Striatal overexpression of β-arrestin2 counteracts L-dopa-induced dyskinesia in 6-hydroxydopamine lesioned Parkinson's disease rats Publication date: December 2019 Source: Neurochemistry International, Volume 131 Author(s): Zeng-Rui Zhang, Xing-Ru Zhang, Xiao-Qian Luan, Xin-Shi Wang, Wen-Wen Wang, Xiao-Yi Wang, Bei Shao, Cheng-Long Xie Abstract Prolonged administration of Levodopa (L-dopa) therapy can generate L-dopa–induced dyskinesia (LID). Accumulating evidence indicates that hyper-activation of the dopamine D1 receptor (D1R) and the cAMP signaling cascade in the medium spiny neurons (MSNs) of the striatum are involved in LID. Previous studies have shown that striatal β-arrestin2 overexpression significantly reduces LID severity and have indicated that β-arrestin2 may play a causal role in the dyskinesia sensitization process. L-dopa-induced changes in the expression of signaling molecules and other proteins in the striatum were examined immunohistochemically and by western blot. A rAAV (recombinant adeno-associated virus) vector was used to overexpress and ablate β-arrestin2. We found that striatal overexpression of AAV-mediated β-arrestin2 produced less severe AIMs (abnormal involuntary movements) in response to L-dopa, whereas selective deletion of β-arrestin2 in the striatal neurons dramatically enhanced the severity of dyskinesia induced by L-dopa. Furthermore, no significant improvements in motor behavior (FFT: forelimb functional test) were seen with the inhibition or overexpression of β-arrestin2. Finally, overexpression of β-arrestin2 diminished L-dopa-induced D1R and phosphor-DARPP32/ERK levels. Viral deletion of β-arrestin2 markedly enhanced the key biochemical markers in the direct pathway. We found that increased availability of β-arrestin2 ameliorated dyskinesia severity with no influence on the anti-Parkinsonian action of L-dopa, suggesting a promising approach for controlling LID in Parkinson's disease. In addition, overexpression of β-Arrestin2 prevented the development of LID by inhibiting G protein-dependent D1R and phosphor-DARPP32/ERK signaling in dyskinetic rats.

The effect of coniferaldehyde on neurite outgrowth in neuroblastoma Neuro2a cells Publication date: December 2019 Source: Neurochemistry International, Volume 131 Author(s): Jieun Jeon, Huiyoung Kwon, Eunbi Cho, Kyung Sook Kim, Jeanho Yun, Young Choon Lee, Dong Hyun Kim Abstract Neurite outgrowth is the differentiation process by which neurons establish synapses. In the dentate gyrus of the hippocampus, new neurons are constantly produced and undergo neurite outgrowth to form synapses, and this process is involved in cognitive ability. Therefore, if an agent could modulate neurite outgrowth, it could potentially be developed as a compound for modulating cognitive ability. In this study, we examined whether coniferaldehyde, a natural compound, regulates neurite outgrowth in Neuro2a cells. We ascertained morphological changes and measured the percentage of neurite-bearing cells and neurite lengths. Coniferaldehyde significantly increased the percentage of neurite-bearing cells, and the length of neurites in a concentration-dependent manner, without inducing cell death. We then have identified that, coniferaldehyde activates the extracellular signals-regulated Kinase 1 and 2 (ERK1/2), and further noted that, U0126, an ERK1/2 inhibitor, blocks coniferaldehyde-facilitated neurite outgrowth. Moreover, Subchronic administration of CA enhanced learning and memory, and increased neurite length of newborn neurons in the hippocampus. These results suggest that coniferaldehyde induces neurite outgrowth by a process possibly mediated by ERK1/2 signaling and enhances learning and memory.

The P2Y14 receptor in the trigeminal ganglion contributes to the maintenance of inflammatory pain Publication date: December 2019 Source: Neurochemistry International, Volume 131 Author(s): Jiu Lin, Yan-yan Zhang, Fei Liu, Xin-yi Fang, Meng-ke Liu, Chao-lan Huang, Hang Wang, Da-qing Liao, Cheng Zhou, Jie-fei Shen Abstract P2Y purinergic receptors expressed in neurons and satellite glial cells (SGCs) of the trigeminal ganglion (TG) contribute to inflammatory and neuropathic pain. P2Y14 receptor expression is reported in the spinal cord, dorsal root ganglion (DRG), and TG. In present study, the role of P2Y14 receptor in the TG in inflammatory orofacial pain of Sprague-Dawley (SD) rats was investigated. Peripheral injection of complete Freund's adjuvant (CFA) induced mechanical hyperalgesia with the rapid upregulation of P2Y14 receptor, glial fibrillary acidic protein (GFAP), interleukin-1β (IL-1β), tumor necrosis factor-α (TNF-α), C–C chemokine CCL2, phosphorylated extracellular signal-regulated kinase 1/2 (p-ERK1/2), and phosphorylated p38 (p-p38) proteins in the TG. Furthermore, immunofluorescence staining confirmed the CFA-induced upregulation of P2Y14 receptor. Double immunostaining showed that P2Y14 receptor colocalized with glutamine synthetase (GS) and neuronal nuclei (NeuN). Finally, trigeminal injection of a selective antagonist (PPTN) of P2Y14 receptor attenuated CFA-induced mechanical hyperalgesia. PPTN also decreased the upregulation of the GFAP, IL-1β, TNF-α, CCL2, p-ERK1/2, and p-p38 proteins. Our findings showed that P2Y14 receptor in TG may contribute to orofacial inflammatory pain via regulating SGCs activation, releasing cytokines (IL-1β, TNF-α, and CCL2), and phosphorylating ERK1/2 and p38.

Phospholipase C-related catalytically inactive protein regulates lipopolysaccharide-induced hypothalamic inflammation-mediated anorexia in mice Publication date: December 2019 Source: Neurochemistry International, Volume 131 Author(s): Yosuke Yamawaki, Satomi Shirawachi, Akiko Mizokami, Kanako Nozaki, Hikaru Ito, Satoshi Asano, Kana Oue, Hidenori Aizawa, Shigeto Yamawaki, Masato Hirata, Takashi Kanematsu Abstract Peripheral lipopolysaccharide (LPS) injection induces systemic inflammation through the activation of the inhibitor of nuclear factor kappa B (NF-κB) kinase (IKK)/NF-κB signaling pathway, which promotes brain dysfunction resulting in conditions including anorexia. LPS-mediated reduction of food intake is associated with activation of NF-κB signaling and phosphorylation of the transcription factor signal transducer and activator of transcription 3 (STAT3) in the hypothalamus. We recently reported phospholipase C-related catalytically inactive protein (PRIP) as a new negative regulator of phosphatidylinositol 3-kinase/AKT signaling. AKT regulates the IKK/NF-κB signaling pathway; therefore, this study aimed to investigate the role of PRIP/AKT signaling in LPS-mediated neuroinflammation-induced anorexia. PRIP gene (Prip1 and Prip2) knockout (Prip-KO) mice intraperitoneally (ip) administered with LPS exhibited increased anorexia responses compared with wild-type (WT) controls. Although few differences were observed between WT and Prip-KO mice in LPS-elicited plasma pro-inflammatory cytokine elevation, hypothalamic pro-inflammatory cytokines were significantly upregulated in Prip-KO rather than WT mice. Hypothalamic AKT and IKK phosphorylation and IκB degradation were significantly increased in Prip-KO rather than WT mice, indicating further promotion of AKT-mediated NF-κB signaling. Consistently, hypothalamic STAT3 was further phosphorylated in Prip-KO rather than WT mice. Furthermore, suppressor of cytokine signaling 3 (Socs3), a negative feedback regulator for STAT3 signaling, and cyclooxogenase-2 (Cox2), a candidate molecule in LPS-induced anorexigenic responses, were upregulated in the hypothalamus in Prip-KO rather than WT mice. Pro-inflammatory cytokines were upregulated in hypothalamic microglia isolated from Prip-KO rather than WT mice. Together, these findings indicate that PRIP negatively regulates LPS-induced anorexia caused by pro-inflammatory cytokine expression in the hypothalamus, which is mediated by AKT-activated NF-κB signaling. Importantly, hypothalamic microglia participate in this PRIP-mediated process. Elucidation of PRIP-mediated neuroinflammatory responses may provide novel insights into the pathophysiology of many brain dysfunctions.

Txnip mediates glucocorticoid-activated NLRP3 inflammatory signaling in mouse microglia Publication date: December 2019 Source: Neurochemistry International, Volume 131 Author(s): Veni Bharti, Hua Tan, Hong Zhou, Jun-Feng Wang Abstract Many studies indicate that chronic stress and excessive stress hormone can cause an inflammatory response. Thioredoxin-interacting protein (Txnip) as an endogenous thioredoxin inhibitor suppresses thioredoxin-produced antioxidant effects. Txnip was also found to interact with nucleotide-binding oligomerization domain-like receptor protein 3 (NLRP3), which activates NLRP3 inflammasome and promotes inflammatory processes. Recently our laboratory found that chronic stress can increase Txnip protein levels in mouse brain, indicating that Txnip may mediate chronic stress-induced inflammation. Microglia play an important role in neuroinflammation. The purpose of this study is to investigate the effect of chronic stress hormone treatment on Txnip and NLRP3 inflammasome signaling in cultured microglia cells. Our result showed that chronic treatment with stress hormone corticosterone increased Txnip protein levels and Txnip-NLRP3 binding in N9 mouse microglia, in primary cultured mouse microglia and in mouse brain. Our result also showed that chronic corticosterone treatment increased procaspase-1 cleavage, caspase-1 activity and interleukin-1β release in N9 microglia. Using CRISPR/Cas9 method we found that knocking out Txnip inhibited corticosterone-increased caspase-1 activity and interleukin-1β release. Our results suggest that chronic corticosterone treatment upregulates Txnip and increases Txnip-NLRP3 binding, which activates NLRP3 inflammasome, resulting in activation of caspase-1 and in further releasing of interleukin-1β. It is therefore likely that Txnip-activated NLRP3 inflammasome contributes to corticosterone-caused neuroinflammation.

Caffeine regulates GABA transport via A1R blockade and cAMP signaling Publication date: December 2019 Source: Neurochemistry International, Volume 131 Author(s): Vladimir Pedro Peralva Borges-Martins, Danielle Dias Pinto Ferreira, Arthur Cardoso Souto, Jessika Geisebel Oliveira Neto, Danniel Pereira-Figueiredo, Karin da Costa Calaza, Karen de Jesus Oliveira, Alex Christian Manhães, Ricardo Augusto de Melo Reis, Regina Célia Cussa Kubrusly Abstract Caffeine is the most consumed psychostimulant drug in the world, acting as a non-selective antagonist of adenosine receptors A1R and A2AR, which are widely expressed in retinal layers. We have previously shown that caffeine, when administered acutely, acts on A1R to potentiate the NMDA receptor-induced GABA release. Now we asked if long-term caffeine exposure also modifies GABA uptake in the avian retina and which mechanisms are involved in this process. Chicken embryos aged E11 were injected with a single dose of caffeine (30 mg/kg) in the air chamber. Retinas were dissected on E15 for ex vivo neurochemical assays. Our results showed that [3H]-GABA uptake was dependent on Na+ and blocked at 4 °C or by NO-711 and caffeine. This decrease was observed after 60 min of [3H]-GABA uptake assay at E15, which is accompanied by an increase in [3H]-GABA release. Caffeine increased the protein levels of A1R without altering ADORA1 mRNA and was devoid of effects on A2AR density or ADORA2A mRNA levels. The decrease of GABA uptake promoted by caffeine was reverted by A1R activation with N6-cyclohexyl adenosine (CHA) but not by A2AR activation with CGS 21680. Caffeine exposure increased cAMP levels and GAT-1 protein levels, which was evenly expressed between E11-E15. As expected, we observed an increase of GABA containing amacrine cells and processes in the IPL, also, cAMP pathway blockage by H-89 decreased caffeine mediated [3H]-GABA uptake. Our data support the idea that chronic injection of caffeine alters GABA transport via A1R during retinal development and that the cAMP/PKA pathway plays an important role in the regulation of GAT-1 function.

Selectivity of (±)-citalopram at nicotinic acetylcholine receptors and different inhibitory mechanisms between habenular α3β4* and α9α10 subtypes Publication date: December 2019 Source: Neurochemistry International, Volume 131 Author(s): Hugo R. Arias, Xiao-Tao Jin, Sofía Gallino, Can Peng, Dominik Feuerbach, Jesús García-Colunga, Ana Belén Elgoyhen, Ryan M. Drenan, Marcelo O. Ortells Abstract The inhibitory activity of (±)-citalopram on human (h) α3β4, α4β2, and α7 nicotinic acetylcholine receptors (AChRs) was determined by Ca2+ influx assays, whereas its effect on rat α9α10 and mouse habenular α3β4* AChRs by electrophysiological recordings. The Ca2+ influx results clearly establish that (±)-citalopram inhibits (IC50's in μM) hα3β4 AChRs (5.1 ± 1.3) with higher potency than that for hα7 (18.8 ± 1.1) and hα4β2 (19.1 ± 4.2) AChRs. This is in agreement with the [3H]imipramine competition binding results indicating that (±)-citalopram binds to imipramine sites at desensitized hα3β4 with >2-fold higher affinity than that for hα4β2. The electrophysiological, molecular docking, and in silico mutation results indicate that (±)-citalopram competitively inhibits rα9α10 AChRs (7.5 ± 0.9) in a voltage-independent manner by interacting mainly with orthosteric sites, whereas it inhibits a homogeneous population of α3β4* AChRs at MHb (VI) neurons (7.6 ± 1.0) in a voltage-dependent manner by interacting mainly with a luminal site located in the middle of the ion channel, overlapping the imipramine site, which suggests an ion channel blocking mechanism. In conclusion, (±)-citalopram inhibits α3β4 and α9α10 AChRs with higher potency compared to other AChRs but by different mechanisms. (±)-Citalopram also inhibits habenular α3β4*AChRs, supporting the notion that these receptors are important endogenous targets related to their anti-addictive activities.

Noradrenaline is crucial for the substantia nigra dopaminergic cell maintenance Publication date: December 2019 Source: Neurochemistry International, Volume 131 Author(s): Sara af Bjerkén, Rasmus Stenmark Persson, Anna Barkander, Nina Karalija, Noelia Pelegrina-Hidalgo, Greg A. Gerhardt, Ana Virel, Ingrid Strömberg Abstract In Parkinson's disease, degeneration of substantia nigra dopaminergic neurons is accompanied by damage on other neuronal systems. A severe denervation is for example seen in the locus coerulean noradrenergic system. Little is known about the relation between noradrenergic and dopaminergic degeneration, and the effects of noradrenergic denervation on the function of the dopaminergic neurons of substantia nigra are not fully understood. In this study, N-(2-chloroethyl)-N-ethyl-2-bromobenzylamine (DSP4) was injected in rats, whereafter behavior, striatal KCl-evoked dopamine and glutamate releases, and immunohistochemistry were monitored at 3 days, 3 months, and 6 months. Quantification of dopamine-beta-hydroxylase-immunoreactive nerve fiber density in the cortex revealed a tendency towards nerve fiber regeneration at 6 months. To sustain a stable noradrenergic denervation throughout the experimental timeline, the animals in the 6-month time point received an additional DSP4 injection (2 months after the first injection). Behavioral examinations utilizing rotarod revealed that DSP4 reduced the time spent on the rotarod at 3 but not at 6 months. KCl-evoked dopamine release was significantly increased at 3 days and 3 months, while the concentrations were normalized at 6 months. DSP4 treatment prolonged both time for onset and reuptake of dopamine release over time. The dopamine degeneration was confirmed by unbiased stereology, demonstrating significant loss of tyrosine hydroxylase-immunoreactive neurons in the substantia nigra. Furthermore, striatal glutamate release was decreased after DSP4. In regards of neuroinflammation, reactive microglia were found over the substantia nigra after DSP4 treatment. In conclusion, long-term noradrenergic denervation reduces the number of dopaminergic neurons in the substantia nigra and affects the functionality of the nigrostriatal system. Thus, locus coeruleus is important for maintenance of nigral dopaminergic neurons.

Norepinephrine upregulates the expression of tyrosine hydroxylase and protects dopaminegic neurons against 6-hydrodopamine toxicity Publication date: December 2019 Source: Neurochemistry International, Volume 131 Author(s): Meng-Yang Zhu, Muhammad U. Raza, Yanqiang Zhan, Yan Fan Abstract As a classic neurotransmitter in the brain, norepinephrine (NE) also is an important modulator to other neuronal systems. Using primary cultures from rat ventral mesencephalon (VM) and dopaminergic cell line MN9D, the present study examined the neuroprotective effects of NE and its effects on the expression of tyrosine hydroxylase (TH). The results showed that NE protected both VM cultures and MN9D cells against 6-hydroxydopamine-caused apoptosis, with possible involvement of adrenal receptors. In addition, treatment with NE upregulated TH protein levels in dose- and time-dependent manner. Further experiments to investigate the potential mechanisms underlying this NE-induced upregulation of TH demonstrated a marked increase in protein levels of the brain-derived neurotrophic factor (BDNF) and the phosphorylated extracellular signal-regulated protein kinase 1 and 2 (pERK1/2) in VM cultures treated with NE. In MN9D cells, a significantly increase of TH and pERK1/2 protein levels were observed after their transfection with BDNF cDNA or exposure to BDNF peptides. Treatment of VM cultures with K252a, an antagonist of the tropomyosin-related kinase B, blocked the upregulatory effects of NE on TH, BDNF and pERK1/2. Administration of MEK1 & MEK2 inhibitors also reversed NE-induced upregulation of TH and pERK1/2. Moreover, ChIP assay showed that treatment with NE or BDNF increased H4 acetylation in the TH promoter. These results suggest that the neuroprotection and modulation of NE on dopaminergic neurons are mediated via BDNF and MAPK/ERK pathways, as well as through epigenetic histone modification, which may have implications for the improvement of therapeutic strategies for Parkinson's disease.

Lercanidipine boosts the efficacy of mesenchymal stem cell therapy in 3-NP-induced Huntington's disease model rats via modulation of the calcium/calcineurin/NFATc4 and Wnt/β-catenin signalling pathways Publication date: December 2019 Source: Neurochemistry International, Volume 131 Author(s): Eman M. Elbaz, Hebatullah S. Helmy, Ayman E. El-Sahar, Muhammed A. Saad, Rabab H. Sayed Abstract 3-Nitropropionic acid (3-NP) induces a spectrum of Huntington's disease (HD)-like neuropathologies in the rat striatum. The present study aimed to demonstrate the neuroprotective effect of lercanidipine (LER) in rats with 3-NP-induced neurotoxicity, address the possible additional protective effect of combined treatment with bone marrow-derived mesenchymal stem cells (BM-MSCs) and LER, and investigate the possible involvement of the Ca2+/calcineurin (CaN)/nuclear factor of activated T cells c4 (NFATc4) and Wnt/β-catenin signalling pathways. Rats were injected with 3-NP (10 mg/kg/day, i.p.) for two weeks and were divided into four subgroups; the first served as the control HD group, the second received a daily dose of LER (0.5 mg/kg, i.p.), the third received a single injection of BM-MSCs (1 x 106/rat, i.v.) and the last received a combination of both BM-MSCs and LER. The combined therapy improved motor and behaviour performance. Meanwhile, this treatment led to a marked reduction in striatal cytosolic Ca2+, CaN, tumour necrosis factor-alpha, and NFATc4 expression and the Bax/Bcl2 ratio. Combined therapy also increased striatal brain-derived neurotrophic factor, FOXP3, Wnt, and β-catenin protein expression. Furthermore, haematoxylin-eosin and Nissl staining revealed an amelioration of striatum tissue injury with the combined treatment. In conclusion, the current study provides evidence for a neuroprotective effect of LER and/or BM-MSCs in 3-NP-induced neurotoxicity in rats. Interestingly, combined LER/BM-MSC therapy was superior to cell therapy alone in inhibiting 3-NP-induced neurological insults via modulation of the Ca2+/CaN/NFATc4 and Wnt/β-catenin signalling pathways. LER/BM-MSC combined therapy may represent a feasible approach for improving the beneficial effects of stem cell therapy in HD.