We have used Drosophila as a model system to gain insight into the mechanism by which the ALS8 associated mutation gives rise to disease. The evolutionary conservation of VAP and the availability of a vast array of genetic tools make the fly a particularly attractive model system with which to dissect pathophysiological mechanisms underlying ALS8. We have compared and assessed the cellular consequences of expression of wild type and ALS8-associated mutant VAP on the development and function of the synapse at the neuromuscular junction. In Drosophila, the synapses at the neuromuscular junction are glutamatergic, providing some similarities to the spinal cord synapse which are affected in ALS patients. Furthermore, the presynaptic neurons are obviously the functional equivalent to the lower motor neurons also affected in ALS patients.
The relationship between mutation and pathogenesis in inherited neurodegenerative disorders is obviously of great interest as it could point towards a mechanistic understanding of the disease and indicate treatment strategies. For genetically dominant disorders, potential mechanisms include toxic gain of function, haploinsufficiency, and dominant negative effects. Pennetta and coworkers concluded that the ALS8 mutation is a toxic gain of function rather than a dominant negative 
. This conclusion was based on the toxic properties of the mutant protein in conjunction with its retention of activity. However, our data and that of others 
argue against retention of VAP activity by the ALS8 mutant, but rather suggest that that the ALS8 mutation in VAMP-associated-protein (VAP) functions through a dominant negative mechanism. Dominant negative mutations are those in which the product of the mutant gene adversely affects function of the normal gene product within the same cell, for example by forming non-functional dimers. Interestingly, members of the VAP family are able to form dimers 
. The following experiments reported here support the notion that the formation of mixed dimers or aggregates between wild type and ALS8 mutant proteins is at least in part responsible for the dominant phenotype. The VAP loss of function phenotype (VAPΔ20
) is characterized by an increase in bouton size and increased microtubule disorganization. Both these phenotypes are observed when VAPP58S
is overexpressed in neurons, indicating that expression of the mutant protein is dominant over the wild type protein. This is not simply due to an excess of mutant protein since VAPP58S
can also suppress the phenotypes generated by overexpression of VAPwt
(). Finally, VAPP58S
forms ubiquitinated aggregates that can also recruit and induce aggregation of wild type protein (), thereby rendering it functionally inactive. The ability of the mutant protein to recruit and induce aggregation of the wild type protein has also been reported for mammalian VAP in vitro 
. The finding that down regulation of mammalian VAP using RNAi techniques is sufficient to induce cell death in motor neurons 
also argues that it is loss of VAP function, rather than the formation of aggregates, that is pathogenic.
VAP was originally identified as a “VAMP-associate protein” in Aplysia
, and antibodies against VAP decrease synaptic transmission 
. Subsequently, VAP was demonstrated to alter the size and number of boutons at the Drosophila
neuromuscular junction 
. Overexpression of VAPwt
leads to a significant decrease in the total number of active zone at the synapse despite an increase in bouton number; fewer nc82 immunoreactive puncta per synapse are present () even though the number of such puncta per unit cross sectional area of the bouton (µm2
) is not significantly different from control and animals expressing mutant VAP. Further research will be necessary in order to determine whether this difference in the total number of active zones might lead to impaired synaptic transmission.
In a recent report, by conducting morophometric analysis on electron micrographs of individual boutons, Pennetta and colleagues show that there is no change in the number of active zones in animals overexpressing VAPwt
in neurons 
. These authors also report locomotory defects, synaptic degeneration and muscle atrophy in larvae expressing VAPP58S
in neurons. We did not observe any of these defects in our experiments. Differences in expression levels of the mutant protein could account for these discrepancies.
Extensive crosstalk between motor neurons and muscle is a conspicuous feature of neuromuscular junction development in Drosophila
, where synapses must accommodate the increase in size of muscles during larval development by increasing bouton number and active zones per bouton. Components of the TGF-β/ BMP signaling pathway are known to regulate neuronal survival, synaptic development and function in vertebrates as well as invertebrates. Recently, reduced TGF-β signaling has been associated with Alzheimers disease and the level of one of the receptors (TGFβRII) is reduced in brain tissue of Alzheimer's disease patients 
, retrograde BMP signaling is required for synaptic growth at the neuromuscular junction 
. At the same time, anterograde BMP signaling in neurons appears to be essential to restore neurotransmission which is significantly lowered in mutants of the BMP pathway. Here, we show using epistasis analysis, the presence of a genetic interaction between VAP and BMP signaling pathways (). We also show that overexpression of VAPwt
potentiates BMP signaling whereas overexpression of VAPP58S
impairs BMP signaling as evidenced by the decrease in intensity of pMAD puncta at the neuromuscular junction () and nuclei of motor neurons (Figure S3
). Expression of VAPP58S
also causes ultrastructural phenotypes reminiscent of mutants in the BMP pathway, e.g., floating T-bars and occasional detached postsynaptic membranes. However, it should be noted that despite the similarities, these phenotypes are by no means identical; BMP mutants show smaller synapses (i.e., fewer boutons) with no significant change in bouton size- a phenotype unlike that observed in animals with neuronal expression of VAPP58S
. This suggests that the aberrations associated with ALS8 are likely to be more complex than simply loss of receptors.
Recent observations that VAP is reduced in ALS patients and mutant SOD transgenic mice 
suggest a potential role for VAP in many forms of ALS. Further genetic analysis using this model as a paradigm for ALS is likely to provide insights into pathways that contribute to motor neuron dysfunction and death in ALS.