has been shown to be the causative gene of late-onset autosomal dominant forms of motor neuron disorders, including typical and atypical ALS and late-onset spinal muscular atrophy (5
). The pathogenic mutation predicts a substitution of a Serine for a conserved Proline (P56). We decided to study the role of hVAPB in MNDs using Drosophila
One of the hallmarks associated with loss-of-function and neuronal overexpression of DVAP-
33A is decreased and increased bouton formation at the NMJ, respectively. Despite this structural alteration, synaptic transmission is maintained within a wt range. At the mechanistic level, muscles respond to a decreased number of boutons and quantal content by upregulating quantal size; conversely muscles compensate an increase in number of boutons and quantal content by downregulating quantal size. Compensatory changes in quantal size during synaptic homeostasis are thought to be determined, largely, by the properties of transmitter receptors. At the Drosophila
NMJ, there are two classes of glutamate receptors: one set containing the subunit IIA and another one containing the subunit IIB (29
). In DVAP-33A
loss-of-function mutations, the increase in quantal size is associated with an increase in the number and average cluster volume of subunit IIA. Conversely, the decrease in quantal size in the oversprouting mutants is accompanied by a decrease in the level of post-synaptic receptor subunit IIA and a reduction in the average cluster volume for several subunits. In agreement with our data, the IIA subunit receptors have been shown to affect quantal size and receptor channel open time (18
). Similar to our oversprouting mutants, in synapses lacking the receptor subunit IIA, a homeostatic increase in neurotransmitter release compensates for the reduction in quantal size and the evoked response is maintained within normal values (18
). These data indicate that expression levels of VAP proteins play a crucial role in synaptic homeostasis by coordinating structural remodeling and post-synaptic sensitivity to neurotransmitter to ensure synaptic efficacy.
Interestingly, expression of hVAPB in neurons rescues lethality, morphological and electrophysiological phenotypes associated with DVAP-33A
loss-of-function mutations. Moreover, neuronal expression of hVAPB in a wt background induces phenotypes similar to the overexpression of DVAP-33A. These data clearly indicate that DVAP-33A and hVAPB perform homologous functions at the synapse and as a consequence, information gained by studying DVAP-33A is expected to be relevant for hVAPB function as well. Surprisingly, neuronal expression of mutant VAP proteins also rescues all phenotypes associated with mutations in DVAP-33A
. Two alternative scenarios could be proposed to explain these data: the mutation is irrelevant for the ALS8 pathogenesis or the mutant allele has a pathogenic effect while retaining certain functional properties of the wt protein. We strongly favor the second hypothesis for the following reasons. First, the P56S mutation in hVAPB has been reported to be causative for an inherited form of MNDs in humans. This mutation affects nine related families totaling 1500 individuals of which 200 suffer from motor neuron disorders (31
). Second, we have generated a genetic model for MNDs where the expression of the aberrant VAP recapitulates major hallmarks of the human disease, clearly indicating that the mutation has a pathogenic effect. Third, our data and data published by others (23
) suggest that both the Drosophila
and the human mutant proteins retain some functional wt properties such as the ability to self-oligomerize. However, neuronal expression of the pathogenic protein induces aggregate formation and depletes the wt protein from its normal localization. These effects are not observed when the wt protein is overexpressed, suggesting that the mutant protein has acquired a new, potentially toxic property.
Indeed, one of the most common features of MNDs and nearly all neurodegenerative diseases is the accumulation of aggregates that are intensively immunoreactive to disease-related proteins (2
). Each disease, however, differs with respect to the anatomical location and morphology of the aggregates. The major component of the aggregates is usually the protein encoded by the gene mutated in the familial forms, which is also unique to each disease. Despite this diversity, a bulk of circumstantial evidence support the hypothesis that aggregates are typical hallmarks of neurodegenerative diseases and have a toxic effect on neurons (32
). While no autopsy material is available for familial cases with the P56S mutation, SOD1-positive inclusions have been reported in human sporadic and familial ALS cases as well as in SOD1 mouse models (28
). We found the presence of aggregates that are intensively immunoreactive for DVAP-33A both in neuronal cell bodies and in nerve fibers of our MND model. Interestingly, hVAPB carrying the pathogenic mutation has also been shown to undergo intracellular aggregation when expressed in a cell culture system (23
). However, similarities between human disease and our fly model are not limited to aggregate formation as flies expressing transgenic VAP proteins carrying the ALS8 mutation, exhibit other hallmarks of the human disease such as neuronal cell death, muscle wasting and defective locomotion behavior.
Although it remains to be established whether the VAP protein in the aggregates represents the mutant protein, the endogenous protein or a mixture of both, we clearly observe a regional decrease in the level of the endogenous protein. The DVAP-33A protein that is normally associated with the plasma membrane in neuronal cell bodies and at the neuromuscular synapses is nearly undetectable in DVAPP58S
transgenic animals. As a consequence of the decrease in synaptic levels of the endogenous protein, a decrease in the number of boutons is observed. We have previously shown that DVAP-33A regulates bouton formation at the synapse in a dosage-dependent manner (9
). Despite these structural alterations a homeostatic mechanism is established to maintain synaptic efficacy within functional boundaries. We speculate that the depletion of the endogenous protein from its normal localization and the formation of aggregates would affect the homeostatic mechanism linking structural remodeling and synaptic efficacy controlled by DVAP-33A. Although not directly tested in our model, experiments in cell culture show that overexpression of mutant hVAPB induces formation of aggregates in which the endogenous wt protein is recruited (23
). This would suggest that the pathogenic allele functions as a dominant negative. However, the depletion of the endogenous protein from its normal localization cannot be the principal mechanism of the disease as mutants lacking DVAP-33A do not develop MND. It is therefore possible that the pathogenic allele has acquired an abnormal, new toxic activity. Similar to what has been proposed for other neurodegenerative diseases, the formation of aggregates may directly interfere with critical cellular processes and/or compromise the ability of the system to keep up with the degradation of aggregated proteins (34
Taken together these data offer experimental support to the hypothesis that VAP proteins play a conserved role in synaptic homeostasis and emphasize the relevance of this fly model in fostering our understanding of the molecular mechanisms underlying VAP-induced motor neuron degeneration in humans.