Substantia Nigra Of MPTP-treated Mice Research Articles

Astrocytic Nrf2 Mediates the Neuroprotective and Anti-Inflammatory Effects of Nootkatone in an MPTP-Induced Parkinson's Disease Mouse Model.

This study aims to investigate the neuroprotective effects of nootkatone (NKT), a sesquiterpenoid compound isolated from grapefruit, in an MPTP-induced Parkinson's disease (PD) mouse model. NKT restored MPTP-induced motor impairment and dopaminergic neuronal loss and increased the expression of neurotrophic factors like BDNF, GDNF, and PGC-1α. In addition, NKT inhibited microglial and astrocyte activation and the expression of pro-inflammatory markers like iNOS, TNF-α, and IL-1β and oxidative stress markers like 4-HNE and 8-OHdG. NKT increased the expression of nuclear factor erythroid 2-related factor 2 (Nrf2)-driven antioxidant enzymes like HO-1 and NQO-1 in astrocytes, but not in neurons or microglia in MPTP-treated mice. To investigate whether Nrf2 mediates the anti-inflammatory, antioxidant, or neuroprotective effects of NKT, mice were pretreated with Nrf2-specific inhibitor brusatol (BT) prior to NKT treatment. BT attenuated the NKT-mediated inhibition of 4-HNE and 8-OHdG and the number of Nrf2+/HO-1+/NQO1+ cells co-localized with GFAP+ astrocytes in the substantia nigra of MPTP-treated mice. In addition, BT reversed the effects of NKT on dopaminergic neuronal cell death, neurotrophic factors, and pro-/anti-inflammatory cytokines in MPTP-treated mice. Collectively, these data suggest that astrocytic Nrf2 and its downstream antioxidant molecules play pivotal roles in mediating the neuroprotective and anti-inflammatory effects of NKT in an MPTP-induced PD mouse model.

Necrosulfonamide exerts neuroprotective effect by inhibiting necroptosis, neuroinflammation, and α-synuclein oligomerization in a subacute MPTP mouse model of Parkinson’s disease

Parkinson’s disease (PD) is an incurable movement disorder characterized by dopaminergic cell loss, neuroinflammation, and α-synuclein pathology. Herein, we investigated the therapeutic effects of necrosulfonamide (NSA), a specific inhibitor of mixed lineage kinase domain-like protein (MLKL), in a subacute 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) mouse model of PD. MLKL is an executor of necroptosis, a programmed cell death pathway that causes inflammation. Repeated administration of NSA resulted in the recovery of impaired motor performance and dopaminergic degeneration. Furthermore, NSA inhibited the phosphorylation, ubiquitylation, and oligomerization of MLKL, all of which are associated with MLKL cell death-inducing activity in dopaminergic cells in the substantia nigra (SN). NSA also inhibited microglial activation and reactive astrogliosis as well as the MPTP-induced expression of proinflammatory molecules such as tumor necrosis factor-α, interleukin-1β, inducible nitric oxide synthase, and cystatin F. Furthermore, NSA inhibited α-synuclein oligomerization and phosphorylation in the SN of MPTP-treated mice by inhibiting the activity of glycogen synthase kinase 3β and matrix metalloproteinase-3. In conclusion, NSA has anti-necroptotic, anti-inflammatory, and anti-synucleinopathic effects on PD pathology. Therefore, NSA is a potential therapeutic candidate for PD.

TRPV4 contributes to ER stress and inflammation: implications for Parkinson\u2019s disease

BackgroundParkinson’s disease (PD) is a progressive neurodegenerative disorder. Its molecular mechanism is still unclear, and pharmacological treatments are unsatisfactory. Transient receptor potential vanilloid 4 (TRPV4) is a nonselective Ca2+ channel. It has recently emerged as a critical risk factor in the pathophysiology of neuronal injuries and cerebral diseases. Our previous study reported that TRPV4 contributed to endoplasmic reticulum (ER) stress in the MPP+-induced cell model of PD. In the present study, we detected the role and the mechanism of TRPV4 in 1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced PD mice.MethodsIntracerebral injection of an adeno-associated virus (AAV) into the substantia nigra (SN) of mice was used to knockdown or upregulate the expression of TRPV4 and intraperitoneal injection of MPTP. Rotarod and pole tests were used to evaluate the locomotor ability of mice. We used immunohistochemistry, Nissl staining and Western blot to detect the alterations in the number of tyrosine hydroxylase (TH)-positive neurons, Nissl-positive neurons, the levels of ER stress-associated molecules and proinflammatory cytokines in the SN.ResultsThe SN was transfected with AAV for 3 weeks and expressed the target protein with green fluorescence. Knockdown of TRPV4 via injection of a constructed AAV-TRPV4 shRNAi into the SN alleviated the movement deficits of PD mice. Upregulation of TRPV4 via injection of a constructed AAV-TRPV4 aggravated the above movement disorders. The expression of TRPV4 was upregulated in the SN of MPTP-treated mice. Injection of AAV-TRPV4 shRNAi into the SN rescued the number of TH-positive and Nissl-positive neurons in the SN decreased by MPTP, while injection of AAV-TRPV4 induced the opposite effect. Moreover, MPTP-decreased Sarco/endoplasmic reticulum Ca2+-ATPase 2 (SERCA2) and pro-cysteinyl aspartate specific proteinase-12 (procaspase-12), MPTP-increased Glucose-regulated protein 78 (GRP78), Glucose-regulated protein 94 (GRP94) and C/EBP homologous protein (CHOP) were inhibited by AAV-TRPV4 shRNAi infection, and enhanced by AAV-TRPV4. In the same way, MPTP-decreased procaspase-1, MPTP-increased Interleukin-18 (IL-18), Cyclooxgenase-2 (COX-2) and 5-Lipoxygenase (5-LOX) were inhibited by AAV-TRPV4 shRNAi, or further exacerbated by AAV-TRPV4.ConclusionsThese results suggest that TRPV4 mediates ER stress and inflammation pathways, contributing to the loss of dopamine (DA) neurons in the SN and movement deficits in PD mice. Moreover, this study provides a new perspective on molecular targets and gene therapies for the treatment of PD in the future.

Inhibition of repulsive guidance molecule-a protects dopaminergic neurons in a mouse model of Parkinson\u2019s disease

Repulsive guidance molecule-a (RGMa), a glycosylphosphatidylinositol-anchored membrane protein, has diverse functions in axon guidance, cell patterning, and cell survival. Inhibition of RGMa attenuates pathological dysfunction in animal models of central nervous system (CNS) diseases including spinal cord injury, multiple sclerosis, and neuromyelitis optica. Here, we examined whether antibody-based inhibition of RGMa had therapeutic effects in a mouse model of Parkinson’s disease (PD). We treated mice with 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) and found increased RGMa expression in the substantia nigra (SN). Intraventricular, as well as intravenous, administration of anti-RGMa antibodies reduced the loss of tyrosine hydroxylase (TH)-positive neurons and accumulation of Iba1-positive microglia/macrophages in the SN of MPTP-treated mice. Selective expression of RGMa in TH-positive neurons in the SN-induced neuronal loss/degeneration and inflammation, resulting in a progressive movement disorder. The pathogenic effects of RGMa overexpression were attenuated by treatment with minocycline, which inhibits microglia and macrophage activation. Increased RGMa expression upregulated pro-inflammatory cytokine expression in microglia. Our observations suggest that the upregulation of RGMa is associated with the PD pathology; furthermore, inhibitory RGMa antibodies are a potential therapeutic option.

Stress increases MHC-I expression in dopaminergic neurons and induces autoimmune activation in Parkinson's disease.

The expression of major histocompatibility complex class I (MHC-I), a key antigen-presenting protein, can be induced in dopaminergic neurons in the substantia nigra, thus indicating its possible involvement in the occurrence and development of Parkinson's disease. However, it remains unclear whether oxidative stress induces Parkinson's disease through the MHC-I pathway. In the present study, polymerase chain reaction and western blot assays were used to determine the expression of MHC-I in 1-methyl-4-phenylpyridinium (MPP+)-treated SH-SY5Y cells and a 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced Parkinson's disease mouse model. The findings revealed that MHC-I was expressed in both models. To detect whether the expression of MHC-I was able to trigger the infiltration of cytotoxic T cells, immunofluorescence staining was used to detect cytotoxic cluster of differentiation 8 (CD8)+ T cell infiltration in the substantia nigra of MPTP-treated mice. The results indicated that the presentation of MHC-I in dopaminergic neurons was indeed accompanied by an increase in the number of CD8+ T cells. Moreover, in MPTP-induced Parkinson's disease model mice, the genetic knockdown of endogenous MHC-I, which was caused by injecting specific adenovirus into the substantia nigra, led to a significant reduction in CD8+ T cell infiltration and alleviated dopaminergic neuronal death. To further investigate the molecular mechanisms of oxidative stress-induced MHC-I presentation, the expression of PTEN-induced kinase 1 (PINK1) was silenced in MPP+-treated SH-SY5Y cells using specific small interfering RNA (siRNA), and there was more presentation of MHC-I in these cells compared with control siRNA-treated cells. Taken together, MPP+-/MPTP-induced oxidative stress can trigger MHC-I presentation and autoimmune activation, thus rendering dopaminergic neurons susceptible to immune cells and degeneration. This may be one of the mechanisms of oxidative stress-induced Parkinson's disease, and implies the potential neuroprotective role of PINK1 in oxidative stress-induced MHC-I presentation. All animal experiments were approved by the Southern Medical University Ethics Committee (No. 81802040, approved on February 25, 2018).

Go-sha-jinki-Gan Alleviates Inflammation in Neurological Disorders via p38-TNF Signaling in the Central Nervous System.

Go-sha-jinki-Gan (GJG) is a traditional Japanese herbal medicine. In clinical practice, GJG is effective against neuropathic pain and hypersensitivity induced by chemotherapy or diabetes. In our previous study using a chronic constriction injury mouse model, we showed that GJG inhibited microglia activation by suppressing the expression of tumor necrosis factor-α (TNF-α) and p38 mitogen-activated protein kinase (p38 MAPK) in the peripheral nervous system. To investigate whether GJG can suppress inflammation in the central nervous system (CNS) in the context of neurological disorders, we examined the effect of GJG on the activation of resident glial cells and on p38-TNF signaling in two mouse models of neurological disorders: the experimental autoimmune encephalomyelitis (EAE) model of multiple sclerosis and the 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) model of Parkinson's disease. GJG administration relieved the severity of clinical EAE symptoms and MPTP-induced inflammation by decreasing the number of microglia and the production of TNF-α in the spinal cord of EAE mice and the substantia nigra of MPTP-treated mice. Accordingly, GJG suppressed the phosphorylation of p38 in glial cells of these two mouse models. We conclude that GJG attenuates inflammation of the CNS by suppressing glial cell activation, followed by a decrease in the production of TNF-α via p38-TNF signaling.

Down-regulation of natural resistance-associated macrophage protein-1 (Nramp1) is associated with 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)/1-methyl-4-phenylpyridinium (MPP+ )-induced α-synuclein accumulation and neurotoxicity.

The accumulation of α-synuclein is a hallmark in the pathogenesis of Parkinson's disease (PD). Natural resistance-associated macrophage protein-1 (Nramp1) was previously shown to contribute to the degradation of extracellular α-synuclein in microglia under conditions of iron overload. This study was aimed at investigating the role of Nramp1 in α-synuclein pathology in the neurone under 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)/1-methyl-4-phenylpyridinium (MPP+ ) treatment. The expression of Nramp1 and pathological features (including iron and α-synuclein accumulation) were examined in the dopaminergic neurones of humans (with and without PD) and of mice [with and without receiving chronic MPTP intoxication]. The effects of Nramp1 expression on low-dose MPP+ -induced α-synuclein expression and neurotoxicity were determined in human dopaminergic neuroblastoma SH-SY5Y cells. Similar to the findings in the substantia nigra of human PD, lower expression of Nramp1 but higher levels of iron and α-synuclein were identified in the dopaminergic neurones of mice receiving chronic MPTP intoxication, compared to controls. In parallel to the loss of dopaminergic neurones, the numbers of glial fibrillary acidic protein- and ionized calcium-binding adapter molecule-1-positive cells were significantly increased in the substantia nigra of MPTP-treated mice. Likewise, in human neuroblastoma SH-SY5Y cells exposed to low-dose MPP+ , Nramp1 expression and cathepsin D activity were decreased, along with an increase in α-synuclein protein expression and aggregation. Overexpression of functional Nramp1 restored cathepsin D activity and attenuated α-synuclein up-regulation and neuronal cell death caused by MPP+ treatment. These data suggest that the neuronal expression of Nramp1 is important for protecting against the development of MPTP/MPP+ -induced α-synuclein pathology and neurotoxicity.

TLR3 contributes to degeneration of dopamine neurons in an MPTP mouse model of Parkinson’s disease

Abstract Toll like receptor 3 (TLR3), a pattern recognition receptor, is highly expressed in the brain. However, its role in neurodegenerative diseases is unknown. The present study investigated the effects of TLR 3 on dopamine (DA) neurons in the 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) mouse model of Parkinson’s disease (PD). Here we show that TLR3-deficient mice are resistant to MPTP neurotoxicity. TH immunohistochemical analysis revealed that number of nigral TH+ neurons and striatal density of TH+ fibers were significantly maintained in MPTP-treated TLR3−/− mice, compared to MPTP-treated TLR3+/+ control mice. The observed neuroprotection was accompanied by reducing blood brain barrier (BBB) leakage and infiltration of immune cells such as CD-3+ T cells in the substantia nigra (SN). Moreover, genetic deletion of IKK-β (IKK-β−/−) in microglia, the downstream signaling of TLR3 and inhibitor of NF-κB kinase subunit beta (IκB) rescued dopamine neurons in the SN of MPTP-treated mice, compared to MPTP-treated IKK-β+/+ control mice. Similar to TLR3−/− mice, number of CD-3+ T cells were significantly attenuated in MPTP-treated IKK-β−/− mice, compared to MPTP-treated IKK-β+/+ control mice. Collectively, these results suggest that TLR 3 via IKK-β signaling pathway may play a crucial role in degeneration of dopamine neurons in the MPTP mouse model of PD.

Long-term Changes in the Nigrostriatal Pathway in the MPTP Mouse Model of Parkinson’s Disease

Parkinson’s disease (PD) is a common and progressive neurodegenerative disorder. The 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) mouse model of PD is widely used to study the progression of this disease. Behavior impairment is closely related to the damage of the dopaminergic system in the basal ganglia. Here, MPTP-induced changes in mouse behavior and glial activation were evaluated at different time points after the treatment and the long-term changes in the nigrostriatal pathway were analyzed. We found that mice exposed to MPTP displayed a full recovery in the rotarod test and the pole test but not in the wire hanging test at 65 days post-injection. A biphasic activation of microglial cells was revealed in the nigrostriatal pathway of MPTP-treated mice. However, activation of astrocytes displayed an approximately bell-shaped kinetics and an approximately S-shaped kinetics in the striatum and the substantia nigra, respectively. In addition, the numbers of complement component 3 (C3)-positive neurotoxic astrocytes in the substantia nigra of MPTP-treated mice increased with time and reached a maximum at 42 days, and declined at 74 days, after the treatment. Three months later, the dopaminergic system was partially recovered from the lesion of MPTP. The time course of pathophysiological events has important implications for the interventions or treatment of PD.

Proteomic change by Korean Red Ginseng in the substantia nigra of a Parkinson's disease mouse model

BackgroundRecent studies have shown that Korean Red Ginseng (KRG) successfully protects against dopaminergic neuronal death in the nigrostriatal pathway of a Parkinson's disease (PD) mouse model induced by 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) administration; however, the mechanism has yet to be identified. Therefore, in this study we used two-dimensional electrophoresis to investigate the effects of KRG on the changes in protein expression in the substantia nigra (SN) of MPTP-treated mice. MethodsMale C57BL/6 mice (9 wk old) were intraperitoneally administered MPTP (20 mg/kg) four times at 2-h intervals, after which KRG (100 mg/kg) was orally administered once a day for 5 d. Two hours after the fifth KRG administration, a pole test was conducted to evaluate motor function, after which the brains were immediately collected. Survival of dopaminergic neurons was measured by immunohistochemistry, and protein expression was measured by two-dimensional electrophoresis and Western blotting. ResultsKRG alleviated MPTP-induced behavioral dysfunction and neuronal toxicity in the SN. Additionally, the expression of eight proteins related to neuronal formation and energy metabolism for survival were shown to have changed significantly in response to MPTP treatment or KRG administration. KRG alleviated the downregulated protein expression following MPTP administration, indicating that it may enhance neuronal development and survival in the SN of MPTP-treated mice. ConclusionThese findings indicate that KRG may have therapeutic potential for the treatment of patients with PD.

Rho kinase II interference by small hairpin RNA ameliorates 1‑methyl‑4‑phenyl‑1,2,3,6‑tetrahydropyridine‑induced parkinsonism in mice.

Novel therapeutic targets are required for the treatment of Parkinson's disease (PD). Previous studies suggest that the Rho/Rho-associated, coiled-coil-containing protein kinases (ROCKs) signaling pathway may be a promising therapeutic target in PD. To elucidate the importance of ROCKII in the pathogenesis of dopaminergic (DA) neuron loss and to investigate the efficacy of ROCK inhibitors in PD, ROCKII expression in the substantia nigra (SN) of mice was silenced through the injection of a lentivirus-based small hairpin RNA system. Empty lentivirus vectors served as controls. Mice were subsequently challenged with 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP). The expression levels and activity of ROCKII were elevated in tyrosine hydroxylase-positive neurons and in cluster of differentiation (CD) 11b-positive microglia within the SN of MPTP-treated mice, which was accompanied by an increased level of expression of inducible nitric oxide synthase (iNOS) and activation of the Toll-like receptor (TLR)2/nuclear factor (NF)-κB signaling pathway in M1 microglia. ROCKII interference (RI) significantly improved movement disorder and attenuated DA neuron loss induced by MPTP. In addition, RI inhibited the activation of M1 microglia in the SN, exhibiting reduced activity of the TLR2/NF-κB signaling pathway and decreased expression levels of iNOS and inflammatory factors, including interleukin (IL)-1β and IL-6. The results of the present study verify that ROCKII participates in the loss of DA neurons induced by MPTP and suggest that ROCKII inhibition may be a promising therapeutic target for PD.

Cold water stress attenuates dopaminergic neurotoxicity induced by 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine in mice

In the present study, we tested the effect of cold water stress (CWS) on dopaminergic neurons in 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced Parkinson's disease (PD) mouse model, and found that CWS pretreatment elicited less MPTP neurotoxicity. To understand the molecular mechanism underlying this phenomenon, we detected the expression of heat shock protein 70 (Hsp70) in the striatum of the experimental mice, and found that CWS pretreatment could significantly increase striatal Hsp70 in MPTP-treated mice. Furthermore, in parallel with the induction of Hsp70, the MPTP-induced increase of striatal α-synuclein was inhibited in the CWS + MPTP-treated mice. CWS pretreatment also significantly inhibited the reduction of anti-apoptotic molecule Bcl-2 expression in the striatum and enhanced Bcl-2 transcription in the substantia nigra of MPTP-treated mice. Taken together, these data indicated that Hsp70 might be an important intermediate for the neuroprotective effect of CWS against MPTP-induced dopaminergic toxicity.

BCG Vaccine-Induced Neuroprotection in a Mouse Model of Parkinson's Disease

There is a growing interest in using vaccination with CNS antigens to induce autoreactive T cell responses that home to damaged areas in the CNS and ameliorate neurodegenerative disease. Neuroprotective vaccine studies have focused on administering oligodendrocyte antigens or Copaxone® in complete Freund's adjuvant (CFA). Theoretical considerations, however, suggest that vaccination with a neuronal antigen may induce more robust neuroprotective immune responses. We assessed the neuroprotective potential of vaccines containing tyrosine hydroxylase (a neuronal protein involved in dopamine synthesis) or Copaxone® in CFA in the 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) mouse model of Parkinson's disease. Surprisingly, we observed that the main beneficial factor in these vaccines was the CFA. Since the major immunogenic component in CFA is Mycobacterium tuberculosis, which closely related to the bacille Calmette-Guérin (BCG) that is used in human vaccines, we tested BCG vaccination in the MPTP mouse model. We observed that BCG vaccination partially preserved markers of striatal dopamine system integrity and prevented an increase in activated microglia in the substantia nigra of MPTP-treated mice. These results support a new neuroprotective vaccine paradigm in which general (nonself-reactive) immune stimulation in the periphery can limit potentially deleterious microglial responses to a neuronal insult and exert a neurorestorative effect in the CNS. Accordingly, BCG vaccination may provide a new strategy to augment current treatments for a wide range of neuropathological conditions.

MPTP-Induced Neuroinflammation Increases the Expression of Pro-Inflammatory Cytokines and Their Receptors in Mouse Brain

Parkinson’s disease (PD) is a common neurodegenerative disease characterised by a slow and progressive degeneration of dopaminergic neurons in the substantia nigra (SN). Despite intensive research, the cause of neuronal loss in PD is poorly understood. Inflammatory mechanisms have been implicated in the pathophysiology of PD. In this study, conducted on an experimental 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) mouse model, we investigated the expression of interleukin (IL)-1β, tumor necrosis factor (TNF)-α, IL-6 and their receptors (IL-1RI, TNF-αRI, IL-6Rα) at the SN and caudate-putamen (CP) levels. In MPTP-treated animals we observed a significant increase in IL-1β, TNF-α and IL-6 mRNA expression levels both in the SN and CP in comparison with untreated mice. In addition, both mRNA and protein levels of IL-1RI, TNF-αRI and IL-6Rα were significantly enhanced in the SN of MPTP-treated mice in comparison to controls, whereas no significant differences were observed in the CP between treated and untreated mice. Overall, these results indicate a role of both pro-inflammatory cytokines and their receptors in the pathogenesis of PD.

CXCR4 and CXCL12 Expression is Increased in the Nigro-Striatal System of Parkinson’s Disease

Except for a handful of inherited cases related to known gene defects, Parkinson's disease (PD) is a sporadic neurodegenerative disease of unknown etiology. There is increasing evidence that inflammation and proliferation of microglia may contribute to the neuronal damage seen in the nigro-striatal dopaminergic system of PD patients. Microglia events that participate in neuronal injury include the release of pro-inflammatory and neurotoxic factors. Characterizing these factors may help to prevent the exacerbation of PD symptoms or to remediate the disease progression. In rodents, the nigro-striatal system exhibits high expression of the chemokine receptor CXCR4. Its natural ligand CXCL12 can promote neuronal apoptosis. Therefore, the present study investigated the expression of CXCR4 and CXCL12 in post-mortem brains of PD and control (non-PD) individuals and in an animal model of PD. In the human substantia nigra (SN), CXCR4 immunoreactivity was high in dopaminergic neurons. Interestingly, the SN of PD subjects exhibited higher expression of CXCR4 expression and CXCL12 than control subjects despite the loss of dopamine (DA) neurons. This effect was accompanied by an increase in activated microglia. However, results from post-mortem brains may not provide indication as to whether CXCL12/CXCR4 can cause the degeneration of DA neurons. To examine the role of these chemokines, we determined the levels of CXCL12 and CXCR4 in the SN of MPTP-treated mice. MPTP produced a time-dependent up-regulation of CXCR4 that preceded the loss of DA neurons. These results suggest that CXCL12/CXCR4 may participate in the etiology of PD and indicate a new possible target molecule for PD.

Fibroblast growth factor 2 enhances striatal and nigral neurogenesis in the acute 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine model of Parkinson's disease

In response to injury, endogenous precursors in the adult brain can proliferate and generate new neurons, which may have the capacity to replace dysfunctional or dead cells. Although injury-induced neurogenesis has been demonstrated in animal models of stroke, Alzheimer's disease (AD) and Huntington's disease (HD), studies of Parkinson's disease (PD) have produced conflicting results. In this study, we investigated the ability of adult mice to generate new neurons in response to the parkinsonian toxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), which causes selective degeneration of nigrostriatal dopamine neurons. MPTP lesions increased the incorporation of 5-bromo-2′-deoxyuridine-5′-monophosphate (BrdU), as well as the number of cells that co-expressed BrdU and the immature neuronal marker doublecortin (DCX), in two neuroproliferative regions—the subgranular zone of the dentate gyrus (DG) and the rostral subventricular zone (SVZ). BrdU-labeled, DCX-expressing cells were not found in the substantia nigra (SN) of MPTP-treated mice, where neuronal cell bodies are destroyed, but were present in increased numbers in the striatum, where SN neurons lost in PD normally project. Fibroblast growth factor-2 (FGF-2), which enhances neurogenesis in a mouse model of HD, also increased the number of BrdU/DCX-immunopositive cells in the SN of MPTP-treated mice. Thus, MPTP-induced brain injury increases striatal neurogenesis and, in combination with FGF-2 treatment, also stimulates neurogenesis in SN.

Fluoro-Jade C can specifically stain the degenerative neurons in the substantia nigra of the 1-methy-4-phenyl-1,2,3,6-tetrahydro pyrindine-treated C57BL/6 mice

Fluoro-Jade C, a new-developed fluorescent dye, has been successfully applied for identification of neuronal degeneration in the substantia nigra of 1-methy-4-phenyl-1,2,3,6-tetrahydro pyrindine (MPTP)-treated mice in the present study. The animal model was first prepared by intraperitoneal injection of neurotoxicant MPTP that can specifically induce degeneration of dopamine neurons in the substantia nigra of C57BL/6 mice. Fluoro-Jade C was then utilized to stain the midbrain sections and semiquantitation analysis was carried out in comparison with controls. It revealed that Fluoro-Jade C-positive cells showed strong green color in neuronal profile and were observed in the substantia nigra of MPTP-treated mice whereas they were not detected in that of controls. The Fluoro-Jade C-positive cells were mostly shrunken or smaller-sized in their cell bodies in comparing with that of normal dopamine neurons of controls. In the midbrain of MPTP-treated mice, Fluoro-Jade C-positive neuronal cells were exclusively distributed in the substantia nigra pars compacta, but rarely seen in the ventral tegemental area where dopamine neurons were numerously distributed. Double-labeling experiments indicated that a population of Fluoro-Jade C-positive cells (23%) exhibited neuron-specific nuclear protein-immunoreactivity and none of them showed immunoreactivity to glial cell marker glial fibrillary acid protein. However, most of Fluoro-Jade C-positive degenerative neurons (98%) lost their immunoreactivity to dopaminergic marker tyrosine hydroxylase in the substantia nigra of MPTP-treated mice. Taken together with previous observations, this study has presented that Fluoro-Jade C can be sensitively and specifically utilized to identify the neuronal degeneration in the substantia nigra of rodent animals receiving MPTP insult.

Iron and α-Synuclein in the Substantia Nigra of MPTP-Treated Mice: Effect of Neuroprotective Drugs R-Apomorphine and Green Tea Polyphenol (-)-Epigallocatechin-3-Gallate

One of the prominent pathological features of Parkinson's disease (PD) is the abnormal accumulation of iron in the substantia nigra pars compacta (SNpc), in the reactive microglia, and in association with neuromelanin, within the melanin-containing dopamine (DA) neurons. Lewy body, the morphological hallmark of PD, is composed of lipids, redox-active iron, and aggregated alpha-synuclein, concentrating in its peripheral halo and ubiquitinated, hyperphosphorylated, neurofilament proteins. The capacity of free iron to enhance and promote the generation of toxic reactive oxygen radicals has been discussed numerous times. Recent observations, that iron induces aggregation of inert alpha-synuclein to toxic aggregates, have reinforced the critical role of iron in oxidative stress-induced pathogenesis of DA neuron degeneration and protein degradation via ubiquitination. N-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)- and 6-hydroxydopamine-induced neurodegeneration in rodents and nonhuman primates is associated with increased presence of iron and alpha-synuclein in the SNpc. The accumulation of iron in MPTP-induced neurodegeneration has been linked to nitric oxide-dependent mechanism, resulting in degradation of prominent iron regulatory proteins by ubiquitination. Radical scavengers such as R-apomorphine and green tea catechin polyphenol (-)-epigallocatechin-3-gallate, as well as the recently developed brain-permeable VK-28 series derivative iron chelators, which are neuroprotective against these neurotoxins in mice and rats, prevent the accumulation of iron and alpha-synuclein in SNpc. This study supports the notion that a combination of iron chelation and antioxidant therapy, as emphasized on several occasions, might be a significant approach to neuroprotection in PD and other neurodegenerative diseases.