Investigating Therapeutic Effects of Indole Derivatives Targeting Inflammation and Oxidative Stress in Neurotoxin-Induced Cell and Mouse Models of Parkinson's Disease.

Neuroinflammation and oxidative stress have been emerging as important pathways contributing to Parkinson's disease (PD) pathogenesis. In PD brains, the activated microglia release inflammatory factors such as interleukin (IL)-ß, IL-6, tumor necrosis factor (TNF)-α, and nitric oxide (NO), which increase oxidative stress and mediate neurodegeneration. Using 1-methyl-4-phenylpyridinium (MPP+)-activated human microglial HMC3 cells and the sub-chronic 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced mouse model of PD, we found the potential of indole derivative NC009-1 against neuroinflammation, oxidative stress, and neurodegeneration for PD. In vitro, NC009-1 alleviated MPP+-induced cytotoxicity, reduced NO, IL-1ß, IL-6, and TNF-α production, and suppressed NLR family pyrin domain containing 3 (NLRP3) inflammasome activation in MPP+-activated HMC3 cells. In vivo, NC009-1 ameliorated motor deficits and non-motor depression, increased dopamine and dopamine transporter levels in the striatum, and reduced oxidative stress as well as microglia and astrocyte reactivity in the ventral midbrain of MPTP-treated mice. These protective effects were achieved by down-regulating NLRP3, CASP1, iNOS, IL-1ß, IL-6, and TNF-α, and up-regulating SOD2, NRF2, and NQO1. These results strengthen the involvement of neuroinflammation and oxidative stress in PD pathogenic mechanism, and indicate NC009-1 as a potential drug candidate for PD treatment.

SUN11602, a bFGF mimetic, modulated neuroinflammation, apoptosis and calcium-binding proteins in an in vivo model of MPTP-induced nigrostriatal degeneration.

BACKGROUND: Parkinson's disease (PD) is the second most frequent neurodegenerative disease. PD etiopathogenesis is multifactorial and not yet fully known, however, the scientific world advised the establishment of neuroinflammation among the possible risk factors. In this field, basic fibroblast growth factor/fibroblast growth factor receptor-1 (bFGF/FGFR1) could be a promising way to treat CNS-mediated inflammation; unfortunately, the use of bFGF as therapeutic agent is limited by its side effects. The novel synthetic compound SUN11602 exhibited neuroprotective activities like bFGF. With this perspective, this study aimed to evaluate the effect of SUN11602 administration in a murine model of MPTP-induced dopaminergic degeneration. METHODS: Specifically, nigrostriatal degeneration was induced by intraperitoneal injection of MPTP (80 mg/kg). SUN11602 (1 mg/kg, 2.5 mg/kg, and 5 mg/kg) was administered daily by oral gavage starting from 24 h after the first administration of MPTP. Mice were killed 7 days after MPTP induction. RESULTS: The results obtained showed that SUN11602 administration significantly reduced the alteration of PD hallmarks, attenuating the neuroinflammatory state via modulation of glial activation, NF-κB pathway, and cytokine overexpression. Furthermore, we demonstrated that SUN11602 treatment rebalanced Ca2+ overload in neurons by regulating Ca2+-binding proteins while inhibiting the apoptotic cascade. CONCLUSION: Therefore, in the light of these findings, SUN11602 could be considered a valuable pharmacological strategy for PD.

RANTES-induced invasion of Th17 cells into substantia nigra potentiates dopaminergic cell loss in MPTP mouse model of Parkinson's disease.

Although Parkinson's disease (PD) is a progressive neurodegenerative disease, the disease does not progress or persist in 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) mouse model, the most common animal model of PD. Recently, we have described that supplementation of regulated on activation, normal T cell expressed and secreted (RANTES), a chemokine known to drive infiltration of T cells, induces persistent nigrostriatal pathology in MPTP mouse model. However, which particular T cell subsets are recruited to the substantia nigra (SN) by RANTES is not known. Here, by adoptive transfer of different subset of T cells from tomato red transgenic mice to MPTP-intoxicated immunodeficient Rag1-/- mice, we describe that invasion of Th17 cells into the SN is stimulated by exogenous RANTES administration. On the other hand, RANTES supplementation remained unable to influence the infiltration of Th1 and Tregs into the SN of MPTP-insulted Rag1-/- mice. Accordingly, RANTES supplementation increased MPTP-induced TH cell loss in Rag1-/-mice receiving Th17, but neither Th1 nor Tregs. RANTES-mediated aggravation of nigral TH neurons also paralleled with significant DA loss in striatum and locomotor deficits in MPTP-intoxicated Rag1-/- mice receiving Th17 cells. Finally, we demonstrate that levels of IL-17 (a Th17-specific cytokine) and RANTES are higher in serum of PD patients than age-matched controls and that RANTES positively correlated with IL-17 in serum of PD patients. Together, these results highlight the importance of RANTES-Th17 pathway in progressive dopaminergic neuronal loss and associated PD pathology.

Synthesis and evaluation of biaryl derivatives for structural characterization of selective monoamine oxidase B inhibitors toward Parkinson's disease therapy.

Benzyloxyphenyl moiety is a common structure of highly potent, selective and reversible inhibitors of monoamine oxidase B (MAO-B), safinamide and sembragiline. We synthesized 4-(benzyloxy)phenyl and biphenyl-4-yl derivatives including halogen substituents on the terminal aryl unit. In addition, we modified the carbon linker between amine group and the biaryl linked unit. Among synthesized compounds, 12c exhibited the most potent and selective MAO-B inhibitory effect (hMAO-B IC50: 8.9 nM; >10,000-fold selectivity over MAO-A) as a competitive inhibitor. In addition, 12c showed greater MAO-B inhibitory activity and selectivity compared to well-known MAO-B inhibitors such as selegiline, safinamide and sembragiline. In the MPTP-induced mouse model of Parkinson's disease (PD), 12c significantly protected the tyrosine hydroxylase (TH)-immunopositive DAergic neurons and attenuated the PD-associated behavioral deficits. This study suggests characteristic structures as a MAO-B inhibitor that may provide a good insight for the development of therapeutic agents for PD.

Methyl Yellow: A Potential Drug Scaffold for Parkinson's Disease.

Parkinson's disease (PD) is an age-related neurodegenerative disease affecting movement. To date, there are no currently available therapeutic agents which can prevent or slow disease progression. Here, we evaluated an azobenzene derivative, methyl yellow (MY), as a potential drug scaffold for PD; its inhibitory activity toward monoamine oxidase B (MAO-B) as well as drug-like properties were investigated. The inhibitory effect of MY on MAO activity was determined by a MAO enzyme inhibition assay. In addition, the in vitro properties of MY as a drug candidate (e.g., blood-brain barrier (BBB) permeability, serum albumin binding, drug efflux through P-glycoprotein (P-gp), drug metabolism by P450, and mitochondrial toxicity) were examined. In vivo effectiveness of MY was also evaluated in the 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) Parkinsonian mouse model. MY selectively inhibited MAO-B in a dose-dependent and reversible manner. MY was BBB-permeable, bound relatively weakly to serum albumin, was an unlikely substrate for both systems of P-gp and P450, and did not cause mitochondrial toxicity. Results from the MPTP Parkinsonian mouse model indicated that, upon treatment with MY, neurotoxicity induced by MPTP was mitigated. Investigations of MY demonstrate its inhibitory activity toward MAO-B, compliant properties for drug consideration, and its neuroprotective capability in the MPTP Parkinsonian mouse model. These data provide insights into potential use, optimization, and new design of azobenzene derivatives for PD treatment.

Discovery of novel benzimidazole derivatives as selective and reversible monoamine oxidase B inhibitors for Parkinson's disease treatment.

Parkinson's disease (PD) is a common neurodegenerative disorder characterized by the loss of dopaminergic neurons in the substantia nigra pars compacta. The development of novel scaffolds for human monoamine oxidase B (hMAO-B) inhibitors with reversible properties represents an important strategy to improve the efficacy and safety for PD treatment. In the current work, we have devised and assessed two innovative derivative series serving as hMAO-B inhibitors. These series have utilized benzimidazole as a scaffold and strategically incorporated a primary amide group, which is recognized as a pivotal pharmacophore in subsequent activity screening and reversible mode of action. Among these compounds, 16d has emerged as the most potent hMAO-B inhibitor with an IC50 value of 67.3 nM, comparable to safinamide (IC50 = 42.6 nM) in vitro. Besides, 16d demonstrated good selectivity towards hMAO-B isoenzyme with a selectivity index over 387. Importantly, in line with the design purpose, 16d inhibited hMAO-B in a competitive and reversible manner (Ki = 82.50 nM). Moreover, 16d exhibited a good safety profile in both cellular and acute toxicity assays in mice. It also displayed ideal pharmacokinetic properties and blood-brain barrier permeability in vivo, essential prerequisites for central nervous system medicines. In the MPTP-induced PD mouse model, 16d significantly alleviated the motor impairment, especially muscle relaxation and motor coordination. Therefore, 16d, serving as a lead compound, holds instructive significance for subsequent investigations regarding its application in the treatment of PD.

Enhancing monoamine oxidase B inhibitory activity via chiral fluorination: Structure-activity relationship, biological evaluation, and molecular docking study.

Parkinson's disease (PD) is a common neurodegenerative disease among the elderly. Currently, monoamine oxidase B (MAO-B) inhibitors are extensively used for PD in clinics. In this work, a series of novel chiral fluorinated pyrrolidine derivatives were designed and synthesized. In vitro biological evaluations revealed that compound D5 was the most potent, selective MAO-B inhibitor (IC50 = 0.019 µM, MAO-A/MAO-B selectivity index = 2440), which was 10-fold than that of miracle drug safinamide (IC50 = 0.163 µM, MAO-A/MAO-B selectivity index = 172). It was verified that the enhanced hydrophobic interaction of D5 improved the activity against MAO-B in molecular docking study. Besides, D5 exhibited excellent metabolic properties and pharmacokinetic profiles in monkeys and rats. Moreover, D5 displayed more efficacious than safinamide in vivo models. In the MPTP-induced PD mouse model, D5 significantly alleviated DA deficits and increased the effect of levodopa on dopamine concentration in the striatum. Meanwhile, D5 produced a prominent reduction in tremulous jaw movements induced by galantamine. Accordingly, we present D5 as a novel, highly potent, and selective MAO-B inhibitor for PD therapy.

AAV-Mediated Expression of Dominant-Negative ULK1 Increases Neuronal Survival and Enhances Motor Performance in the MPTP Mouse Model of Parkinson's Disease.

Loss of nigrostriatal projections by axonal degeneration is a key early event in Parkinson's disease (PD) pathophysiology, being accountable for the lack of dopamine in the nigrostriatal system and resulting in motor symptoms such as bradykinesia, rigidity, and tremor. Since autophagy is an important mechanism contributing to axonal degeneration, we aimed to evaluate the effects of competitive autophagy inhibition in the 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) mouse model of PD in vivo. Adeno-associated viral vector (AAV)-mediated overexpression of a dominant-negative form of the unc-51 like autophagy-initiating kinase (ULK1.DN) in the substantia nigra was induced 3 weeks before MPTP treatment. Analysis of motor behavior demonstrated a significant improvement of ULK1.DN expressing mice after MPTP treatment. Immunohistochemical analyses of dopaminergic nigral neurons and nigrostriatal projections revealed a significant protection from MPTP-induced neurotoxicity after ULK1.DN expression. Western blot analysis linked these findings to an activation of mTOR signaling. Taken together, our results indicate that expression of ULK1.DN can attenuate MPTP-induced axonal neurodegeneration, suggesting that ULK1 could be a promising novel target in the treatment of PD.

Dopamine Receptor D3 Expression Is Altered in CD4+ T-Cells From Parkinson's Disease Patients and Its Pharmacologic Inhibition Attenuates the Motor Impairment in a Mouse Model.

Neuroinflammation constitutes a fundamental process involved in Parkinson's disease (PD). Microglial cells play a central role in the outcome of neuroinflammation and consequent neurodegeneration of dopaminergic neurons in the substantia nigra. Current evidence indicates that CD4+ T-cells infiltrate the brain in PD, where they play a critical role determining the functional phenotype of microglia, thus regulating the progression of the disease. We previously demonstrated that mice bearing dopamine receptor D3 (DRD3)-deficient CD4+ T-cells are completely refractory to neuroinflammation and consequent neurodegeneration induced by the administration of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP). In this study we aimed to determine whether DRD3-signalling is altered in peripheral blood CD4+ T-cells obtained from PD patients in comparison to healthy controls (HC). Furthermore, we evaluated the therapeutic potential of targeting DRD3 confined to CD4+ T-cells by inducing the pharmacologic antagonism or the transcriptional inhibition of DRD3-signalling in a mouse model of PD induced by the chronic administration of MPTP and probenecid (MPTPp). In vitro analyses performed in human cells showed that the frequency of peripheral blood Th1 and Th17 cells, two phenotypes favoured by DRD3-signalling, were significantly increased in PD patients. Moreover, naïve CD4+ T-cells obtained from PD patients displayed a significant higher Th1-biased differentiation in comparison with those naïve CD4+ T-cells obtained from HC. Nevertheless, DRD3 expression was selectively reduced in CD4+ T-cells obtained from PD patients. The results obtained from in vivo experiments performed in mice show that the transference of CD4+ T-cells treated ex vivo with the DRD3-selective antagonist PG01037 into MPTPp-mice resulted in a significant reduction of motor impairment, although without significant effect in neurodegeneration. Conversely, the transference of CD4+ T-cells transduced ex vivo with retroviral particles codifying for an shRNA for DRD3 into MPTPp-mice had no effects neither in motor impairment nor in neurodegeneration. Notably, the systemic antagonism of DRD3 significantly reduced both motor impairment and neurodegeneration in MPTPp mice. Our findings show a selective alteration of DRD3-signalling in CD4+ T-cells from PD patients and indicate that the selective DRD3-antagonism in this subset of lymphocytes exerts a therapeutic effect in parkinsonian animals dampening motor impairment.

Neuroprotective Effect of a New 7,8-Dihydroxycoumarin-Based Fe2+/Cu2+ Chelator in Cell and Animal Models of Parkinson's Disease.

Disturbed iron homeostasis, often coupled to mitochondrial dysfunction, plays an important role in the progression of common neurodegenerative diseases such as Parkinson's disease (PD). Recent studies have underlined the relevance of iron chelation therapy for the treatment of these diseases. Here we describe the synthesis, chemical, and biological characterization of the multifunctional chelator 7,8-dihydroxy-4-((methylamino)methyl)-2H-chromen-2-one (DHC12). Metal selectivity of DHC12 was Cu2+ ∼ Fe2+ > Zn2+ > Fe3+. No binding capacity was detected for Hg2+, Co2+, Ca2+, Mn2+, Mg2+, Ni2+, Pb2+, or Cd2+. DHC12 accessed cells colocalizing with Mitotracker Orange, an indication of mitochondrial targeting. In addition, DHC12 chelated mitochondrial and cytoplasmic labile iron. Upon mitochondrial complex I inhibition, DHC12 protected plasma membrane and mitochondria against lipid peroxidation, as detected by the reduced formation of 4-hydroxynonenal adducts and oxidation of C11-BODIPY581/591. DHC12 also blocked the decrease in mitochondrial membrane potential, detected by tetramethylrhodamine distribution. DHC12 inhibited MAO-A and MAO-B activity. Oral administration of DHC12 to mice (0.25 mg/kg body weight) protected substantia nigra pars compacta (SNpc) neurons against MPTP-induced death. Taken together, our results support the concept that DHC12 is a mitochondrial-targeted neuroprotective iron-copper chelator and MAO-B inhibitor with potent antioxidant and mitochondria protective activities. Oral administration of low doses of DHC12 is a promising therapeutic strategy for the treatment of diseases with a mitochondrial iron accumulation component, such as PD.

Validity of the MPTP-Treated Mouse as a Model for Parkinson's Disease.

Parkinson's disease (PD) is characterized by dopaminergic (DA) neuron death in the substantia nigra (SN) and subsequent striatal adaptations. Mice treated with the neurotoxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyrimidine (MPTP) are widely used as a model for PD. To assess the validity of the MPTP mouse model for PD pathogenesis, we here identify the biological processes that are dysregulated in both human PD and MPTP-treated mice. Gene enrichment analysis of published differentially expressed messenger RNAs (mRNAs) in the SN of PD patients and MPTP-treated mice revealed an enrichment of gene categories related to motor dysfunction and neurodegeneration. In the PD striatum, a similar enrichment was found, whereas in the striatum of MPTP mice, acute processes linked to epilepsy were selectively enriched shortly following MPTP treatment. More importantly, we integrated the proteins encoded by the differentially expressed mRNAs into molecular landscapes showing PD pathogenesis-implicated processes only in the SN, including vesicular trafficking, exocytosis, mitochondrial apoptosis, and DA neuron-specific transcription, but not in the striatum. We conclude that the current use of the MPTP mouse as a model for studying the molecular processes in PD pathogenesis is more valid for SN than striatal mechanisms in PD. This novel insight has important practical implications for future studies using this model to investigate PD pathogenesis and evaluate the efficacy of new treatments.