Targeting neuronal nitric oxide synthase as a valuable strategy for the therapy of neurological disorders

The management of neurological disorders have huge and increasing human and economic costs. De-spite this, there is a scarcity of effective therapeutics, and there is an extreme urgency for new and real treatments. In this short review we analyze some promising advancements in the search of new bioactive molecules targeting neuronal nitric oxide synthase (nNOS), an enzyme deputed to the biosynthesis of nitric oxide (NO). In different conditions of neuronal damages, this molecule is overproduced, contribut-ing to the pathogenesis and progression of neuronal diseases. Two main approaches to modulate nNOS are discussed: a ifrst one consisting in the direct inhibition of the enzyme by means of small organic molecules, which can be also active against other different targets involved in such diseases. A second section is dedi-cated to molecules able to prevent the formation of the ternary complex N-methyl-D-aspartate (NMDA)-type glutamate receptors, postsynaptic density-95 (PSD95) protein-nNOS, which is necessary to activate the latter for the biosynthesis of NO.     

Parasitaemia and gametocytaemia after treatment with chloroquine, pyrimethamine/sulfadoxine, and pyrimethamine/sulfadoxine combined with artesunate in young Gambians with uncomplicated malaria

As part of a study to assess the infectivity of gametocytes after treatment with four antimalarial regimens, the efficacy of each treatment was also determined. From September to December 1998, 598 children with uncomplicated malaria were treated; 135 received chloroquine (CQ) alone, 276 received pyrimethamine/sulfadoxine (Fansidar, PSD) alone, 113 received PSD with a single dose of artesunate (PSD + 1ART) and 74 received PSD combined with three doses of artesunate (PSD + 3ART). On day 28 19/63 (30.2%; 95% C.I. 19.2% to 43.1%) of children treated with CQ alone, 5/134 (3.7%; 95% C.I. 1.2% to 8.5%) treated with PSD alone, 1/71 (1.4%, 95% C.I. 0.0% to 7.9%) treated with PSD + 1ART and 0/45 (0.0%; 95% C.I. 0.0% to 7.9%) treated with PSD + 3ART were parasitaemic. The proportion of children with gametocytes on day 7 after treatment with CQ alone was 16/89 (18.0%; 95% C.I. 10.6% to 27.6%), 98/174 (56.3%; 95% C.I. 48.6% to 63.8%) after treatment with PSD alone, 8/70 (11.4%; 95% C.I. 5.1% to 21.3%) after treatment with PSD + 1ART and 4/46 (8.7%; 95% C.I., 2.4% to 20.8%) after treatment with PSD + 3ART. CQ thus has a lower efficacy than PSD or either of the PSD and artesunate combinations. Use of PSD alone as an alternative first line treatment results in a very high post-treatment gametocyte prevalence that is likely to enhance transmission. There would be greater and more sustainable benefits from using PSD and artesunate combinations.     

Organization of TNIK in dendritic spines

Tumor necrosis factor receptor‐associated factor 2 (TRAF2)‐ and noncatalytic region of tyrosine kinase (NCK)‐interacting kinase (TNIK) has been identified as an interactor in the psychiatric risk factor, Disrupted in Schizophrenia 1 (DISC1). As a step toward deciphering its function in the brain, we performed high‐resolution light and electron microscopic immunocytochemistry. We demonstrate here that TNIK is expressed in neurons throughout the adult mouse brain. In striatum and cerebral cortex, TNIK concentrates in dendritic spines, especially in the vicinity of the lateral edge of the synapse. Thus, TNIK is highly enriched at a microdomain critical for glutamatergic signaling. J. Comp. Neurol. 523:1913–1924, 2015 © 2015 Wiley Periodicals, Inc.     

Molecular interactions of the plasma membrane calcium ATPase 2 at pre- and post-synaptic sites in rat cerebellum

The plasma membrane calcium extrusion mechanism, PMCA (plasma membrane calcium ATPase) isoform 2 is richly expressed in the brain and particularly the cerebellum. Whilst PMCA2 is known to interact with a variety of proteins to participate in important signalling events [Strehler EE, Filoteo AG, Penniston JT, Caride AJ (2007) Plasma-membrane Ca(2+) pumps: structural diversity as the basis for functional versatility. Biochem Soc Trans 35 (Pt 5):919–922], its molecular interactions in brain synapse tissue are not well understood. An initial proteomics screen and a biochemical fractionation approach identified PMCA2 and potential partners at both pre- and post-synaptic sites in synapse-enriched brain tissue from rat. Reciprocal immunoprecipitation and GST pull-down approaches confirmed that PMCA2 interacts with the post-synaptic proteins PSD95 and the NMDA glutamate receptor subunits NR1 and NR2a, via its C-terminal PDZ (PSD95/Dlg/ZO-1) binding domain. Since PSD95 is a well-known partner for the NMDA receptor this raises the exciting possibility that all three interactions occur within the same post-synaptic signalling complex. At the pre-synapse, where PMCA2 was present in the pre-synapse web, reciprocal immunoprecipitation and GST pull-down approaches identified the pre-synaptic membrane protein syntaxin-1A, a member of the SNARE complex, as a potential partner for PMCA2. Both PSD95–PMCA2 and syntaxin-1A–PMCA2 interactions were also detected in the molecular and granule cell layers of rat cerebellar sagittal slices by immunohistochemistry. These specific molecular interactions at cerebellar synapses may allow PMCA2 to closely control local calcium dynamics as part of pre- and post-synaptic signalling complexes.     

Organization of amyloid‐β protein precursor intracellular domain‐associated protein‐1 in the rat brain

Sustained activity‐dependent synaptic modifications require protein synthesis. Although proteins can be synthesized locally in dendrites, long‐term changes also require nuclear signaling. Amyloid‐β protein precursor intracellular domain‐associated protein‐1 (AIDA‐1), an abundant component of the biochemical postsynaptic density fraction, contains a nuclear localization sequence, making it a plausible candidate for synapse‐to‐nucleus signaling. We used immunohistochemistry to study the regional, cellular, and subcellular distribution of AIDA‐1. Immunostaining was prominent in the hippocampus, cerebral cortex, and neostriatum. Along with diffuse staining of neuropil, fluorescence microscopy revealed immunostaining of excitatory synapses throughout the forebrain, and immunoreactive puncta within and directly outside the nucleus. Presynaptic staining was conspicuous in hippocampal mossy fibers. Electron microscopic analysis of material processed for postembedding immunogold revealed AIDA‐1 label within postsynaptic densities in both hippocampus and cortex. Together with previous work, these data suggest that AIDA‐1 serves as a direct signaling link between synapses and the nucleus in adult rat brain. J. Comp. Neurol. 518:3221–3236, 2010. © 2010 Wiley‐Liss, Inc.     

米氮平与万拉法新治疗精神分裂症后抑郁对照研究

目的探讨米氮平与万拉法新治疗精神分裂症后抑郁障碍的临床疗效及安全性。方法将59例精神分裂症后抑郁障碍患者随机分为研究组31例,对照组28例,两组均维持原用抗精神病药物治疗的种类、剂量、用法不变,在此基础上研究组口服米氮平治疗,对照组口服万拉法新治疗。观察4周。于治疗前及治疗2周、4周末采用汉密顿抑郁量表评定抑郁状况,简明精神病量表评定精神症状,副反应量表评定不良反应。结果治疗后两组汉密顿抑郁量表总分、各因子分及简明精神病量表总分均较治疗前有显著下降（P〈0．01）;治疗4周末,研究组显效率70．97％、总有效率93．55％,对照组分别为71．43％、92．86％,两组差异均无显著性（P〉0．05）。两组不良反应主要表现为体重增加、口干、出汗、恶心、头晕等,且程度均轻微。结论精神分裂症后抑郁障碍患者在应用抗精神病药物治疗的基础上,联合新型抗抑郁剂米氮平与万拉法新治疗起效快、疗效显著、安全性高、依从性好。     

小檗碱对三转基因阿尔茨海默病小鼠的学习记忆和海马PSD95突触相关蛋白表达水平的影响

目的 探讨小檗碱(BBR)对三转基因阿尔茨海默病(AD)小鼠的学习记忆及海马组织PSD95突触蛋白表达水平的影响。方法 将30只三转基因(APP/Tau/PS1)AD小鼠按随机数字表法分成3组,即AD对照组、AD+25 mgBBR组、AD+50 mgBBR,每组各10只,后2组以灌胃方式且剂量分别为25 mg·kg^-1·d^-1、50 mg·kg^-1·d^-1,对照组给予等剂量生理盐水连续3个月灌胃处理; 采用Morris水迷宫方法探测各组AD小鼠行为学改变、空间记忆及探索情况; 免疫荧光染色检测各组小鼠海马组织突触后致密蛋白95(PSD95)阳性表达水平; Western blotting(WB)法检测各组三转基因AD小鼠海马脑组织PSD95蛋白、磷酸化蛋白激酶B(p-Akt)和磷酸化雷帕霉素靶蛋白(p-mTOR)表达水平及微管相关蛋白轻链3-Ⅱ(LC3-Ⅱ)自噬水平。结果 AD+25 mgBBR组的逃避潜伏期的学习记忆能力、免疫荧光PSD95表达水平以及PSD995、LC3-Ⅱ、p-Akt、p-mTOR蛋白表达水平与AD对照组比较均有明显差异(P<0.05); AD+50 mgBBR组逃避潜伏期的学习记忆能力、免疫荧光PSD95表达水平以及LC3-Ⅱ、p-Akt、p-mTOR表达水平与AD对照组比较差异均更明显(P<0.05,P<0.01)。结论 应用50 mg小檗碱能较好改善三转基因AD小鼠的学习记忆、空间探索能力,其机制可能是通过增加自噬水平LC3-Ⅱ调控Akt/mTOR信号通路,增加突触蛋白PSD95的表达水平及突触数量,以改善AD相关临床症状。     

TGF‐β2 and TGF‐β3 from cultured β‐amyloid‐treated or 3xTg‐AD‐derived astrocytes may mediate astrocyte–neuron communication

Astrocytes participate in the development and resolution of neuroinflammation in numerous ways, including the release of cytokines and growth factors. Among many, astrocytes release transforming growth factors beta (TGF‐β) TGF‐β1, TGF‐β2 and TGF‐β3. TGF‐β1 is the most studied isoform, while production and release of TGF‐β2 and TGF‐β3 by astrocytes have been poorly characterized. Here, we report that purified cultures of hippocampal astrocytes produce mainly TGF‐β3 followed by TGF‐β2 and TGF‐β1. Furthermore, astrocytes release principally the active form of TGF‐β3 over the other two. Changes in release of TGF‐β were sensitive to the calcineurin (CaN) inhibitor FK506. Starvation had no effect on TGF‐β1 and TGF‐β3 while TGF‐β2 mRNA was significantly up‐regulated in a CaN‐dependent manner. We further investigated production and release of astroglial TGF‐β in Alzheimer's disease‐related conditions. Oligomeric β‐amyloid (Aβ) down‐regulated TGF‐β1, while up‐regulating TGF‐β2 and TGF‐β3, in a CaN‐dependent manner. In cultured hippocampal astrocytes from 3xTg‐AD mice, TGF‐β2 and TGF‐β3, but not TGF‐β1, were up‐regulated, and this was CaN‐independent. In hippocampal tissues from symptomatic 3xTg‐AD mice, TGF‐β2 was up‐regulated with respect to control mice. Finally, treatment with recombinant TGF‐βs showed that TGF‐β2 and TGF‐β3 significantly reduced PSD95 protein in cultured hippocampal neurons, and this effect was paralleled by conditioned media from Aβ‐treated astrocytes or from astrocytes from 3xTg‐AD mice. Taken together, our data suggest that TGF‐β2 and TGF‐β3 are produced by astrocytes in a CaN‐dependent manner and should be investigated further in the context of astrocyte‐mediated neurodegeneration.     

Pallidal low β-low γ phase-amplitude coupling inversely correlates with Parkinson disease symptoms

ObjectiveRecent discoveries suggest that it is most likely the coupling of β oscillations (13–30 Hz) and not merely their power that relates to Parkinson disease (PD) pathophysiology.MethodsWe analyzed power and phase amplitude coupling (PAC) in local field potentials (LFP) recorded from Pallidum after placement of deep brain stimulation (DBS) leads in nineteen PD patients and three patients with dystonia.ResultsWithin GPi, we identified PAC between phase of β and amplitude of high frequency oscillations (200–300 Hz) and distinct β-low γ (40–80 Hz) PAC both modulated by contralateral movement. Resting β-low γ PAC, also present in dystonia patients, inversely correlated with severity of rigidity and bradykinesia (R = −0.44, P = 0.028). These findings were specific to the low β band, suggesting a differential role for the two β sub-bands.ConclusionsPAC is present across distinct frequency bands within the GPi. Given the presence of low β-low γ PAC in dystonia and the inverse correlation with symptom severity, we propose that this PAC may be a normal pallidal signal.SignificanceThis study provides new evidence on the pathophysiological contribution of local pallidal coupling and suggests similar and distinct patterns of coupling within GPi and STN in PD.