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.<b>Abbreviations</b>: 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.