Elia Di Schiavi et al. (2005) International Worm Meeting "KAL-1 function in C.elegans"

Elia Di Schiavi, Claudia Vicario, Salvatore Arbucci, Anna Sollo, Paolo Bazzicalupo

[International Worm Meeting, 2005]

kal-1 is the C. elegans homolog of Kal1 the gene responsible in human for X-linked Kallmann Syndrome (KS). KS is an inherited disorder defined by the association of hypogonadism and anosmia. The syndrome is genetically heterogeneous and three modes of inheritance have been described: autosomal recessive, autosomal dominant and X-linked. Kal1 encodes a secreted protein involved in olfactory axon guidance. C. elegans is the first animal for which a mutant completely lacking the function of the Kal1 gene has been produced. The gene is expressed in a subset of C. elegans neurons. The phenotypes presented by loss of function and overexpression mutants indicate that kal-1 modulates neurite branching in vivo (Rugarli et al., 2002; Bulow et al., 2002). In addition kal-1 is part of a redundant mechanism by which neurons influence epidermal cells morphogenesis in ventral enclosure, seam cells arrangement and male tail formation. The nematode and human genes are functionally conserved such that the human protein can compensate for the loss of the nematode one (Rugarli et al., 2002). In order to comprehend the still elusive role played by kal-1 in the ECM and to find candidate genes for the autosomal forms of KS, we have looked for interactors. We showed that in C. elegans, and possibly in humans, semaphorin pathway genes act in part on the same developmental processes in which kal-1 is involved and have thus begun to analyze the genetic interaction between kal-1 and these genes. To identify other interactors we are following different approaches: i) appropriate genetic screens for enhancer and suppressors of the neurite branching defects shown by kal-1 mutants; ii) a yeast two hybrid approach; iii) an in vivo structure/function relationship study of the different domains of which KAL-1 is composed. The understanding of the cellular and molecular mechanisms of kal-1 action and the identification of other genes involved in the processes in which KAL1 is involved might lead to the discovery of the genes responsible for the other genetic forms of the disease and to the elucidation of the still elusive mechanism of action in vertebrates.

Elia Di Schiavi et al. (2003) International Worm Meeting "kal-1 in epithelial morphogenesis and interaction with semaphorins."

Elia Di Schiavi, Paolo Bazzicalupo, Salvatore Arbucci, Maria Lettieri

[International Worm Meeting, 2003]

The molecular mechanisms through which cells and neuronal growth cones migrate, adhere to the substrate and find their targets are varied, appear to often work in redundant pathways and appear to be conserved in evolution. Many molecules involved in these processes still need to be discovered, especially the components of the extra cellular matrix that play a role in the final steps of morphogenesis. We have focused our study on kal-1, the C. elegans homolog of the X-linked Kallmann Syndrome gene. KS is an inherited disorder defined by the association of anosmia and hypogonadism, owing to impaired targeting and migration of olfactory axons and gonadotropin-releasing hormone secreting neurons. The gene responsible for the X-linked form of KS, Kal1, encodes a secreted protein which has been proposed to be involved in some aspects of olfactory axon guidance. We have identified the C. elegans homolog, kal-1, and have documented its function in the nematode studying its expression pattern and using loss of function and overexpression mutants. We have shown that kal-1 is part of a redundant mechanism by which neurons influence migration and adhesion of epidermal cells morphogenesis in ventral enclosure, seam cells arrangement and male tail formation. We have also shown that kal-1 affects neurite outgrowth in vivo by modulating branching. In order to comprehend the still elusive role played by kal-1 in the ECM and to find candidate genes for the autosomal forms of KS, we have looked for interactors. We have choosen mutants in genes which represent good candidates on the basis either of vertebrate studies and/or of the similarity of the phenotypes they generate in the worm. Among these genes we observed interesting results with the semaphorins. The semaphorins are a family of secreted proteins mainly involved in axon guidance in vertebrates. In C. elegans mutants show minor defects in the nervous system, but alterations in cell migration and adhesion with a set of phenotypes similar to that of kal-1 mutants. The study of epithelial morphogenesis in various genetic combination has indeed shown a complex but specific genetic interaction between kal-1 and both type of semaphorins. The fact that these multi-domain proteins are secreted and are interacting in the extracellular space and on the cell surface with many other molecules complicates the interpretation of the mechanisms involved. Its noteworthy that the expression of semaphorin genes is widespread and includes epidermal cells affected in kal-1 mutants, while kal-1 is produced only by underlying neurons, suggesting that KAL-1 may play a role as modulator of the semaphorins action.

Elia Di Schiavi et al. (2008) European Worm Meeting "kal-1 role in neuronal spine formation"

Elia Di Schiavi, Paolo Bazzicalupo, Giuliana Gelsomino, Silvana Castro

[European Worm Meeting, 2008]

kal-1 is the C. elegans homolog of Kal1 the gene responsible in human for X-. linked Kallmann Syndrome. Kallmann Syndrome is an inherited disorder caused. by defects of axonal outgrowth in the Olfactory System. Kal1 encodes a. secreted protein whose role in olfactory axon guidance is still elusive. C.. elegans is the first and only animal for which a mutant completely lacking. the function of the Kal gene has been produced. The gene is expressed in a. subset of C. elegans neurons. Main phenotypes presented by loss of function. and overexpression mutants indicate that kal-1 is part of a redundant. mechanism by which neurons influence epidermal cells morphogenesis. In. addition kal-1 modulates neurite branching in vivo (Rugarli et al., 2002).. In order to better understand the role played by kal-1 in neuronal. development we used the loss of function mutant gb503 and a panel of neuron. specific GFP markers. The most interesting results were obtained studying. HSN neuron development. kal-1 mutants present a posteriorly directed spine,. coming out from the HSN cellular body and parallel to the antero-posterior. axis of the worm. In order to determine the cell from which KAL-1 has to be. secreted to control HSN spine formation, we used a modification of the RNA-. interference method, recently developed in the lab (Esposito G. et al.,. 2007). Using this approach we were able to reduce the function of kal-1 in. selected neurons at the midbody of the animal and the results obtained. suggest that although KAL-1 is a secreted protein, its range of action is. limited in space.. Interestingly, the HSN-spine phenotype is completely suppressed by. mutations in hst-6, an enzyme that modifies proteoglycans. This result is. quite surprising because mutations in this gene were originally identified. as suppressors of neuronal branching defects due to the overexpression of. KAL-1 (Bulow et al., 2002). The possible mechanism by which kal-1 and hst-6. cooperate to affect branch/spine formation and how this could help. elucidate the still elusive role played by kal-1 in neuronal development,. will be discussed.

Elia Di Schiavi et al. (2002) European Worm Meeting "Functional conservation between the human and nematode KAL proteins"

Elia Di Schiavi, Paolo Bazzicalupo, Salvatore Arbucci

[European Worm Meeting, 2002]

The molecular mechanisms through which cells and neuronal growth cones migrate, adhere to the substrate and find their targets are varied, appear to often work in redundant pathways, and appear to be conserved in evolution. Although many molecules involved in these processes have already been identified and studied, many other still need to be discovered, especially the components of the extra cellular matrix (ECM) that play a role in the final steps of morphogenesis. We have focused our study on kal-1, the C. elegans homolog of the X-linked Kallmann syndrome gene. Kallmann syndrome is an inherited disorder defined by the association of anosmia and hypogonadism, owing to impaired targeting and migration of olfactory axons and gonadotropin-releasing hormone secreting neurons. The gene responsible for the X-linked form of Kallmann syndrome, KAL-1, encodes a secreted protein which has been proposed to be involved in some aspects of olfactory axon guidance. We have identified the C. elegans homolog, kal-1, and have documented its function in the nematode studying its expression pattern and using loss of function and overexpression mutants. We have shown that kal-1 is part of a mechanism by which neurons influence migration and adhesion of epidermal cells undergoing morphogenesis during ventral enclosure and male tail formation. We also have shown that kal-1 affects neurite outgrowth in vivo by modulating branching (Rugarli et al., 2002). This last finding has been recently confirmed by work on organotypic and primary cell cultures of rodents (Soussi-Yanicostas et al., 2002). Our findings add a new player to the set of molecules, which appear to underlie both morphogenesis and axonal/neuronal navigation in vertebrates and invertebrates. We show that human KAL-1 cDNA can compensate for the loss of worm kal-1 and that overexpression of worm or human KAL-1 cDNAs in the nematode results in the same sets of phenotypes. These data indicate that, despite the fact that on a residue to residue basis the identity between the human and the nematode protein is only 22% and that overall similarity is about 35%, there is significant functional conservation between these proteins. In addition these results definitely establish C. elegans as a powerful organism in which to investigate KAL function in vivo.

Elia Di Schiavi et al. (2008) C.elegans Neuronal Development Meeting "Cell Specific Knock-down of Gene Function in Targeted C. elegans Neurons"

Ivan Gallotta, Monica Vitale, Giovanni Esposito, Elia Di Schiavi, Paolo Bazzicalupo

[C.elegans Neuronal Development Meeting, 2008]

In C. elegans the available approaches to identify the neuron(s) where the function of a gene is required for a given trait (e.g. developmental, behavioral, etc.) are time consuming and largely restricted to non essential genes for which mutants are available. We have described a simple reverse genetics approach for reducing, in chosen C. elegans neurons, the function of genes (Esposito et al., 2007). The method is based on the expression, under cell specific promoters, of sense and antisense RNA corresponding to a gene of interest. We do not clone the knock-down constructs but directly inject in worms a mixture of two separate PCR fusion reactions. In each fusion a promoter specifically active in the neuron(s) we want to target drives the transcription, either in the sense or in the antisense orientation, of a CDS fragment of the gene we want to knock-down. By targeting the Green Fluorescent Protein gene, gfp, and the genes osm-10 and osm-6, we showed that this approach leads to efficient, heritable and cell autonomous knock-downs of gene function, even in neurons usually refractory to classic RNA interference (RNAi). By targeting the essential and ubiquitously expressed gene, gpb-1, which encodes a G protein beta-subunit, we identified two distinct sets of neurons in which the function of gpb-1 is required to regulate two distinct behavior: egg-laying and avoidance of repellents. The cell specific knock-downs obtained with this approach provide information (is the gene necessary?) that is complementary to that provided by the cell specific rescue of loss of function mutations (is the gene sufficient?). We will present examples to show how this approach can be used: i) to address questions in neural development (kal-1); ii) to identify the neuron(s) of a circuit where the function of a gene is required for a given behavior (gpb-1); iii) to study the neuronal function of pleiotropic and/or essential genes (gpb-1, unc-70, smn-1). We will also discuss the limitations of the method and what we still do not know about the mechanism and the features of the gene function knock-down that this procedure elicits.

Elia Di Schiavi et al. (2007) International Worm Meeting "Insights into the mechanism of action of kal-1, the C.elegans, homolog of the gene responsible for X-linked Kallmann Syndrome."

Paolo Bazzicalupo, Elia Di Schiavi, Giuliana Gelsomino, Claudia Vicario

[International Worm Meeting, 2007]

kal-1 is the C. elegans homolog of Kal1 the gene responsible in human for X-linked Kallmann Syndrome (KS). KS is an inherited disorder caused by defects of axonal outgrowth in the Olfactory System. The syndrome is genetically heterogeneous and three modes of inheritance have been described: autosomal recessive, autosomal dominant and X-linked. Kal1 encodes a secreted protein involved in olfactory axon guidance. C. elegans is the first animal for which a mutant completely lacking the function of the Kal gene has been produced. The gene is expressed in a subset of C. elegans neurons. The phenotypes presented by loss of function and overexpression mutants indicate that kal-1 modulates neurite branching in vivo (Rugarli et al., 2002; Bulow et al., 2002). In addition kal-1 is part of a redundant mechanism by which neurons influence epidermal cells morphogenesis in ventral enclosure, seam cells arrangement and male tail formation. The nematode and human genes are functionally conserved such that the human protein can compensate for the loss of the nematode one (Rugarli et al., 2002). In order to find candidate genes for the autosomal forms of KS, we have looked for interactors and we will show that semaphorin pathway genes act on the same developmental processes in which kal-1 is involved. With regard to neuronal branching we will describe the role, not studied before, played by kal-1 in HSN neuron development. The possible mechanism by which kal-1 acts to affect HSN cell body shape and axon branching will be discussed. In addition, recently identified modifiers of the kal-1 neurite branching phenotype will be described and could help elucidate the still elusive role of kal-1 in neuronal development. Finally, we used a modification of the RNA-interference method, recently developed in the lab, to reduce the function of kal-1 in selected neurons at the posterior end of the animal (Esposito G. et al., 2007). With this approach we were able to determine that the kal-1 expressing neurons DVA and PDB are responsible for two of the kal-1 male tail phenotypes, Ray 1 anteriorization and SET cell detachment, but that do not influence other defects (e.g Ray 5 and 6 inversion). At the same time we could also exclude that the kal-1 expressing neuron, PVW has a role in either Ray 1 anteriorization or SET cell detachment. These results suggest that although KAL-1 is a secreted protein, its range of action is limited in space. This is probably due to the fact that, unlike in other species, CeKAL-1 is anchored to the membrane via GPI.

Elia Di Schiavi et al. (2001) International C. elegans Meeting "kal-1, the C. elegans homolog of the X-linked Kallmann syndrome gene, is involved in epidermal morphogenesis and neuronal growth."

Paolo Bazzicalupo, Elena I Rugarli, Salvatore Arbucci, Elia Di Schiavi, Massimo A Hilliard, Andrea Ballabio, Anna Facciolli

[International C. elegans Meeting, 2001]

The molecular mechanisms through which cells and neuronal growth cones migrate, adhere to the substrate and find their targets are varied, appear to often work in redundant pathways, and appear to be conserved in evolution. Although many molecules have already been identified and studied, especially using the invertebrate model systems, it is clear that many other molecules involved in these processes still need to be discovered, especially the components of the extra cellular matrix (ECM) that play a role the final steps of morphogenesis. We have focused our study on kal-1 , the C. elegans homolog of the X-linked Kallmann syndrome gene. The syndrome is characterized by anosmia and hypogonadism and is apparently due to a failure in the migration or proper targeting of olfactory axons in the olfactory bulbs. The human gene responsible for the X-linked form of the disease has been identified. It codes for a secreted protein of still elusive function that contains a cysteine rich N-terminal domain, a four disulfide core domain, WAP, and three fibronectin type III domains. The C. elegans gene is composed of 6 exons and codes for a 700 aa protein with a domain topology similar to that of vertebrates. Using reporters we have determined that kal-1 is expressed by a subset of neuroblasts/neurones beginning early in embryogenesis. To understand the function of kal-1 in C. elegans , we generated and studied a null deletion mutant, kal-1(gb503) , as well as worms overexpressing KAL-1 from transgenic extrachromosomal arrays. The phenotypes shown by kal-1 mutants include: i ) male tail defects; in general the whole structure appears scrawny or distorted and the sensory rays are variously altered with reductions and losses of rays, presence of extra rays, fusions and inversions of the position of rays. The male tail defects of the loss of function mutant gb503 are recessive and represent the most penetrant phenotype observed. The defects are rescued in gb503 worms carrying a wild type copy of the gene as a transgene on an extra-chromosomal array. ii ) embryonic lethality and morphological abnormalities of newly hatched larvae; affected kal-1 mutant embryos are defective in ventral enclosure and rupture ventrally with cells protruding out of the embryonic mass. Some embryos appear to present later enclosure defects resulting in head and tail abnormalities of the hatched larvae. iii ) incompletely penetrant neuronal growth defects with extra-branching of processes. Experiments using specific reagents to visualize epithelial cell boundaries in mutant worms indicate that the defects, caused by a reduction or an increase of kal-1 function, are mediated by dramatic effects on shape, position, adhesion and migration of epidermal cells undergoing active morphogenesis in the proximity of kal-1 expressing neurons. Since kal-1 reporters are expressed in neuronal cells while the cells most affected by mutants are epithelial cells, we propose that CeKAL-1 acts non-cell-autonomously to modulate, in concert with other molecules, the adhesion of cells and growth cones to the matrix and to other cells. The combination of phenotypes we have observed in kal-1 mutants is strongly reminiscent of that presented by mutants in other genes which affect epithelial morphogenesis and axonal growth, such as mab-20 (Ce-Sema-2a), vab-2 and vab-1 (ephrin and ephrin receptor) and others. It is possible that kal-1 acts in the same pathways in which these genes function.

Bertapelle, Carla et al. (2017) International Worm Meeting "A C.elegans model to study LDL-Related Proteins involvement in Alzheimer's disease."

Russo, Claudio, Cocco, Federica, Di Schiavi, Elia, Bertapelle, Carla

[International Worm Meeting, 2017]

The Alzheimer's disease (AD) is the most common neurodegenerative disorder and its symptoms include dementia, loss of memory and motor dysfunction. The main neuropathologic hallmarks of AD are the extracellular deposits of beta -amyloid (A beta ) plaques, insoluble aggregates of beta-amyloid peptide, and the intraneuronal fibrillary tangles, resulting from the hyperphosphorylation of the microtubule associated protein Tau. These aggregates both contribute to the loss of synapse functions and the consequent neuronal death. The major genetic risk factor in sporadic AD is represented by the E4 isoform of Apolipoprotein E (ApoE), ligand of the Low Density Lipoprotein-Related Proteins Receptors (LRPs), involved in a variety of functions, including cholesterol metabolism and cell signaling. LRPs intramembrane proteolysis is regulated by the ?-secretase cleavage and similarly the beta-amyloid peptide is the product of the Amyloid Precursor Protein (APP) ?-secretase cleavage. Moreover, Apo E affects A beta production and clearance and colocalizes with A beta in amyloid plaques. C.elegans presents the orthologs of human APP, ?-secretase presenilins and Tau (respectively apl-1, sel-12, hop-1 and ptl-1), and it has been extensively used to study AD, with several neurodegeneration models generated to investigate APP processing, A beta aggregation and Tau hyperphosphorylation. Neurodegeneration results in defects progressively increasing with aging, such as neuronal abnormalities (inclusions and varicosities in cell body), axonal degeneration (bulges, dilatation and collapse of axonal membrane), altered neurotransmission and uncoordinated movement (Kraemer et al., 2003). Considering the lack of informations about LRPs and its involvement in AD, we propose a new C.elegans model to study the correlation of human LRP8, the ApoE receptor, to APP and Tau. Several data led us to hypothesize that LRPs could affect APP processing and signaling (and vice versa) through ?-secretase, resulting in alteration of Tau phosphorylation levels. To test this hypothesis we generated transgenic worms expressing human LRP8 in all neurons and analyzed their phenotypes to determine whether LRP8 overexpression caused any defect similar to the one caused by Tau hyperphosphorylation or APP overexpression. Our preliminary results suggest that huLRP8 overexpression affects nematode locomotion, a defect that progressively worsen in aged worms, and lifespan. These results allow us to hypothesize an involvement of LRP8 in neuronal function and to investigate huLRP8 processing and its genetic interaction with APP.

La Rocca, Federica et al. (2021) International Worm Meeting "hnRNPQ/hrp-2 role in splicing and neurodegeneration."

Cieri, Federica, Di Schiavi, Elia, La Rocca, Federica, Santonicola, Pamela, Zampi, Giuseppina

[International Worm Meeting, 2021]

A correct splicing of mRNA is globally required in all cells, but neurons seem highly sensitive to perturbations with numerous neurological diseases caused by splicing defects, including spinal muscular atrophy (SMA), amyotrophic lateral sclerosis (ALS), and dementias. Neurons have features that make them vulnerable to mis-splicing events, such as their postmitotic state and longevity. However, the reason of this peculiar sensitivity of neurons to splicing alterations, why only subclasses of neurons are affected and which step of RNA processing is impaired in these diseases is still debated. We are investigating the molecular mechanisms underlying the neurodegeneration caused by splicing defects in SMA, using a neuron specific silencing approach in C.elegans. SMA is a motor neurons (MNs) specific disorder caused by mutations in the Survival Motor Neuron (SMN) gene. RNA-sequencing of iPS-MNs from SMA patients and healthy people allowed us to identify differentially expressed/spliced genes, enriched in RNA motif 7 (CAAAAAG). This motif is specifically bound by hnRNPQ, a protein interacting with SMN in the SMN complex. hrp-2 is the C.elegans homolog of hnRNPQ and we determined that, similarly to smn-1(KO), hrp-2 (ok1278) null mutants arrest as larvae, have a severe decrease in lifespan and locomotion defects. hrp-2 mutants show a reduction of visible D-type MNs and axonal defects, suggesting a specific role in neuron survival. hrp-2 overexpression in D-type MNs rescued the degeneration of MNs and the defects in locomotion observed in smn-1 silenced animals. These data suggest a specific involvement of hrp-2 in MNs survival and in smn-1 pathway, supporting the role of hrp-2 as an smn-1 modifier and as a possible therapeutic target for the disease. Further experiments are ongoing to better understand the genetic interaction between the two genes in MNs and to identify the role of mis-spliced targets in neurodegeneration.

Cieri, Federica et al. (2021) International Worm Meeting "B-Raf contribution to motoneuron degeneration"

Cieri, Federica, Di Schiavi, Elia, Claus, Peter, Santonicola, Pamela, Hensel, Niko, Zampi, Giuseppina

[International Worm Meeting, 2021]

The B-Raf orthologue lin-45 belongs to MAPK/ERK pathway, is involved in the regulation of cellular proliferation and differentiation and shows a Ras GTPase binding activity. Beside its well-known role in vulva formation, lin-45 is also expressed in neurons, including motoneurons, and is critical for chemo- and thermo-sensory behaviors and locomotion. Using a phospho-antibody array combined with a network-biology approach on Spinal Muscular Atrophy (SMA) mice, we identified B-Raf as a signaling hub among different dysregulated pathways involved in motor neuron degeneration. In SMA, motor neurons degenerate because of mutations in the Survival Motor Neuron 1 (SMN1) gene. We investigated in C. elegans the role of B-Raf/lin-45 in degenerating motoneurons (MNs) and observed that lin-45 expression is reduced in pre-symptomatic smn-1(KO) animals. We rescued the neurodegeneration and locomotion defects caused by smn-1 silencing in D-type MNs by re-expressing lin-45 specifically in MNs. We thus demonstrated that lin-45 can play a cell-autonomous neuroprotective role when smn-1 is downregulated. Consistently, a hyperactive isoform of lin-45 (S312A), which is devoid of an inhibitory phospho-site, maximized the rescue effects. The neurodegeneration caused by smn-1 silencing increases with animal ageing and we were able to rescue it by expressing lin-45 from L2 stage, through inducible transgenics. This is important since at L2 stage the degeneration has already started and all 19 D-type motoneurons should have been generated, thus suggesting that lin-45 protects from motoneuron loss rather than interfering with neurogenesis and can block the degeneration when it is already started. Using MAPK pathway drug inhibitors and mek-2 mutants we abrogated the rescue obtained after lin-45 overexpression. Thus, genetic and pharmacological approaches showed that lin-45 rescue is mediated by the MAPK/ERK pathway. The central role of B-Raf was confirmed in a SMA model of SMA and in patient cells and strongly support a role of B-RAF in neurodegeneration and in particular in the Smn1 pathway.