SK2 Channels as Novel Neuroprotective Targets Against Cerebral Ischemia

描述（由申请人提供）：心脏骤停/心肺复苏术（CA/CPR）引起缺血，神经元兴奋毒性和认知能力下降。尽管付出了巨大的努力，但结果仍然很差。兴奋性毒性是由于谷氨酸神经传递增加，以及由此引起的过量Ca2+通过nmda型谷氨酸受体（NMDAr）内流。海马CA1神经元对学习和记忆很重要，对兴奋性毒性非常敏感。我们已经证明，小电导Ca2+激活的K+通道，2型（SK2通道）与NMDAr一起在海马CA1神经元的棘上表达，在那里它们通过NMDAr减弱Ca2+内流。此外，SK2通道在模式活动后从突触中移除，无论是正常的长期增强（LTP）诱导，还是CA/CPR后的异常。突触SK2通道的缺失消除了SK通道对Ca2+通过NMDAr内流的“刹车”，这是由于SK2通道的蛋白激酶A磷酸化。我们的研究结果进一步表明，增加SK2通道活性可显著提高CA/CPR后的神经元存活。因此，我们将使用一个综合的技术曲目来检验这些特定的假设：遗传或药物增强SK2通道活性可保护CA1神经元并改善认知结果。我们将使用遗传小鼠模型和SK增强药物来确定i) CA1神经元的存活和ii)认知表现。2. CA/ cpr诱导的缺血导致CA1神经元突触SK2通道的延迟和延长损失，增加nmda依赖的Ca2+瞬态，导致兴奋性毒性。CA/CPR后保持突触SK2通道活性保护CA1神经元。我们将测量缺血对SK2和NMDAr对谷氨酸传递（EPSP）的贡献的时间过程和影响，以及NMDAr介导的Ca2+瞬态。3. CA/ cpr诱导的缺血引起脊柱SK2通道PKA磷酸化，诱导通道内吞作用。pka免疫SK2通道的表达将使SK2和NMDAr对EPSP （NMDAr依赖的Ca2+瞬态）的贡献正常化，并保护CA1神经元免于兴奋性毒性细胞死亡。我们将使用表达pka免疫SK2通道的对照小鼠或小鼠来确定：i) SK2通道在脊柱下的分布；ii) SK2和NMDAr对EPSP的贡献；iii)脊柱Ca2+瞬态；iv) CA1活力。4. 异常持续缺血诱导的突触SK2通道丢失导致缺血性LTP （iLTP）移位？M，修正阈值，对更高的刺激频率，并削弱进一步的增强。维持功能性突触SK2通道的表达可阻止iLTP并使其正常化。我们将测量CA/ cpr诱导的缺血对突触可塑性的长期影响。公共卫生相关性：心脏病发作和随之而来的脑缺血是美国死亡和残疾的主要原因之一，不幸的是，目前没有可用的药物可以改善需要心肺复苏的严重心脏病发作后的结果。SK2通道是一种Ca2+激活的K+通道，在解剖学和功能上可以改善中风后的脑损伤。拟议的研究将证明SK2通道的神经保护作用，并提出新的干预策略来保护心脏病发作后的大脑，提高生存率，减少记忆缺陷，提高生活质量。

DESCRIPTION (provided by applicant): Cardiac arrest/cardiopulmonary resuscitation (CA/CPR) causes ischemia, neuronal excitotoxicity and cognitive decline. Despite intensive efforts, outcome remains poor. Excitotoxicity results from increased glutamate neurotransmission, and the consequent excessive Ca2+ influx through NMDA-type glutamate receptors (NMDAr). Hippocampal CA1 neurons are important to learning and memory and are acutely sensitive to excitotoxicity. We have shown that small conductance Ca2+-activated K+ channels, type 2 (SK2 channels) are expressed together with NMDAr in the spines on hippocampal CA1 neurons where they act to attenuate Ca2+ influx through NMDAr. In addition, SK2 channels are removed from synapses following patterned activity, either normally as for the induction of long term potentiation (LTP), or abnormally after CA/CPR. The loss of synaptic SK2 channels removes the SK channel 'brake' on Ca2+ influx through NMDAr and is due to protein kinase A phosphorylation of the SK2 channels. Our results further show that increasing SK2 channel activity substantially improves neuronal survival after CA/CPR. Therefore, we will use an integrated technical repertoire to test these specific hypotheses: 1. Genetic or pharmacologic enhancement of SK2 channel activity protects CA1 neurons and improves cognitive outcome. We will use genetic mouse models and SK enhancing drugs to determine the i) survival of CA1 neurons and, ii) cognitive performance. 2. CA/CPR-induced ischemia causes a delayed and prolonged loss of synaptic SK2 channels in CA1 neurons, increasing the NMDAr-dependent Ca2+ transient that causes excitotoxicity. Preserving synaptic SK2 channel activity after CA/CPR protects CA1 neurons. We will measure the time course and effects of ischemia on the SK2 and NMDAr contributions to glutamate transmission (EPSP), and NMDAr-mediated Ca2+ transients. 3. CA/CPR-induced ischemia causes PKA phosphorylation of spine SK2 channels, inducing channel endocytosis. Expression of PKA-immune SK2 channels will normalize the SK2 and NMDAr contributions to the EPSP, the NMDAr-dependent Ca2+ transient, and protect CA1 neurons from excitotoxic cell death. We will use control mice or mice expressing PKA-immune SK2 channels to determine: i) the sub-spine distribution of SK2 channels; ii) the SK2 and NMDAr contributions to the EPSP; iii) the spine Ca2+ transient; iv) CA1 viability. 4. The aberrantly sustained ischemia-induced loss of synaptic SK2 channels results in ischemic LTP (iLTP) that shifts ?m, the modification threshold, to higher stimulus frequencies and impairs further potentiation. Maintained expression of functional synaptic SK2 channels prevents iLTP and normalizes ?m. We will measure the long-term effects of CA/CPR-induced ischemia on synaptic plasticity. PUBLIC HEALTH RELEVANCE: Heart attack and the consequent cerebral ischemia is one of the leading causes of death and disability in the United States and, unfortunately, there are currently no drugs available that improve outcome following severe heart attack requiring cardio-pulmonary resuscitation. SK2 channels, one type of Ca2+- activated K+ channel, are anatomically and functionally poised to ameliorate brain damage following stroke. The proposed studies will demonstrate the neuroprotective role of SK2 channels and suggest novel interventional strategies to protect the brain following heart attack, improving survival, diminishing memory deficits, and improving quality of life.