SORLA-mediated trafficking of TrkB enhances the response of neurons to BDNF

Abstract

Stimulation of neurons with brain-derived neurotrophic factor (BDNF) results in robust induction of SORLA, an intracellular sorting receptor of the VPS10P domain receptor gene family. However, the relevance of SORLA for BDNF-induced neuronal responses has not previously been investigated. We now demonstrate that SORLA is a sorting factor for the tropomyosin-related kinase receptor B (TrkB) that facilitates trafficking of this BDNF receptor between synaptic plasma membranes, post-synaptic densities, and cell soma, a step critical for neuronal signal transduction. Loss of SORLA expression results in impaired neuritic transport of TrkB and in blunted response to BDNF in primary neurons; and it aggravates neuromotoric deficits caused by low BDNF activity in a mouse model of Huntington's disease. Thus, our studies revealed a key role for SORLA in mediating BDNF trophic signaling by regulating the intracellular location of TrkB.

Figure 1. The global BDNF-dependent proteome response is altered in SORLA-deficient neurons.

(A) A prototypic two-dimensional polyacrylamid gel of proteins from Sorl1^−/− primary cortical neurons stained with silver nitrate. Significant protein spot alterations in response to BDNF treatment (150 ng/ml for 2 days) in wild type neurons (red circles), in SORLA-deficient neurons (blue circles) or in both genotypes (green circles) as compared to untreated samples are indicated. (n = 6 biological replicates per genotype; Student’s t-test p<0.05). (B) Total number of protein spots altered in primary cortical neurons of the indicated genotypes in response to BNDF treatment as exemplified in panel A. (n = 6 biological replicates per genotype; Student’s t-test p<0.05).

Figure 2. Reduced BDNF-dependent activation of TrkB in SORLA-deficient neurons.

(A, B) Quantification of phosphorylated (p) forms of TrkB, Akt, and ERK in primary cortical neurons either non-treated (−) or treated with 150 ng/ml BDNF for 20 min (+) using Western blotting (A) and densitometric scanning of replicate blots (B). Sorl1^−/− neurons show reduced levels of pTrkB, pAKT, and pERK compared with Sorl1^+/+ cells (n = 6–12, Mann-Whitney U test). (C, D) Quantification of total levels of TrkB, Akt, and ERK in primary neurons either non-treated (−) or treated with 150 ng/ml BDNF (+) for 20 min using Western blot (C) and densitometric scanning of replicate blots (D). Detection of tubulin (tub.) served as loading controls in A and C.

Figure 3. Interaction with SORLA controls trafficking and synaptic exposure of TrkB.

(A) Colocalization of endogenous SORLA (red) and TrkB (green) in primary cortical neurons as shown by confocal immunofluorescence microscopy. Scale bar: 10 µm. (B) Co-immunoprecipitation of endogenous SORLA and TrkB from brain lysates is seen using anti-SORLA (IP anti-SORLA; lane 2) and anti-Trk antisera (IP anti-TrkB; lane 3). Panel Input (lane 1) indicates presence of endogenous SORLA and TrkB in brain lysate prior to co-immunoprecipitation. Lane 4 indicates lack co-immunoprecipitation in the absence of primary antibody (No IgG). (C) SH-SY5Y neuroblastoma cells stably overexpressing SORLA (SY5Y-S) or parental control cells (SY5Y) were transfected with expression constructs for TrkB. Subsequently, proteins at the cell surface were biotinylated and immunoprecipitated using streptavidin beads. Western blot analysis documents reduced levels of biotinylated TrkB at the cell surface in SY5Y-S compared to SY5Y cells (panel Surface). Panel Input represents levels of TrkB and SORLA in cell lysates prior to precipitation. Detection of tubulin (tub.), β-integrin (β -integ.), and PDGF-β receptor (PDGF-R) served as controls for loading and immunoprecipitation, respectively. (D) Densitometric quantification of replicate Western blots as exemplified in (C) (n = 6, Student’s t-test). (E) Subcellullar fractionations of wild type and SORLA-deficient mouse brain extracts were probed for the indicated proteins using Western blot analysis. Elevated levels of TrkB receptors (pan-Trk antibody) are seen in the synaptosomal plasma membrane (pm) fraction in SORLA-deficient (lanes 7 and 8) as compared to control brains (lanes 5 and 6). In contrast, levels of Trk receptors in the postsynaptic density (PSD) are reduced in receptor-deficient (lane 10) compared with wild type brains (lane 9). Levels of Trk in total brain lysates prior to subcellular fractionation are similar between genotypes (lanes 1–4). Detection of synaptophysin (Synapt), AMPA receptors, and PSD95 served as respective loading controls. (F) Densitometric quantification of replicate Western blots as exemplified in (E) (n = 4–6, Student’s t-test).

Figure 4. Loss of SORLA impairs vesicular trafficking of TrkB.

(A) Movement of TrkB-EGFP in neurites of cultured hippocampal neurons. Arrows indicate anterograde (red) and retrograde movement (yellow), or no movement (blue) of vesicles. Images were captured every 2 sec. (B) The cumulative distance travelled by TrkB-EGFP fusion proteins either anterogradely or retrogradely is significantly reduced in Sorl1^−/− neurons as compared to controls (n = 14–16, Student’s t-test). (C) The cumulative anterograde or retrograde speed of the TrkB-EGFP fusion protein is significantly reduced in Sorl1^−/− neurons as compared to Sorl1^+/+ cells (n = 14–16, Student’s t-test).

Figure 5. Loss of SORLA aggravates neuromotoric deficits in Huntington’s disease mice.

(A) At 10 weeks of age, Huntington’s disease mice deficient for SORLA (HD82xSorl1^−/−) display aggravated hind limb clasping compared with HD82 control littermates (HD82xSorl1^+/+). (B, C) Worsening of neuromotoric deficits due to loss of SORLA is also seen when comparing (HD82xSorl1^−/−) animals with (HD82, Sorl1^+/+) controls of either sex at 10 (B) and 18 weeks of age (C) during consecutive days of rotarod performance test (n = 8–9, Mann-Whitney-U). (D) No difference in rotarod performance is seen comparing 10 weeks-old Sorl1^+/+ and Sorl1^−/− mice matched for age and sex (n = 10, Mann-Whitney-U).