Methamphetamine, mitochondria, and neurodegeneration

摘要 甲基苯丙胺（Methamphetamine）是一种成瘾性精神兴奋剂。我们的初步研究表明，慢性冰毒导致 黑质黑部多巴胺蓝斑去甲肾上腺素 (NE)而对小鼠腹侧被盖区（VTA）DA和中缝背核（DR）5-HT神经元无明显影响。DA，NE， 和5-HT是在轴突隔室中合成并包装成囊泡的单胺神经递质。 甲基增加胞质单胺和单胺氧化酶依赖的线粒体压力在SNc轴突，但不是索马。 初步研究表明，MAO抑制防止甲基诱导的SNc变性。这些数据表明 退化是由MAO依赖性轴突线粒体应激驱动的。然而，试点数据显示，冰毒也 增加LC、VTA和DR轴突线粒体应激，但慢性甲安非他明诱导SNc和LC，而不是VTA或 DR变性。我们在SNc中发现了L型钙通道（LCC）依赖的线粒体应激，但在VTA中没有发现 轴突我们提出了一个“两次打击”假说，其中MAO和LCC依赖的轴突线粒体应激 是退化所必需的变性的“触发器”是甲基苯丙胺诱导的单胺氧化酶依赖性轴突线粒体 压力和脆弱性是轴突LCC依赖性线粒体压力的结果。自MAO-和LCC- 依赖应力集中在轴突，我们提出轴突损失先于躯体损失的“回死”模式 退化的迹象。试点数据表明，SNc轴突线粒体压力增加禁欲期间。我们 假设这种应激是单胺氧化酶依赖性的，并在禁欲期间促成退化。最后，冰毒-- 预期诱导的退化会损害依赖于新物体的识别和恐惧条件反射， 分别在DA和NE上。将使用脑中的双光子激光扫描显微镜来检验假设 切片，遗传编码的生物传感器，MAO/LCC的药理学和遗传学操作，以及 免疫组化目的1：确定单胺能轴突中线粒体应激的机制， 慢性冰毒对线粒体的影响，以及线粒体压力是否在 禁欲我们假设SNc、VTA、LC和DR轴突都具有甲基诱导的MAO依赖性。 然而，SNc和LC而不是VTA或DR轴突将具有LCC诱导的线粒体应激。 我们还假设，轴突应力将保持升高，整个戒断从慢性冰毒， 依赖于MAO。目的2：确定MAO和LCC在慢性甲基苯丙胺诱导的变性中的作用 和行为后果。我们假设，抑制或基因删除单胺氧化酶， 或LCC将防止慢性甲基诱导的脆弱（SNc和LC）神经元变性，并删除 来自抗性（VTA和DR）神经元的Ca 2+缓冲蛋白钙结合蛋白将使它们易受 退化我们进一步假设，在禁欲和MAO期间，退化将是渐进的- 依赖。最后，我们假设SNc和LC变性将导致认知障碍。

ABSTRACT Methamphetamine (meth) is an addictive psychostimulant. Our pilot studies show that chronic meth resulted in degeneration of substantia nigra pars compacta (SNc) dopamine (DA) and locus coeruleus (LC) norepinephrine (NE) but not ventral tegmental area (VTA) DA or dorsal raphe (DR) serotonin (5-HT) neurons in mice. DA, NE, and 5-HT are monoamine neurotransmitters synthesized and packaged into vesicles in axonal compartments. Meth increases cytosolic monoamines and MAO-dependent mitochondrial stress in SNc axons but not the soma. Pilot studies show that MAO inhibition prevented meth-induced SNc degeneration. These data suggest that degeneration is driven by MAO-dependent axonal mitochondrial stress. However, pilot data show meth also increased LC, VTA, and DR axonal mitochondrial stress yet chronic meth induced SNc and LC but not VTA or DR degeneration. We found L-type Ca2+ channel (LCC)-dependent mitochondrial stress in SNc but not VTA axons. We propose a `two-hit' hypothesis wherein both MAO- and LCC-dependent axonal mitochondrial stress are required for degeneration. The degeneration `trigger' is meth-induced MAO-dependent axonal mitochondrial stress and vulnerability is a consequence of axonal LCC-dependent mitochondrial stress. Since MAO- and LCC- dependent stress converge in axons, we propose that axonal loss precedes somatic loss in a `dying-back' pattern of degeneration. Pilot data indicate that SNc axonal mitochondrial stress was increased during abstinence. We hypothesize this stress is MAO-dependent and contributes to degeneration during abstinence. Lastly, meth- induced degeneration is expected to impair novel object recognition and fear conditioning which are dependent on DA and NE, respectively. Hypotheses will be tested using two-photon laser scanning microscopy in brain slices, genetically encoded biosensors, pharmacological and genetic manipulation of MAO/LCCs, and immunohistochemistry. Aim 1: Determine mechanisms of mitochondrial stress in monoaminergic axons, consequences of chronic meth on mitochondria, and whether mitochondrial stress continues during abstinence. We hypothesize that SNc, VTA, LC, and DR axons all have meth-induced MAO-dependent mitochondrial stress whereas SNc and LC but not VTA or DR axons will have LCC-induced mitochondrial stress. We also hypothesize that axonal stress will remain elevated throughout abstinence from chronic meth and will be MAO-dependent. Aim 2: Determine the roles of MAO and LCCs in chronic meth-induced degeneration and behavioral consequences. We hypothesize that pharmacologically inhibiting or genetically deleting MAO or LCCs will prevent chronic meth-induced degeneration of vulnerable (SNc and LC) neurons and deleting the Ca2+ buffering protein calbindin from resistant (VTA and DR) neurons will render them vulnerable to degeneration. We further hypothesize that degeneration will be progressive throughout abstinence and MAO- dependent. Lastly, we hypothesize that SNc and LC degeneration will result in cognitive impairments.