Trans-synaptic signaling through GRID1/glutamate receptor delta-1 and CBLN1/cerebellin-1 facilitates autophagic flux in central amygdala and prevents chronic pain

Central amygdala (CeA) is a component of the spino-parabrachio-amygdala nociceptive pathway. Neuroplasticity in this pathway, such as increased cellular excitability and excitatory neurotransmission, play a key role in the development and persistence of chronic pain. However, the underlying mechanisms of neuroplastic changes in the CeA remain poorly understood. We recently demonstrated that GRID1/GluD1 (glutamate ionotropic receptor delta type subunit 1) and its binding partner CBLN1 (cerebellin 1 precursor) are downregulated in models of inflammatory and neuropathic pain. Furthermore, we have shown that GRID1-CBLN1 signaling regulates autophagy in multiple brain regions. Here, we tested the causal relationship between GRID1-CBLN1 downregulation, macroautophagy impairment, and subsequent hyperalgesia, using models of inflammatory and neuropathic pain. During pain, the downregulation of GRID1 and CBLN1 was accompanied by neuroplastic changes, as evidenced by an increase in excitatory neurotransmission and AMPA receptor (AMPAR) expression. In addition, significant downregulation of BECN1 and upregulation of SQSTM1 and MAP1LC3B, demonstrating impaired autophagic flux, were observed in the pain state. These changes appear to be cell type-specific, as observed by the higher localization of BECN1 and LAMP1, a marker for autolysosomes, to PRKCD+ neurons, in which GRID1 is preferentially expressed. Using a GRID1 C-terminal-derived peptide (Tat-HRSPN), we demonstrate that GRID1 directly facilitates autophagy and subsequently reduces AMPAR expression in normal animals. This effect may be attributable to the direct interaction of GRID1 with mediators of autophagy, such as neuronal (n)GOPC/nPIST, BECN1 and LAMP1. Together, these results identified a novel GRID1/GluD1-dependent trans-synaptic autophagy mechanism, the deficit of which drives chronic pain. Abbreviations: aCSF: artificial cerebro spinal fluid; AMPARs: α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors; ATG: autophagy related; BAPTA: 1,2-bis(o-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid; BECN1: beclin 1; CBLN1: cerebellin 1 precursor; CeA: central amygdala; CFA: complete Freund’s adjuvant; CRH/CRF: corticotropin releasing hormone; CTD: carboxy-terminal domain; DHPG: dihydroxy phenyl glycine; GOPC/PIST: golgi associated PDZ and coiled-coil motif containing; GRID1/GluD1: glutamate ionotropic receptor delta type subunit 1; GRIN/NMDAR: glutamate ionotropic receptor NMDA type; iGluRs: ionotropic glutamate receptors; KO: knockout; LA/BLA; lateral amygdala/baso-lateral amygdala; LAMP1: lysosomal associated membrane protein 1; LTD: long-term depression; MAP1LC3: microtubule associated protein 1 light chain 3; mEPSCs: miniature excitatory post-synaptic currents; GRM: glutamate metabotropic receptor; MTOR: mechanistic target of rapamycin kinase; nGOPC/nPIST: neuronal GOPC/PIST; NRXN1a: neurexin 1 alpha; PB: parabrachial nucleus; PBS: phosphate-buffered saline; PB-CeLC: parabrachio-central laterocapsular amygdala; PI3K: phosphoinositide 3-kinase; PRKCD+: protein kinase C delta positive; rCBLN1: recombinant CBLN1; SDS-PAGE: sodium dodecyl sulfate-polyacrylamide gel electrophoresis; SQSTM1: sequestosome 1; SNL: spinal nerve ligation; SST+: somatostatin positive; TBD11: Tat-beclin 1 D11; TBST: Tris-buffered saline with Tween 20; WT: wild type.中央杏仁核（CeA）是脊髓-臂旁-杏仁核伤害感受通路的组成部分。该通路的神经可塑性（如细胞兴奋性提升与兴奋性神经传递增强）在慢性疼痛的发生与维持过程中发挥关键作用。然而，中央杏仁核内神经可塑性变化的潜在机制仍未被充分阐明。我们此前的研究证实，GRID1/GluD1（离子型谷氨酸受体δ亚基1，glutamate ionotropic receptor delta type subunit 1）与其结合蛋白CBLN1（小脑肽1前体，cerebellin 1 precursor）在炎症性与神经性疼痛模型中均表达下调。此外，我们的研究发现，GRID1-CBLN1信号通路可调控多个脑区的自噬过程。本研究借助炎症性与神经性疼痛模型，探究了GRID1-CBLN1表达下调、巨自噬受损与后续痛觉超敏之间的因果关系。 在疼痛状态下，GRID1与CBLN1的表达下调伴随神经可塑性变化，具体表现为兴奋性神经传递增强与AMPA受体（AMPAR，α-氨基-3-羟基-5-甲基-4-异恶唑丙酸受体）表达上调。此外，疼痛状态下可观察到BECN1表达显著下调，而SQSTM1与MAP1LC3B表达上调，该现象提示自噬流受损。上述变化呈现细胞类型特异性：GRID1优先表达于PRKCD+（蛋白激酶Cδ阳性）神经元，而BECN1与自噬溶酶体标志物LAMP1（溶酶体相关膜蛋白1，lysosomal associated membrane protein 1）在该类神经元中的定位显著升高，印证了这一特异性。 我们通过GRID1 C端衍生肽（Tat-HRSPN）实验证实，在正常动物体内，GRID1可直接促进自噬，并随后降低AMPA受体的表达水平。该效应可能源于GRID1与自噬调控因子——例如神经元型GOPC/nPIST（neuronal GOPC/PIST）、BECN1及LAMP1——的直接相互作用。综上，本研究揭示了一种全新的、依赖GRID1/GluD1的跨突触自噬机制，该机制的缺陷可诱发慢性疼痛。 缩写说明： aCSF：人工脑脊液（artificial cerebrospinal fluid） AMPARs：α-氨基-3-羟基-5-甲基-4-异恶唑丙酸受体（α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors） ATG：自噬相关基因（autophagy related） BAPTA：1,2-双(邻氨基苯氧基)乙烷-N,N,N′,N′-四乙酸（1,2-bis(o-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid） BECN1：Beclin 1 CBLN1：小脑肽1前体（cerebellin 1 precursor） CeA：中央杏仁核（central amygdala） CFA：完全弗氏佐剂（complete Freund’s adjuvant） CRH/CRF：促肾上腺皮质激素释放激素（corticotropin releasing hormone） CTD：羧基末端结构域（carboxy-terminal domain） DHPG：二羟基苯甘氨酸（dihydroxy phenyl glycine） GOPC/PIST：含PDZ与卷曲螺旋基序的高尔基体相关蛋白（golgi associated PDZ and coiled-coil motif containing） GRID1/GluD1：离子型谷氨酸受体δ亚基1（glutamate ionotropic receptor delta type subunit 1） GRIN/NMDAR：离子型谷氨酸受体NMDA型（glutamate ionotropic receptor NMDA type） iGluRs：离子型谷氨酸受体（ionotropic glutamate receptors） KO：基因敲除（knockout） LA/BLA：外侧杏仁核/基底外侧杏仁核（lateral amygdala/baso-lateral amygdala） LAMP1：溶酶体相关膜蛋白1（lysosomal associated membrane protein 1） LTD：长时程抑制（long-term depression） MAP1LC3：微管相关蛋白1轻链3（microtubule associated protein 1 light chain 3） mEPSCs：微小兴奋性突触后电流（miniature excitatory post-synaptic currents） GRM：代谢型谷氨酸受体（glutamate metabotropic receptor） MTOR：雷帕霉素机制靶蛋白激酶（mechanistic target of rapamycin kinase） nGOPC/nPIST：神经元型GOPC/PIST（neuronal GOPC/PIST） NRXN1a：神经连接蛋白1α（neurexin 1 alpha） PB：臂旁核（parabrachial nucleus） PBS：磷酸盐缓冲液（phosphate-buffered saline） PB-CeLC：臂旁核-中央杏仁核外侧壳部（parabrachio-central laterocapsular amygdala） PI3K：磷脂酰肌醇3-激酶（phosphoinositide 3-kinase） PRKCD+：蛋白激酶Cδ阳性（protein kinase C delta positive） rCBLN1：重组CBLN1（recombinant CBLN1） SDS-PAGE：十二烷基硫酸钠-聚丙烯酰胺凝胶电泳（sodium dodecyl sulfate-polyacrylamide gel electrophoresis） SQSTM1：SQSTM1（自噬相关蛋白，sequestosome 1） SNL：脊髓神经结扎（spinal nerve ligation） TBD11：Tat-Beclin 1 D11 TBST：含Tween 20的Tris缓冲液（Tris-buffered saline with Tween 20） WT：野生型（wild type）