Neuroprotective and regenerative effects of small molecules

小分子的神经保护和再生作用

• 批准号: 8148515
• 负责人: Yun Wang
• 金额: $ 55.78万
• 依托单位: NATIONAL INSTITUTE ON DRUG ABUSE
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/8148515
• 关键词: regenerative; small molecule

(1) Diadenosine tetraphosphate (AP4A). The purpose of this study was to determine whether AP4A is protective against methamphetamine (MA) -mediated toxicity in dopaminergic neurons in vitro and in vivo. Primary neuronal cultures were prepared from rat embryonic (E15) ventral mesencephalic tissue. Cells were treated with 2 mM MA for 48 hours. Application of MA increased LDH levels, decreased TH immunoreactivity, and increased TUNEL. All these changes were reduced by pretreatment with AP4A. The protective effect of AP4A was also examined in vivo. Adult Sprague Dawley rats were injected with AP4A or vehicle intracerebroventricularly followed by 4 doses of MA (5mg/kg). Administration of MA increased caspase-3 immunoreactivity in striatum and cortex. Pretreatment with AP4A significantly reduced the density of caspase-3 (+) cells. Using microdialysis, dopamine (DA) release was monitored in dorsal striatum in freely moving rats. AP4A did not acutely alter MA-evoked DA release, suggesting that AP4A -induced protection is not directly mediated through a change in DA overflow. Taken together, these data show that AP4A has protective effects against MA-mediated neuronal injury both in vitro and in vivo. The mechanism of action may involve suppression of MA -induced apoptosis. (2) Astaxanthin (ATX). We found that ATX has protective effects against ischemic brain injury. ATX or vehicle was administered intracerebroventricularly 10-20 minutes prior to a 60-min middle cerebral artery occlusion (MCAo) in adult rats. At 2 days after MCAo, rats that received ATX had an increase in locomotor activity. ATX significantly reduced cerebral infarction and TUNEL labeling in ischemic brain. ATX antagonized ischemia -mediated loss of aconitase activity, an indirect marker for the production of reactive oxygen species and reduced glutamate release, lipid peroxidation, translocation of chrome C, as well as TUNEL labeling in the ischemic cortex. ATX did not alter physiological parameters, such as blood gases, blood pH, blood pressure, body temperature, brain temperature and cerebral blood flow. Collectively, our data suggest that astaxanthin has protective effects against free radical toxicity and ischemia-related injury in vivo through the inhibition of oxidative stress, inhibition of glutamate release, and anti-apoptosis. (3) pifithrin-a (PFT-a): (A) We found that PFTa has protective against MA mediated injury in dopaminergic neurons. High dose of MA reduced TH immunoreactivity and fiber density in primary dopaminergic neuronal culture. Co-treatment with PFTa significantly attenuated these degenerative changes. Our data suggest that treatment with a p53 inhibitor or suppression of p53 expression limits neuronal dysfunction and cell death induced by exposure to dopaminergic neurotoxins. (B) PFT-a enhanced the survival of neural progenitor cells (NPCs) both in vivo and in vitro. The enhancement of survival and proliferation of NPCs was first examined in SVZ neurospheres in tissue culture. PFT-a dose-dependently increased the number and size of new neurosphere formation. Delayed treatment (i.e. days 6 to 9 after injury) with PFT-a enhanced survival of endogenous neural progenitor cells (NPCs) in the subventricular zone in adult rats. PFTa enhanced proliferation, survival, migration, and differentiation of endogenous NPCs and enhanced the functional recovery in locomotor behavior in stroke animals. PFT- inhibited the expression of a p53-dependent pro-apoptotic gene, termed PUMA (p53-upregulated modulator of apoptosis), within the SVZ of stroke animals. Our data suggest that delayed treatment with a p53 inhibitor PFT-a is able to modify endogenous neurogenesis and improve the functional recovery after brain injury. (c) Systemic administration of PFT- enhanced the survival of dopaminergic neuronal transplants in vivo. PFT- treatment suppressed cell death, increased survival of TH (+) cells, enhanced TH neurite outgrowth from VM grafts, and augmented behavioral recovery in Parkinsonian rats. In conclusion, our data suggest that activation of p53 is an important mediator for cell death after injury or during neural repair. Transient suppression of p53 may increase the survival of neurons or NPCs after brain injury. (4) Retinoic acid (RA): Pretreatment with 9 cis RA (9cRA) increased locomotor activity, attenuated neurological deficits, and reduced cerebral infarction and TUNEL labeling in stroke rats. 9cRA increased the expression of bone morphogenetic protein 7 (BMP7) in brain. The protective response of 9cRA can be antagonized by BMP antagonist noggin. Our data suggest that RA can induce protective responses in cultured cells and in vivo, possibly through BMP7 mechanism. (5) (-)Naloxone: Neurodegeneration can occur through the activation of inflammatory processes. From experiments conducted this past year, we have observed improved behavioral recovery (body asymmetery and neurological score) following stroke when (+)naloxone was administered daily for 1 week starting one day after middle cerebral artery occlusion in rats. We are currently following up these studies comparing (-)naloxone as well as examining changes in markers of inflammation that are upregulated following ischemia.

(1)四磷酸二腺苷(AP4A)。本研究的目的是确定AP4A在体外和体内对甲基苯丙胺(MA)介导的多巴胺能神经元毒性是否具有保护作用。原代培养的神经元取自大鼠胚胎(E15)腹侧中脑组织。用2 mM MA处理细胞48h。应用MA可增加LDH水平，降低TH免疫反应性，增加TUNEL。AP4A可使上述改变减轻。对AP4A的体内保护作用进行了研究。成年SD大鼠脑室注射AP4A或赋形剂后，给予4个剂量的MA(5 mg/kg)。给予MA后，纹状体和皮质的caspase-3免疫反应增强。AP4A可显著降低caspase-3(+)细胞的密度。用微透析法监测自由活动大鼠背侧纹状体多巴胺(DA)的释放。AP4A没有明显改变MA诱导的DA释放，表明AP4A诱导的保护不是通过改变DA溢出直接介导的。综上所述，这些数据表明，AP4A在体外和体内都对MA介导的神经元损伤具有保护作用。其作用机制可能与抑制MA诱导的细胞凋亡有关。 (2)虾青素(ATX)。我们发现ATX对缺血性脑损伤有保护作用。成年大鼠在大脑中动脉闭塞(MCAO)60min前10-20分钟侧脑室注射ATX或赋形剂。在MCAO后2天，接受ATX治疗的大鼠的运动活动增加。ATX可显著减少脑梗塞和缺血脑组织的TUNEL标记。ATX可拮抗缺血所致的乌头酸酶活性丧失，而乌头酸酶活性是产生活性氧的间接标志，并可减少缺血皮质谷氨酸释放、脂质过氧化、铬C转位以及TUNEL标记。ATX没有改变生理参数，如血气、血液pH值、血压、体温、脑温度和脑血流量。综上所述，我们的研究结果表明，虾青素通过抑制氧化应激、抑制谷氨酸释放和抗细胞凋亡，对体内自由基毒性和缺血相关损伤具有保护作用。 (3)PFT-a：(A)我们发现PFTa对MA所致的多巴胺能神经元损伤具有保护作用。大剂量MA可降低原代培养的多巴胺能神经元的TH免疫反应性和纤维密度。与PFTA联合治疗显著减轻了这些退行性改变。我们的数据表明，使用P53抑制剂或抑制P53表达可以限制因暴露于多巴胺能神经毒素而导致的神经元功能障碍和细胞死亡。(B)PFT-a可提高神经前体细胞在体内和体外的存活。在组织培养中首次检测了SVZ神经球对神经干细胞存活和增殖的促进作用。PFT-a呈剂量依赖性地增加新神经球形成的数量和大小。延迟治疗(即损伤后6至9天)PFT可提高成年大鼠脑室下区内源性神经前体细胞(NPC)的存活。PFTA促进中风动物内源性神经前体细胞的增殖、存活、迁移和分化，并促进运动行为的功能恢复。PFT-抑制卒中动物SVZ中一种被称为PUMA(P53上调的细胞凋亡调节因子)的p53依赖的促凋亡基因的表达。我们的数据表明，延迟使用P53抑制剂PFT-a能够改变内源性神经发生，促进脑损伤后的功能恢复。(C)全身应用PFT可提高体内多巴胺能神经元移植的存活率。PFT治疗抑制了帕金森病大鼠的细胞死亡，增加了TH(+)细胞的存活率，促进了移植物TH突起的生长，并促进了行为恢复。总之，我们的数据表明，P53的激活是损伤后或神经修复过程中细胞死亡的重要中介。短暂抑制P53可能会增加脑损伤后神经元或神经前体细胞的存活率。 (4)维甲酸(RA)：9顺式维甲酸(9cRA)可增加卒中大鼠的运动能力，减轻神经功能缺失，减少脑梗塞和TUNEL标记。9cRA可增加脑内骨形态发生蛋白7(BMP7)的表达。BMP拮抗剂noggin可拮抗9cRA的保护作用。我们的研究结果提示，RA可以通过BMP7机制在培养细胞和体内诱导保护性反应。 (5)(-)纳洛酮：神经变性可通过激活炎症过程发生。从过去一年进行的实验中，我们观察到，在大鼠大脑中动脉闭塞后的一天开始，每天服用(+)纳洛酮1周，可以改善中风后的行为恢复(身体不对称和神经评分)。我们目前正在跟进这些研究，比较(-)纳洛酮，并检查缺血后上调的炎症标志物的变化。