Drosophila Neurotrophins Reveal a Common Mechanism for Nervous System Formation

Neurotrophic interactions occur in Drosophila, but to date, no neurotrophic factor had been found. Neurotrophins are the main vertebrate secreted signalling molecules that link nervous system structure and function: they regulate neuronal survival, targeting, synaptic plasticity, memory and cognition. We have identified a neurotrophic factor in flies, Drosophila Neurotrophin (DNT1), structurally related to all known neurotrophins and highly conserved in insects.By investigating with genetics the consequences of removing DNT1 or adding it in excess, we show that DNT1 maintains neuronal survival, as more neurons die in DNT1 mutants and expression of DNT1 rescues naturally occurring cell death, and it enables targeting by motor neurons. We show that Spa¨ tzle and a further fly neurotrophin superfamily member, DNT2, also have neurotrophic functions in flies. Our findings imply that most likely a neurotrophin was present in the common ancestor of all bilateral organisms, giving rise to invertebrate and vertebrate neurotrophins through gene or whole-genome duplications. This work provides a missing link between aspects of neuronal function in flies and vertebrates, and it opens the opportunity to use Drosophila to investigate further aspects of neurotrophin function and to model related diseases

Molecular regulation of somatosensory neuron development

The somatosensory system of vertebrates perceives and transmits a variety of information from both external and internal environments to the central nervous system where an integrated response is established leading to adaptive outcome. Specific classes of sensory neurons convey the information consisting of touch, muscle stretch, temperature, itch, and pain. Each type of sensory neuron expresses a group of specific markers or proteins in order to perform a specialized function. However, the mechanisms that ensure the acquisition of various molecular traits by somatosensory neurons during development is still not fully understood. This doctoral thesis explores several early developmental events for different types of somatosensory neurons at molecular and cellular levels in order to reduce the gap of knowledge in this field. In Paper I and II, we investigated the neuronal specification of nociceptive neurons, which were derived from specific waves of neurogenesis. We found that PRDM12, an epigenetic regulator, was necessary for the entire nociceptive lineage to develop. In the absence of PRDM12, neural crest precursors failed to generate all of the nociceptive neurons. We also found that the key transcription factor RUNX1, which plays an important role in the diversification of nociceptive neurons, was induced by factors released by early born neurons, emphasizing the important influence of the environment created by early postmitotic neurons on the fate of later born neurons. In Paper III, we proposed a new cell selection model in the early cell death of sensory neurons using the proprioceptive neurons population as a model system. The canonical neurotrophic theory suggests similarity of neurons when competing for target-derived neurotrophins for their survival. However, our data showed that early proprioceptive neurons exhibit a molecular heterogeneity code leading to different capacities to survive already before the cell death period. Further, this capacity was intrinsically regulated by the transcription factor RUNX3 whose expression was defined by the surrounding morphogen retinoic acid. Finally, in Paper IV, we showed that the transcription factor RUNX3 controls the axonal growth rate of developing sensory neurons in a strict temporal and spatial manner. Taking advantage of both chicken embryos and mouse genetics, we observed that the difference in peripheral nerve growth at different axial levels was encoded by RUNX3 expression. In summary, the data collected in this thesis describes several new insights into the molecular regulation during the step-wise development of somatosensory neurons, including neurogenesis, neuronal specification, early cell death, and axonal growth. This knowledge will help us to the better understanding of the development of the somatosensory system as well as provide new knowledge that might help improving approaches of treatment for patients with somatosensory disorders such as congenital insensitivity to pain

Cyclotraxin-B, the First Highly Potent and Selective TrkB Inhibitor, Has Anxiolytic Properties in Mice

In the last decades, few mechanistically novel therapeutic agents have been developed to treat mental and neurodegenerative disorders. Numerous studies suggest that targeting BDNF and its TrkB receptor could be a promising therapeutic strategy for the treatment of brain disorders. However, the development of potent small ligands for the TrkB receptor has proven to be difficult. By using a peptidomimetic approach, we developed a highly potent and selective TrkB inhibitor, cyclotraxin-B, capable of altering TrkB-dependent molecular and physiological processes such as synaptic plasticity, neuronal differentiation and BDNF-induced neurotoxicity. Cyclotraxin-B allosterically alters the conformation of TrkB, which leads to the inhibition of both BDNF-dependent and -independent (basal) activities. Finally, systemic administration of cyclotraxin-B to mice results in TrkB inhibition in the brain with specific anxiolytic-like behavioral effects and no antidepressant-like activity. This study demonstrates that cyclotraxin-B might not only be a powerful tool to investigate the role of BDNF and TrkB in physiology and pathology, but also represents a lead compound for the development of new therapeutic strategies to treat brain disorders

Molecular modulation of brain development and function: lessons from “old” and “new”

Dissertation presented to obtain the Ph.D degree in BiologyUnderstanding how protein families interact to coordinate the development and function of the central nervous system (CNS)is critical for the understanding of this structure in normal conditions as well as in disease.(...

Molecular control of local translation in axon development and maintenance.

The tips of axons are often far away from the cell soma where most proteins are synthesized. Recent work has revealed that axonal mRNA transport and localised translation are key regulatory mechanisms that allow these distant outposts of the cell to respond rapidly to extrinsic factors and maintain axonal homeostasis. Here, we review recent evidence pointing to an increasingly broad role for local protein synthesis in controlling axon shape, synaptogenesis and axon survival by regulating diverse cellular processes such as vesicle trafficking, cytoskeletal remodelling and mitochondrial integrity. We further highlight current research on the regulatory mechanisms that coordinate the localization and translation of functionally linked mRNAs in axons

The role of basic fibroblast growth factor in the survival of rostral and caudal cells in the developing cerebral cortex of the rat: An in vitro study

The cerebral cortex is generated through the proliferation of the epithelium lining the lateral ventricles of the telencephalon. Although the ventricular epithelium (VE) has a uniform appearance, evidence is mounting to suggest that the VE is heterogeneous population of cells. In support of this, we have demonstrated that dissociated cell cultures prepared from rostral regions of the embryonic cortex undergo cell death whilst their caudal counterparts survive for longer periods. Furthermore, our experiments have shown that in co-cultures, caudally derived cells are able to rescue rostral sister cells. This suggests that a factor is present in caudal cortical cells that is able to promote the survival of rostral cells. Evidence from basic fibroblast growth factor (bFGF) knockout animals has shown that there is a significant reduction in the thickness of the cerebral cortex indicating that bFGF may be one such signalling molecule. In an effort to identify neurotrophic factors involved in the survival of these cortical cells, we applied a range of growth factors and neurotrophins including bFGF to rostral cultures derived from embryonic day 17 cortices. Only bFGF significantly rescued the rostral cortical cells indicating that bFGF is a candidate factor produced by caudal cells. Caudal cells treated with genistein, to block the effects of bFGF, survived for shorter period than the untreated control cultures. We have described the distribution of FGFR 1 receptor and bFGF in rostral and caudal cortical cultures using immunocytochemistry. Western blotting and in situ hybridisation. We propose that caudal cells produce bFGF which acts in an autocrine manner to maintain their survival and is also the factor likely to be affecting rostral cells in vivo

Immunohistochemical investigation of caspase-3 in neuronal apoptosis after experimental closed head trauma

The aim of this study was to investigate the caspase-3 activity in neuronal apoptosis after experimental closed head trauma model in rats. Twenty adult male rats were randomly divided into two groups: control and trauma groups. In trauma group, a cranial impact was delivered to the skull from a height of 7 cm at a point just in front of the coronal suture and over the right hemisphere. Rats were sacrificed at 12 hours after the onset of injury. Brain tissues were removed for histopathological investigation. In the trauma group, the neurons became extensively dark and degenerated into picnotic nuclei. The number of apoptotic neurons in frontal cortex tissue of trauma group was significantly more than control groups. In conclusion, the caspase 3 immunopositivity was increased in degenerating neurons of the frontal cortex tissue following trauma. The present results indicate that closed head trauma caused degenerative changes and increased caspase 3 immonupositivity in neurons. © 2011 OMU All rights reserved

The gut-brain axis, BDNF, NMDA and CNS disorders

Gastro-intestinal (GI) microbiota and the ‘gut-brain axis’ are proving to be increasingly relevant to early brain development and the emergence of psychiatric disorders. This review focuses on the influence of the GI tract on Brain-Derived Neurotrophic Factor (BDNF) and its relationship with receptors for N-methyl-d-aspartate (NMDAR), as these are believed to be involved in synaptic plasticity and cognitive function. NMDAR may be associated with the development of schizophrenia and a range of other psychopathologies including neurodegenerative disorders, depression and dementias. An analysis of the routes and mechanisms by which the GI microbiota contribute to the pathophysiology of BDNF-induced NMDAR dysfunction could yield new insights relevant to developing novel therapeutics for schizophrenia and related disorders. In the absence of GI microbes, central BDNF levels are reduced and this inhibits the maintenance of NMDAR production. A reduction of NMDAR input onto GABA inhibitory interneurons causes disinhibition of glutamatergic output which disrupts the central signal-to-noise ratio and leads to aberrant synaptic behaviour and cognitive deficits. Gut microbiota can modulate BDNF function in the CNS, via changes in neurotransmitter function by affecting modulatory mechanisms such as the kynurenine pathway, or by changes in the availability and actions of short chain fatty acids (SCFAs) in the brain. Interrupting these cycles by inducing changes in the gut microbiota using probiotics, prebiotics or antimicrobial drugs has been found promising as a preventative or therapeutic measure to counteract behavioural deficits and these may be useful to supplement the actions of drugs in the treatment of CNS disorders

Interaction between neurons, glia and target field cells in regulating the survival of cranial sensory neurons

1. AIMS: During embryonic development, most cutaneous sensory neurons depend for their survival on a supply of NGF synthesised in the skin. NGF promotes survival by binding to the trkA receptor tyrosine kinase whose signalling is modulated by the common neurotrophin receptor p75. trkA is expressed in trigeminal neurons shortly after axons reach their targets and NGF expression begins with the arrival of the earliest axons. This thesis was aimed at investigating interactions between neurons and targets using the trigeminal ganglion and its maxillary target field. Specifically, it assessed a. whether the induction and subsequent developmental changes in trkA mRNA seen in the ganglion in vivo are intrinsically regulated or dependent upon extrinsic signals, and whether N regulation ofNGF expression in the target field is influenced by the innervating ganglion. Further, it was aimed at c. assessing the importance of the trkA, trkB (BDNF-receptor) and full-length and truncated p75 neurotrophin receptors in promoting survival in the developing trigeminal ganglion, and d. determining the role of non target-related survival-mechanisms by Schwann cell precursors on trigeminal ganglion neurons and other cranial sensory ganglia, namely the nodose, dorsal root and superior cervical ganglia.2. METHODOLOGY: a. and b. Ganglion-target interactions and their effect on trkA and NGF expression were assessed using cultures of trigeminal ganglia and its target fields alone or in combination. Complementary approaches used knockout mice that increased or decreased the neuronal population in the trigeminal ganglion in vivo. c. The role of the neurotrophin receptors trkA, trkB and p75 in trigeminal neuron survival was assessed using knockout mice, including double knockout mice for trkA and trkB. d. The role of Schwann cell precursors in the survival of different populations of cranial sensory neurons was assessed using ErbB3 knockouts, which lack these cells.3. MAIN FINDINGS: a. Upregulation of trkA mRNA expression in the trigeminal ganglion appears to follow an intrinsic programme, with in vitro expression levels mimicking levels in vivo. However, extrinsic signals from the target-fields have a negative effect on trkA expression in vitro, b. Early target field NGF mRNA expression was positively influenced by ganglion innervation in vitro, and was significantly lower in the early target fields of embryos lacking trigeminal neurons early in development in vivo. c. Double trkA/trkB knockouts displayed neuronal death in the trigeminal ganglion, in a pattern suggesting that during certain phases in development there are subsets of neurons, which can survive with either one or the other receptor, whereas at other developmental stages both receptors are required. Neuronal losses in different p75 mutant embryos suggest a survival-promoting effect of p75 early in embryonic development, with truncated p75 having a role earlier in development than fulllength p75. d. Neuronal deficiencies in ErbB3-t- embryos support the idea that populations of cranial sensory neurons differ in their survival-requirement for Schwann cell precursors early in development, with early trigeminal and dorsal root neurons being more dependent on this support than early nodose neurons.4. SYNTHESIS AND CONCLUSIONS: a. and b. The results suggest that in addition to intrinsic mechanisms of regulation, trkA and NGF expression are subject to complex reciprocal interactions between the trigeminal ganglion and its target fields early in development. The control of survival of neurons during development may thus involve more than the restricted supply of survival factor from the target field, c, Sequential dependence of sensory neurons on one or more survival factor probably serves to increase survival to maximize the 'choice' of the target field during naturally occurring cell death, and to establish heterogeneity in the ganglion, d. Differences in the sensitivity of cranial sensory neurons to trophic support by Schwann cell precursors during early development are presumably related to the distance different populations grow to their target fields. Thus, in addition to survival provided by the target field, neurons appear to depend on survival signals from surrounding cells between the ganglion and the target field.Overall, these data support modifications to the way we should think about the way target derived signals regulate the survival of peripheral neurons. Rather than being a passive receipt of a restricted supply of NGF from the target field, it appears that complex target-ganglion interactions are involved, as well as input from other neurotrophic factors, either separately or in synergy with NGF, and input from non-target cells, such as Schwann cell precursors