Tiziana Cotrufo

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.

β-amyloid controls altered Reelin expression and processing in Alzheimer's disease

Reelin is a glycoprotein that modulates synaptic function and plasticity in the mature brain, thereby favouring memory formation. We recently reported altered cerebral Reelin expression in Alzheimers disease (AD). Here we demonstrate pronounced Reelin changes at protein and mRNA levels in the frontal cortex in adult Downs syndrome (DS), where the extra copy of chromosome 21 leads to overexpression of beta-amyloid. In cortical extracts of fetal DS samples we detected increased levels of the full-length Reelin and the 310-kDa fragment. Overexpression of mutant human amyloid precursor protein also led to an increase in levels of Reelin fragments in Tg2576 transgenic mice for human beta-amyloid. Finally, in vitro Abeta42 treatment of SH-SY5Y neuroblastoma cells led to increased Reelin levels. An altered pattern of Reelin glycosylation was detected in extracts from the frontal cortex of AD patients and in Abeta42-treated SH-SY5Y cells, supporting the notion that beta-amyloid triggers altered Reelin processing. These results provide evidence that Reelin expression and processing is altered in several amyloid conditions.

Ribose 1-phosphate and inosine activate uracil salvage in rat brain

The purpose of this study was to determine the mechanism by which inosine activates pyrimidine salvage in CNS. The levels of cerebral inosine, hypoxanthine, uridine, uracil, ribose 1-phosphate and inorganic phosphate were determined, to evaluate the Gibbs free energy changes (deltaG) of the reactions catalyzed by purine nucleoside phosphorylase and uridine phosphorylase, respectively. A deltaG value of 0.59 kcal/mol for the combined reaction inosine+uracil <==> uridine+hypoxanthine was obtained, suggesting that at least in anoxic brain the system may readily respond to metabolite fluctuations. If purine nucleoside phosphorolysis and uridine phosphorolysis are coupled to uridine phosphorylation, catalyzed by uridine kinase, whose activity is relatively high in brain, the three enzyme activities will constitute a pyrimidine salvage pathway in which ribose 1-phosphate plays a pivotal role. CTP, presumably the last product of the pathway, and, to a lesser extent, UTP, exert inhibition on rat brain uridine nucleotides salvage synthesis, most likely at the level of the kinase reaction. On the contrary ATP and GTP are specific phosphate donors.

Effects of dark rearing on phosphorylation of neurotrophin Trk receptors

Total lack of visual experience (dark rearing, DR) is known to affect development of mammalian visual cortex (VC) and to prolong the critical period of visual cortical plasticity. Neurotrophins (NTs) have been proposed to play a relevant role in activity dependent processes important for the final shaping of cortical visual connections. Neurotrophin supply or antagonism of endogenous NT action profoundly affect visual cortical development and plasticity; in particular, exogenous supply of NTs counteracts DR effects on VC development. However, the effects of DR on NT expression are still debated and mounting evidence reports a mismatch between BDNF mRNA and protein expression in DR animals. To gain insight into the effects of DR on expression of nerve growth factor (NGF) and the functional state of NT signalling pathways, we assessed the phosphorylation state of Trk receptors in light‐reared animals (LR), in dark‐reared animals (DR), in DR animals briefly exposed to light and in DR animals with exogenous supply of NTs [NGF, brain‐derived neurotrophic factor (BDNF) and NT‐4] in the VC. We report that DR increases the expression of NGF but reduces the phosphorylation of TrkA and TrkB receptors with respect to LR; normal phosphorylation is rapidly rescued by a brief exposure to light. Exogenous supply of NGF, BDNF or NT4 in DR animals also rescues the phosphorylation of their receptors.

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.

Regulation of Patterned Dynamics of Local Exocytosis in Growth Cones by Netrin-1

Axonal guidance and synaptic specification depends on specific signaling mechanisms that occur in growth cones. While several signaling pathways implicated in cone navigation have been identified, membrane dynamics in growth cones remains largely unknown. We took advantage of SynaptopHluorin and high-speed optical recordings to monitor the patterns of membrane dynamics in rat hippocampal growth cones. We show that exocytosis occurs both at the peripheral and central domains, including filopodia, and that SynaptopHluorin signals occur as spontaneous patterned peaks. Such transients average approximately two per minute and last ∼30 s. We also demonstrate that the chemoattractant Netrin-1 elicits increases in the frequency and slopes of these transients, with peaks averaging up to six per minute in the peripheral domain. Netrin-1-dependent regulation of exocytotic events requires the activation of the Erk1/2 and SFK pathways. Furthermore, we show that domains with high SynaptopHluorin signals correlate with high local calcium concentrations and that local, spontaneous calcium increases are associated with higher SynaptopHluorin signals. These findings demonstrate highly stereotyped, spontaneous transients of local exocytosis in growth cones and that these transients are positively regulated by chemoattractant molecules such as Netrin-1.

A role for the tyrosine kinase ACK1 in neurotrophin signaling and neuronal extension and branching.

Neurotrophins are involved in many crucial cellular functions, including neurite outgrowth, synapse formation, and plasticity. Although these events have long been known, the molecular determinants underlying neuritogenesis have not been fully characterized. Ack1 (activated Cdc42-associated tyrosine kinase) is a non-receptor tyrosine kinase that is highly expressed in the brain. Here, we demonstrate that Ack1 is a molecular constituent of neurotrophin signaling cascades in neurons and PC12 cells. We report that Ack1 interacts with Trk receptors and becomes tyrosine phosphorylated and its kinase activity is increased in response to neurotrophins. Moreover, our data indicate that Ack1 acts upstream of the Akt and MAPK pathways. We show that Ack1 overexpression induces neuritic outgrowth and promotes branching in neurotrophin-treated neuronal cells, whereas the expression of Ack1 dominant negatives or short-hairpin RNAs counteract neurotrophin-stimulated differentiation. Our results identify Ack1 as a novel regulator of neurotrophin-mediated events in primary neurons and in PC12 cells.

Blockade of the SNARE protein syntaxin 1 inhibits glioblastoma tumor growth.

Glioblastoma (GBM) is the most prevalent adult brain tumor, with virtually no cure, and with a median overall survival of 15 months from diagnosis despite of the treatment. SNARE proteins mediate membrane fusion events in cells and are essential for many cellular processes including exocytosis and neurotransmission, intracellular trafficking and cell migration. Here we show that the blockade of the SNARE protein Syntaxin 1 (Stx1) function impairs GBM cell proliferation. We show that Stx1 loss-of-function in GBM cells, through ShRNA lentiviral transduction, a Stx1 dominant negative and botulinum toxins, dramatically reduces the growth of GBM after grafting U373 cells into the brain of immune compromised mice. Interestingly, Stx1 role on GBM progression may not be restricted just to cell proliferation since the blockade of Stx1 also reduces in vitro GBM cell invasiveness suggesting a role in several processes relevant for tumor progression. Altogether, our findings indicate that the blockade of SNARE proteins may represent a novel therapeutic tool against GBM.

SNARE proteins play a role in motor axon guidance in vertebrates and invertebrates

Axonal growth and guidance rely on correct growth cone responses to guidance cues, both in the central nervous system (CNS) and in the periphery. Unlike the signaling cascades that link axonal growth to cytoskeletal dynamics, little is known about the cross‐talk mechanisms between guidance and membrane dynamics and turnover in the axon. Our studies have shown that Netrin‐1/deleted in colorectal cancer signaling triggers exocytosis through the SNARE Syntaxin‐1 (STX‐1) during the formation of commissural pathways. However, limited in vivo evidence is available about the role of SNARE proteins in motor axonal guidance. Here we show that loss‐of‐function of SNARE complex members results in motor axon guidance defects in fly and chick embryos. Knock‐down of Syntaxin‐1, VAMP‐2, and SNAP‐25 leads to abnormalities in the motor axon routes out of the CNS. Our data point to an evolutionarily conserved role of the SNARE complex proteins in motor axon guidance, thereby pinpointing an important function of SNARE proteins in axonal navigation in vivo.

Neural Damage in Experimental Trypanosoma brucei gambiense: Infection: Hypothalamic Peptidergic Sleep and Wake-Regulatory Neurons.

Neuron populations of the lateral hypothalamus which synthesize the orexin (OX)/hypocretin or melanin-concentrating hormone (MCH) peptides play crucial, reciprocal roles in regulating wake stability and sleep. The disease human African trypanosomiasis (HAT), also called sleeping sickness, caused by extracellular Trypanosoma brucei (T. b.) parasites, leads to characteristic sleep-wake cycle disruption and narcoleptic-like alterations of the sleep structure. Previous studies have revealed damage of OX and MCH neurons during systemic infection of laboratory rodents with the non-human pathogenic T. b. brucei subspecies. No information is available, however, on these peptidergic neurons after systemic infection with T. b. gambiense, the etiological agent of 97% of HAT cases. The present study was aimed at the investigation of immunohistochemically characterized OX and MCH neurons after T. b. gambiense or T. b. brucei infection of a susceptible rodent, the multimammate mouse, Mastomys natalensis. Cell counts and evaluation of OX fiber density were performed at 4 and 8 weeks post-infection, when parasites had entered the brain parenchyma from the periphery. A significant decrease of OX neurons (about 44% reduction) and MCH neurons (about 54% reduction) was found in the lateral hypothalamus and perifornical area at 8 weeks in T. b. gambiense-infected M. natalensis. A moderate decrease (21% and 24% reduction, respectively), which did not reach statistical significance, was found after T. b. brucei infection. In two key targets of diencephalic orexinergic innervation, the peri-suprachiasmatic nucleus (SCN) region and the thalamic paraventricular nucleus (PVT), densitometric analyses showed a significant progressive decrease in the density of orexinergic fibers in both infection paradigms, and especially during T. b. gambiense infection. Altogether the findings provide novel information showing that OX and MCH neurons are highly vulnerable to chronic neuroinflammatory signaling caused by the infection of human-pathogenic African trypanosomes.