Munc18 and Munc13 regulate early neurite outgrowth

Abstract

Background information: During development, growth cones of outgrowing neurons express proteins involved in vesicular secretion, such as SNARE (soluble N-ethylmaleimide-sensitive fusion protein-attachment protein receptor) proteins, Munc13 and Munc18. Vesicles are known to fuse in growth cones prior to synapse formation, which may contribute to outgrowth.

Results: We tested this possibility in dissociated cell cultures and organotypic slice cultures of two release-deficient mice (Munc18-1 null and Munc13-1/2 double null). Both types of release-deficient neurons have a decreased outgrowth speed and therefore have a smaller total neurite length during early development [DIV1-4 (day in vitro 1-4)]. In addition, more filopodia per growth cone were observed in Munc18-1 null, but not WT (wild-type) or Munc13-1/2 double null neurons. The smaller total neurite length during early development was no longer observed after synaptogenesis (DIV14-23).

Conclusion: These data suggest that the inability of vesicle fusion in the growth cone affects outgrowth during the initial phases when outgrowth speed is high, but not during/after synaptogenesis. Overall, the outgrowth speed is probably not rate-limiting during neuronal network formation, at least in vitro. In addition, Munc18, but not Munc13, regulates growth cone filopodia, potentially via its previously observed effect on filamentous actin.

Figure 1. Munc18-1 null neurons are deficient in vesicle recycling

In order to investigate the endocytosis of release-deficient Munc18-1 null neurons, an FM4-64 dye uptake experiment was done. After loading the cells using stimulation with 60 mM KCl and washing for 10 min with Tyrode's solution, WT neurons show punctuate staining in the growth cone (top row), which is not the case for the M18 null growth cone (bottom row). Scale bar, 5 μm.

Figure 2. Developmental time course shows lagging of release-deficient neurites

(A) Neurite length was visualized by tracing the neurites at the indicated time points; showing decreased overall length and decreased arbour complexity in release-deficient M18 null and M13 null neurons. (B) Quantification of the neurite length showed that, during development, release-deficient neurons lagged behind in overall neurite length from DIV3 onwards. Means±S.E.M. are plotted. DIV1 WT: n=285; M18: n=211; M13: n=404; DIV2 WT: n=146; M18: n=124; M13: n=212; DIV3 WT: n=135; M18: n=62; M13: n=169; DIV4 WT: n=83; M18: n=34; M13: n=136.

Figure 3. Vesicle release is involved in outgrowth speed

Quantification of outgrowth speed in slices at DIV3 shows differences between release-deficient and WT neurons. (A) Five frames taken from a 20-min time-lapse series at the indicated time points (top) showing the progression of a growth cone in an organotypic slice culture into the surrounding matrix. The red line indicates the starting position of the growth cone at t=0 min. (B) Quantification of the speed of outgrowth showing a large decrease for M18 null (46%) and M13 null (42%) as compared with WT. (C) The average accumulated distance during the 20 min time-lapse was severely reduced in both release-deficient genotypes (blue and red curves) as compared with WT control (black curve). Scale bar in (A) is 3 μm. Means±S.E.M. are plotted. ***P<0.001; numbers in bars indicate the number of growth cones.

Figure 4. Growth cone morphology is altered in M18-1 null neurons

Shown is an overlay of the first frame (red) and a projection of its consecutive frames (green) in a 60-min time-lapse. (A) Projected time-lapse series of the indicated genotypes showing distinct differences in morphology in M18 null growth cones. (B) Quantification of the growth cone palm area showed a decrease in M13 null. (C) Compared with WT, M18 null had an increased number of filopodia per growth cone, but this was not seen in M13 null. (D) The average length per filopodium showed no apparent differences. The scale bar in (A) is 2 μm. Means±S.E.M. are plotted. *P<0.05; numbers in bars represent the number of growth cones (B, C) or the number of filopodia (D).

Figure 5. Munc18-1 influences the actin cytoskeleton

Staining for actin (green) and tubulin (red) in WT and Munc18-1 null cultures was used to quantify the effect of Munc18 protein on the actin cytoskeleton (A). The area of actin and tubulin was measured and the ratio was taken. Compared with WT, Munc18-1 null growth cones had a 35% increase in actin content (B). Bar in overview is 10 μm, zoomed picture is 5 μm. Means±S.E.M. are plotted.

Figure 6. Decreased outgrowth recovers before synaptogenesis

All MAP2-positive neurites in a field of view were traced and the total dendrite length was divided by the number of somata. (A) Multiple fields of view were taken from WT (left) and M13 null (right) neurons at DIV14, showing a dense network of MAP2-positive neurites (green) originating from several somata (red asterisks). Furthermore, synapse staining (synapsin1, red) shows similar numbers of putative synapses in WT and Munc13-1/2 null neurons. (B) Quantification of dendrite length at DIV14, showing no significant difference (t test; WT: n=15, M13: n=92; P=0.48) between WT and M13 null neurons. (C) Examples of neurons from WT and M13 null cultures at DIV23, showing comparable neurite length in kRas-EGFP labelled neurites. Scale bars: (A) 20 μm and (C) 100 μm. Means±S.E.M. are plotted.