The neuronal ceroid lipofuscinosis protein Cln7 functions in the postsynaptic cell to regulate synapse development.

The neuronal ceroid lipofuscinoses (NCLs) are a group of fatal, monogenic neurodegenerative disorders with an early onset in infancy or childhood. Despite identification of the genes disrupted in each form of the disease, their normal cellular role and how their deficits lead to disease pathology is not fully understood. Cln7, a major facilitator superfamily domain-containing protein, is affected in a late infantile-onset form of NCL. Cln7 is conserved across species suggesting a common function. Here we demonstrate that Cln7 is required for the normal growth of synapses at the Drosophila larval neuromuscular junction. In a Cln7 mutant, synapses fail to develop fully leading to reduced function and behavioral changes with dysregulation of TOR activity. Cln7 expression is restricted to the post-synaptic cell and the protein localizes to vesicles immediately adjacent to the post-synaptic membrane. Our data suggest an involvement for Cln7 in regulating trans-synaptic communication necessary for normal synapse development.

A dual-strategy expression screen for candidate connectivity labels in the developing thalamus.

The thalamus or "inner chamber" of the brain is divided into ~30 discrete nuclei, with highly specific patterns of afferent and efferent connectivity. To identify genes that may direct these patterns of connectivity, we used two strategies. First, we used a bioinformatics pipeline to survey the predicted proteomes of nematode, fruitfly, mouse and human for extracellular proteins containing any of a list of motifs found in known guidance or connectivity molecules. Second, we performed clustering analyses on the Allen Developing Mouse Brain Atlas data to identify genes encoding surface proteins expressed with temporal profiles similar to known guidance or connectivity molecules. In both cases, we then screened the resultant genes for selective expression patterns in the developing thalamus. These approaches identified 82 candidate connectivity labels in the developing thalamus. These molecules include many members of the Ephrin, Eph-receptor, cadherin, protocadherin, semaphorin, plexin, Odz/teneurin, Neto, cerebellin, calsyntenin and Netrin-G families, as well as diverse members of the immunoglobulin (Ig) and leucine-rich receptor (LRR) superfamilies, receptor tyrosine kinases and phosphatases, a variety of growth factors and receptors, and a large number of miscellaneous membrane-associated or secreted proteins not previously implicated in axonal guidance or neuronal connectivity. The diversity of their expression patterns indicates that thalamic nuclei are highly differentiated from each other, with each one displaying a unique repertoire of these molecules, consistent with a combinatorial logic to the specification of thalamic connectivity.

in vivo localization of the neuronal ceroid lipofuscinosis proteins, CLN3 and CLN7, at endogenous expression levels.

The neuronal ceroid lipofuscinoses are a group of recessively inherited, childhood-onset neurodegenerative conditions. Several forms are caused by mutations in genes encoding putative lysosomal membrane proteins. Studies of the cell biology underpinning these disorders are hampered by the poor antigenicity of the membrane proteins, which makes visualization of the endogenous proteins difficult. We have used Drosophila to generate knock-in YFP-fusions for two of the NCL membrane proteins: CLN7 and CLN3. The YFP-fusions are expressed at endogenous levels and the proteins can be visualized live without the need for overexpression. Unexpectedly, both CLN7 and CLN3 have restricted expression in the CNS of Drosophila larva and are predominantly expressed in the glia that form the insect blood-brain-barrier. CLN7 is also expressed in neurons in the developing visual system. Analogous with murine CLN3, Drosophila CLN3 is strongly expressed in the excretory and osmoregulatory Malpighian tubules, but the knock-in also reveals unexpected localization of the protein to the apical domain adjacent to the lumen. In addition, some CLN3 protein in the tubules is localized within mitochondria. Our in vivo imaging of CLN7 and CLN3 suggests new possibilities for function and promotes new ideas about the cell biology of the NCLs.

Mushroom body defect is required in parallel to Netrin for midline axon guidance in Drosophila.

The outgrowth of many neurons within the central nervous system is initially directed towards or away from the cells lying at the midline. Recent genetic evidence suggests that a simple model of differential sensitivity to the conserved Netrin attractants and Slit repellents is insufficient to explain the guidance of all axons at the midline. In the Drosophila embryonic ventral nerve cord, many axons still cross the midline in the absence of the Netrin genes (NetA and NetB) or their receptor frazzled. Here we show that mutation of mushroom body defect (mud) dramatically enhances the phenotype of Netrin or frazzled mutants, resulting in many more axons failing to cross the midline, although mutations in mud alone have little effect. This suggests that mud, which encodes a microtubule-binding coiled-coil protein homologous to NuMA and LIN-5, is an essential component of a Netrin-independent pathway that acts in parallel to promote midline crossing. We demonstrate that this novel role of Mud in axon guidance is independent of its previously described role in neural precursor development. These studies identify a parallel pathway controlling midline guidance in Drosophila and highlight a novel role for Mud potentially acting downstream of Frizzled to aid axon guidance.

Modification of the Drosophila model of in vivo Tau toxicity reveals protective phosphorylation by GSK3ß.

Hyperphosphorylation of the microtubule associated protein, Tau, is the hallmark of a group of neurodegenerative disorders known as the tauopathies which includes Alzheimer's disease. Precisely how and why Tau phosphorylation is increased in disease is not fully understood, nor how individual sites modify Tau function. Several groups have used the Drosophila visual system as an in vivo model to examine how the toxicity of Tau varies with phosphorylation status. This system relies on overexpression of Tau from transgenes but is susceptible to position effects altering expression and activity of the transgenes. We have refined the system by eliminating position effects through the use of site-specific integration. By standardising Tau expression levels we have been able to compare directly the toxicity of different isoforms of Tau and Tau point mutants that abolish important phosphorylation events. We have also examined the importance of human kinases in modulating Tau toxicity in vivo. We were able to confirm that human GSK3ß phosphorylates Tau and increases toxicity but, unexpectedly, we identified that preventing phosphorylation of Ser404 is a protective event. When phosphorylation at this site is prevented, Tau toxicity in the Drosophila visual system is increased in the presence of GSK3ß. Our data suggest that not all phosphorylation events on Tau are associated with toxicity.

Commissureless regulation of axon outgrowth across the midline is independent of Rab function.

Nervous system function requires that neurons within neural circuits are connected together precisely. These connections form during the process of axon guidance whereby each neuron extends an axon that migrates, often large distances, through a complex environment to reach its synaptic target. This task can be simplified by utilising intermediate targets to divide the route into smaller sections. This requires that axons adapt their behaviour as they migrate towards and away from intermediate targets. In the central nervous system the midline acts as an intermediate target for commissural axons. In Drosophila commissural axons switch from attraction towards to extension away from the midline by regulating the levels of the Roundabout receptor on their cell surface. This is achieved by Commissureless which directs Roundabout to an intracellular compartment in the soma prior to reaching the midline. Once across the midline Roundabout is allowed to reach the surface and acts as a receptor for the repellent ligand Slit that is secreted by cells at the midline. Here we investigated candidate intracellular mechanisms that may facilitate the intracellular targeting of Commissureless and Roundabout within the soma of commissural neurons. Using modified forms of Commissureless or Rabs we show that neither ubiquitination nor Rab activity are necessary for the intracellular targeting of Commissureless. In addition we reveal that axon outgrowth of many populations of neurons within the Drosophila central nervous system is also independent of Rab activity.

Use of model organisms for the study of neuronal ceroid lipofuscinosis.

Neuronal ceroid lipofuscinoses are a group of fatal progressive neurodegenerative diseases predominantly affecting children. Identification of mutations that cause neuronal ceroid lipofuscinosis, and subsequent functional and pathological studies of the affected genes, underpins efforts to investigate disease mechanisms and identify and test potential therapeutic strategies. These functional studies and pre-clinical trials necessitate the use of model organisms in addition to cell and tissue culture models as they enable the study of protein function within a complex organ such as the brain and the testing of therapies on a whole organism. To this end, a large number of disease models and genetic tools have been identified or created in a variety of model organisms. In this review, we will discuss the ethical issues associated with experiments using model organisms, the factors underlying the choice of model organism, the disease models and genetic tools available, and the contributions of those disease models and tools to neuronal ceroid lipofuscinosis research. This article is part of a Special Issue entitled: The Neuronal Ceroid Lipofuscinoses or Batten Disease.

The Batten disease gene CLN3 is required for the response to oxidative stress.

Mutations in the CLN3 gene cause juvenile neuronal ceroid lipofuscinosis (JNCL or Batten disease), an early onset neurodegenerative disorder. JNCL is the most common of the NCLs, a group of disorders with infant or childhood onset that are caused by single gene mutations. The NCLs, although relatively rare, share many pathological and clinical similarities with the more common late-onset neurodegenerative disorders, while their simple genetic basis makes them an excellent paradigm. The early onset and rapid disease progression in the NCLs suggests that one or more key cellular processes are severely compromised. To identify the functional pathways compromised in JNCL, we have performed a gain-of-function modifier screen in Drosophila. We find that CLN3 interacts genetically with the core stress signalling pathways and components of stress granules, suggesting a function in stress responses. In support of this, we find that Drosophila lacking CLN3 function are hypersensitive to oxidative stress yet they respond normally to other physiological stresses. Overexpression of CLN3 is sufficient to confer increased resistance to oxidative stress. We find that CLN3 mutant flies perceive conditions of increased oxidative stress correctly but are unable to detoxify reactive oxygen species, suggesting that their ability to respond is compromised. Together, our data suggest that the lack of CLN3 function leads to a failure to manage the response to oxidative stress and this may be the key deficit in JNCL that leads to neuronal degeneration.

Interactions between the juvenile Batten disease gene, CLN3, and the Notch and JNK signalling pathways.

Mutations in the gene CLN3 are responsible for the neurodegenerative disorder juvenile neuronal ceroid lipofuscinosis or Batten disease. CLN3 encodes a multi-spanning and hydrophobic transmembrane protein whose function is unclear. As a consequence, the cell biology that underlies the pathology of the disease is not well understood. We have developed a genetic gain-of-function system in Drosophila to identify functional pathways and interactions for CLN3. We have identified previously unknown interactions between CLN3 and the Notch and Jun N-terminal kinase signalling pathways and have uncovered a potential role for the RNA splicing and localization machinery in regulating CLN3 function.

Dynein branches out.

Individual neurons form specific elaborate dendritic structures that receive presynaptic information. The pattern of dendritic branching is regulated by the microtubule-associated motor protein dynein, which is responsible for the transport of essential endosomes and other organelles into the dendrites.

The extracellular leucine-rich repeat superfamily; a comparative survey and analysis of evolutionary relationships and expression patterns.

BACKGROUND: Leucine-rich repeats (LRRs) are highly versatile and evolvable protein-ligand interaction motifs found in a large number of proteins with diverse functions, including innate immunity and nervous system development. Here we catalogue all of the extracellular LRR (eLRR) proteins in worms, flies, mice and humans. We use convergent evidence from several transmembrane-prediction and motif-detection programs, including a customised algorithm, LRRscan, to identify eLRR proteins, and a hierarchical clustering method based on TribeMCL to establish their evolutionary relationships. RESULTS: This yields a total of 369 proteins (29 in worm, 66 in fly, 135 in mouse and 139 in human), many of them of unknown function. We group eLRR proteins into several classes: those with only LRRs, those that cluster with Toll-like receptors (Tlrs), those with immunoglobulin or fibronectin-type 3 (FN3) domains and those with some other domain. These groups show differential patterns of expansion and diversification across species. Our analyses reveal several clusters of novel genes, including two Elfn genes, encoding transmembrane proteins with eLRRs and an FN3 domain, and six genes encoding transmembrane proteins with eLRRs only (the Elron cluster). Many of these are expressed in discrete patterns in the developing mouse brain, notably in the thalamus and cortex. We have also identified a number of novel fly eLRR proteins with discrete expression in the embryonic nervous system. CONCLUSION: This study provides the necessary foundation for a systematic analysis of the functions of this class of genes, which are likely to include prominently innate immunity, inflammation and neural development, especially the specification of neuronal connectivity.

Drosophila as a genetic and cellular model for studies on axonal growth.

One of the most fascinating processes during nervous system development is the establishment of stereotypic neuronal networks. An essential step in this process is the outgrowth and precise navigation (pathfinding) of axons and dendrites towards their synaptic partner cells. This phenomenon was first described more than a century ago and, over the past decades, increasing insights have been gained into the cellular and molecular mechanisms regulating neuronal growth and navigation. Progress in this area has been greatly assisted by the use of simple and genetically tractable invertebrate model systems, such as the fruit fly Drosophila melanogaster. This review is dedicated to Drosophila as a genetic and cellular model to study axonal growth and demonstrates how it can and has been used for this research. We describe the various cellular systems of Drosophila used for such studies, insights into axonal growth cones and their cytoskeletal dynamics, and summarise identified molecular signalling pathways required for growth cone navigation, with particular focus on pathfinding decisions in the ventral nerve cord of Drosophila embryos. These Drosophila-specific aspects are viewed in the general context of our current knowledge about neuronal growth.

Getting axons onto the right path: the role of transcription factors in axon guidance.

The normal function of the nervous system requires that the constituent neurons are precisely 'wired together'. During embryogenesis, each neuron extends an axonal process, which can navigate a considerable distance to its target. Although a number of the receptors and guidance signals that direct axonal growth have been identified, less is known about the transcription factors that regulate the expression of these molecules within the neuron and its environment. This review examines recent studies in vertebrates and Drosophila that address the identity of the transcription factors that either control the repertoire of guidance receptors and signals that permits an axon to take a particular trajectory or act themselves as novel extracellular guidance factors.

The Drosophila microtubule associated protein Futsch is phosphorylated by Shaggy/Zeste-white 3 at an homologous GSK3beta phosphorylation site in MAP1B.

The Drosophila homologue of the microtubule associated protein MAP1B is encoded by the futsch locus. The deduced protein Futsch is about twice the size of MAP1B and shows high homology in the N- and C-terminal domains. The central part of Futsch is characterized by a highly repetitive structure based on a 37 amino acid motif. Futsch, like MAP1B, colocalizes with microtubules and is necessary for the organization of the microtubule cytoskeleton during axonal growth and synaptogenesis. To further analyze the functional relevance of Futsch as a MAP1B-like protein, we performed a molecular analysis of the conserved protein domains. Using a number of antisera, we show that, unlike the MAP1B polyprotein, which is cleaved to generate a heavy and light chain, Futsch is expressed as a single protein. The function of MAP1B is in part regulated by phosphorylation mediated by kinases that include casein kinase 2 and glycogen synthase kinase 3beta (GSK3beta). We show here that at least one GSK3beta phosphorylation site of MAP1B is conserved in Futsch and that this site can be phosphorylated by GSK3beta and its Drosophila homologue, Shaggy/Zeste-white 3. To test the functional relevance of these findings we generated a number of minigenes and assayed their ability to rescue the phenotype of futsch mutants. Our data highlight some differences between MAP1B and Futsch but demonstrate that important structural and functional aspects are conserved between fly and vertebrate members of this protein family.

Functions of the segment polarity genes midline and H15 in Drosophila melanogaster neurogenesis.

The Drosophila melanogaster ventral nerve cord derives from neural progenitor cells called neuroblasts. Individual neuroblasts have unique gene expression profiles and give rise to distinct clones of neurons and glia. The specification of neuroblast identity provides a cell intrinsic mechanism which ultimately results in the generation of progeny which are different from each other. Segment polarity genes have a dual function in early neurogenesis: within distinct regions of the neuroectoderm, they are required both for neuroblast formation and for the specification of neuroblast identity. Previous studies of segment polarity gene function largely focused on neuroblasts that arise within the posterior part of the segment. Here we show that the segment polarity gene midline is required for neuroblast formation in the anterior-most part of the segment. Moreover, midline contributes to the specification of anterior neuroblast identity by negatively regulating the expression of Wingless and positively regulating the expression of Mirror. In the posterior-most part of the segment, midline and its paralog, H15, have partially redundant functions in the regulation of the NB marker Eagle. Hence, the segment polarity genes midline and H15 play an important role in the development of the ventral nerve cord in the anterior- and posterior-most part of the segment.

mummy/cystic encodes an enzyme required for chitin and glycan synthesis, involved in trachea, embryonic cuticle and CNS development--analysis of its role in Drosophila tracheal morphogenesis.

Tracheal and nervous system development are two model systems for the study of organogenesis in Drosophila. In two independent screens, we identified three alleles of a gene involved in tracheal, cuticle and CNS development. Here, we show that these alleles, and the previously identified cystic and mummy, all belong to the same complementation group. These are mutants of a gene encoding the UDP-N-acetylglucosamine diphosphorylase, an enzyme responsible for the production of UDP-N-acetylglucosamine, an important intermediate in chitin and glycan biosynthesis. cyst was originally singled out as a gene required for the regulation of tracheal tube diameter. We characterized the cyst/mmy tracheal phenotype and upon histological examination concluded that mmy mutant embryos lack chitin-containing structures, such as the procuticle at the epidermis and the taenidial folds in the tracheal lumen. While most of their tracheal morphogenesis defects can be attributed to the lack of chitin, when compared to krotzkopf verkehrt (kkv) chitin-synthase mutants, mmy mutants showed a stronger phenotype, suggesting that some of the mmy phenotypes, like the axon guidance defects, are chitin-independent. We discuss the implications of these new data in the mechanism of size control in the Drosophila trachea.

glaikit is essential for the formation of epithelial polarity and neuronal development.

Epithelial cells have a distinctive polarity based on the restricted distribution of proteins and junctional complexes along an apical-basal axis. Studying the formation of the polarized ectoderm of the Drosophila embryo has identified a number of the molecules that establish this polarity. The Crumbs (Crb) complex is one of three separate complexes that cooperate to control epithelial polarity and the formation of zonula adherens. Here we show that glaikit (gkt), a member of the phospholipase D superfamily, is essential for the formation of epithelial polarity and for neuronal development during Drosophila embryogenesis. In epithelial cells, gkt acts to localize the Crb complex of proteins to the apical lateral membrane. Loss of gkt during neuronal development leads to a severe CNS architecture disruption that is not dependent on the Crb pathway but probably results from the disrupted localization of other membrane proteins. A mutation in the human homolog of gkt causes the neurodegenerative disease spinocerebellar ataxia with neuropathy (SCAN1), making it possible that a failure of membrane protein localization is a cause of this disease.

Drosophila T box proteins break the symmetry of hedgehog-dependent activation of wingless.

BACKGROUND: Segmentation of the Drosophila embryo is a classic paradigm for pattern formation during development. The Wnt-1 homolog Wingless (Wg) is a key player in the establishment of a segmentally reiterated pattern of cell type specification. The intrasegmental polarity of this pattern depends on the precise positioning of the Wg signaling source anterior to the Engrailed (En)/Hedgehog (Hh) domain. Proper polarity of epidermal segments requires an asymmetric response to the bidirectional Hh signal: wg is activated in cells anterior to the Hh signaling source and is restricted from cells posterior to this signaling source. RESULTS: Here we report that Midline (Mid) and H15, two highly related T box proteins representing the orthologs of zebrafish hrT and mouse Tbx20, are novel negative regulators of wg transcription and act to break the symmetry of Hh signaling. Loss of mid and H15 results in the symmetric outcome of Hh signaling: the establishment of wg domains anterior and posterior to the signaling source predominantly, but not exclusively, in odd-numbered segments. Accordingly, loss of mid and H15 produces defects that mimic a wg gain-of-function phenotype. Misexpression of mid represses wg and produces a weak/moderate wg loss-of-function phenocopy. Furthermore, we show that loss of mid and H15 results in an anterior expansion of the expression of serrate (ser) in every segment, representing a second instance of target gene repression downstream of Hh signaling in the establishment of segment polarity. CONCLUSIONS: The data we present here indicate that mid and H15 are important components in pattern formation in the ventral epidermis. In odd-numbered abdominal segments, Mid/H15 activity plays an important role in restricting the expression of Wg to a single domain.

Neuroglian and FasciclinII can promote neurite outgrowth via the FGF receptor Heartless.

To further investigate the role of the Drosophila cell adhesion molecules (CAMs), we have developed an in vitro assay that allows us to test the contribution individual CAMs make to promote outgrowth of specific Drosophila neurons. The extension of primary cultured neurons on a substrate of purified recombinant CAM is measured. We show that both FasciclinII and Neuroglian are able to promote outgrowth of FasciclinII or Neuroglian expressing neurons, respectively. Furthermore, this growth promotion activity is provided when the CAMs are presented both in a substrate bound or soluble form. We also show that the signal provided by the CAMs acts via the Heartless fibroblast growth factor receptor (FGFR) as outgrowth is reduced to basal levels in the presence of an FGFR inhibitor or if Heartless function is missing from the neurons.

Dynamic expression patterns of Robo (Robo1 and Robo2) in the developing murine central nervous system.

The Robo family of molecules is important for axon guidance across the midline during central nervous system (CNS) development in invertebrates and vertebrates. Here we describe the patterns of Robo protein expression in the developing mouse CNS from embryonic day (E) 9.5 to postnatal day (P) 4, as determined by immunohistochemical labeling with an antibody (S3) raised against a common epitope present in the Robo ectodomain of Robos 1 and 2. In the spinal cord, midline-crossing axons are initially (at E11) S3-positive. At later times, midline Robo expression disappears, but is strongly upregulated in longitudinally running postcrossing axons. It is also strongly expressed in noncrossing longitudinal axons. Differential expression of Robo along axons was also found in axons cultured from E14 spinal cord. These findings resemble those from the Drosophila ventral nerve cord and indicate that in vertebrates a low level of Robo expression occurs in the initial crossing of the midline, while a high level of expression in the postcrossing fibers prevents recrossing. Likewise, Robo-positive ipsilateral axons are prevented from crossing at all. However, in the brain different rules appear to apply. Most commissural axons including those of the corpus callosum are strongly S3-positive along their whole length from their time of formation to postnatal life, but some have more complex age-dependent expression patterns. S3 labeling of the optic pathway is also complex, being initially strong in the retinal ganglion cells, optic tract, and chiasma but thereafter being lost except in a proportion of postchiasmal axons. The corticospinal tract is strongly positive throughout its course at all stages examined, including its decussation, formed at about P2 in the central part of the medulla oblongata.