Defects in vesicular trafficking appear to play an important role in the development of ALS and motor neuron diseases (MND). Recently, a missense mutation in vesicle-associated membrane protein/synaptobrevin-associated membrane protein B (VAPB) have been identified to cause ALS8, a dominant form of familial ALS. VAPB is likely involved in vesicular transport between ER and Golgi networks. The missense mutation in the conserved major sperm protein (MSP) domain of VAPB, which results in a Proline (P) to Serine (S) substitution at the residue 56 (P56S), likely affects the structure and function of VAPB and interferes with the normal ER-Golgi trafficking in motor neurons. However, how the P56S affects the function of VAPB and contributes to the motor neuron degeneration remains elusive. Therefore, our specific aims for this project are:? Aim 1: To investigate the pathogenic mechanisms of VAPB P56S mutation in vivo by generation and characterization of VAPB wild-type (WT) and P56S mutant transgenic mice;? Aim 2: To determine the normal function of VAPB by generation and characterization of VAPB knockout (KO) mice;? Aim 3: To examine the intracellular vesicular transport pathways affected by VAPB or the VAPB P56S mutation in the spinal motor neurons of VAPB KO or P56S transgenic mice.? ? 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 have obtained 3 independent lines of both VAPB P56S and WT transgenic mice. We have also developed a rabbit polyclonal antibody specifically against VAPB, but not its close homologous protein, VAPA. The expression of VAPB mRNA and protein in the brain was quantified by real-time RT-PCR and Western blot. We found that the steady level of VAPB P56S mutant protein is significantly less compared to that of WT protein, indicating that the P56S mutation may destabilize VAPB protein in neurons. We chose higher expression lines of VAPB P56S and WT transgenic mice for the downstream neuropathological and motor behavioral analyses. Both VAPB P56S mutant and WT transgenic mice develop normally. However, only the VAPB P56S mutant mice displayed gait abnormalities starting as early as 2 months of ages. We will continue to closely monitor the alterations of motor behaviors in both WT and mutant transgenic mice and examine the appearance of any potential neuropathological abnormalities after the mice have developed more severe motor phenotypes.? ? We have also obtained multiple lines of VAPB KO chimeric mice. These mice will be used to generate VAPB heterozygous and homozygous KO mice. We will document the behavioral and neuropathological phenotypes of VAPB KO mice. This study will allow us to investigate the nature of P56S mutation in VAPB through either a gain or loss of function mechanism.? ? To examine the intracellular vesicular transport pathways affected by VAPB P56S mutation, we focus our studies on the subcellular localization of WT and mutant 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 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. Furthermore, when we closely examined the subcellular location of mutant VAPB, we found a small fraction of VAPB was specifically targeted to the postsynaptic sites of cistern bouttons (C-boutons) in SMN. C-boutons, formed by flat and extended postsynaptic cistern, are restricted to the somata and proximal dendrites of SMN. C-boutons receive cholinergic innervation from a group of cholinergic interneurons near the central canal of spinal cord. 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. It is likely that the mutant VAPB may cause the dysfunction of motor neurons through affecting the normal function of C-boutons. ? ? In the future, we will continue to closely monitor any motor and neuropathological abnormalities in aged VAPB transgenic and KO mice. We will also study the structural and functional alterations of C-bouton at the single synapse level by FM dye staining in organotypic spinal cord slice culture and by electrophysiological measurements. In addition, we will study 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.
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