Lissencephaly-1 dependent axonal retrograde transport of L1-type CAM Neuroglian in the adult drosophila central nervous system

Abstract

Here, we established the Drosophila Giant Fiber neurons (GF) as a novel model to study axonal trafficking of L1-type Cell Adhesion Molecules (CAM) Neuroglian (Nrg) in the adult CNS using live imaging. L1-type CAMs are well known for their importance in nervous system development and we previously demonstrated a role for Nrg in GF synapse formation. However, in the adult they have also been implicated in synaptic plasticity and regeneration. In addition, to its canonical role in organizing cytoskeletal elements at the plasma membrane, vertebrate L1CAM has also been shown to regulate transcription indirectly as well as directly via its import to the nucleus. Here, we intend to determine if the sole L1CAM homolog Nrg is retrogradley transported and thus has the potential to relay signals from the synapse to the soma. Live imaging of c-terminally tagged Nrg in the GF revealed that there are at least two populations of retrograde vesicles that differ in speed, and either move with consistent or varying velocity. To determine if endogenous Nrg is retrogradely transported, we inhibited two key regulators, Lissencephaly-1 (Lis1) and Dynactin, of the retrograde motor protein Dynein. Similar to previously described phenotypes for expression of poisonous subunits of Dynactin, we found that developmental knock down of Lis1 disrupted GF synaptic terminal growth and that Nrg vesicles accumulated inside the stunted terminals in both mutant backgrounds. Moreover, post mitotic Lis1 knock down in mature GFs by either RNAi or Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) induced mutations, resulted in normal length terminals with fully functional GF synapses which also exhibited severe accumulation of endogenous Nrg vesicles. Thus, our data suggests that accumulation of Nrg vesicles is due to failure of retrograde transport rather than a failure of terminal development. Together with the finding that post mitotic knock down of Lis1 also disrupted retrograde transport of tagged Nrg vesicles in GF axons, it demonstrates that endogenous Nrg protein is transported from the synapse to the soma in the adult central nervous system in a Lis1-dependent manner.

Fig 1. Trafficking of tagged-Nrg vesicles in the GF axon.

(a) Schematic representation of the GFC in the nervous system. Both GFs as well as the downstream circuit with the TTMns, the PSIs and the dorsal longitudinal motor neurons (DLMns) for one GF are depicted. DLMns and TTMns synapse onto the dorsal longitudinal muscles (DLMs) and TTMs, respectively. Live imaging and dye injections of GF axon for visualization of the GF anatomy were performed in the cervical connective (CvC). Areas of anatomical analysis by confocal microscopy in the ventral nerve cord (VNC) are highlighted by a gray square. Placement of stimulation and recording electrodes for electrophysiological analysis are shown. (b) Kymographs of mCherry- and eGFP-tagged Nrg vesicles in GF axons of homozygous UAS-Nrg^eGFP;R68A06 and UAS-Nrg^mCherry;R68A06 flies. Axons were imaged in the CvC at 4 frames/second. A small region of the axon was photobleached before acquisition to better visualize vesicle trafficking. Ascending and descending slopes represent retrograde and anterograde vesicles, respectively. Arrows point to bright, slow moving, while arrowheads indicate fast moving retrograde vesicles. Scale bar is 10 μm. (c) Directionality of anterograde and retrograde Nrg^eGFP and Nrg^mCherry vesicles. Numbers of vesicles (n) statistically (Mann-Whitney Rank Sum Test, * p = 0.013 and **p = 0.007) analyzed in ten axons were expressed in percent of total vesicles. (d) Frequency distribution of velocities of anterograde and retrograde mCherry- and eGFP-tagged Nrg vesicles. The numbers of vesicles (n) for velocity increments of 0.2 μm/sec in five axons was calculated in percent.

Fig 2. Lis1 phenotypes and Nrg localization in wild type and lis1 mutant backgrounds.

(a) Maximum intensity projection views of confocal image stacks showing representative GF terminals. GF synapse anatomy was visualized with neurobiotin (top panel) and TRITC-dextran (middle panel, magenta). Neuronal Nrg¹⁸⁰ (green) was immunohistochemically labeled with monoclonal antibody BP104 (middle and lower panel). Neurobiotin dye-coupling of the GFs to the post-synaptic TTMns demonstrates the presence of synaptic connections between the medial TTMn dendrites and GF-TTMn. Accumulation of vesicular Nrg¹⁸⁰ clusters in the synaptic terminal and in the axon at the PSI contact region are indicated with arrows. Scale bar is 10 μm. (b) Sample trace of electrophysiological recordings of the GF to TTM pathway from control animals and lis1 mutants. The response latency of wild type animals is approximately 0.8 ms (dashed line). Ten stimuli given at 100 Hz and the lack of some responses in mutant animals are indicated with asterisks. (c) Maximum intensity projection view of confocal image stacks of GF terminal expression a poisonous subunit of Drosophila dynactin p150 Glued protein. GF was dye injected with TRITC-dextran (magenta) and Nrg¹⁸⁰ (green) was immunohistochemically labeled with monoclonal antibody BP104.

Fig 3. Rescue of Lis1 phenotypes.

(a, b) Rescue of Lis RNAi phenotypes with co-expression of Lis1^GFP. Co-expression of UAS-CD8-GFP with UAS-Lis1^RNAiH was used as a negative control. (c, d) Rescue of phenotypes in hypomorphic lis1^k11702/lis1^G10.14 with transgenic expression of wild type Lis1 in the GF and its target neurons or in the GF but not its target neurons with the A307 and R91H05 Gal4-drivers, respectively. A307, lis1^G10.14/lis1^k11702 without the UAS-Lis1 served as a negative control. (a, c) Giant fiber morphology and Nrg localization. All images show maximum intensity projections of confocal image stacks. GFs of adult flies were labeled by TRITC-Dextran injections (magenta) and displayed together with immunohistochemically labeled Nrg¹⁸⁰ (anti-BP104 with goat anti-mouse-Cy5, pseudo-colored in green) in the VNC (upper row). Co-localization of both labels appears white. The lower row displays immunohistochemically labeled Nrg separately as gray scale images. Lis-GFP and CD8-GFP in (a) were present but are not shown. Scale bars are 20 μm. (b, d) The function of the GF synapse was determined with average following frequency and average response latency. Error bars indicate standard error of the mean and sample sizes are shown in the bars. Significant differences are indicated with asterisks (*** = p<0.001, Mann-Whitney Rank Sum Test).

Fig 4. Phenotypes of GFs with post developmentally knocked down Lis1.

Comparison of phenotypes when Lis1 was knocked down in the GF during its development (R91H05) or after its development (GF-Split-Gal4) with RNAi (UAS-Lis1^RNAiH) and CRISPR (UAS-Lis1^gRNA). All images show maximum intensity projections of confocal image stacks. GFs of adult flies were labeled by TRITC-dextran injections (magenta) and displayed together with immunohistochemically labeled Nrg¹⁸⁰ (green) in the VNC (upper rows). Co-localization of both labels appears white. The lower rows display immunohistochemically labeled Nrg¹⁸⁰ separately as gray scale images. Scale bars are 20 μm. Localization of Nrg¹⁸⁰ (anti-Nrg-BP104, green) in wild type and lis1 mutant backgrounds. Vesicular accumulations of Nrg¹⁸⁰ in stunted and normal sized terminals are indicated by arrows. A large vacuole is indicated by an arrowhead.

Fig 5. Retrograde trafficking of tagged Nrg in Lis1 knock down and Glued^Δ96B mutant background.

(a) Axonal transport of Nrg^eGFP vesicles in GFs with postdevelopmental Lis1 knock down. Kymographs showing trajectory of Nrg^eGFP vesicles in a photobleached area flanked by unbleached areas in the GF axon over a 10 min time period. Videos were obtained at one frame per second. Ascending and descending slopes represent retrograde and anterograde vesicles, respectively. Vertical lines are stationary vesicles. Scale bar is 4 μm. (b) Quantification of Nrg^eGFP vesicles in wild type and in flies expressing UAS-Lis1^RNAiH with GF-Split-Gal4. The lower expression, magnification and one frame per second imaging rate allows to quantify only slow (< 1μm/sec) moving Nrg^eGFP vesicles. The average flux (left graph) are the average numbers of retrograde vesicles that entered the photobleached area per minute, with n indicating the number of GFs analyzed. The maximum net displacement of retrograde Nrg^eGFP vesicles during the imaging period was analyzed and the average travel velocity was expressed in μm/second. Ten and 15 axons were assessed in wild type and Lis1 knock down background, respectively, with n indicating the number of vesicles analyzed. The average number of stationary vesicles per micron axon length was assessed in the unbleached regions. A total axon length of 169 μm and 180 μm was assessed in wild type and Lis1 knock down background, respectively, with n indicating the number axons analyzed. Error bars show standard error of the mean and significant differences are indicated with asterisks (*** = p<0.001, Kruskal-Wallis One Way Analysis Of Variance and Mann-Whitney Rank Sum Test). (c) Expression of Nrg^mCherry without and with UAS-Lis1^RNAiH and UAS-Glued^Δ96B using the R68A06 driver line. Left panel, quantification of fast (> 1μm/sec) retrograde vesicles in eight (n) GF axons of each genotype. Error bars show standard error of the mean and significant differences are indicated with asterisks (*** = p<0.001, Kruskal-Wallis One Way Analysis Of Variance and Mann-Whitney Rank Sum Test). Right panel, kymographs of Nrg^mCherry vesicles without and with Lis1^RNAiH and Glued^Δ96B expression. Videos were obtained at 4 frames per second and a 2 minute time period after phototobleaching is depicted. Some fast retrograde vesicles are indicated with arrows. Scale bar is 10 μm.