For Aim 1, we have successfully generated human VAPB P56S mutant and WT transgenic mice under the Thy1.2 promoter. The Thy1.2 promoter has been used previously to drive the expression of transgene in many different types of neurons, including spinal motor neurons (SMN). We also inserted an IRES-GFP reporter construct immediately after the translation stop codon of VAPB, which will facilitate the downstream gene expression analysis in transgenic mice. We have obtained 3 independent lines of both VAPB P56S and WT transgenic mice. We have developed a rabbit polyclonal antibody specifically against VAPB, but not its close homologous protein, VAPA. The expression of VAPB mRNA in the brain was quantified by real-time RT-PCR. VAPB mRNA was over-expressed by 8 to 28 folds in various lines. However, the steady level of VAPB P56S mutant protein is significantly less compared to WT protein with a similar expression level of mRNA, indicating that the P56S mutation may destabilize VAPB protein in neurons. We focus on two higher expression lines of VAPB P56S (25 folds vs. endogenous) and WT (28 folds vs. endogenous) transgenic mice for the downstream neuropathological and motor behavioral analyses. Both VAPB P56S mutant and WT transgenic mice develop normally and have yet to show any obvious motor dysfunction up to 12 months of age. However, VAPB P56S mutant mice started to display shorter stride in the TreadScan Gait analysis at 2 months of ages. We will continue to closely monitor the alterations of motor behaviors in mutant mice and examine the appearance of any potential neuropathological abnormalities after these mice have developed more severe motor phenotypes.
For Aim 2 to examine the intracellular vesicular transport pathways affected by VAPB P56S mutation, we focus our studies on the subcellular localization of VAPB in SMN. Endogenous VAPB is highly expressed by SMN as shown by immuno-fluorescent staining. Within SMN, VAPB is primarily located in some vesicular structures in somata. Immuno-EM studies indicate that VAPB is mainly associated with ER and multi-vesicular body (MVB). Over-expression of WT VAPB does not alter the overall cellular localization of VAPB. In contrast, over-expression of VAPB P56S mutation in the D3 line leads to formation of aggresome-like inclusion bodies in somata of motor neurons. Consistently, more mutant VAPB protein was detected in Triton-X100-insoluble fraction by Western blot. These observations are in line with previous in vitro work (Kanekura et al., 2006;Teuling et al., 2007). However, when we closely examined the cellular location of mutant VAPB, we found a small fraction of VAPB was specifically targeted to the postsynaptic sites of cistern boutons (C-boutons) in SMN (Fig. 2C-D). C-boutons, formed by flat and extended postsynaptic cistern, are large (37 um in diameter) and restricted to the somata and proximal dendrites of SMN (Hellstrom et al., 2003). C-boutons receive cholinergic innervation from a group of cholinergic interneurons near the central canal of spinal cord (Miles et al., 2007). The type 2 muscarinic (M2) receptor-mediated postsynaptic response by C-boutons modulates the excitability of spinal motor neurons and influences the motor neuron output (Miles et al., 2007). To investigate whether mutant VAPB affects the structure of C-boutons, we quantified the density and size of C-boutons in SMN of non-transgenic (NTg), WT, and P56S transgenic mice. A significant reduction in density and size of C-boutons was observed in VAPB P56 transgenic mice. Consistent with these morphological studies, we observed a significant decrease of SMN activities in an electrophysiological study of whole mount spinal cord. Therefore, our studies in transgenic mice clearly demonstrate an apparent gain-of-function mechanism of the P56S mutation in VAPB, which leads to a selective targeting of VAPB to C-boutons, resulting in disruption of the normal morphology and function of C-boutons in SMN. Conclusions and Future Directions: Our present studies indicated that the P56S mutation in VAPB led to protein aggregation in somata of SMN and a part of mutant protein specifically located in the subsurface of C-boutons of SMNs. The P56S mutation in VAPB changed the morphology of C-boutons and reduced the number and size of presynaptic C-type nerve terminals on spinal motor neurons, which may compromise the normal function of these terminals. We postulate that a reduction in cholinergic input to spinal motor neurons might be the early event preceding the dysfunction and potential loss of motor neurons.
For Aim 1, we will continue to closely monitor any motor abnormalities in mutant mice and run a series of motor behavioral tests at 12 and 18 months of ages. We will also examine the existence of any potential neuropathological abnormalities, particularly related to spinal motor neurons at 12 and 18 months of ages.
For Aim 2, we will study the functional alteration of C-bouton at the single synapse level by FM dye staining in organotypic spinal cord slice culture and by electrophysiological measurements. We will also investigate the molecular mechanism that underlies the selective targeting of mutant VAPB to the C-boutons. As an extension of this study on mutant VAPB, we will examine the morphology of C-boutons in other ALS mouse models and in ALS patients. It will be interesting to learn if the dysfunction of C-boutons serves as a general pathological mechanism for ALS and other MND.
