The Role Of Locus Coeruleus- Norepinephrine In The Progression Of Parkinson’s Disease

ObjectiveParkinson’s disease (PD) is the second most common neurodegenerative disorder, characterized by progressive movement disorders, including resting tremor, rigidity, bradykinesia, and gait disturbance. It affected approximately seven million people globally. In China, the Prevalence rate of PD is about 2.05% of people over 65 years old. The costs of PD to society are high as it was shown that the cost per patient per year in the U.S. is probably around$10,000. In addition to economic costs, PD reduces the life quality of the patients and their caregivers. The exact etiology of PD remains unclear. Parkinson’s disease in most people is idiopathic. The known causes of PD include genetics, environmental causes, or a combination of the two. The epidemiological studies indicated that only less than 10% of PD is caused by genetic mutations, while more than 90% of PD patients are sporadic. Currently, there is no cure available for PD. The main families of drugs useful for treating motor symptoms are levodopa, dopamine agonists and MAO-B inhibitors, which could only relieve the symptoms but cannot halt or reverse the disease progression.The pathological change of PD includes loss of dopaminergic neurons, depletion of striatal dopamine and lewy body formation, among which the most important character of PD is the progressive nature. In the normal people, a small amount of DA neuron loss is observed in the brain in aging process. Most of the PD patients were diagnosed when obvious symptoms appear as more than 60% of DA neurons were lost. Although the nigrostriatal pathway remains a major focus, emerging information demonstrates the participation of both the mesocortical and cortico-limbic pathways in this disease process. In addition to being a disorder of the dopamine projection system, glutamatergic, cholinergic, tryptaminergic, GABAergic, noradrenergic, and adrenergic damage has also been reported in PD. In 2003, a Germany pathologist Dr. Braak addressed that PD is not only a movement disorder, a series of non-motor symptoms happened from the onset of the disease to the end at various stages based on staining of α-synuclein in post-mortem PD brains.Recent studies reveal that loss of norepinephrine (NE) neurons in the locus coeruleus (LC) occurs prior to loss of dopaminergic (DA) neuronal in the substantia nigra (SN) and these sequential neuronal losses are associated with pre-motor and motor symptoms, respectively in PD patients. However, progress of research on PD premotor symptoms has been hampered by the lack of an animal model. The previous studies in our lab have established a lipopolysaccharide (LPS)-mediated rodent PD model showing the progressive dopaminergic neuronal loss of SN as well as the motor deficits. However, as we have all known that PD is not only a motor-change disease and the neuronal loss of PD patients is not limited to the SNpc brain region, it’s critical to ascertain the pathological change of other brains regions and also the non-motor symptoms in the LPS-mediated PD model. Thus, in the present study, instead of focusing on substantia nigra. we had tried to examine the neuronal loss in the lower brainstem, especially locus coeruleus (LC), which is well-documented for its earlier neuronal degeneration in PD patients.The research goals of this study are to ascertain the sequential neuronal loss and motor/non-motor symptoms in the inflammation-mediated rodent PD model and to elucidate the role of Locus Coeruleus-Norepinephrine (LC-NE) in the progression of PD as well as the underlying cellular/molecular mechanisms. We hypothesized that the earlier adrenergic neuronal loss in LC facilitated the progression of Parkinson’s disease. The aims of our research includes:1) To ascertain the sequential neuronal loss in the inflammation-mediated rodent PD model, especially LC.2) To determine whether earlier loss of NE secreted by LC is associated with the subsequent sequential neurodegeneration in adrenergic innervated brain regions.3) To create a novel PD model showing ascending, sequential neurodegeneration in different brain regions and progressive motor and non-motor symptoms.4) To elucidate the mechanism how LC-NE regulates inflammation-mediated neurodegeneration.5) To develop novel glia-based, disease-modifying therapies for PD.Methods1. Animal treatment:Three-month old male C57BL/6 mice were injected with a single systemic LPS (5 mg/kg, i.p), DSP-4 (50 mg/kg. i.p) or combined injection in different regimens based on the purpose of the experiment design. At desired time points (1,2,4,7,10, and 12-month after injection, respectively),5-8 mice from each group were euthanized, and brains were removed and post-fixed in 4% paraformaldehyde overnight at 4℃. Brains were then placed into 30% sucrose/PBS solution at 4℃ until the brains sank to the bottom of the container. Coronal sections of brainstem containing LC, midbrain containing SN, hippocampus, and cortex were cut on a horizontal sliding microtome into 35 μm transverse free-floating sections.2. Immuno-staining:The free-floating brain sections were immune-blocked with 4-10% goat serum and then incubated with polyclonal rabbit anti-tyrosine hydroxylase (TH) antibody (1:2,000-5,000 dilution), or neuronal nuclear (NeuN) antibody (1:1,000 dilution) for 48h at 4℃, respectively. Antibody binding was visualized using a Vectastain ABC Kit and diaminobenzidine substrate or with confocal double-label immunofluorescence staining.3. Neuronal counting:The number of TH-immunoreactive (THir) neurons in the SNpc was estimated by stereology counting using an optical fractionator method that systematically randomizes unbiased counting frames (100μm×100μm) within defined boundaries of the SN. The number of NeuN positive neurons in the hippocampus granule layer, cortex and caudate putamen in DAB staining and the neuron or microglial numbers in the double-label immunofluorescence staining were measured by automated counting of single color images using ImageJ software.4. Monoamine detection:The levels of norepinephrine, dopamine, serotonin and their major metabolites in cortex, hippocampus, caudate putamen, midbrain and brainstem were analyzed using high-performance liquid chromatography (HPLC).5. The mRNA analysis of frozen tissue was performed by Real-time PCR. Briefly, total RNA was extracted from brains with RNeasy Mini kit (Qiagen) and reverse transcribed with an oligodT primer. Real-time PCR amplification was performed using SYBR Green PCR Master Mix (Applied Biosystems) and Applied Biosystems 7900HT Fast Real-Time PCR System according to manufacturer’s protocols.6. Behavior tests:At the time point of 12 months after DSP-4 or/and LPS injection,8 mice from each group (saline control, DSP-4 alone, DSP-4+LPS, and LPS alone) were randomly selected from each group for the motor and non-motor behavior test. The motor behavior testes included wire hang test, accelerating rotarod, open field test, as well as gait disturbance analysis. And the non-motor behavior tests covered marble-burying assay for olfactory function, anxiety test by elevated plus maze, one hour stool collection for constipation test, acoustic startle and prepulse inhibition response as well as the acquisition and reversal learning in the Morris water maze.7. Primary cultures preparation and treatment:Primary neuron-glia and mix-glia cultures were prepared for the underlying cellular and molecular mechanism studies for neuroprotective or anti-inflammatory effect of NE. The preparation of the primary cultures was as described previously. In brief, dissociated cells from the ventral mesencephalon of embryonic day 14±0.5 Fischer 334 rats, C57BL/6 or transgenic mice were seeded at 5.5 x 105 cells/well (rat) or 6.5×105 cells/well (mice) in poly-D-lysine-coated 24-well plates, respectively. The cultures were maintained at 37℃ in the incubator with 5% CO2 and 95% air in minimum essential medium. The cultures were ready for experiments at desired time points.8. Cell lines:the transfected monkey kidney COS7-PHOX and COS7 cell lines are used in the study. And the rat microgliosis cell line HAPI was also employed in this study.9. Dopaminergic neuron function test:[3H]dopamine (DA) uptake assays were performed for the functional evaluation of the dopaminergic neurons in the primary neuron-glia culture. Briefly, cells were incubated for 21 minutes at 37℃ with 1μM [3H]DA in Krebs-Ringer buffer. Cells were washed with ice-cold Krebs-Ringer buffer three times, and then were collected in 1 N NaOH. Radioactivity was determined by liquid scintillation counting. Nonspecific DA uptake observed in the presence of mazindol (10μM) was subtracted.10. Inflammatory factors detection:The production of nitric oxide (NO) was determined by measuring accumulated levels of nitrite in the supernatant with Griess reagent, and the release of tumor necrosis factor-a (TNF-a) was measured with a TNF-a ELISA kit. The production of superoxide was assessed by measuring the SOD-inhibitable reduction of the tetrazolium salt WST-1. To determine whether NE acts as a superoxide scavenger, the superoxide-generating xanthine/xanthine oxidase system was used.11. Membrane fractionation and western blot analysis:HAPI microglia were lysed in hypotonic lysis buffer and then subjected to Dounce homogenization (20-25 stokes, tight pestle A). The lysates were centrifuged at 1,600 x g for 15 min; the supernatant was centrifuged at 100,000×g for 30 minutes. The pellets solubilized in 1% Nonidet P-40 hypotonic lysis buffer were used as membranous fraction and ready for western blot analysis.12. Blood analysis:Twenty-one days after the first injection of clozapine or clozapine-N-oxide, the mice were euthanized and eyeball blood was collected in a 1.5 ml heparin tube. After a fully vortex, the number of different types of cells in the blood was counted by an Automatic Blood cell Counter.13. Statistical analysis:All group data are expressed as mean±SEM. Group means were compared using one-or two-way ANOVA with treatment as the independent variable. When ANOVA showed a significant difference, pair wise comparisons between group means were examined by Dunnett post hoc test. Statistical analysis was performed using GraphPad Prism version 6.00 for Windows with two sided a of 0.05.Results1. Sequential, ascending, progressive neuron loss in different brain regions of systemic LPS-injected mice1.1 Adrenergic neurodegeneration of LC was 3 months earlier than dopaminergic neurons of SN in LPS-induced PD mouse models.After a single systemic LPS injection in C57BL/6 mice, neuron loss was initially detected of adrenergic neurons in LC after 4 months of LPS injection as shown by 30% decrease of THir cells. NE neuron loss was continually observed up to 52% loss after 10 months of injection, indicating chronic progressive degeneration. The dopaminergic neuron loss of SNpc was detected in midbrain after 7 months of treatment with about 32% decrease. This result was further demonstrated by human A53T transgenic mice showing that the adrenergic neuronal loss started at 1 month after LPS injection whereas the dopaminergic neurons started significant loss at 3 months later.1.2 Sequential, ascending, progressive loss of neurons in different brain regions of systemic LPS-injected C57BL/6 miceAfter 10 months of LPS treatment, neurodegeneration was continually extended to cortex and hippocampus as shown by decrease of NeuN positive cells in these areas. Altogether, we demonstrated that the neurodegeneration in this inflammation-mediated mouse model exhibited a progressive and caudo-rostral pattern that ascends from lower brainstem to frontal cortex.1.3 LC-NE depletion enhanced the loss of nigral dopaminergic neurons in LPS-injected mice.C57BL/6 mice received another 4 injections of DSP-4 (50 mg/kg; i.p.) at biweekly intervals after 6-month of a single systemic LPS injection. Depletion of NE by DSP-4 enhanced the LPS-induced loss of nigral dopaminergic neurons up to 50% compared to the 30% of LPS alone group.2. Locus coeruleus-norepinephrine deficiency potentiated neurodegeneration of adrenergic neuron-innervated brain regions2.1 NE depletion elicited progressive neurodegeneration that ascends caudo-rostrally in brain.After the LC-NE was depleted by DSP-4, a delayed and progressive loss of THir neurons in SNpc was first detected. The significant loss of THir neurons started to manifest between 4 (32.4% loss) and 10 (49.9% loss) months after DSP-4 injection. Further, neurodegeneration induced by DSP-4 extended to cortex and hippocampus. Analysis of Neu-N-immunoreactive neuron revealed 27.7% loss in cortex and 30.0% loss in hippocampus granule layer (DG), respectively, after 10 months of DSP-4 injection (p< 0.01 compared with vehicle controls). In contrast, neurons in the Caudate Putamen (Cpu) (without LC-NE projection) were completely spared from DSP-4-induced degeneration up to 10 months of treatment.2.2 NE depletion by DSP-4 caused sustained microglial activation in the noradrenergic neuron-innervated brain regions.To determine whether the depletion of NE could elicit microglial activation, we stained microglia using two microglial markers (alpha chain of β-2 intergrin receptor, CD11b and ionized calcium binding adaptor molecule 1, Iba-1) in SN, hippocampus and cortex of DSP-4-treated mice. In all these noradrenergic-innervated brain regions, microglial activation characterized by hypertrophied morphology and intensified CDllb and Ibal staining was observed as early as 7 days after DSP-4 treatment and sustained up to 10 months. Compared with time-matched vehicle controls (set as 100%), DSP-4 treatment resulted in 153.5% to 193.81% ncrease of CD11b density in SN, hippocampus (DG) and cortex after 7 days of treatment, which was elevated to ～250% increase 10 months after injection. Iba-1 density showed a similar pattern. In contrast, microglia in the Cpu and LC remained unchanged in DSP-4-treatedd mice as shown by similar morphology and density of CD11b and/or Iba-1 staining compared with time-matched vehicle controls.2.3 DBH knockout induced neurodegeneration and microglial activation in LC noradrenergic neuron-innervated brain regions.To verify the role of NE deficiency in neurodegeneration and neuroinflammation, transgenic mice lacking DBH, one enzyme responsible for NE synthesis, was employed. Consistent with the alterations of NE depletion induced neurodegeneration by DSP-4, neurodegeneration in LC noradrenergic neuron-innervated brain regions including SN, VTA, hippocampus and cortex was observed in DBH-/- mice compared to DBH-/- mice. In addition to eliciting neurodegeneration, DBH-/- mice also exhibited microglial activation in brain in the consistent pattern with the microglial activation found in DSP-4 injected mice.3. The motor and non-motor behavior changes in the (DSP-4+LPS) two-hit mouse model of PD3.1 Prior treatment of DSP-4 to LPS enhanced neurodegeneration of adrenergic neuron-projected brain regions.NE depletion by DSP-4 increased neuronal loss in SN, hippocampus and cortex after 12 months DSP-4/LPS injection in some degree but not significant.3.2 NE depletion by DSP-4 potentiated the appearance of pre-motor symptoms of Parkinson’s disease in LPS-treated mice.Compared to the saline control or LPS alone group, prior NE depletion by DSP-4 potentiated the olfactory discrimination deficits, appearance of anxiety-like and depressive phenotypes, and gastrointestinal dysfunction shown as constipation in inflammation-mediated PD model.3.3 NE depletion by DSP-4 potentiated the appearance of motor symptoms of PD in LPS-treated mice.Compared to saline control group, mice exhibited significant shorter wire-hang latency in DSP-4 or/and LPS injected mice. Impaired performance on the rotarod test was significantly preceded and potentiated by DSP-4 compared to LPS alone group. The locomotor activities in mice with DSP-4 and/or LPS injection decreased compared to saline control, as shown by the decrease of ambulatory activity and distance traveled. In the unforced moving gait analysis system, mice in DSP-4+ LPS group clearly showed severe gait disturbance, characterized by shorten stride length and increased paw angle to keep balance (increased hindpaw width without change of forepaw width) compared to age-matched saline, DSP-4-only, and LPS-only mice. Besides, the moving track of the mice showed shorter mean stride lengths variability, duck flippers feet, dragging steps, and twisted walking track in our two-hit mice after 12 months treatment.3.4 NE depletion by DSP-4 potentiated the startle response in LPS-treated mice.Mice exposed to both DSP-4 and LPS demonstrated increased prepulse inhibition and exaggerated startle responses at 12 months after LPS/DSP-4 injection, in comparison to the age-matched vehicle-treated mice (p<0.05). No significant group differences were found between mice exposed only to LPS compared to vehicle controls. Overall, these results suggest that the combined DSP-4/LPS challenge led to hyper-reactivity to acoustic stimuli.3.5 NE depletion by DSP-4 potentiated the appearance of cognition deficit in LPS-treated mice.Treatment with DSP-4, LPS or DSP-4+LPS led to impaired spatial learning in the Morris water maze (MWM) test. However, mice treated with DSP-4 or LPS or the combination of DSP-4 and LPS did not demonstrate selective quadrant search. Further, post-hoc tests indicated that all these treatment groups spent less time in the target quadrant than the mice given vehicle (p<0.01).4. The mechanisms underlying NE deficiency-related neuroinflammation and neurodegeneration4.1 Sub-micromolar concentrations of NE protected dopaminergic neurons against LPS-induced neurotoxicity in midbrain neuron-glia cultures.The [3H]DA uptake assay showed that NE pretreatment restored the culture DA uptake capacity in a dose-dependent manner (10-6-10-9 μM), indicating that NE significantly attenuated LPS-induced dopaminergic neurotoxicity. This conclusion was further corroborated with the results from cell count, which revealed that pretreatment of NE dose-dependently mitigated the LPS-induced decrease in the number of TH-ir neurons. In addition to restoring the numbers of dopaminergic neurons, NE also rescued the dopaminergic neurite retraction and degradation associated with inflammation-mediated dopaminergic neurotoxicity. Taken together, these two measurements clearly demonstrated the neuroprotective effects of NE on LPS-induced DA neurotoxicity.4.2 Microglia were essential for sub-micromolar NE-mediated dopaminergic neuroprotection.Treatment with NE (10-7 M) reversed the MPP+-induced [3H]DA uptake decrease by 20% in neuron-glia culture, but showed no protective effect in microglia-depleted neuron-glia cultures. This finding suggests that the partial NE-mediated dopaminergic neuroprotection required the presence of microglia, most likely through the inhibition of reactive microgliosis.4.3 Sub-micromolar NE attenuated LPS-induced microglial activation and pro-inflammatory factor release.Immunocytochemistry of Iba-1 showed that 24 hours after NE exposure, microglia reverted from hypertrophied cells (i.e., fewer projections, enlarged soma) in response to LPS activation to a ramified morphology (i.e., numerous projections, small soma). Cellular quantification showed that NE pretreatment significantly suppressed the up-regulated expression of Iba-1 and thus reduced the number of Iba-1-positive cell after LPS stimulation. In addition to morphological observations, the inhibitory effects of NE on the release of pro-inflammatory factors from microglia were measured. Pre-treatment of NE markedly reduced the production of TNF-α and inhibited the release of both nitric oxide (measured as nitrite) and superoxide in a dose-dependent manner.4.4 Beta2-AR was not the sole mechanism mediating the anti-inflammatory and neuroprotective effects of sub-micromolar NE.Compared to wild type, the NE-elicited inhibitory effect of LPS-induced TNF-α release was only partially reversed in the β2-AR-/- culture (40% in WT vs.20% in β2-AR-/-), suggesting that another signaling pathway may mediate the NE-elicited reduction in LPS-induced TNF-α. Consistently, measurement of [3H]DA uptake capacity in midbrain neuron-glia cultures also confirmed that the neuroprotective effect of NE was only partially affected by β2-AR deletion. LPS caused more than a 50% reduction in DA uptake capacity in both WT and β2-AR-/- cultures, which was reversed by NE to 80% in WT, but only 65% in β2-AR-/- cultures.4.5 Sub-micromolar NE inhibited NOX2-generated superoxide in an adrenergic receptor-independent manner.In LPS-treated mixed-glia cultures, (+)-NE and (-)-NE were equi-potent in inhibiting LPS-induced superoxide production. This adrenergic receptor-independent inhibitory action of NE on superoxide production was further demonstrated in COS7 cells, since both NE isomers displayed similar potency in inhibiting phorbol myristate acetate (PMA)-induced superoxide in COS7 cells transfected with all the essential subunits of NOX2. Moreover, NE exerted a similar potency of superoxide inhibitory effect in mixed-glia cultures prepared from both wild type control and β2-AR-/- mice. Pretreatment with non-selective al (phentolamine) or β1 (propranolol)-AR antagonists either alone or in combination failed to influence the NE elicited inhibition of superoxide production in LPS-stimulated mix-glia cultures, suggesting other types of adrenergic receptors besides the β2-AR were not involved in mediating NE-elicited superoxide production.4.6 NE prevented LPS-induced membrane translocation of the cytosolic subunit p47phox.Western analysis using HAPI microglia cells showed that LPS significantly reduced the immunoreactivity of p47phox in the cytosol fraction, but increased the amount in the membrane, indicating an increase in the translocation of cytosolic subunits of NOX2. This increase in membrane translocation was prevented by the pretreatment with NE.4.7 NOX2 mediated β2-AR-independent anti-inflammatory/neuroprotective effects of sub-micromolar NE.To determine whether NOX2 is the putative β2-AR-independent target for NE-elicited anti-inflammatory/neuroprotective effect, NOX2 inhibitor diphenyliodonium (DPI) was added to neuron-glia cultures prepared from β2-AR-/- mice. The inhibitory effects of DPI on LPS-induced TNF-a production in β2-AR-/- culture were comparable to those of NE and no further reduction was observed when DPI and NE were combined. And the [3H]DA uptake measurement confirmed that the neuroprotective effect of DPI on LPS-induced neurotoxicity were comparable to those of NE and no additive protection was observed when DPI and NE were combined.The TNF-a production results showed that blockade of the β2-AR by the antagonist ICI-118,551 partially reversed the inhibitory effect of NE in C57BL/6 neuron-glia culture, whereas a total reversal of the inhibitory effect of NE occurred in CYBB neuron-glia culture. Similar results were obtained in [3H]DA uptake studies, i.e., the β2-AR antagonist partially decreased the neuroprotective effect of NE in the C57BL/6 neuron-glia culture. In contrast, in the presence of the β2-AR antagonist, the neuroprotective effect of NE in CYBB neuron-glia culture was totally prevented. Altogether, these data suggest that β2-AR-independent anti-inflammatory effects of NE were indeed mediated through microglial NOX2.4.8 NADPH oxidase inhibitor DPI post-treatment attenuated microglial activation in NE-deficient mice.DSP-4-injected mice were infused with DPI of 10 ng/kg/day at 4 days after DSP-4 injection. DPI treatment markedly attenuated DSP-4-induced microglial activation in LC noradrenergic neuron-innervated brain regions as shown by a ramified morphology and a reduced expression of CD11b versus the DSP-4 alone group. Analysis of CDllb density showed significant decrease in SN, hippocampus and cortex of DSP-4/DPI-treated mice compared with DSP-4 alone group.5. Clozapine metabolites protect dopaminergic neurons through inhibition of microglial NADPH oxidase5.1 Clozapine metabolites (CNO and NDC) protected DA neurons from LPS-induced neurotoxicity.Pretreatment with CNO or NDC protected DA neurons against LPS-induced reduction of uptake capacity in a bell-shaped curve. CNO and NDC were most effective at the concentration of 0.01 μM and 0.1 μM respectively, in restoring DA uptake capacity (from 47.4% of LPS alone back to 79.9% and 77.1% by CNO and NDC, respectively) and ameliorating loss of THir neurons (from 57.5% of LPS alone back to 85.2% and 84.2% by CNO and NDC, respectively).5.2 Microglia were essential in CNO-and NDC-elicited neuroprotection.CNO or NDC was shown to afford their neuroprotective effects in neuron-glia culture, but not in microglia-depleted neuron-astrocyte culture from MPP+-mediated neurotoxicity. Results showed that LPS stimulation resulted in increased cell number and size of Iba-1 positive microglia and CNO or NDC pretreatment mitigated these changes. Western blot analysis of Ibal and CD11b showed that pre-treatment of CNO or NDC effectively inhibited the increased expression of Iba-1 and CD11b induced by LPS treatment. Pre-treatment with CNO or NDC reduced the release of extracellular superoxide, TNF-a and NO in LPS-challenged neuron-glia cultures.5.3 Microglial NOX2 was essential in mediating the anti-inflammatory and neuroprotective effects of CNO and NDC.Pretreatment with CNO or NDC significantly attenuated LPS-induced TNF-a release and decreased DA uptake capacity in gp91phox+/+ cultures. In contrast, in gp91phox-/- culture, CNO and NDC failed to show any effect on LPS-induced production of TNF-a and reduction of [3H] DA uptake capacity. Western blot analysis showed that the levels of p47phox was decreased in the cytosolic fraction but increased in membrane fractions in LPS-stimulated HAPI microglia cells, indicating membrane translocation of p47phox. Quantitative analysis supported our observation by restoring p47phox levels in both cytosolic (from 70.62% of LPS alone back to 95.65% and 89.01% by CNO and NDC, respectively) and membrane fractions (from 154.2% of LPS alone back to 122.18% and 118.83% by CNO and NDC, respectively). These results suggest that CNO and NDC inhibited NOX2 activation in microglia by interfering with the membrane translocation of NOX2 cytosolic subunits.5.4 CNO attenuated MPTP-induced dopaminergic neuron damage and motor deficits without showing granulocyte toxicity.MPTP injection reduced the rotarod activity in mice by about 43% of latency time on the accelerating rod. Both CNO and Clozapine treatment significantly ameliorated MPTP-induced deficits of rotarod activity with equal-potency. In addition to ameliorating motor deficits, CNO and clozapine treatment displayed potent efficacy in attenuating MPTP-induced dopaminergic neurodegeneration. MPTP treatment caused 50% loss of THir neurons in the SNpc, which was reduced to 28% in mice pretreated with CNO and clozapine. To detect the neutropenia of both clozapine and CNO, the number of WBC in whole blood samples prepared from mice treated with CNO and clozapine. Unlike parent compound clozapine, CNO had no significant toxicity on neutrophils even combined with MPTP up to consecutive 21 days treatment.5.5 CNO attenuated MPTP-induced reactive microgliosis.In MPTP-treated mice, activated microglia characterized by a hypertrophied morphology and intensified CDllb staining were observed throughout the nigral reticulate area. Analysis of CD11b density supported these morphological observations. Compared with the MPTP alone group, both clozapine and CNO treatment markedly attenuated microglial activation, as shown by a reduced density of CD11b staining.Conclusion1. Adrenergic neuron loss of LC is earlier than that of dopaminergic neuron of SN in the inflammation-mediated mice PD model.2. LC-NE deficiency initiated the chronic progressive neurodegeneration of adrenergic neuron-innervated brain regions by a caudo-rostrally ascending pattern across the brain.3. Prior NE depletion by DSP-4 potentiated the onset of motor and non-motor symptoms in the inflammation-mediated PD model.4. Extra-synaptic NE played neuroprotective effects by regulating immune-homeostasis via microglia.5. Extra-synaptic NE played anti-inflammatory effect through a novel mechanism targeting on microglial NOX2.6. By inhibiting microglial activation through NOX2, clozapine metabolites CNO and NDC protect dopaminergic neurons from inflammation-mediated dopaminergic neurodegeneration.