Roles of SN27 in regulating glutamate receptors during neurodegeneration

描述（由申请人提供）：谷氨酸受体（GluRs）的失调已经在各种神经退行性疾病中被观察到，如阿尔茨海默病（AD）和唐氏综合症（DS）。然而，导致GluR变化的潜在机制以及GluR失调对神经退行性变的贡献在很大程度上仍然是难以捉摸的。在我们的初步研究中，我们发现分类连接蛋白27 （SNX27）可以与GluRs相互作用，并控制其运输到质膜进行再循环。SNX27在大脑中大量表达，SNX27纯合敲除（KO）小鼠表现出与AD和DS患者相似的严重神经病变。SNX27杂合型KO小鼠存在记忆缺陷，其神经元GluR水平降低和突触功能障碍。退行性痴呆是由21号染色体的额外拷贝引起的，这条染色体上的基因过量。我们发现，在DS患者的大脑中，21号染色体上的一个负调控转录因子C/EBPβ的microRNA基因miR-155上调，同时伴有C/EBPβ和SNX27的减少。我们还发现C/EBPβ正调控SNX27的表达。这些结果揭示了miR-155/C/EBPβ/SNX27通路在DS中导致GluR失调和突触功能障碍。由于晚期退行性痴呆患者出现AD样病理和认知缺陷，我们研究了SNX27对β-淀粉样蛋白（Aβ）生成的影响。β-淀粉样蛋白是AD发病的元凶，是由β-淀粉样蛋白前体蛋白通过β-和γ-分泌酶的序列切割而产生的。我们发现SNX27的过表达降低了Aβ的产生，也降低了γ-分泌酶的水平和活性。因此，我们假设SNX27在调节GluRs和突触功能中发挥重要作用，并且SNX27缺乏可能导致DS和AD的神经退行性变。在本研究中，我们将通过以下方法进一步确定SNX27在DS中的参与作用：(1)证实SNX27及其调节剂（miR155和C/EBPβ）在初级神经元中对GluRs的调控；(2)利用病毒系统调节SNX27缺陷小鼠和Ts65Dn小鼠的miR155、C/EBPβ和SNX27水平，研究是否可以改善这些小鼠的受损表型；(3)生成脑特异性SNX27转基因（Tg）小鼠，与Ts65Dn小鼠杂交，研究SNX27过表达对病理的改善作用。此外，我们将探讨SNX27在AD中的作用。我们将：(1)调节SNX27的水平，并确定γ-分泌酶水平/运输/活性和APP加工/Aβ生成是否以及如何受到影响；(2)研究SNX27是否受a β介导的神经毒性的影响和参与，并检测AD脑组织中SNX27的变化；(3) SNX27 Tg小鼠与Tg2576 AD小鼠杂交，试图挽救AD小鼠受损的表型。这些结果将阐明SNX27在DS和AD中调控GluRs和突触功能的作用，为疾病的发病/病理提供新的分子机制。

DESCRIPTION (provided by applicant): Dysregulation of glutamate receptors (GluRs) has been observed in various neurodegerative diseases such as Alzheimer's disease (AD) and Down Syndrome (DS). However, the underlying mechanisms responsible for the changes in GluRs and the contribution of GluR dysregulation to neurodegeneration have remained largely elusive. In our preliminary studies, we found that the Sorting Nexin 27 (SNX27) protein can interact with GluRs and govern their trafficking to the plasma membrane for recycling. SNX27 is abundantly expressed in the brain and SNX27 homozygous knockout (KO) mice exhibit severe neuropathologies resembling those found in AD and DS patients. SNX27 heterozygous KO mice have memory deficits and their neurons have reduced GluR levels and synaptic dysfunction. DS is caused by an extra copy of chromosome 21 which results in over- dosage of the genes on this chromosome. We found that in DS patient brains, a microRNA gene on chromosome 21, miR-155, which negatively regulates the transcription factor C/EBPβ, was upregulated, accompanied by a concomitant reduction of C/EBPβ and SNX27. We also show that C/EBPβ positively regulates SNX27 expression. These results reveal a miR-155/C/EBPβ/SNX27 pathway that leads to GluR dysregulation and synaptic dysfunction in DS. Since late stage DS patients develop AD-like pathologies and cognitive deficits, we studied the effect of SNX27 on the generation of β-amyloid (Aβ), which is the primary culprit in AD pathogenesis and is derived from β-amyloid precursor protein through sequential cleavages by β- and γ-secretases. We found that overexpression of SNX27 reduces Aβ production and the level and activity of γ-secretase as well. Therefore, we hypothesize that SNX27 plays an important role in regulating GluRs and thus synaptic function, and that SNX27 deficiency may contribute to neurodegeneration in DS and AD. In this proposal, we will further ascertain the participation of SNX27 in DS by: (1) corroborating the regulation of GluRs by SNX27 and its modulators (miR155 and C/EBPβ) in primary neurons; (2) using virus systems to modulate miR155, C/EBPβ, and SNX27 levels in SNX27 deficient mice and Ts65Dn mice, and study whether the impaired phenotypes in these mice can be ameliorated; and (3) generating brain-specific SNX27 transgenic (Tg) mice and crossing them with Ts65Dn mice to study any amelioration of pathology through SNX27 overexpression. Moreover, we will explore the role of SNX27 in AD. We will: (1) modulate the levels of SNX27 and determine whether and how γ-secretase level/trafficking/activity and APP processing/Aβ generation are affected; (2) study whether SNX27 is affected by and involved in Aβ-mediated neurotoxicity and examine any changes in SNX27 in AD brains; and (3) cross SNX27 Tg mice with Tg2576 AD mice and attempt to rescue impaired phenotypes in AD mice. These results will elucidate the role of SNX27 in regulating GluRs and synaptic functions in DS and AD, providing novel molecular mechanisms for disease pathogenesis/pathology.