Sarm1, a negative regulator of innate immunity, interacts with syndecan-2 and regulates neuronal morphology

Abstract

Dendritic arborization is a critical neuronal differentiation process. Here, we demonstrate that syndecan-2 (Sdc2), a synaptic heparan sulfate proteoglycan that triggers dendritic filopodia and spine formation, regulates dendritic arborization in cultured hippocampal neurons. This process is controlled by sterile α and TIR motif-containing 1 protein (Sarm1), a negative regulator of Toll-like receptor 3 (TLR3) in innate immunity signaling. We show that Sarm1 interacts with and receives signal from Sdc2 and controls dendritic arborization through the MKK4-JNK pathway. In Sarm1 knockdown mice, dendritic arbors of neurons were less complex than those of wild-type littermates. In addition to acting downstream of Sdc2, Sarm1 is expressed earlier than Sdc2, which suggests that it has multiple roles in neuronal morphogenesis. Specifically, it is required for proper initiation and elongation of dendrites, axonal outgrowth, and neuronal polarization. These functions likely involve Sarm1-mediated regulation of microtubule stability, as Sarm1 influenced tubulin acetylation. This study thus reveals the molecular mechanism underlying the action of Sarm1 in neuronal morphogenesis.

Figure 1.

Sdc2 regulates dendritic arborization. (A) Knockdown of Sdc-2 in mature neurons reduces dendritic arborization. At 13 DIV, cultured hippocampal neurons were transfected with either an Sdc2 shRNA (Sdc2i) or a vector control (pSuper.neo+GFP, abbreviated as pSuper). Dendritic arbors were analyzed at 17 DIV. Because pSuper co-expresses the GFP–neo fusion and the shRNA, GFP signals were used to outline cell morphology. (B) Sdc2 expression induces dendrite outgrowth in young neurons. Sdc2 and GFP were cotransfected into hippocampal neurons at 2 DIV, and cell morphology was analyzed at 5 DIV. Only GFP signal is shown here. The number of primary dendrites and the total dendritic length of transfected neurons were measured in three independent experiments. Equal numbers of transfected neurons were analyzed. Mean values ± SEM are shown (error bars). **, P < 0.005; ***, P < 0.0005. Bars, 30 µm.

Figure 2.

Sarm1 interacts with the cytoplasmic domain of Sdc2. (A) GST–Sdc2 pull-down assay with extract prepared from P21 mouse brain and immunoblotted (IB) with Sarm1 antibody. GST alone was used as a control. (B) Co-immunoprecipitation of Sdc2 and Sarm1 from mouse brain. Sdc2 antibodies and nonimmune rabbit IgG were used in the immunoprecipitation. The precipitates were immunoblotted with Sarm1 and Sdc2 antibodies as indicated. The arrowhead indicates the position of Sarm1. Because of the heterogeneity of glycosylation, SDS-PAGE resulted in a ladder of Sdc2-immunoreactive bands. (C) Schematic of Sdc2 constructs. TM, transmembrane domain; C1, conserved region 1; V, variable region; C2, conserved region 2; Myr, myristoylation modification. (D) Sdc2 directly interacts with Sarm1. Sdc2-GST fusion proteins were used to pull down purified MBP-Sarm1 fusion protein in the presence of glutathione agarose. The presence of Sarm1 in the precipitate was examined by immunoblotting with MBP antibody. The relative amounts of GST fusions used in the pull-down assay are indicated by Coomassie blue staining. Sdc2 forms an SDS-resistant dimer through its transmembrane domain. The Sdc2 dimer and monomer are marked by the arrowhead and arrows, respectively. (E) The cytoplasmic domain of Sdc2 is sufficient for the interaction with Sarm1. HEK293T cells were cotransfected with HA-G-S2C, Myc-tagged Sarm1, and vector control, as indicated. Immunoprecipitation was performed using a Myc-tag antibody. The precipitates were then analyzed by immunoblotting with Myc and HA antibodies. The positions of HA-G-S2C and immunoglobulin light chain (IgL) are indicated. (F) Three regions (C1, V, and C2) are involved in the interaction with Sarm1. Various GST–Sdc2 fusion proteins were used to pull down Sarm1 from mouse extract. The results were immunoblotted with Sarm1 antibody. Coomassie blue stain indicated the relative amounts of GST fusions used in the pull-down assay. Molecular mass standards (kD) are indicated next to the gel blots.

Figure 3.

Sarm1 is widely expressed in rodent brain and neurons. (A) Immunoblot of Sarm1 in different mouse organs. GAPDH is used as an internal control. (B) Regional distribution of Sarm1 in mouse brain. Cx, cerebral cortex; Hi, hippocampus; St, striatum; Th, thalamus; Cb, cerebellum; BS, brain stem. α-Tubulin was used as an internal control. (C) Staining patterns of Sarm1 in mouse brain. The top right shows the merged image of the MAP2/Sarm1 double stain in the CA1 region of the hippocampus. The top left and bottom panels depict the Sarm1 patterns in brain regions including layer five of the somatosensory cortex (Cx), the posterior thalamic nuclear group (Th), and the caudate putamen of the striatum (St). 2-mo-old mice were used in A–C. (D) Developmental expression profile of Sarm1. The plotted relative Sarm1 protein expression levels were obtained by normalization to the corresponding α-tubulin protein amounts. The results are the means of three independent experiments. Error bars indicate SEM. (E) Distribution of Sarm1 protein in biochemical subcellular fractions of adult mouse brain. H, total homogenate; P1, nuclei and cell debris; S1, supernatant of P1; P2, crude synaptosomal fraction; S2, supernatant of P2; LP1, lysed synaptosomal membrane; LS1, supernatant of LP1; P3, light membrane fraction; S3, soluble cytosolic fraction. PSD-95 enriched in the P2 and LP1 fractions was used as a quality control of fraction preparation. Molecular mass standards (kD) are indicated next to the gel blots. (F) Distribution of PSD-95 (red) and Sarm1 (green) in cultured hippocampal neurons at 21 DIV. Representative high-magnification images are shown on the top right. Arrowheads indicate the Sarm1 puncta overlapping with PSD-95; arrows indicate the Sarm1 puncta adjacent to PSD-95 puncta. The percentage of overlapped Sarm1 and PSD-95 is shown on the bottom right. The original images and the overlays shifted for 1 and 1.66 µm were analyzed. Error bars indicate mean values ± SEM. **, P < 0.01; ***, P < 0.005. Bars: (C) 30 µm; (F, left) 20 µm (F, top right) 2 µm.

Figure 4.

Sarm1 is critical for dendritic arborization in cultured hippocampal neurons. (A) Sarm1 knockdown in COS-1 cells via cotransfection of Sarm1i¹, Sarm1i², or pSuper control and Myc-tagged wild-type Sarm1 or specific silent mutants resistant to Sarm1i¹ and Sarm1i² (mt1 and mt2). Immunoblotting was performed with Myc tag and α-tubulin antibodies. Molecular mass standards (kD) are indicated next to the gel blots. (B and C) Knockdown of Sarm1 in cultured hippocampal neurons. Neurons were transfected with the indicated plasmids at 0 (B) or 5 DIV (C) and immunostained with Sarm1 and GFP antibodies at 2 (B) or 9 DIV (C). Arrows point to transfected neurons. (D) Sarm1 knockdown affects dendritic arbors. At 13 DIV, cultured hippocampal neurons were transfected using the plasmids indicated. Neuronal morphology was monitored by GFP signals at 17 DIV. (E) Sholl analysis of the effect of Sarm1 knockdown. ***, P < 0.001. (F) Total dendrite length. (G) The number of primary dendrites. ***, P < 0.0005. (H) The specificity of the effect of Sarm1 knockdown on dendrite outgrowth was confirmed by two additional controls. The experiment was performed as described in D, except that both shCtrl and shLuc were also included. Representative images and analysis of total dendrite length are shown. Equal numbers of transfected neurons, as indicated, were analyzed. Means ± SEM are shown (error bars). Experiments were performed in triplicate. Bars, 30 µm.

Figure 5.

Sarm1 knockdown influences spine morphology and density in mature neurons. (A) Dendritic spine morphology. At 13 DIV, cultured hippocampal neurons were transfected with Sarm1i¹ or pSuper vector control. 4 d later, cells were fixed and stained with GFP antibodies. A region indicated by the arrows in the whole cell image is shown enlarged in on the bottom. Bars: (top) 30 µm; (bottom) 5 µm. (B) Quantification of the spine density, the width of the spine head, and the length of spines. Error bars indicate mean values ± SEM. ***, P < 0.0005.

Figure 6.

Time-lapse study of the effect of Sarm1 knockdown on dendrite morphology. Cultured hippocampal neurons were transfected with Sarm1i¹ or pSuper at 13 DIV. The images were recorded 10 min/frame for 24 h starting from 15 DIV. (A) Representative images of a control neuron (pSuper; top) and a Sarm1 knockdown neuron (Sarm1i; bottom). Bar, 30 µm. (B) Enlarged local images of neurons transfected with Sarm1i¹. Arrowheads indicate a continuously retracting dendrite, whereas the arrows denote a dendrite that outgrew first and then retracted. Bar, 5 µm. (C) The relative dendrite length of individual neurons during the recording period. (D) The growth and pruning rates of individual neurons; the rates of individual neurons are connected with a line. (E) The means of the ratio of growth rate to pruning rate (n = 5). Error bars indicate mean values ± SEM. **, P < 0.005.

Figure 7.

Sarm1 regulates development of dendrites and axons. Cultured hippocampal neurons were transfected with vector control, Sarm1i¹, or Sarm1 at 2 (A), 5 (B), 1 (D), or 0 DIV (E), and harvested for immunostaining at 6 (A), 9 (B), 3 (D), or 2 (E) DIV. (A) Sarm1 regulates dendritic initiation. (B) Sarm1 contributes to dendrite elongation, as indicated by the total dendrite length and primary dendrite number. (C) Sarm1 is expressed in neurites. At DIV 2, cultured hippocampal neurons were immunostained with Sarm1 (viewed by Alexa Fluor 488) and Tuj1 (viewed by Alexa Fluor 555) antibodies. (D) Sarm1 knockdown shortens the length of the longest neurites. (E and F) Sarm1 regulates neuronal polarity. 4 h after plating, cultured hippocampal neurons were transfected with Sarm1i¹ or vector control (E) or Sarm1i¹, shCtrl, shLuc, or pSuper vector control (F), and the percentage of neurons that did not develop a dominant neurite (axon) was determined (see Materials and methods for details). Mean values ± SEM (error bars) are shown from three independent experiments in which equal numbers of transfected neurons were analyzed. Bars: (A, B, D, and E) 30 µm; (C) 20 µm. *, P < 0.05; ***, P < 0.0005.

Figure 8.

Sdc2 regulates dendrite outgrowth through Sarm1. (A) Cultured hippocampal neurons were transfected with the indicated plasmids at 2 DIV and then fixed for immunostaining with GFP and Sdc2 antibodies at 5 DIV. pSuper and pGW1 are the vectors for RNAi knockdown and gene expression, respectively. Insets (enlarged from the boxed regions) show the filopodia induced by Sdc2. Bar, 30 µm. (B) Total dendrite lengths. (C) Number of primary dendrites. (D) Density of dendritic filopodia. Because the vector control and Sarm1i¹ alone did not obviously induce filopodia formation, only neurons transfected with Sdc2 alone or both Sdc2 and Sarm1i¹ were subjected to quantitative analysis of filopodia. Thirty transfected neurons were collected randomly from each group. The experiments were repeated three times; data represent mean values ± SEM (error bar). *, P < 0.05; ***, P < 0.0005.

Figure 9.

The JNK pathway and microtubule stability are downstream of Sarm1 and Sdc2. (A) Sarm1 overexpression increases the acetylation levels of tubulin. COS-1 cells were transfected with vector control or Sarm1 and were immunoblotted with acetyl-tubulin and α-tubulin antibodies. The levels of tubulin acetylation were normalized to total α-tubulin. (B) Sarm1 knockdown reduces tubulin acetylation in mouse neuroblastoma Neuro-2A cells. 2 d after transfection, the acetylation levels of tubulin were determined by immunoblotting. (C) A JNK kinase dead mutant (JNK^KR) and an MKK4 dominant-negative mutant (MKK4^DN) suppress the effect of Sarm1 overexpression in dendritic arborization. (D) Sarm1-expressing neurons treated with JNK inhibitor SP600125 (SP) have shorter dendrites compared with Sarm1-expressing cells. In C and D, transfection was performed at 9 DIV and cells were harvested for immunostaining at 12 DIV. (E) Co-expression of JNK^KR abolishes Sdc2–induced dendrite outgrowth. At 2 DIV, cultured hippocampal neurons were transfected with the indicated constructs, then fixed for immunostaining at 5 DIV. Three independent experiments were performed. Equal numbers of transfected neurons were analyzed for each experiment. The data represent mean values ± SEM (error bars). *, P < 0.05; **, P < 0.005; ***, P < 0.0005. Bars, 30 µm.

Figure 10.

Reduction of Sarm1 in vivo impairs dendritic arborization. (A) Strategy used to generate Sarm1 knockdown mice; details are described in Materials and methods. (B) GFP expression in P5 Sarm1 knockdown transgenic (Tg) mice. (C) Reduction of Sarm1 protein levels in three independent Tg mouse lines. Immunoblotting was used to determine the protein levels of Sarm1 and GFP in the extracts containing forebrain and subcortical regions. For each line, two GFP-positive Tg and two wild-type (WT) littermates were examined. GAPDH was used as an internal control. Molecular mass standards (kD) are indicated next to the gel blots. (D) The relative brain weights of Sarm1 knockdown mice and WT littermates. P14, P28, and P60 mice were analyzed. *, P < 0.05; **, P < 0.005. (E) Total brain area of P14 mice. Sums of the total areas of a series of brain sections were determined to compare the brain size of Sarm1 knockdown mice and WT littermates. **, P < 0.005. (F) Cell morphology of CA1 neurons. 2-mo-old mice were used for Golgi-Cox staining. Camera lucida was then performed to examine neuronal morphology in the brain. Bar, 50 µm. (G) Sholl analysis of CA1 neurons. *, P < 0.05; **, P < 0.01; ***, P < 0.001. (H) Total length of basal and apical dendrites of CA1 neurons. **, P < 0.005; ***, P < 0.0005. The data represent mean values ± SEM (error bars).