Recent studies revealed that the recycling endosomal compartment is involved in a variety of membrane trafficking events and might thereby control cellular processes ranging from cellular polarization over cytokinesis to cell fate specification (van Ijzendoorn, 2006). REs can also serve as an intermediate sorting station for proteins from the TGN en route to the plasma membrane as well as back to the TGN. They might also be a sorting station for proteins, such as the 46 kDa cation-independent mannose-6-phosphate receptor (MPR46), destined for the late endosomal/ lysosomal pathway and back (Lin et al., 2004). Furthermore, in neurons it has been shown that REs are crucial for postsynaptic AMPA receptor trafficking and the regulation of synaptic plasticity (Park et al., 2004) as well as the delivery of membrane material for the growth of dendritic spines (Park et al., 2006). We thus speculated that recycling endosomes might be involved in the delivery of membrane bounded organelles for neurite outgrowth, a process that is highly dependent on the redistribution of membranes and associated proteins towards the site of membrane addition, in this case the growth cones of neurites. A possible source of membrane material are REs. As γ- BAR is associated with and might regulate the transport of REs, it could well be involved in the process of neurite outgrowth and/ or axon formation in developing neurons. In fact, in a microscopy based screen to identify novel proteins involved in neurite formation and extension, γ-BAR was found to influence the length of neurites formed by PC12 cells upon stimulation with NGF (Laketa et al., 2007). Thus, we analyzed the effect of γ-BAR overexpression or knockdown on neurite outgrowth in primary hippocampal neurons. We transfected neurons prepared from E18 rat embryos directly after preparation using the Amaxa Nucleofector technology before plating them onto coverslips. Neurite length was analyzed 26 and 54 hours after transfection (stage 2 and 3 of neuronal development) by measuring the length of the longest neurite that would eventually become the axon.
Overexpression of eGFP-tagged γ-BAR full length reduced neurite length by about 30% after 54 hours compared to cells expressing eGFP. These data parallel the effects observed in NGF-stimulated PC12 cells (Laketa et al., 2007). A kinesin binding deficient γ-BAR truncation mutant (aa 1-140) had a less pronounced effect and reduced neurite length by about 15%.
Results 89
Figure 3-33: γ-BAR overexpression reduces the length of growing neurites
Primary hippocampal neurons prepared from E18 rat embryos were transfected with constructs encoding the indicated proteins (eGFP, eGFP-tagged γ-BAR full length and aa 1-140) directly after preparation using the Amaxa Nucleofector technology and plated onto coverslips. Cells were fixed 26 and 54 hours after transfection and immunostained with an antibody against eGFP to enhance signal intensities. The length of the longest neurite of each transfected cell was measured in bright field images (phase) by drawing a freehand line along the neurite and using the measure function of the software (Scion Image, Scion Corporation). The average neurite length of eGFP transfected control cells after 54 hours was set to 100%. n represents the number of cells analyzed from three independent experiments (for γ-BAR aa 1- 140 only two experiments were performed). Error bars correspond to s.e.m.. The differences of the means between eGFP and γ-BAR FL were statistically significant in all three experiments (unpaired t-test, two- tailed P value: P<0.0001).
In order to complement these data, we used siRNA-mediated knockdown to inhibit γ-BAR expression in hippocampal neurons and analyzed neurite length as described above. 21-mer siRNAs against rat γ-BAR were synthesized. These siRNAs were highly efficient as assessed by their ability to knockdown expression of eGFP-tagged rat γ-BAR in transfected Cos7 cells (Figure 3-34, panel C). We chose a siRNA exhibiting nearly 100% knockdown efficiency towards the co-transfected rat protein and used a scrambled siRNA as control. Hippocampal neurons prepared from E18 rat embryos were transfected with synthetic siRNAs directly after preparation (Amaxa Nucleofector), plated and analyzed 54 hours after transfection as described above. Knockdown of γ-BAR resulted in about 50% longer neurites compared to control siRNA treated cells (Figure 3-34, panel B). It should be noted that the absolute length of the control siRNA treated neurons was significantly shorter than normally seen for
cultured hippocampal neurons at this stage of development, i.e. 54 hours after preparation (Frank Bradke, personal communication). This phenomenon can not be explained yet, but might be due to the siRNA transfection procedure or fixation artifacts. Further experiments analyzing the effects of other γ-BAR and control siRNAs are clearly necessary to clarify this point.
Figure 3-34: γ-BAR knockdown increases the length of growing neurites
(A) Synthetic siRNAs against rat γ-BAR or a control siRNA (scrambled) were transfected into primary
hippocampal neurons directly after preparation from E18 rat embryos. The cells were plated onto coverslips and analyzed 54 hours after transfection by immunostaining using antibodies against γ-BAR and γ-adaptin. Cells that showed no or reduced staining of γ-BAR after treatment with the γ-BAR siRNA were chosen for analysis and the length of the longest neurite (see phase images) was measured as described. Insets represent 3-fold magnification of boxed areas.
(B) Quantification of the neurite length in scrambled (control) vs. γ-BAR siRNA treated neurons. The
average neurite length of control treated cells was set to 100%. n indicates the number of cells analyzed from two independent experiments. Error bars represent s.e.m. The difference of the means is statistically significant (unpaired t-test, two-tailed P value: P<0.0001).
Results 91
(C) Cos7 cells were co-transfected with plasmids encoding eGFP-tagged rat γ-BAR and siRNAs against
rat γ-BAR or a control siRNA (scrambled) in order to test knockdown efficiency of the siRNAs used in (A). Triton X-100 extracts were prepared from the cells 24 hours after transfection and subjected to SDS- PAGE and immunoblotting using antibodies against GFP (detection of transfected rat γ-BAR) as well as γ-adaptin (AP-1) and actin as controls. The rat γ-BAR siRNA efficiently suppressed the expression of co- transfected rat γ-BAR.
Discussion 93