3.5.1 ELUCIDATION OF THE ROLE OF LNP-1 IN C. ELEGANS
The main objective of this work is to use the well-defined model system C. elegans to evaluate the Lnp-1 involvement in the nervous system. Understanding the role of Lnp-1 in the C. elegans nervous system could also provide novel insights into nervous system development and function of this model organism. In addition, it should also help the identification of conserved molecules in more complex organisms and determine whether they belong to conserved functional pathways.
We investigated the function of LNP-1 in nervous system by using reverse genetic approaches, analysing two deletion lnp-1 mutants and its RNAi knockdown by feeding procedure. Our data show that in C. elegans, lnp-1 is expressed early during development and its expression is enriched in neural tissues specifically in the ventral nerve cord. LNP-1 sub-cellular localization is dependent of the UNC-104/kinesin protein. Analyses on lnp-1 loss-of-function animals suggest that it could be involved in the neural circuitry, which controls worm locomotion. In C. elegans, assessing the neurological function of a given gene employs standardized behavioural and pharmacological tools, as described in the Introduction, Chapter 3.2. We utilized several behavioural and pharmacological tools related to presumed neurological function to determine the role of lnp-1. Deletion mutations in lnp-1 lead to increased resistance to aldicarb, an acetylcholinesterase inhibitor, and in slight locomotor and egg laying defects. However, sensitivity to levamisole, a nicotinic agonist that unlike aldicarb only affects postsynaptic function, was similar to that of wild-type animals, suggesting a presynaptic function for LNP-1 in acetylcholine-dependent neurotransmission. To characterize the role of LNP-1 in neuronal trafficking we took advantage of already known GFP transgenics of vesicular markers. The mislocalization of presynaptic proteins, such as Synaptobrevin-1 or RAB-3, in lnp-1 mutants further supports the involvement of LNP-1 in synaptic trafficking.
3.5.2 IDENTIFICATION OF LNP-1 PARTNERS
Assessing the function of lnp-1 also requires the identification of putative partners and the investigation of its involvement in known molecular pathways. Consequently, the putative partners of LNP-1 participation in the nervous system function should also be considered. The identification of LNP-1-interacting proteins can provide important clues about its function by enabling an integrative view of LNP-1 position into the cellular and molecular pathways. In a yeast two hybrid screen we have identified a putative interaction partner, coding for the ortholog protein of the Drosophila seven in absentia (SINA) protein, which has been shown to play an
important role in neuronal cell fate determination. In C. elegans siah-1 (sina homologue) expression pattern is related to that of lnp-1. Broad and possibly ubiquitous expression is first visible during gastrulation. In larvae and adults, LNP-1 and SIAH-1 reporters are expressed in numerous cells bodies along the ventral cord, around the pharynx and tail and most of them are neuronal cells. Deletion mutants and RNAi knockdown of SIAH-1 were compared to LNP-1 results to establish the relationship between these two proteins. Analysis of SNB-1 and RAB-3 transgenic strains revealed localization defects of SNB-1 and RAB-3 in dssiah-1 fed animals similar with in lnp-1 mutants.
The results obtained suggested a role for LNP-1 in synaptic vesicle trafficking in the pre-synaptic element and neurotransmission. To determine if LNP-1/Siah-1 couple is involved in more general vesicle transport or is specific for presynaptic vesicle transport we tested a known marker for the post-synaptic element: glutamate receptor 1 (GLR-1). Previously was shown that GLR-1 localization and recycling are dependent of ubiquitination. In this work, we analyse the requirement of LNP-1 for the ubiquitin-dependent turnover of GLR-1 and the involvement of ubiquitin in SNB-1 turnover regulation. Altogether, our data points toward a very tempting hypothesis: LNP-1 regulates the synaptic strength through its interaction with ubiquitin/proteasome system.
3.5.3 ANALYZING THE CONSERVATION OF LNP-1 THROUGH EVOLUTION
As lnp-1 gene was first identified in mouse, is of interest to study whether the intracellular localization, interaction partners, and consequently its function are evolutionarily conserved. Using different cell lines and intracellular markers we started in this work to characterize the LNP protein in mammals.
We studied the sublocalisation of Lnp in mammalian cell cultures (3T3 and C3H fibroblast and NGF-treated PC12 cell lines) using specific antibodies we generated against the mouse Lnp. We performed colocalisation experiments with known intracellular structure or organelle markers like tubulin, actin, vinculin, stage-specific endosomal markers. We could show that in both fibroblast and NGF-differentiated PC12 cells, Lnp revealed punctuate staining pattern in cell bodies and neuritic processes. Partial colocalisation was found with synaptophysin, a synaptic vesicle marker and kinesin, a motor. It has been reported that Siah, like Lnp, partially colocalises with synaptophysin in NGF-differentiated PC12 cells (Wheeler et al., 2002). To further analyse the putative interaction between these two proteins in vivo, we performed immunohistochemistry in mammalian cell cultures. We showed that in NGF-differentiated PC12 cells, Siah and Lnp colocalised in the cell body and neuritic processes, to punctuate structures, some of which are positive for the synaptic vesicle marker synaptophysin.
Elucidating the molecular mechanisms by which LNP-1/ubiquitin proteaseome system regulates synaptogenesis could be an essential aspect of understanding the pathological processes that underlie some central nervous system disorders in humans. It is interesting to note that parkin – a mutated gene responsible for Parkinson’s disease, encodes an ubiquitin E3 ligase. It is very likely that in a near future defects in this system will be linked to a growing number of diseases.