Methamphetamine, mitochondria, and neurodegeneration

摘要 甲基苯丙胺（meth）是一种令人上瘾的精神兴奋剂。我们的试点研究表明，长期吸食冰毒会导致 黑质致密部 (SNc) 多巴胺 (DA) 和蓝斑 (LC) 去甲肾上腺素变性 (NE) 但不是小鼠腹侧被盖区 (VTA) DA 或中缝背侧 (DR) 血清素 (5-HT) 神经元。 DA、NE、 5-HT 和 5-HT 是合成并包装到轴突室囊泡中的单胺神经递质。 冰毒会增加 SNc 轴突中的胞质单胺和 MAO 依赖性线粒体应激，但不会增加体细胞。 初步研究表明，MAO 抑制可防止甲基苯丙胺诱导的 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 LC、VTA 和 DR 轴突线粒体应激增加，但慢性甲基苯丙胺诱导 SNc 和 LC，但不诱导 VTA 或 DR退化。我们在 SNc 中发现 L 型 Ca2 通道 (LCC) 依赖性线粒体应激，但在 VTA 中没有发现 轴突。我们提出了“双重打击”假说，其中 MAO 和 LCC 依赖的轴突线粒体应激 是退化所必需的。退化“触发因素”是甲基苯丙胺诱导的 MAO 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。由于 MAO- 和 LCC- 依赖性应力集中在轴突中，我们认为轴突损失先于体细胞损失，呈“死亡”模式 的退化。试验数据表明，禁欲期间 SNc 轴突线粒体应激增加。我们 假设这种压力是 MAO 依赖性的，并导致禁欲期间的退化。最后，方法—— 诱发退化预计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。假设将使用双光子激光扫描显微镜在大脑中进行测试 切片、基因编码生物传感器、MAO/LCC 的药理学和基因操作，以及 免疫组织化学。目标 1：确定单胺能轴突中线粒体应激的机制， 慢性甲基苯丙胺对线粒体的影响，以及线粒体应激是否持续 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 线粒体应激，而 SNc 和 LC 但不是 VTA 或 DR 轴突将有 LCC 诱导的线粒体应激。 我们还假设，在戒除慢性冰毒期间，轴突应力将保持升高，并且将 MAO 依赖性。目标 2：确定 MAO 和 LCC 在慢性甲基致性退化中的作用 和行为后果。我们假设药物抑制或基因删除 MAO 或 LCC 将防止慢性甲基诱导的脆弱神经元（SNc 和 LC）变性并删除 来自抗性（VTA 和 DR）神经元的 Ca2 缓冲蛋白钙结合蛋白将使它们容易受到 退化。我们进一步假设，在禁欲和 MAO 过程中，退化将会逐渐进展。 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.