Rosa Andrés

Multiple Roles for Hepatocyte Growth Factor in Sympathetic Neuron Development

We have studied the role of hepatocyte growth factor (HGF)/Met signaling in the development of sympathetic neuroblasts and neurons. Anti-HGF antibodies reduced the number of sympathetic neuroblasts that differentiated into neurons, but neither anti-HGF antibodies nor HGF affected neuroblast proliferation. Anti-HGF antibodies also reduced the survival of neuroblasts but not sympathetic neurons. HGF greatly enhanced the neurite outgrowth of NGF-dependent sympathetic neurons throughout development. These in vitro effects of anti-HGF antibodies and HGF were abolished by a disabling mutation of Met, the HGF receptor tyrosine kinase. The Met mutation also increased sympathetic neuroblast apoptosis in vivo. Because Met and HGF are expressed in sympathetic ganglia throughout development, it is possible that the multiple effects of HGF/Met signaling on sympathetic neuroblasts and neurons occur in part by an autocrine mechanism.

Coupling Met to Specific Pathways Results in Distinct Developmental Outcomes

Receptor tyrosine kinases (RTKs) mediate distinct biological responses by stimulating similar intracellular signaling pathways. Whether the specificity of the response is determined by qualitative or quantitative differences in signaling output is not known. We addressed this question in vivo by replacing the multifunctional docking sites of Met, the receptor for hepatocyte growth factor, with specific binding motifs for phosphatidylinositol-3 kinase, Src tyrosine kinase, or Grb2 (Met(2P), Met(2S), and Met(2G), respectively). All three mutants retained normal signaling through the multiadaptor Gab1, but differentially recruited specific effectors. While Met(2G) mice developed normally, Met(2P) and Met(2S) mice were loss-of-function mutants displaying different phenotypes and rescue of distinct tissues. These data indicate that RTK-mediated activation of specific signaling pathways is required to fulfill cell-specific functions in vivo.

Reelin delays amyloid-beta fibril formation and rescues cognitive deficits in a model of Alzheimer’s disease

Reelin is an extracellular matrix protein that is crucial for neural development and adult brain plasticity. While the Reelin signalling cascade has been reported to be associated with Alzheimers disease (AD), the role of Reelin in this pathology is not understood. Here we use an in vitro approach to show that Reelin interacts with amyloid-β (Aβ42) soluble species, delays Aβ42 fibril formation and is recruited into amyloid fibrils. Furthermore, Reelin protects against both the neuronal death and dendritic spine loss induced by Aβ42 oligomers. In mice carrying the APP(Swe/Ind) mutation (J20 mice), Reelin overexpression delays amyloid plaque formation and rescues the recognition memory deficits. Our results indicate that by interacting with Aβ42 soluble species, delaying Aβ plaque formation, protecting against neuronal death and dendritic spine loss and preventing AD cognitive deficits, the Reelin pathway deserves consideration as a therapeutic target for the treatment of AD pathogenesis.

A Signaling Mechanism Coupling Netrin-1/Deleted in Colorectal Cancer Chemoattraction to SNARE-Mediated Exocytosis in Axonal Growth Cones

Directed cell migration and axonal guidance are essential steps in neural development. Both processes are controlled by specific guidance cues that activate the signaling cascades that ultimately control cytoskeletal dynamics. Another essential step in migration and axonal guidance is the regulation of plasmalemma turnover and exocytosis in leading edges and growth cones. However, the cross talk mechanisms linking guidance receptors and membrane exocytosis are not understood. Netrin-1 is a chemoattractive cue required for the formation of commissural pathways. Here, we show that the Netrin-1 receptor deleted in colorectal cancer (DCC) forms a protein complex with the t-SNARE (target SNARE) protein Syntaxin-1 (Sytx1). This interaction is Netrin-1 dependent both in vitro and in vivo, and requires specific Sytx1 and DCC domains. Blockade of Sytx1 function by using botulinum toxins abolished Netrin-1-dependent chemoattraction of axons in mouse neuronal cultures. Similar loss-of-function experiments in the chicken spinal cord in vivo using dominant-negative Sytx1 constructs or RNAi led to defects in commissural axon pathfinding reminiscent to those described in Netrin-1 and DCC loss-of-function models. We also show that Netrin-1 elicits exocytosis at growth cones in a Sytx1-dependent manner. Moreover, we demonstrate that the Sytx1/DCC complex associates with the v-SNARE (vesicle SNARE) tetanus neurotoxin-insensitive vesicle-associated membrane protein (TI-VAMP) and that knockdown of TI-VAMP in the commissural pathway in the spinal cord results in aberrant axonal guidance phenotypes. Our data provide evidence of a new signaling mechanism that couples chemotropic Netrin-1/DCC axonal guidance and Sytx1/TI-VAMP SNARE proteins regulating membrane turnover and exocytosis.

Podocalyxin Is a Novel Polysialylated Neural Adhesion Protein with Multiple Roles in Neural Development and Synapse Formation

Neural development and plasticity are regulated by neural adhesion proteins, including the polysialylated form of NCAM (PSA-NCAM). Podocalyxin (PC) is a renal PSA-containing protein that has been reported to function as an anti-adhesin in kidney podocytes. Here we show that PC is widely expressed in neurons during neural development. Neural PC interacts with the ERM protein family, and with NHERF1/2 and RhoA/G. Experiments in vitro and phenotypic analyses of podxl-deficient mice indicate that PC is involved in neurite growth, branching and axonal fasciculation, and that PC loss-of-function reduces the number of synapses in the CNS and in the neuromuscular system. We also show that whereas some of the brain PC functions require PSA, others depend on PC per se. Our results show that PC, the second highly sialylated neural adhesion protein, plays multiple roles in neural development.

Syntaxin 1 is required for DCC/Netrin‐1‐dependent chemoattraction of migrating neurons from the lower rhombic lip

Directed cell migration and axonal guidance are essential steps in neural development that share many molecular mechanisms. The guidance of developing axons and migrating neurons is likely to depend on the precise control of plasmalemma turnover in selected regions of leading edges and growth cones, respectively. Previous results provided evidence of a signaling mechanism that couples chemotropic deleted in colorectal cancer (DCC)/Netrin‐1 axonal guidance and exocytosis through Syntaxin1(Sytx1)/TI‐VAMP SNARE proteins. Here we studied whether Netrin‐1‐dependent neuronal migration relies on a similar SNARE mechanism. We show that migrating neurons in the lower rhombic lip (LRL) express several SNARE proteins, and that DCC co‐associates with Sytx1 and TI‐VAMP in these cells. We also demonstrate that cleavage of Sytx1 by botulinum toxin C1 (BoNT/C1) abolishes Netrin‐1‐dependent chemoattraction of migrating neurons, and that interference of Sytx1 functions with shRNAs or Sytx1‐dominant negatives disrupts Netrin‐1‐dependent chemoattraction of LRL neurons. These findings indicate that a Sytx1/DCC interaction is required for Netrin‐1 guidance of migrating neurons, thereby highlighting a relationship between guidance signaling and SNARE proteins that regulate membrane turnover.

Signalling effect of NIR pulsed lasers on axonal growth

In this work we show that a pulsed laser light placed at a distance is able to modulate the growth of axons of primary neuronal cell cultures. In our experiments continuous wave (CW), chopped CW and modelocked fs (FS) laser light was focused through a microscope objective to a point placed at a distance of about 15 microm from the growth cone. We found that CW light does not produce any significant influence on the axon growth. In contrast, when using pulsed light (chopped CW light or FS pulses), the beam was able to modify the trajectory of the axons, attracting approximately 45% of the observed cases to the beam spot. Such effect could possibly indicate the capacity of neurons to interpret the pulsating NIR light as the source of other nearby cells, resulting in extension of processes in the direction of the source.

Novel Candidate Genes and a Wide Spectrum of Structural and Point Mutations Responsible for Inherited Retinal Dystrophies Revealed by Exome Sequencing.

Background NGS-based genetic diagnosis has completely revolutionized the human genetics field. In this study, we have aimed to identify new genes and mutations by Whole Exome Sequencing (WES) responsible for inherited retinal dystrophies (IRD). Methods A cohort of 33 pedigrees affected with a variety of retinal disorders was analysed by WES. Initial prioritization analysis included around 300 IRD-associated genes. In non-diagnosed families a search for pathogenic mutations in novel genes was undertaken. Results Genetic diagnosis was attained in 18 families. Moreover, a plausible candidate is proposed for 10 more cases. Two thirds of the mutations were novel, including 4 chromosomal rearrangements, which expand the IRD allelic heterogeneity and highlight the contribution of private mutations. Our results prompted clinical re-evaluation of some patients resulting in assignment to a syndromic instead of non-syndromic IRD. Notably, WES unveiled four new candidates for non-syndromic IRD: SEMA6B, CEP78, CEP250, SCLT1, the two latter previously associated to syndromic disorders. We provide functional data supporting that missense mutations in CEP250 alter cilia formation. Conclusion The diagnostic efficiency of WES, and strictly following the ACMG/AMP criteria is 55% in reported causative genes or functionally supported new candidates, plus 30% families in which likely pathogenic or VGUS/VUS variants were identified in plausible candidates. Our results highlight the clinical utility of WES for molecular diagnosis of IRD, provide a wider spectrum of mutations and concomitant genetic variants, and challenge our view on syndromic vs non-syndromic, and causative vs modifier genes.

FAIM-L regulation of XIAP degradation modulates synaptic long-term depression and axon degeneration

Caspases have recently emerged as key regulators of axonal pruning and degeneration and of long-term depression (LTD), a long-lasting form of synaptic plasticity. However, the mechanism underlying these functions remains unclear. In this context, XIAP has been shown to modulate these processes. The neuron-specific form of FAIM protein (FAIM-L) is a death receptor antagonist that stabilizes XIAP protein levels, thus preventing death receptor-induced neuronal apoptosis. Here we show that FAIM-L modulates synaptic transmission, prevents chemical-LTD induction in hippocampal neurons, and thwarts axon degeneration after nerve growth factor (NGF) withdrawal. Additionally, we demonstrate that the participation of FAIM-L in these two processes is dependent on its capacity to stabilize XIAP protein levels. Our data reveal FAIM-L as a regulator of axonal degeneration and synaptic plasticity.

Ultra-short pulses to signal neuronal growth cone machinery

Measurable change in the sensory motor machinery of growth cones are induced by non contact femtosecond laser. The focused laser beam with an average power of 3 mW was positioned at some distance away from the closest fillopodia of cortical neurons from primary cell cultures (mice E15). By identifying a set of preliminary parameters we were able to statistically analyze the phenomenological behavior of the fillopodia and classify the effects different conditions of laser light has on the growth cone. Results show that fillopodia become significantly biased towards the focused femtosecond laser light. The same experiment performed with continuous wave (CW) produced results which were indistinguishable from the case where there is no laser light present (placebo condition) indicating no clear effects of the CW laser light on the fillopodia at a distance. These findings show the potential for ultrashort pulsed light to become a new type of pathfinding cue for neuronal growth cones.