Connecting reticulophagy and neuronal NTRK2/TrkB signaling

ABSTRACT

Tightly regulated cell surface expression of NTRK2/TrkB provides a mechanism for fine-tuning cellular responses to the neurotrophic factor BDNF. Recently, the degradation of NTRK2 by reticulophagy has been identified as a mechanism to limit its availability for trafficking to the cell membrane. The ER-chaperone CANX (calnexin) delivers NTRK2 to the reticulophagy receptor RETREG1/Fam134b for lysosomal degradation. Upon phosphorylation of CANX, NTRK2 is released from this complex, which facilitates its cell surface transport. These results identify a novel role for CANX in regulating the cell surface expression of NTRK2 and imply a function for reticulophagy that goes beyond regulating the degradation of misfolded proteins within the ER.

Signaling in the nervous system is highly controlled at the cellular level to meet the needs of synaptic plasticity. Such plasticity mechanisms include the regulated transport of neurotransmitter receptor components to synaptic sites on the cell surface, but also the removal of such molecules to extra- and intracellular sites. Several lines of evidence indicate that NTRK2 (neurotrophic receptor tyrosine kinase 2), the receptor for BDNF (brain derived neurotrophic factor), a major signaling molecule for synaptic plasticity, is regulated by precise mechanisms that determine the level and location of this receptor at the cell surface. For example, medium spiny neurons in the striatum elevate NTRK2 cell surface levels when they receive dopaminergic input via DRD1 (dopamine receptor D1) signaling. Without dopaminergic input, NTRK2 remains in the endoplasmic reticulum (ER). This raises the question of how NTRK2 is removed from the ER under conditions of long-term deprivation of dopaminergic input, for example in Parkinson disease, or during development when the NTRK2 function in establishing cortical neuronal networks switches toward a function in regulating plasticity at individual synapses.

Transactivation of NTRK receptors is a cellular mechanism to activate an intracellular pool of these receptors in the absence of their ligands. Our previous work established a critical role for the transactivation of NTRK2 by EGF (epidermal growth factor) during cerebral development in mice. EGF-mediated transactivation of NTRK2 has an essential function for the migration of early cortical neurons at developmental stages when BDNF, the ligand for NTRK2 is not yet expressed at the high levels that are found in the adult nervous system. Transactivation by EGFR (epidermal growth factor receptor) signaling also triggers the rapid trafficking of intracellular NTRK2 from the ER to the cell surface, increasing the availability of NTRK2 for BDNF. Recently, we identified a novel role for the ER chaperone CANX regulating the transport of NTRK2 to the cell surface [1]. Upon EGFR stimulation, CANX is phosphorylated, which results in the release of NTRK2, enabling the trafficking to the cell membrane. In the absence of signaling by EGFR, CANX delivers NTRK2 to RETREG1 for lysosomal turnover by reticulophagy (Figure 1).

Figure 1.

Scheme of the proposed mechanism for the reticulophagy of NTRK2. In the absence of signaling by EGFR, CANX and RETREG1 sequester NTRK2 for lysosomal degradation (left side). Upon stimulation with EGF, MAPK phosphorylates CANX, which leads to the release of NTRK2 and its subsequent transport to the cell surface (right side). Limiting the cell surface transport of NTRK2 by reticulophagy represents a mechanism to fine-tune the sensitivity for BDNF.

Reticulophagy is a specialized form of macroautophagy/autophagy, defined by the constitutive or regulated clearance of ER portions by lysosomal degradation. Reticulophagy depends on reticulophagy receptors located in the ER membrane linking the ER fragment designated for degradation to LC3s, GABARAPs, or Atg8 localized at the limiting membrane of phagophores. Previous work suggested a central role for CANX in directing misfolded proteins to ER-to-lysosome-associated degradation/ERLAD by acting as a cargo receptor for the reticulophagy receptor RETREG1. Due to its lack of an ER lumenal region, RETREG1 requires the interaction with an additional membrane-embedded protein that directs selected proteins within the ER lumen to reticulophagy-mediated degradation. Our recent study extends the role of CANX as a cargo receptor, which delivers NTRK2 to ER-to-lysosome-associated degradation.

In CANX-depleted cortical precursor cells, we found an enhanced responsiveness to EGF, leading to increased NTRK2 transactivation. This effect occurs at first as a consequence of elevated EGFR cell surface levels, resulting in an increase of phosphorylated EGFR and its downstream signaling intermediates. Despite the increased transactivation of NTRK2, we found an impaired EGF-induced cell surface transport of NTRK2 in CANX-depleted cells, leading to a clustering of NTRK2 in ER structures. Expression of wild-type CANX and the phosphorylation mutant CANX^S543A,S544A, but not CANX^S563A, restores the cell surface transport of NTRK2. This phosphorylation site in the cytosolic tail of CANX is a known target of MAPK that is activated downstream of EGFR activation. These results suggest that downstream signaling from EGFR triggers the phosphorylation of CANX, which causes the release of NTRK2 and its subsequent cell surface translocation.

The lack of NTRK2 surface translocation in CANX-deficient cells raises the question of whether NTRK2 accumulates in the ER or is processed for degradation in a separate compartment. Based on the clustering of NTRK2 in CANX-deficient neuronal precursor cells and the recently described role of CANX as a cargo receptor for reticulophagy we hypothesized that NTRK2 might get sequestered to lysosomal degradation by reticulophagy. In wild-type cells, triple labeling of LC3, ER, and NTRK2 revealed that a subset of intracellular NTRK2 clusters stains positive for LC3 and ER markers, suggesting that NTRK2 is sequestered from the ER to autophagosomes. In addition, blocking lysosomal turnover by bafilomycin A₁ treatment results in a marked enrichment of total NTRK2 in protein lysates, further supporting the notion that NTRK2 is delivered to lysosomes. In CANX-deficient cells, NTRK2 appears close to LC3⁺ structures and we detect a reduced number of NTRK2⁺ autolysosomes upon blocking lysosomal function. These results indicate an impaired delivery of NTRK2 to autophagosomal/lysosomal degradation in the absence of CANX. However, we do not detect an increase in the total levels of NTRK2 in CANX-deficient cells, which we expected under conditions with defective degradation. Instead, we find reduced mRNA expression levels of NTRK2 in CANX-deficient cells, providing evidence for a possible compensatory mechanism preventing the accumulation of NTRK2 during conditions with a defective turnover. Mechanistically, we show that CANX delivers NTRK2 to RETREG1 for reticulophagy-mediated lysosomal degradation under basal conditions. Upon stimulation with EGF, phosphorylation of CANX by MAPK leads to the release of NTRK2, which enables the transport to the cell membrane.

In summary, our data demonstrate that reticulophagy represents a mechanism to fine-tune the availability of NTRK2 for its ligand BDNF, which ensures a spatially and temporally well-defined switch from NTRK2 transactivation to signaling via BDNF in early cortical neurons during cerebral development. Similarly, reticulophagy might contribute to early synaptic defects in neurodegenerative disorders such as Parkinson disease. These findings also imply an additional role of CANX, going beyond the degradation of misfolded protein. Depending on the physiological demand, CANX “decides” whether intact proteins are sorted toward autophagosomal and lysosomal processing and degradation or rapid transport to the cell surface.

Funding Statement

P.L. was supported by Deutsche Forschungsgemeinschaft (DFG) grant DFG LU 2347/3-1. M.S. received support from the DFG through project 424778381 (SFB-TRR 295, A05) and projects SE 697/5-2 and SE 697/7-1.

Disclosure statement

No potential conflict of interest was reported by the author(s).