neurodegeneration in 6-OHDA-Induced Parkinson's Disease Rat Models.

Parkinson's disease (PD) is one of the most common neurodegenerative diseases worldwide. Although dopamine replacement therapy mitigates motor dysfunction in PD patients, there are no therapeutics that are currently available to reverse neuronal cell death in the substantia nigra pars compacta (SNc), which is the main region for dopamine loss in PD patients. The protein concentration of the Pilose antler extracts (PAEs) was estimated using the Bradford Protein Assay Kit. Hematoxylin and eosin (HE) staining was used to evaluate the protective effect of PAEs on 6-OHDA induced cell death in PD model rats. Immunohistochemistry (IHC) was used to detect the tyrosine hydroxylase (TH) positive neuronal cell in SNc. HPLC-MS was used to detect dopamine (DA), 3,4-Dihydroxyphenylacetic acid (DOPAC), homovanillic acid (HVA), 5-hydroxytryptamine (5-HT), 5-hydroxyindoleacetic acid (5-HIAA), and glutamate (Glu) levels in the striatum and cerebrospinal fluid (CSF). The amino acid level in the striatum and CSF was measured by HPLC-FLD. Protein expression of growth associated protein-43 (GAP-43) and neurofilament heavy polypeptide (NF-H) was measured using western blotting. The components of PAEs through blood vessels were detected by HPLC/MS/MS. In this study, PAEs with proteins ranging from 10 kDa to 250 kDa molecular weight was administered to 6-OHDA-induced PD rats. We found that PAEs inhibited 6-OHDA-induced neuronal cell death and TH-positive neuronal loss in SNc. PAEs administration also increased the levels of DA, DOPAC, and 5-HT, in addition to DOPAC/DA and HVA/DA indexes in the CSF and Striatum of 6-OHDA induced rats. Conversely, PAEs decreased the levels of Glu and GABA. Treatment with PAEs and Madopar increased GAP-43 and NF-H expression in the SNc and striatum. Proteomic analysis using LC/MS/MS indicated that 11 components of PAEs may have neuropharmacological effects. These results demonstrate that PAEs protects against 6-OHDA induced toxic effects in the PD rat models. Intragastric administration of PAEs may be a novel therapeutic strategy for neurodegenerative disorders like PD.

Animal models of synucleinopathies: prion-like propagation of alpha-synuclein in wild-type animals

Accumulation of insoluble alpha-synuclein (αS) is a pathological hallmark of some progressive neurodegenerative diseases including Parkinson's disease, dementia with Lewy bodies, and multiple system atrophy, collectively termed synucleinopathies. In diseased brain, αS forms β-sheet-rich amyloid fibrils and it is accumulated in neurons or glial cells. A growing body of evidence suggests that spreading of αS pathology occur by prion-like propagation mechanisms. Our study revealed that intracerebral injection of synthetic αS amyloid fibrils into wild-type mice induced prion-like propagation of αS pathology at 1 month post injection, while injection of soluble αS did not induce αS pathology. Furthermore, injection of αS amyloid fibrils into αS knockout mice failed to induce any pathologies. We also have demonstrated that intracerebral injection of αS amyloid fibrils into small primates, adult common marmosets, resulted in spreading of αS pathologies and loss of TH-positive neurons. These in vivo experiments clearly indicate that αS amyloid fibrils has prion-like properties and it propagates through neural networks. The underlying mechanisms of αS propagation are poorly understood, however, αS propagation model animals would be useful in elucidating pathogenetic mechanisms and developing disease-modifying drugs for sporadic synucleinopathies.

Expression of magnesium transporter SLC41A1 in the striatum of 6-hydroxydopamine-induced parkinsonian rats

ObjectivesThe aim of this study was to investigate the expression of the mammalian Mg2+ transporter solute carrier family 41, member 1 (SLC41A1) in the striatum (STR) of a 6-hydroxydopamine (6-OHDA)-induced rat model, and its response to magnesium before the degenerative process commenced. Materials and methodsA unilateral parkinsonian rat model was induced by injection of 6-OHDA into the right medial forebrain bundle. Some rats received MgSO4 (90 mg/kg/day, intraperitoneal injection) for 7 or 14 days starting from the day following the 6-OHDA injection. The extent of dopamine depletion was determined by assessing the numbers of tyrosine hydroxylase (TH)-immunoreactive neurons in the substantia nigra pars compacta (SNc) and the apomorphine (APO)-induced rotational behavior. The mRNA and protein expression of SLC41A1 in STR were evaluated by real-time RT-PCR and western blotting respectively. ResultsAPO-induced rotations increased significantly in the 6-OHDA lesioned rats compared with the controls, with MgSO4 administration significantly improving the behavior. The numbers of TH-immunoreactive neurons in SNc were significantly lower in the lesioned side than in the unlesioned side. Administration of MgSO4 for 14 days partly ameliorated the loss of TH-positive neurons. The mRNA and protein expressions of SLC41A1 in the lesioned STR were lower in the 6-OHDA lesioned rats than in the controls at both 7 and 14 days post-lesion induction. MgSO4 supplementation partly reversed the mRNA and protein expressions of SLC41A1. ConclusionThe regulation of SLC41A1 expression is responsive to magnesium in a 6-OHDA-induced rat model, wherein 6-OHDA may alter magnesium transport in the brain.

Therapeutic Potential of a Prolyl Hydroxylase Inhibitor FG-4592 for Parkinson's Diseases in Vitro and in Vivo: Regulation of Redox Biology and Mitochondrial Function.

As the main transcription factor that regulates the cellular responses to hypoxia, Hypoxia-inducible factor-1α (HIF-1α) plays an important role in the pathogenesis of Parkinson’s disease (PD). HIF-1α is normally degraded through ubiquitination after hydroxylation by prolyl hydroxylases (PHD). Emerging evidence has suggested that HIF PHD inhibitors (HIF-PHI) may have neuroprotective effects on PD through increasing HIF-1α levels. However, the therapeutic benefit of HIF-PHI for PD remains poorly explored due to the lack of proper clinical compounds and understanding of the underlying molecular mechanisms. In this study, we examined the therapeutic benefit of a new HIF-PHI, FG-4592, which is currently in phase 3 clinical trials to treat anemia in patients with chronic kidney diseases (CKD) in PD models. FG-4592 attenuates MPP+ -induced apoptosis and loss of tyrosine hydroxylase (TH) in SH-SY5Y cells. Pretreatment with FG-4592 mitigates MPP+-induced loss of mitochondrial membrane potential (MMP), mitochondrial oxygen consumption rate (OCR), production of reactive oxygen species (ROS) and ATP. Furthermore, FG-4592 counterbalances the oxidative stress through up-regulating nuclear factor erythroid 2 p45-related factor 2 (Nrf-2), heme oxygenase-1 (HO-1) and superoxide dismutase 2 (SOD2). FG-4592 treatment also induces the expression of Peroxisome proliferator-activated receptor-γ coactivator-1α (PGC-1α) through increasing the phosphorylation of AMP-activated protein kinase (AMPK). In MPTP-treated mice, FG-4592 protects against MPTP-induced loss of TH-positive neurons of substantia nigra and attenuates behavioral impairments. Collectively, our study demonstrates that FG-4592 is a promising therapeutic strategy for PD through improving the mitochondrial function under oxidative stress.

1-Methyl-4-Phenylpyridinium-Induced Death of Differentiated SH-SY5Y Neurons Is Potentiated by Cholesterol

Background/Aims: Hypercholesterolemia is recently considered a risk factor for Parkinson’s disease (PD), the most consistent neurodegenerative movement disorder. The study aimed to investigate the effect of exogenous cholesterol on 1-methyl-4-phenylpyridinium (MPP⁺) parkinsonian neurotoxin-induced cell death, loss of mitochondrial membrane potential, and dopaminergic deficiency in a cellular model of PD. Methods: Cholesterol (50 μM) when added in the culture media alone or in combination with MPP⁺ was studied in SH-SY5Y neuroblastoma cells. There were 4 groups that were studied; SH-SY5Y cells treated with vehicle (control), cells that were treated with 1 mM MPP⁺ (MPP⁺), or cholesterol (chol) or both (M + chol). The loss of cell survival was measured by 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide assay. Dopamine depletion, microtubule-associated protein 2 (MAP-2), and tyrosine hydroxylase (TH)-positive neuronal loss were determined by HPLC-electrochemical detection and TH immunocytochemistry respectively. Mitochondrial membrane potential in cells stained by tetramethylrhodamine methyl ester dye was analysed by flow cytometry. Results: Cholesterol treatment potentiated a reduction of neuronal viability with loss of TH-positive neurons in cultures. MPP⁺-induced depletion of dopamine level in the post-mitotic MAP-2 immunoreactive neurons and loss of mitochondrial membrane potential were also heightened by cholesterol. Conclusion: Apparently, changes in neuronal cholesterol content significantly influenced the neurotoxicity and the direct mitochondrial mechanisms involved in MPP⁺-induced cell death. Our observations demonstrate that high cholesterol incorporated into the differentiated human neuroblastoma cells worsened dopaminergic neuronal survivability through increased depolarization of mitochondrial membrane potential, which is a known mechanism of dopaminergic cell death by MPP⁺. The present findings support the hypothesis that hypercholesterolemia could be a risk factor for PD.

Metformin Prevented Dopaminergic Neurotoxicity Induced by 3,4-Methylenedioxymethamphetamine Administration.

Metformin, a well-known antidiabetic drug, has recently been proposed to promote neurogenesis and to have a neuroprotective effect on the neurodegenerative processes induced by the dopaminergic neurotoxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) in models of Parkinson's disease. Interestingly, metformin has antioxidant properties and is involved in regulating the production of cytokines released during the neuroinflammatory process. Several studies have reported that 3,4-methylenedioxymethamphetamine (MDMA), a recreational drug mostly consumed by young adults, produces a persistent loss of dopaminergic neurons in the substantia nigra pars compacta (SNc) and caudate putamen (CPu) of mice. The aim of this study was to investigate the potential neuroprotective effect of metformin against short- and long-term neurotoxicity induced by MDMA and its role on MDMA-induced hyperthermia. Adult mice received metformin (2×200mg/kg, 11-h intervals, administered orally), MDMA (4×20mg/kg, 2-h interval, administered intraperitoneally), or MDMA plus metformin (2×200mg/kg, 1h before the first MDMA administration and 4h after the last). On the second and third day, mice were treated with vehicle or metformin (1×200mg/kg) and sacrificed 48h and 7days after the last MDMA administration. The neuroprotective effect of metformin on MDMA-induced dopaminergic damage was evaluated by dopamine transporter (DAT) and tyrosine hydroxylase (TH) immunohistochemistry in SNc and CPu. Metformin prevented the MDMA-induced loss of TH-positive neurons in the SNc and TH- and DAT-positive fibers in CPu, both at 48h and 7days after the last MDMA administration. These results show that metformin is neuroprotective against the short- and long-lasting dopaminergic neurodegeneration induced by MDMA.

Single injection of small-molecule amyloid accelerator results in cell death of nigral dopamine neurons in mice.

The assembly process of α-synuclein toward amyloid fibers is linked to neurodegeneration in Parkinson’s disease. In the present study, we capitalized on the in vitro discovery of a small-molecule accelerator of α-synuclein amyloid formation and assessed its effects when injected in brains of normal mice. An accelerator and an inhibitor of α-synuclein amyloid formation, as well as vehicle only, were injected into the striatum of normal mice and followed by behavioral evaluation, immunohistochemistry, and metabolomics up to six months later. The effects of molecules injected into the substantia nigra of normal and α-synuclein knock-out mice were also analyzed. When accelerator or inhibitor was injected into the brain of normal mice no acute compound toxicity was found. However, 6 months after single striatal injection of accelerator, mice sensorimotor functions were impaired, whereas mice injected with inhibitor had no dysfunctions. Injection of accelerator (but not inhibitor or vehicle) into the substantia nigra revealed significant loss of tyrosine hydroxylase (TH)-positive neurons after 3 months. No loss of TH-positive neurons was found in α-synuclein knock-out mice injected with accelerator into the substantia nigra. Metabolic serum profiles from accelerator-injected normal mice matched those of newly diagnosed Parkinson’s disease patients, whereas the profiles from inhibitor-injected normal mice matched controls. Single inoculation of a small-molecule amyloid accelerator may be a new approach for studies of early events during dopamine neurodegeneration in mice.

Berberine chloride pretreatment exhibits neuroprotective effect against 6-hydroxydopamine-induced neuronal insult in rat.

Parkinson's disease (PD) is a rather common movement disorder as a result of the degeneration of dopaminergic neurons within the substantianigra. Current treatments for PD afford symptomatic relief with no prevention of disease progression. Due to the neuroprotective and anti-apoptotic potential of the isoquinoline alkaloid berberine (BBR), this study was conducted to assess whether BBR pretreatment could attenuate behavioral and neuronal derangement in 6-hydroxydopamine (6-OHDA)-induced model of PD in the rat. Unilateral intrastriatal 6-OHDA-lesioned rats received BBR at doses of 25 and/or 50 mg/kg (i.p.) three times at an interval of 24 h, started 2 days before the surgery. After 1 week, apomorphine caused significant contralateral rotations and a significant reduction in the number of Nissl-stained and tyrosine-hydroxylase (TH)-positive neurons on the left side of the substantianigra. BBR pretreatment at a dose of 50 mg/kg significantly reduced rotations and prevented loss of TH-positive neurons. These results indicate pre-lesion administration of BBR could protect against 6-OHDA toxicity and this may be of benefit besides other available therapies in PD.

Transgenic supplementation of SIRT1 fails to alleviate acute loss of nigrostriatal dopamine neurons and gliosis in a mouse model of MPTP-induced parkinsonism

Background Dopamine (DA) neuron-selective uptake and toxicity of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) causes parkinsonism in humans. Loss of DA neurons via mitochondrial damage and oxidative stress is reproduced by systemic injection of MPTP in animals, which serves as models of parkinsonism and Parkinson’s disease (PD). This study aimed to test whether pan-neural supplementation of the longevity-related, pleiotropic deacetylase SIRT1, which confers partial tolerance to at least three models of stroke and neurodegeneration, could also alleviate MPTP-induced acute pathological changes in nigrostriatal DA neurons and neighboring glia. Results We employed a line of prion promoter-driven Sirt1-transgenic (Sirt1Tg) mice that chronically overexpress murine SIRT1 in the brain and spinal cord. Sirt1Tg and wild-type (WT) male littermates (3‒4 months old) were subjected to intraperitoneal injection of MPTP. Acute histopathological changes in the midbrain and striatum (caudoputamen) were assessed with serial coronal sections triply labeled for tyrosine hydroxylase (TH), glial fibrillary acidic protein (GFAP), and nuclear DNA. In the substantia nigra pars compacta (SNpc) of the midbrain, the number of TH-positive neurons and the reactive gliosis were comparable between the Sirt1Tg and WT littermates. In the striatum, the relative fluorescence intensity of TH-positive nerve terminals and the level of gliosis did not differ by the genotypes. Conclusions Sirt1Tg and WT littermate mice exhibited comparable acute histopathological reactions to the systemic injection of MPTP, loss of TH-positive neurons and reactive gliosis. Thus, the genetic supplementation of SIRT1 does not confer histologically recognizable protection on nigrostriatal DA neurons against acute toxicity of MPTP.

Topographical Distribution of Morphological Changes in a Partial Model of Parkinson's Disease--Effects of Nanoencapsulated Neurotrophic Factors Administration.

Administration of various neurotrophic factors is a promising strategy against Parkinson's disease (PD). An intrastriatal infusion of 6-hydroxidopamine (6-OHDA) in rats is a suitable model to study PD. This work aims to describe stereological parameters regarding rostro-caudal gradient, in order to characterize the model and verify its suitability for elucidating the benefits of therapeutic strategies. Administration of 6-OHDA induced a reduction in tyrosine hidroxylase (TH) reactivity in the dorsolateral part of the striatum, being higher in the caudal section than in the rostral one. Loss of TH-positive neurons and axodendritic network was highly significant in the external third of substantia nigra (e-SN) in the 6-OHDA group versus the saline one. After the administration of nanospheres loaded with neurotrophic factors (NTF: vascular endothelial growth factor (VEGF) + glial cell line-derived neurotrophic factor (GDNF)), parkinsonized rats showed more TH-positive fibers than those of control groups; this recovery taking place chiefly in the rostral sections. Neuronal density and axodendritic network in e-SN was more significant than in the entire SN; the topographical analysis showed that the highest difference between NTF versus control group was attained in the middle section. A high number of bromodeoxyuridine (BrdU)-positive cells were found in sub- and periventricular areas in the group receiving NTF, where most of them co-expressed doublecortin. Measurements on the e-SN achieved more specific and significant results than in the entire SN. This difference in rostro-caudal gradients underpins the usefulness of a topological approach to the assessment of the lesion and therapeutic strategies. Findings confirmed the neurorestorative, neurogenic, and synergistic effects of VEGF+GDNF administration.