Anxiolytic effect of antidiabetic metformin is mediated by AMPK activation in mPFC inhibitory neurons.

Diabetic patients receiving the antidiabetic drug metformin have been observed to exhibit a lower prevalence of anxiety disorders, yet the precise mechanism behind this phenomenon is unclear. In our study, we found that anxiety induces a region-specific reduction in AMPK activity in the medial prefrontal cortex (mPFC). Concurrently, transgenic mice with brain-specific AMPK knockout displayed abnormal anxiety-like behaviors. Treatment with metformin or the overexpression of AMPK restored normal AMPK activity in the mPFC and mitigated social stress-induced anxiety-like behaviors. Furthermore, the specific genetic deletion of AMPK in the mPFC not only instigated anxiety in mice but also nullified the anxiolytic effects of metformin. Brain slice recordings revealed that GABAergic excitation and the resulting inhibitory inputs to mPFC pyramidal neurons were selectively diminished in stressed mice. This reduction led to an excitation-inhibition imbalance, which was effectively reversed by metformin treatment or AMPK overexpression. Moreover, the genetic deletion of AMPK in the mPFC resulted in a similar defect in GABAergic inhibitory transmission and a consequent hypo-inhibition of mPFC pyramidal neurons. We also generated a mouse model with AMPK knockout specific to GABAergic neurons. The anxiety-like behaviors in this transgenic mouse demonstrated the unique role of AMPK in the GABAergic system in relation to anxiety. Therefore, our findings suggest that the activation of AMPK in mPFC inhibitory neurons underlies the anxiolytic effects of metformin, highlighting the potential of this primary antidiabetic drug as a therapeutic option for treating anxiety disorders.

Addition of α-synuclein aggregates to the intestinal environment recapitulates Parkinsonian symptoms in model systems.

The gut-brain axis plays a vital role in Parkinson's disease (PD). The mechanisms of gut-brain transmission mainly focus on α-synuclein deposition, intestinal inflammation and microbiota function. A few studies have shown the trigger of PD pathology in the gut. α-Synuclein is highly conserved in food products, which was able to form ß-folded aggregates and to infect the intestinal mucosa. In this study we investigated whether α-synuclein-preformed fibril (PFF) exposure could modulate the intestinal environment and induce rodent models replicating PD pathology. We first showed that PFF could be internalized into co-cultured Caco-2/HT29/Raji b cells in vitro. Furthermore, we demonstrated that PFF perfusion caused the intestinal inflammation and activation of enteric glial cells in an ex vivo intestinal organ culture and in an in vivo intestinal mouse coloclysis model. Moreover, we found that PFF exposure through regular coloclysis induced PD pathology in wild-type (WT) and A53T α-synuclein transgenic mice with various phenotypes. Particularly in A53T mice, PFF induced significant behavioral disorders, intestinal inflammation, α-synuclein deposition, microbiota dysbiosis, glial activation as well as degeneration of dopaminergic neurons in the substantia nigra. In WT mice, however, the PFF induced only mild behavioral abnormalities, intestinal inflammation, α-synuclein deposition, and glial activation, without significant changes in microbiota and dopaminergic neurons. Our results reveal the possibility of α-synuclein aggregates binding to the intestinal mucosa and modeling PD in mice. This study may shed light on the investigation and early intervention of the gut-origin hypothesis in neurodegenerative diseases.

Pharmacological characterization of the small molecule 03A10 as an inhibitor of α-synuclein aggregation for Parkinson's disease treatment.

Aggregation of α-synuclein, a component of Lewy bodies (LBs) or Lewy neurites in Parkinson's disease (PD), is strongly linked with disease development, making it an attractive therapeutic target. Inhibiting aggregation can slow or prevent the neurodegenerative process. However, the bottleneck towards achieving this goal is the lack of such inhibitors. In the current study, we established a high-throughput screening platform to identify candidate compounds for preventing the aggregation of α-synuclein among the natural products in our in-house compound library. We found that a small molecule, 03A10, i.e., (+)-desdimethylpinoresinol, which is present in the fruits of Vernicia fordii (Euphorbiaceae), modulated aggregated α-synuclein, but not monomeric α-synuclein, to prevent further elongation of α-synuclein fibrils. In α-synuclein-overexpressing cell lines, 03A10 (10 µM) efficiently prevented α-synuclein aggregation and markedly ameliorated the cellular toxicity of α-synuclein fibril seeds. In the MPTP/probenecid (MPTP/p) mouse model, oral administration of 03A10 (0.3 mg· kg-1 ·d-1, 1 mg ·kg-1 ·d-1, for 35 days) significantly alleviated behavioral deficits, tyrosine hydroxylase (TH) neuron degeneration and p-α-synuclein aggregation in the substantia nigra (SN). As the Braak hypothesis postulates that the prevailing site of early PD pathology is the gastrointestinal tract, we inoculated α-synuclein preformed fibrils (PFFs) into the mouse colon. We demonstrated that α-synuclein PFF inoculation promoted α-synuclein pathology and neuroinflammation in the gut and brain; oral administration of 03A10 (5 mg· kg-1 ·d-1, for 4 months) significantly attenuated olfactory deficits, α-synuclein accumulation and neuroinflammation in the olfactory bulb and SN. We conclude that 03A10 might be a promising drug candidate for the treatment of PD. 03A10 might be a novel drug candidate for PD treatment, as it inhibits α-synuclein aggregation by modulating aggregated α-synuclein rather than monomeric α-synuclein to prevent further elongation of α-synuclein fibrils and prevent α-synuclein toxicity in vitro, in an MPTP/p mouse model, and PFF-inoculated mice.

MCC950 ameliorates the dementia symptom at the early age of line M83 mouse and reduces hippocampal α-synuclein accumulation.

Dementia with Lewy bodies (DLB) is the second most common type of neurodegenerative dementia after Alzheimer's disease (AD). Neuroinflammation plays an important role in neurodegenerative diseases. It is urgent to unravel the pathogenesis of DLB and find potential therapeutic drugs. Here, we investigated the pharmacological effects of the NLRP3 inflammasome inhibitor MCC950 in A53T α-synuclein transgenic line M83 mice aged 4 months. The behavioral tests including Y-maze, Barnes maze, nest building and Rotarod showed that MCC950 significantly improved the cognitive dysfunction symptom without affecting the motor coordination after consecutive intragastric administration every day for 5 weeks. Furthermore, immunostaining or immunoblotting experiments on the hippocampal tissue were performed, and the results suggested that MCC950 not only inhibited the expression of NLRP3, and suppressed the activation of astrocytes and microglia, but also promoted the mTOR-mediated autophagy pathway to reduce human α-synuclein accumulation. Our findings further demonstrate that line M83 mice may be used as an animal model for DLB research, and can provide preclinical evidences for the development of MCC950 as a promising therapeutic drug.

Paeoniflorin ameliorates ischemic injury in rat brain via inhibiting cytochrome c/caspase3/HDAC4 pathway.

Paeoniflorin (PF), a bioactive monoterpene glucoside, has shown a variety of pharmacological effects such as anti-inflammation and autophagy modulation etc. In this study, we investigated whether and how PF exerted a protective effect against ischemic brain injury in vivo and in vitro. Primary rat cortical neurons underwent oxygen/glucose deprivation/reperfusion (OGD/R) for 90 min. We showed that after OGD/R, a short fragment of histone deacetylase 4 (HDAC4) produced by caspase3-mediated degradation was markedly accumulated in the nucleus and the activity of caspase3 was increased. Treatment with PF (100 nM, 1 µM) significantly improved the viability of cortical neurons after OGD/R. Furthermore, PF treatment could maintain HDAC4 intrinsic subcellular localization and reduce the caspase3 activity without changing the HDAC4 at the transcriptional level. PF treatment significantly reduced OGD/R-caused inhibition of transcriptional factor MEF2 expression and increased the expression of downstream proteins such as GDNF, BDNF, and Bcl-xl, thus exerting a great anti-apoptosis effect as revealed by TUNEL staining. The beneficial effects of PF were almost canceled in HDAC4 (D289E)-transfected PC12 cells after OGD/R. In addition, PF treatment reduced the caspase9 activity, rescued the release of cytochrome c from mitochondria, and maintained the integrity of mitochondria membrane. We conducted in vivo experiments in 90-min-middle cerebral artery occlusion (MCAO) rat model. The rats were administered PF (20, 40 mg/kg, ip, 3 times at the reperfusion, 24 h and 48 h after the surgery). We showed that PF administration dose-dependently reduced infarction area, improved neurological symptoms, and maintained HDAC4 localization in rats after MCAO. These results demonstrate that PF is effective in protecting against ischemic brain injury and inhibit apoptosis through inhibiting the cytochrome c/caspase3/HDAC4 pathway.

TPN672: A Novel Serotonin-Dopamine Receptor Modulator for the Treatment of Schizophrenia.

TPN672 [7-(2-(4-(benzothiophen-4-yl) piperazin-1-yl)ethyl)quinolin-2(1H)-one maleate] is a novel antipsychotic candidate with high affinity for serotonin and dopamine receptors that is currently in clinical trial for the treatment of psychiatric disorders. In vitro binding study showed that TPN672 exhibited extremely high affinity for serotonin 1A receptor (5-HT1AR) (K i = 0.23 nM) and 5-HT2AR (K i = 2.58 nM) as well as moderate affinity for D3R (K i = 11.55 nM) and D2R (K i = 17.91 nM). In vitro functional assays demonstrated that TPN672 acted as a potent 5-HT1AR agonist, D2R/D3R partial agonist, and 5-HT2AR antagonist. TPN672 displayed robust antipsychotic efficacy in rodent models (e.g., blocking phencyclidine-induced hyperactivity), significantly better than aripiprazole, and ameliorated negative symptoms and cognitive deficits in the sociability test, dark avoidance response, Morris water maze test, and novel object recognition test. The results of electrophysiological experiments showed that TPN672 might inhibit the excitability of the glutamate system through activating 5-HT1AR in medial prefrontal cortex, thereby improving cognitive and negative symptoms. Moreover, the safety margin (the ratio of minimum catalepsy-inducing dose to minimum effective dose) of TPN672 was about 10-fold, which was superior to aripiprazole. In conclusion, TPN672 is a promising new drug candidate for the treatment of schizophrenia and has been shown to be more effective in attenuating negative symptoms and cognitive deficits while having lower risk of extrapyramidal symptoms and hyperprolactinemia. SIGNIFICANCE STATEMENT: TPN672 is a promising new drug candidate for the treatment of schizophrenia and has been shown to be more effective in attenuating negative symptoms and cognitive deficits while having a lower risk of extrapyramidal symptoms and hyperprolactinemia. A phase I clinical trial is now under way to test its tolerance, pharmacokinetics, and pharmacodynamic effects in human volunteers. Accordingly, the present results will have significant impact on the development of new antischizophrenia drugs.

Aha1 Exhibits Distinctive Dynamics Behavior and Chaperone-Like Activity.

Aha1 is the only co-chaperone known to strongly stimulate the ATPase activity of Hsp90. Meanwhile, besides the well-studied co-chaperone function, human Aha1 has also been demonstrated to exhibit chaperoning activity against stress-denatured proteins. To provide structural insights for a better understanding of Aha1's co-chaperone and chaperone-like activities, nuclear magnetic resonance (NMR) techniques were used to reveal the unique structure and internal dynamics features of full-length human Aha1. We then found that, in solution, both the two domains of Aha1 presented distinctive thermal stabilities and dynamics behaviors defined by their primary sequences and three-dimensional structures. The low thermal stability (melting temperature of Aha128-162: 54.45 °C) and the internal dynamics featured with slow motions on the µs-ms time scale were detected for Aha1's N-terminal domain (Aha1N). The aforementioned experimental results suggest that Aha1N is in an energy-unfavorable state, which would therefore thermostatically favor the interaction of Aha1N with its partner proteins such as Hsp90's middle domain. Differently from Aha1N, Aha1C (Aha1's C-terminal domain) exhibited enhanced thermal stability (melting temperature of Aha1204-335: 72.41 °C) and the internal dynamics featured with intermediate motions on the ps-ns time scale. Aha1C's thermal and structural stabilities make it competent for the stabilization of the exposed hydrophobic groove of dimerized Hsp90's N-terminal domain. Of note, according to the NMR data and the thermal shift results, although the very N-terminal region (M1-W27) and the C-terminal relaxin-like factor (RLF) motif showed no tight contacts with the remaining parts of human Aha1, they were identified to play important roles in the recognition of intrinsically disordered pathological α-synuclein.

A Novel 2-Phenylamino-Quinazoline-Based Compound Expands the Neural Stem Cell Pool and Promotes the Hippocampal Neurogenesis and the Cognitive Ability of Adult Mice.

The adult neurogenesis occurs throughout the life of the mammalian hippocampus and is found to be essential for learning and memory. Identifying new ways to manipulate the number of neural stem cells (NSCs) and enhance endogenous neurogenesis in adults is very important. Here we found that a novel compound, N2-(4-isopropylphenyl)-5-(3-methoxyphenoxy)quinazoline-2,4-diamine (code-named Yhhu-3792), enhanced the self-renewal capability of NSCs in vitro and in vivo. In vitro, Yhhu-3792 increased the ratio of 5-Bromo-2-deoxyuridine+ /4'-6-diamidino-2-phenylindole+ embryonic NSCs and accelerated the growth of neurospheres significantly. We demonstrated that Yhhu-3792 activated Notch signaling pathway and promoted the expression of Notch target genes, Hes3 and Hes5. And the Notch signaling inhibitor DAPT could inhibit its function. Thus, we concluded Yhhu-3792 increased the number of embryonic NSCs via activating the Notch signaling pathway. We measured the effect of Yhhu-3792 on epidermal growth factor receptor signaling, which demonstrated Yhhu-3792 act via a different mechanism with the quinazoline parent chemical group. In the eight-week-old male C57BL/6 mice, chronic Yhhu-3792 administration expanded the NSCs pool and promoted endogenous neurogenesis in the hippocampal dentate gyrus (DG). It also increased the spatial and episodic memory abilities of mice, when evaluated with the Morris water maze and Fear conditioning tests. In conclusion, Yhhu-3792 could be a novel drug candidate to promote the self-renew of NSCs and adult neurogenesis. And it may have therapeutic potential in the impairment of learning and memory associated DG dysfunction. Stem Cells 2018;36:1273-1285.

Caffeic acid reduces A53T α-synuclein by activating JNK/Bcl-2-mediated autophagy in vitro and improves behaviour and protects dopaminergic neurons in a mouse model of Parkinson's disease.

The human A53T mutant of α-synuclein tends to aggregate and leads to neurotoxicity in familial Parkinson's disease (PD). The aggregation of α-synuclein is also found in sporadic PD. Thus, targeting α-synuclein clearance could be used as a drug-discovery strategy for PD treatment. Caffeic acid (CA) has shown neuroprotection in Alzheimer's disease or cerebral ischaemia; however, it is unclear whether CA confers neuroprotection in α-synuclein-induced PD models. Here we focus on whether and how A53T α-synuclein is affected by CA. We assessed the effect of CA on cell viability in SH-SY5Y cells overexpressing A53T α-synuclein. Pathway-related inhibitors were used to identify the autophagy mechanisms. Seven-month-old A53T α-synuclein transgenic mice (A53T Tg mice) received CA daily for eight consecutive weeks. Behaviour tests including the buried food pellet test, the pole test, the Rotarod test, open field analysis, and gait analysis were used to evaluate the neuroprotective effect of CA. Tyrosine hydroxylase and α-synuclein were assessed by immunohistochemistry or western blot in the substantia nigra (SN). We found that CA alleviated the cell damage induced by overexpressing A53T α-synuclein and that CA reduced A53T α-synuclein by activating the JNK/Bcl-2-mediated autophagy pathway. The efficacy of CA on A53T α-synuclein degradation was reversed by the autophagy inhibitor bafilomycin A1 and the JNK inhibitor SP600125. In A53T Tg mice, CA improved behavioural impairments, attenuated loss of dopaminergic neurons, enhanced autophagy and reduced α-synuclein in the SN. Thus, the results provide scientific evidence for the neuroprotective effect of CA in PD. Our work lays the foundation for CA clinical trials to treat PD in the future.

Protein kinase A rescues microtubule affinity-regulating kinase 2-induced microtubule instability and neurite disruption by phosphorylating serine 409.

Microtubule affinity-regulating kinase 2 (MARK2)/PAR-1b and protein kinase A (PKA) are both involved in the regulation of microtubule stability and neurite outgrowth, but whether a direct cross-talk exists between them remains unclear. Here, we found the disruption of microtubule and neurite outgrowth induced by MARK2 overexpression was blocked by active PKA. The interaction between PKA and MARK2 was confirmed by coimmunoprecipitation and immunocytochemistry both in vitro and in vivo. PKA was found to inhibit MARK2 kinase activity by phosphorylating a novel site, serine 409. PKA could not reverse the microtubule disruption effect induced by a serine 409 to alanine (Ala) mutant of MARK2 (MARK2 S409A). In contrast, mutation of MARK2 serine 409 to glutamic acid (Glu) (MARK2 S409E) did not affect microtubule stability and neurite outgrowth. We propose that PKA functions as an upstream inhibitor of MARK2 in regulating microtubule stability and neurite outgrowth by directly interacting and phosphorylating MARK2.

Anti-inflammatory Inositol Derivatives from the Whole Plant of Inula cappa.

Twelve new inositol derivatives, classified into myoinositol (1-6) and l-inositol (10-15) types, along with five known analogues were isolated from the whole plant of Inula cappa. The structures of the new compounds were established by extensive analysis of mass spectrometric and 1D and 2D NMR spectroscopic data. All the tested compounds showed anti-inflammatory activities against the production of NO in RAW264.7 macrophages stimulated by lipopolysaccharide, with IC50 values ranging from 7 to 23 µM.

Connexin 43 stabilizes astrocytes in a stroke-like milieu to facilitate neuronal recovery.

AIM: Connexin 43 (Cx43) is a member of connexin family mainly expressed in astrocytes, which forms gap junctions and hemichannels and maintains the normal shape and function of astrocytes. In this study we investigated the role of Cx43 in astrocytes in facilitating neuronal recovery during ischemic stroke. METHODS: Primary culture of astrocytes or a mixed culture of astrocytes and cortical neurons was subjected to oxygen glucose deprivation and reperfusion (OGD/R). The expression of Cx43 and Ephrin-A4 in astrocytes was detected using immunocytochemical staining and Western blot assays. Intercellular Ca(2+) concentration was determined with Fluo-4 AM fluorescent staining. Middle cerebral artery occlusion (MCAO) model rats were used for in vivo studies. RESULTS: OGD/R treatment of cultured astrocytes caused a decrement of Cx43 expression and translocation of Cx43 from cell membrane to cytoplasm, accompanied by cell retraction. Furthermore, OGD/R increased intracellular Ca(2+) concentration, activated CaMKII/CREB pathways and upregulated expression of Ephrin-A4 in the astrocytes. All these changes in OGD/R-treated astrocytes were alleviated by overexpression of Cx43. In the cortical neurons cultured with astrocytes, OGD/R inhibited the neurite growth, whereas overexpression of Cx43 or knockdown of Ephrin-A4 in astrocytes restored the neurite growth. In MCAO model rats, neuronal recovery was found to be correlated with the recuperation of Cx43 and Ephrin-A4 in astrocytes. CONCLUSION: Cx43 can stabilize astrocytes and facilitate the resistance to the deleterious effects of a stroke-like milieu and promote neuronal recovery.

Paeoniflorin ameliorates ischemic neuronal damage in vitro via adenosine A1 receptor-mediated transactivation of epidermal growth factor receptor.

AIM: Paeoniflorin from Chinese herb Paeoniae Radix has been shown to ameliorate middle cerebral artery occlusion-induced ischemia in rats. The aim of this study was to investigate the mechanisms underlying the neuroprotective action of PF in cultured rat cortical neurons. METHODS: Primary cultured cortical neurons of rats were subjected to oxygen-glucose deprivation and reoxygenation (OGD/R) insult. Cell survival was determined using MTT assay. HEK293 cells stably transfected with A1R (HEK293/A1R) were used for detailed analysis. Phosphorylation of the signaling proteins was evaluated by Western blot or immunoprecipitation. Receptor interactions were identified using co-immunoprecipitation and immunofluorescence staining. RESULTS: Paeoniflorin (10 nmol/L to 1 µmol/L) increased the survival of neurons subjected to OGD/R. Furthermore, paeoniflorin increased the phosphorylation of Akt and ERK1/2 in these neurons. These effects were blocked by PI3K inhibitor wortmannin or MEK inhibitor U0126. Paeoniflorin also increased the phosphorylation of Akt and ERK1/2 in HEK293/A1R cells. Both A1R antagonist DPCPX and EGFR inhibitor AG1478 not only blocked paeoniflorin-induced phosphorylation of ERK1/2 and Akt in HEK293/A1R cells, but also paeoniflorin-increased survival of neurons subjected to OGD/R. In addition, paeoniflorin increased the phosphorylation of Src kinase and activation of MMP-2 in HEK293/A1R cells. Both Src inhibitor PP2 and MMP-2/MMP-9 inhibitor BiPs not only blocked paeoniflorin-induced phosphorylation of ERK1/2 (and Akt) in HEK293/A1R cells, but also paeoniflorin-increased survival of neurons subjected to OGD/R. CONCLUSION: Paeoniflorin promotes the survival of cultured cortical neurons by increasing Akt and ERK1/2 phosphorylation via A1R-mediated transactivation of EGFR.

Modulation of A2a receptor antagonist on D2 receptor internalization and ERK phosphorylation.

AIM: To explore the effects of heterodimerization of D2 receptor/A2a receptor (D2R/A2aR) on D2R internalization and D2R downstream signaling in primary cultured striatal neurons and HEK293 cells co-expressing A2aR and D2R in vitro. METHODS: Primary cultured rat striatal neurons and HEK293 cells co-expressing A2aR and D2R were treated with A2aR- or D2R-specific agonists. D2R internalization was detected using a biotinylation assay and confocal microscopy. ERK, Src kinase and ß-arrestin were measured using Western blotting. The interaction between A2aR and D2R was detected using bioluminescence resonance energy transfer (BRET) and immunoprecipitation. RESULTS: D2R and A2aR were co-localized and formed complexes in striatal neurons, while both the receptors formed heterodimers in the HEK293 cells. In striatal neurons and the HEK293 cells, the D2R agonist quinpirole (1 µmol/L) marked increased Src phosphorylation and ß-arrestin recruitment, thereby D2R internalization. Co-treatment with the A2aR antagonist ZM241385 (100 nmol/L) significantly attenuated these D2R-mediated changes. Furthermore, both ZM241385 (100 nmol/L) and the specific Src kinase inhibitor PP2 (5 µmol/L) blocked D2R-mediated ERK phosphorylation. Moreover, expression of the mutant ß-arrestin (319-418) significantly attenuated D2R-mediated ERK phosphorylation in HEK293 cells expressing both D2R and A2aR, but not in those expressing D2R alone. CONCLUSION: A2aR antagonist ZM241385 significantly attenuates D2R internalization and D2R-mediated ERK phosphorylation in striatal neurons, involving Src kinase and ß-arrestin. Thus, A2aR/D2R heterodimerization plays important roles in D2R downstream signaling.

Design, synthesis and biological evaluation of bivalent ligands against A(1)-D(1) receptor heteromers.

AIM: To design and synthesize bivalent ligands for adenosine A1-dopamine D1 receptor heteromers (A1-D1R), and evaluate their pharmacological activities. METHODS: Bivalent ligands and their corresponding A1R monovalent ligands were designed and synthesized. The affinities of the bivalent ligands for A1R and D1R in rat brain membrane preparation were examined using radiolabeled binding assays. To demonstrate the formation of A1-D1R, fluorescence resonance energy transfer (FRET) was conducted in HEK293 cells transfected with D1-CFP and A1-YFP. Molecular modeling was used to analyze the possible mode of protein-protein and protein-ligand interactions. RESULTS: Two bivalent ligands for A1R and D1R (20a, 20b), as well as the corresponding A1R monovalent ligands (21a, 21b) were synthesized. In radiolabeled binding assays, the bivalent ligands showed affinities for A1R 10-100 times higher than those of the corresponding monovalent ligands. In FRET experiments, the bivalent ligands significantly increased the heterodimerization of A1R and D1R compared with the corresponding monovalent ligands. A heterodimer model with the interface of helixes 3, 4, 5 of A1R and helixes 1, 6, 7 from D1R was established with molecular modeling. The distance between the two ligand binding sites in the heterodimer model was approximately 48.4 Å, which was shorter than the length of the bivalent ligands. CONCLUSION: This study demonstrates the existence of A1-D1R in situ and a simultaneous interaction of bivalent ligands with both the receptors.

Oral [60]fullerene reduces neuroinflammation to alleviate Parkinson's disease via regulating gut microbiome.

Neuroinflammation is considered to drive the pathogenic process of neuronal degeneration in Parkinson's disease (PD). However, effective anti-neuroinflammation therapeutics for PD still remain dissatisfactory. Here we explore a robust therapeutic strategy for PD using anti-neuroinflammatory fullerenes. Methods: Oral fullerene was prepared by a ball-milling method. 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced PD mouse model was used to investigate the therapeutic effects and mechanisms of it. The gut microenvironment was evaluated by 16S rRNA gene sequencing, gas chromatography-mass spectrometry, quantitative polymerase chain reaction (Q-PCR), and western blot (WB). The neuroinflammation and neurodegeneration were evaluated by pathological analysis, Elisa kits, transmission electron microscopy, Q-PCR, WB and so on. Toxicity was assessed by weight, blood test and hematoxylin-eosin (HE) staining. Results: Oral fullerene therapeutic system that dissolved [60]fullerene into olive oil (abbreviated as OFO) was dexterously designed, which could reduce neuroinflammation via regulating the diversity of gut microbiome, increasing the contents of short chain fatty acids (SCFAs) and recovering the integrity of gut barrier. Accordingly, the reduction of neuroinflammation prevented dopaminergic neuronal degeneration. And thus, OFO significantly ameliorated motor deficits and fundamentally reversed dopamine (DA) loss in MPTP-induced PD mice. Of note, OFO exhibited low toxicity towards the living body. Conclusion: Our findings suggest that OFO is a safe-to-use, easy-to-apply, and prospective candidate for PD treatment in clinic, opening a therapeutic window for neuroinflammation-triggered neurodegeneration.

Transferrin-modified c[RGDfK]-paclitaxel loaded hybrid micelle for sequential blood-brain barrier penetration and glioma targeting therapy.

The effective chemotherapy for glioblastoma multiform (GBM) requires a nanomedicine that can both penetrate the blood-brain barrier (BBB) and target the glioma cells subsequently. In this study, Transferrin (Tf) modified cyclo-[Arg-Gly-Asp-d-Phe-Lys] (c[RGDfK])-paclitaxel conjugate (RP) loaded micelle (TRPM) was prepared and evaluated for its targeting efficiency, antiglioma activity, and toxicity in vitro and in vivo. Tf modification significantly enhanced the cellular uptake of TRPM by primary brain microvascular endothelial cells (BMEC) to 2.4-fold of RP loaded micelle (RPM) through Tf receptor mediated endocytosis, resulting in a high drug accumulation in the brain after intravenous injection.The c[RGDfK] modified paclitaxel (PTX) was released from micelle subsequently and targeted to integrin overexpressed glioma cells in vitro, and showed significantly prolonged retention in glioma tumor and peritumoral tissue. Most importantly, TRPM exhibited the strongest antiglioma activity, as the mean survival time of mice bearing intracranial U-87 MG glioma treated with TRPM (42.8 days) was significantly longer than those treated with Tf modified PTX loaded micelle (TPM) (39.5 days), PTX loaded micelle (PM) (34.8 days), Taxol (33.6 days), and saline (34.5 days). Noteworthy, TRPM did not lead to body weight loss compared with saline and was less toxic than TPM. These results indicated that TRPM could be a promising nanomedicine for glioma chemotherapy.

LLDT-67 attenuates MPTP-induced neurotoxicity in mice by up-regulating NGF expression.

AIM: To investigate the neuroprotective effects of LLDT-67, a novel derivative of triptolide, in MPTP-induced mouse Parkinson's disease (PD) models and in primary cultured astrocytes, and to elucidate the mechanisms of the action. METHODS: In order to induce PD, C57BL/6 mice were injected MPTP (30 mg/kg, ip) daily from d 2 to d 6. MPTP-induced behavioral changes in the mice were examined using pole test, swimming test and open field test. The mice were administered LLDT-67 (1, 2, or 4 mg/kg, po) daily from d 1 to d 11. On d 12, the mice were decapitated and brains were collected for immunohistochemistry study and measuring monoamine levels in the striatum. Primary cultured astrocytes from the cortices of neonatal C57BL/6 mouse pups were prepared for in vitro study. RESULTS: In MPTP-treated mice, administration of LLDT-67 significantly reduced the loss of tyrosine hydroxylase-positive neurons in the substantia nigra, and ameliorated the behavioral changes. LLDT-67 (4 mg/kg) significantly increased the expression of NGF in astrocytes in the substantia nigra and striatum of the mice. Furthermore, administration of LLDT-67 caused approximately 2-fold increases in the phosphorylation of TrkA at tyrosine 751, and marked increases in the phosphorylation of AKT at serine 473 as compared with the mice model group. In the cultured astrocytes, LLDT-67 (1 and 10 nmol/L) increased the NGF levels in the culture medium by 179% and 160%, respectively. CONCLUSION: The neuroprotective effect of LLDT-67 can be mostly attributed to its ability to enhance NGF synthesis in astrocytes in the midbrain and to rescue dopaminergic neurons indirectly through TrkA activation.

Synthesis and biological evaluation of (--)-linarinic acid derivatives as neuroprotective agents against OGD-induced cell damage.

A series of novel (-)-1,2,3,9-tetrahydropyrrolo[2,1-b]quinazoline-1-carboxylic acid derivatives were designed and synthesized. All of the prepared compounds were screened for their neuroprotective effects using an in vitro oxygen glucose deprivation (OGD) model of ischemic stroke. Some of the target compounds exhibited moderate to excellent protective potency. In particular, compounds 9d, 9e, 9g, and 9h showed significant protective effects in the SH-SY(5) Y cell line at all three concentrations tested.

A53T α-synuclein induces neurogenesis impairment and cognitive dysfunction in line M83 transgenic mice and reduces the proliferation of embryonic neural stem cells.

Dementia with Lewy body (DLB) is the second most common degenerative dementia after Alzheimer's disease. There is no therapeutic drug for DLB currently. It's urgent for us to understand the pathological mechanism of dementia mediated by α-synuclein, as the main component of Lewy body. Here, we found that the A53T α-synuclein transgenic mice showed decreased nesting behavior starting from the age of 1 month. The results in Morris water maze test suggested that the 6-month-old mice had learning memory deficits. Golgi staining indicated that the apical neuronal dendritic spines of hippocampal CA1 neurons were significantly reduced in 6-month-old homozygotes and heterozygotes, although MAP2 protein expression revealed no significant difference in the hippocampus among wild-type mice, homozygotes and heterozygotes. In vitro, we proved mutant A53T α-synuclein decreased the dendritic branches and dendrite spines on the embryonic mice hippocampal neurons. Furthermore, Ki67 immunofluorescence staining identified that the Ki67-positive cells of the hippocampal dentate gyrus and subventricular zone were significantly reduced in 6-month-old homozygotes and heterozygotes, compared with age-matched wild-type mice. Similarly, when 6-month-old mice were injected with BrdU for one day, the immunostaining results also confirmed that BrdU-positive cells were significantly reduced in homozygous and heterozygous mice. Lastly, we transfected primary embryonic hippocampal neural stem cells with lentivirus vector expressing A53T α-synuclein in vitro. Both BrdU staining and Western blotting showed that A53T α-synuclein significantly decreased the proliferation of embryonic neural stem cells. Taken together, these data suggest that A53T α-synuclein can induce adult neurogenesis impairment and cognitive dysfunction. The A53T α-synuclein transgenic mice may be used as an animal model for DLB. Promoting adult neurogenesis may be a promising approach to treat DLB pathogenesis.