SorCS1-mediated sorting in dendrites maintains neurexin axonal surface polarization required for synaptic function

Abstract

The pre- and postsynaptic membranes comprising the synaptic junction differ in protein composition. The membrane trafficking mechanisms by which neurons control surface polarization of synaptic receptors remain poorly understood. The sorting receptor Sortilin-related CNS expressed 1 (SorCS1) is a critical regulator of trafficking of neuronal receptors, including the presynaptic adhesion molecule neurexin (Nrxn), an essential synaptic organizer. Here, we show that SorCS1 maintains a balance between axonal and dendritic Nrxn surface levels in the same neuron. Newly synthesized Nrxn1α traffics to the dendritic surface, where it is endocytosed. Endosomal SorCS1 interacts with the Rab11 GTPase effector Rab11 family-interacting protein 5 (Rab11FIP5)/Rab11 interacting protein (Rip11) to facilitate the transition of internalized Nrxn1α from early to recycling endosomes and bias Nrxn1α surface polarization towards the axon. In the absence of SorCS1, Nrxn1α accumulates in early endosomes and mispolarizes to the dendritic surface, impairing presynaptic differentiation and function. Thus, SorCS1-mediated sorting in dendritic endosomes controls Nrxn axonal surface polarization required for proper synapse development and function.

Fig 1. SorCS1-mediated sorting controls an axonal–dendritic surface balance of Nrxn1α.

(A) DIV9 Sorcs1^HA mouse cortical neurons immunostained for endogenous (endog.) HA-SorCS1 (grayscale and green) and MAP2 (blue). Red arrowheads indicate the axon, and the blue asterisk marks the cell body. High-zoom images show dendritic (dotted blue box) and axonal (dotted red box) distribution of HA-SorCS1. (B) DIV10 WT mouse hippocampal neurons transfected with HA-SorCS1 and immunostained for HA (grayscale). (C) Quantification of panels A and B: dendritic versus axonal distribution (D:A–polarity index) of endogenous (DIV9, n = 26 neurons and DIV14, n = 26) and exogenous (exog.; n = 14) HA-SorCS1 from 3 and 2 independent experiments, respectively. (D) DIV8 to DIV10 Sorcs1^flox/flox mouse cortical neurons electroporated with EGFP (Ctr), Cre-EGFP, Cre-EGFP-T2A-SorCS1^WT, or Cre-EGFP-T2A-SorCS1^Y1132A and transfected with HA-Nrxn1α, immunostained for surface (s.) HA-Nrxn1α (grayscale and green) and MAP2 (blue). (E) Quantification of panel D: surface HA-Nrxn1α fluorescence intensity in axon and dendrites relative to total surface levels and normalized to cells expressing EGFP, and ratio of axonal–dendritic surface HA intensity. Ctr (n = 27 neurons); Cre (n = 29); Cre_SorCS1^WT (n = 28); Cre_SorCS1^Y1132A (n = 28). ***P < 0.001 (Kruskal-Wallis test followed by Dunn’s multiple comparisons test, 3 independent experiments). Underlying numerical values can be found in S1 Data. Graphs show mean ± SEM. Scale bars, 20 μm (panel B); 5 μm (panel A [high-zoom], panel D). Cre, Cre recombinase; Ctr, control; DIV, days in vitro; EGFP, enhanced green fluorescent protein; HA, hemagglutinin; MAP2, microtubule-associated protein 2; Nrxn, neurexin; SorCS1, Sortilin-related CNS expressed 1; WT, wild type.

Fig 2. Indirect axonal trafficking of Nrxn1α in mature neurons.

(A) Nonpermeabilized DIV3, DIV7, and DIV11 Nrxn1α^HA cortical neurons live-labeled with an HA antibody to visualize endogenous surface (s.) HA-Nrxn1α (grayscale) in axon and dendrites. Red arrowheads indicate the axon, and the blue asterisk marks the cell body. (B) Quantification of panel A: surface HA-Nrxn1α fluorescence intensity in axon and dendrites normalized to DIV3 neurons and the ratio of axonal–dendritic surface HA-Nrxn1α intensity. DIV3 (n = 30 neurons); DIV7 (n = 29); DIV11 (n = 24). **P < 0.01; ***P < 0.001 (Kruskal-Wallis test followed by Dunn’s multiple comparisons test, 3 independent cultures). (C) Schematic representation of streptavidin-KDEL (ER hook) and reporters (Nrxn1α and TfR) used in RUSH experiments. Addition of biotin dissociates the reporters from the ER hook, inducing synchronous release from the ER and transport through the secretory pathway. (D) DIV9 WT mouse cortical neuron co-expressing SBP-EGFP-Nrxn1α and streptavidin-KDEL immunostained for MAP2 (blue), Ankyrin-G (red), and EGFP-Nrxn1α (grayscale and green) at t = 0 before adding biotin. (E) Live-cell imaging in DIV8 to DIV10 WT rat cortical neurons co-expressing SBP-EGFP-Nrxn1α and ER hook. After 24 to 31 h of expression, neurons were imaged every 5 min for 2.5 h. Biotin was added 10 min after the beginning of the imaging session. Shown are representative images of SBP-EGFP-Nrxn1α fluorescence in dendrites and axon before (t0) and 30, 60, 90 and 120 min after adding biotin. Red arrowheads indicate axon and black arrows indicate SBP-EGFP-Nrxn1α-positive puncta. See also S1 Movie. (F–I) Live-cell imaging in DIV8 to DIV10 WT rat cortical neurons co-expressing SBP-EGFP-Nrxn1α and ER hook. After 21 to 30 h of expression, neurons were imaged every second for 60 to 120 s either 20 to 34 min or 1 to 2 h after adding biotin. See also S2 Movie and S3 Movie. (F) Kymographs illustrating EGFP-Nrxn1α vesicle dynamics over a 60 s period in dendrites (D) from neurons treated with biotin for either 25 min or 2 h. (G) Mean number of motile and nonmotile EGFP-Nrxn1α vesicles in dendrites from neurons treated with biotin for either 20 to 34 min (n = 17 neurons) or 1 to 2 h (n = 16) in 2 and 3 independent experiments, respectively. *P < 0.05 (Mann-Whitney test). (H) Kymographs illustrating EGFP-Nrxn1α vesicle dynamics over a 60 s period in axons (A) from neurons treated with biotin for either 25 min or 2 h. (I) Mean number of motile and nonmotile EGFP-Nrxn1α vesicles in axons. ***P < 0.001 (Mann-Whitney test). Underlying numerical values can be found in S1 Data. Graphs show mean ± SEM. Scale bars, 20 μm (panels A and D); 10 μm (panel E). AnkG, Ankyrin-G; DIV, days in vitro; ER, endoplasmic reticulum; EGFP, enhanced green fluorescent protein; HA, hemagglutinin; KDEL, endoplasmic reticulum retention signal KDEL; KI, knock in; MAP2, microtubule-associated protein 2; Nrxn, neurexin; RUSH, retention using selective hooks; SBP, streptavidin-binding protein; TfR, transferrin receptor; WT, wild type.

Fig 3. SorCS1 interacts with Rab11FIP5/Rip11 to regulate Nrxn1α transition from EEs to REs.

(A) DIV8 to DIV10 Sorcs1^flox/flox cortical neurons electroporated with mCherry (Ctr) or Cre-T2A-mCherry and transfected with HA-Nrxn1α (pulse-chased for 20 min), labeled for internalized (int.) HA-Nrxn1α (grayscale and green) and EEA1 (grayscale and red). (B) Quantification of panel A: number of internalized Nrxn1α- and EEA1-double-positive puncta normalized to cells expressing mCherry and intensity of internalized Nrxn1α fluorescence in the double-positive puncta normalized to cells expressing mCherry. Ctr (n = 26 neurons); Cre (n = 25). *P < 0.05; ***P < 0.001 (Mann-Whitney test, 3 independent experiments). (C) DIV8 to DIV10 Sorcs1^flox/flox cortical neurons electroporated with mCherry (Ctr) or Cre-T2A-mCherry and co-transfected with HA-Nrxn1α and EGFP-Rab4 (pulse-chased for 25 min), labeled for internalized HA-Nrxn1α (grayscale and green) and EGFP-Rab4 (grayscale and red). (D) Quantification of panel C; Ctr (n = 28 neurons); Cre (n = 26). **P < 0.01 (Mann-Whitney test, 3 independent experiments). (E) DIV8 to DIV10 Sorcs1^flox/flox cortical neurons electroporated with mCherry (Ctr) or Cre-T2A-mCherry and co-transfected with HA-Nrxn1α and EGFP-Rab11 (pulse-chased for 40 min), labeled for internalized HA-Nrxn1α (grayscale and green) and EGFP-Rab11 (grayscale and red). (F) Quantification of panel E; Ctr (n = 29 neurons); Cre (n = 23). *P < 0.05 (Mann-Whitney test, 3 independent experiments). (G) DIV8 to DIV10 Sorcs1^flox/flox cortical neurons electroporated with mCherry (Ctr) or Cre-T2A-mCherry and co-transfected with HA-Nrxn1α and EGFP-Rab11 (pulse-chased for 90 min), labeled for internalized HA-Nrxn1α (grayscale and green) and EGFP-Rab11 (grayscale and red). (H) Quantification of panel G; Ctr (n = 28 neurons); Cre (n = 26). ***P < 0.001 (Mann-Whitney test, 3 independent experiments). (I) Western blot for the recovery of Rab11FIP5/Rip11 in immunoprecipitated HA-SorCS1 complexes from P21–P28 Sorcs1^HA cortical prey extracts. See S9 Fig for raw uncropped blots. (J) DIV9 to DIV10 WT cortical neurons co-expressing EGFP-Rip11 and SorCS1cβ-myc immunostained for EGFP-Rip11 (grayscale and green), SorCS1-myc (grayscale and red) and MAP2 (blue). (K) Quantification of the colocalization of Rip11 with SorCS1 expressed as Manders coefficient (n = 20 neurons) in 2 independent experiments. (L) DIV9 WT cortical neurons co-expressing HA-Nrxn1α and EGFP (Ctr), WT EGFP-Rip11 or DN EGFP-Rip11, and immunostained for surface (s.) HA-Nrxn1α (grayscale). Red arrowheads indicate the axon, and the blue asterisk marks the cell body. (M) Quantification of panel L: surface HA-Nrxn1α fluorescence intensity in axon and dendrites relative to total surface levels and normalized to cells expressing EGFP and ratio of axonal–dendritic surface HA intensity (n = 30 neurons for each group). *P < 0.05; ***P < 0.001 (Kruskal-Wallis test followed by Dunn’s multiple comparisons test, 3 independent experiments). Underlying numerical values can be found in S1 Data. Graphs show mean ± SEM. Scale bars, 5 μm (panels G and J); 20 μm (panel L). Cre, Cre recombinase; Ctr, control; DIV, days in vitro; DN, dominant negative; EE, early endosome; EEA1, early endosome antigen 1; EGFP, enhanced green fluorescent protein; HA, hemagglutinin; MAP2, microtubule-associated protein 2; Nrxn, neurexin; Rab, Rab GTPase; RE, recycling endosome; Rip11, Rab11 interacting protein; SorCS1, Sortilin-related CNS expressed 1; WT, wild type.

Fig 4. SorCS1-mediated axonal surface polarization of Nrxn is required for presynaptic differentiation.

(A) DIV8 to DIV10 WT mouse cortical neurons transfected with WT HA-Nrxn1α and a cytoplasmic deletion mutant lacking the 4.1-binding motif (HA-Nrxn1α Δ4.1) and immunostained for surface (s.) HA-Nrxn1α (grayscale). Red arrowheads indicate the axon, and the blue asterisk marks the cell body. High-zoom images show dendritic (D, dotted blue box) and axonal (A, dotted red box) HA-Nrxn1α. (B) Quantification of panel A: surface HA-Nrxn1α fluorescence intensity in axon and dendrites relative to total surface levels and normalized to cells expressing WT-Nrxn1α and ratio of axonal–dendritic surface HA intensity. WT (n = 30 neurons); Δ4.1 (n = 29). ***P < 0.001 (Mann-Whitney test, 3 independent experiments). (C) DIV8 to DIV10 mouse Sorcs1^flox/flox cortical neurons electroporated with EGFP (Ctr) or Cre-EGFP (Cre) and transfected with WT HA-Nrxn1α or HA-Nrxn1α Δ4.1. Neurons were immunostained for surface HA-Nrxn1α (grayscale). (D) Quantification of panel C: ratio of axonal–dendritic surface HA-Nrxn1α fluorescence intensity. Ctr_WT (n = 46 neurons); Ctr_Δ4.1 (n = 25); Cre_WT (n = 30); Cre_Δ4.1 (n = 27). **P < 0.01; ***P < 0.001 (Kruskal-Wallis test followed by Dunn’s multiple comparisons test, at least 3 independent experiments). (E) DIV8 to DIV10 WT mouse cortical neurons transfected with WT HA-Nrxn1α or HA-Nrxn1α Δ4.1 and treated with DMSO (vehicle) or Dynasore. Neurons were immunostained 18 h after treatment for surface HA-Nrxn1α (grayscale). (F) Quantification of panel E: ratio of axonal–dendritic surface HA fluorescence intensity. WT_DMSO (n = 40 neurons); Δ4.1_DMSO (n = 40); Δ4.1_Dynasore (n = 28). ***P < 0.001 (Kruskal-Wallis test followed by Dunn’s multiple comparisons test, at least 3 independent experiments). (G) HEK293T-cells expressing FLAG-Nlgn1 co-cultured with DIV10 Sorcs1^flox/flox cortical neurons infected with LV expressing mCherry (Control), Cre-T2A-mCherry, Cre-T2A-mCherry-T2A-WT Nrxn1α, Cre-T2A-mCherry-T2A-Nrxn1α Δ4.1, or Nrxn TKD; immunostained for FLAG (blue) and Synapsin1 (grayscale and green). (H) Quantification of panel G: the area of Synapsin1 clustering on the surface of Nlgn1-expressing HEK cells and normalized to cells expressing mCherry. Control (n = 29 neurons); Cre (n = 30); Cre-WT (n = 30); Cre-Δ4.1 (n = 30); TKD Nrxns (n = 30). *P < 0.05; ***P < 0.001 (Kruskal-Wallis test followed by Dunn’s multiple comparisons test, 3 independent experiments). (I) HEK293T-cells expressing NGL-3-EGFP co-cultured with DIV10 Sorcs1^flox/flox cortical neurons infected with LV expressing mCherry (Control) or Cre-T2A-mCherry and immunostained for EGFP (blue) and Synapsin1 (grayscale and green). (J) Quantification of panel I. Control (n = 30 neurons); Cre (n = 30). Underlying numerical values can be found in S1 Data. Graphs show mean ± SEM. Scale bars, 20 μm (panels A, C, and E); 5 μm (panels A [high-zoom], G, and I). Cre, Cre recombinase; Ctr, control; DIV, days in vitro; EGFP, enhanced green fluorescent protein; HA, hemagglutinin; HEK293T, human embryonic kidney cells; LV, lentivirus; NGL-3, Netrin-G Ligand 3; Nrxn, neurexin; NS, nonsignificant; SorCS1, Sortilin-related CNS expressed 1; TKD, triple knock down; WT, wild type.

Fig 5. SorCS1 is required for presynaptic function.

(A) Example traces of mEPSCs recorded from DIV14 to DIV16 Sorcs1^flox/flox autaptic cortical neurons electroporated with EGFP (Control) or Cre-EGFP (Cre). (B) mEPSC frequency, but not amplitude and decay time, is decreased in Sorcs1 KO neurons. Control (n = 18 neurons); Cre (n = 15). ***P < 0.001 (Mann-Whitney test, 3 independent experiments). (C) Example traces of eEPSCs recorded from DIV14 to DIV16 Sorcs1^flox/flox autaptic cortical neurons electroporated with EGFP or Cre-EGFP. (D) eEPSC amplitude and eEPSC charge are decreased in Sorcs1 KO neurons. Control (n = 17 neurons); Cre (n = 14). *P < 0.05 (Mann-Whitney test, 3 independent experiments). (E) Example traces of sucrose responses recorded from DIV14 Sorcs1^flox/flox autaptic cortical neurons electroporated with EGFP or Cre-EGFP. (F–H) Decreased eEPSC amplitude, eEPSC charge (F) and RRP size (G) in Sorcs1 KO neurons, but unaltered Pves (H). Control (n = 17 neurons); Cre (n = 21). *P < 0.05; **P < 0.01 (Mann-Whitney test, 3 independent experiments). (I) Example traces of train stimulations (10 Hz) recorded from DIV14 to DIV16 Sorcs1^flox/flox autaptic cortical neurons electroporated with EGFP or Cre. (J) Increased depression during 10 Hz stimulation in Sorcs1 KO neurons. Control (n = 18 neurons); Cre (n = 13); 3 independent experiments. (K) Increased paired-pulse depression in Sorcs1 KO neurons throughout different interstimulation intervals. Control (n = 18 neurons); Cre (n = 14). *P < 0.05 (two-tailed t test, 3 independent experiments). (L) Estimation of the RRP size used during neuronal activity by back-extrapolating a linear fit of the steady state current towards the ordinate axis intercept, which represents the initial RRP size before train stimulations (10 Hz). RRP size of active synapses was reduced in DIV14 to DIV16 Sorcs1 KO neurons. Control (n = 18 neurons); Cre (n = 13). **P < 0.01 (Mann-Whitney test, 3 independent experiments). Underlying numerical values can be found in S1 Data. Graphs show mean ± SEM. Cre, Cre recombinase; DIV, days in vitro; eEPSC, evoked excitatory postsynaptic current; EGFP, enhanced green fluorescent protein; KO, knock out; mEPSC, miniature excitatory postsynaptic current; RRP, readily releasable pool; SorCS1, Sortilin-related CNS expressed 1.