Molecular Mechanisms of Assembly and Long-term Maintenance of Neuronal Architecture: A Dissertation

Nervous system function is closely tied to its structure, which ensures proper connectivity and neural activity. Neuronal architecture is assembled by a series of morphogenetic events, including the coordinated migrations of neurons and axons during development. Subsequently, the neuronal architecture established earlier must persist in the face of further growth, maturation of the nervous system, and the mechanical stress of body movements. In this work, we have shed light on the molecular mechanisms governing both the initial assembly of the nervous system and the long-term maintenance of neural circuits. In particular, we identified heparan sulfate proteoglycans (HSPGs) as regulators of neuronal migrations. Our discovery and analysis of viable mutations in the two subunits of the heparan sulfate co-polymerase reveals the importance of the coordinated and dynamic action of HSPGs in neuronal and axon guidance during development. Furthermore, we uncovered that the HSPG LON-2/glypican functions as a modulator of UNC-6/netrin signaling through interactions with the UNC-40/DCC receptor. During larval and adult life, molecules such as the protein SAX-7, homologous to mammalian L1CAM, function to protect the integrity of nervous system architecture. Indeed, loss of sax-7 leads to progressive disorganization of neuronal architecture. Through a forward genetic screen, we identified LON-1 as a novel maintenance molecule that functions post-embryonically with SAX-7 to maintain the architecture of the nervous system. Together, our work highlights the importance of extracellular interactions to modulate signaling events during the initial development of the nervous system, and to subsequently maintain neuronal architecture for the long-term

The Secreted Immunoglobulin Domain Proteins ZIG-5 and ZIG-8 Cooperate with L1CAM/SAX-7 to Maintain Nervous System Integrity

During nervous system development, neuronal cell bodies and their axodendritic projections are precisely positioned through transiently expressed patterning cues. We show here that two neuronally expressed, secreted immunoglobulin (Ig) domain-containing proteins, ZIG-5 and ZIG-8, have no detectable role during embryonic nervous system development of the nematode Caenorhabditis elegans but are jointly required for neuronal soma and ventral cord axons to maintain their correct position throughout postembryonic life of the animal. The maintenance defects observed upon removal of zig-5 and zig-8 are similar to those observed upon complete loss of the SAX-7 protein, the C. elegans ortholog of the L1CAM family of adhesion proteins, which have been implicated in several neurological diseases. SAX-7 exists in two isoforms: a canonical, long isoform (SAX-7L) and a more adhesive shorter isoform lacking the first two Ig domains (SAX-7S). Unexpectedly, the normally essential function of ZIG-5 and ZIG-8 in maintaining neuronal soma and axon position is completely suppressed by genetic removal of the long SAX-7L isoform. Overexpression of the short isoform SAX-7S also abrogates the need for ZIG-5 and ZIG-8. Conversely, overexpression of the long isoform disrupts adhesion, irrespective of the presence of the ZIG proteins. These findings suggest an unexpected interdependency of distinct Ig domain proteins, with one isoform of SAX-7, SAX-7L, inhibiting the function of the most adhesive isoform, SAX-7S, and this inhibition being relieved by ZIG-5 and ZIG-8. Apart from extending our understanding of dedicated neuronal maintenance mechanisms, these findings provide novel insights into adhesive and anti-adhesive functions of IgCAM proteins

Neuronal post-developmentally acting SAX-7S/L1CAM can function as cleaved fragments to maintain neuronal architecture in C. elegans [preprint]

Whereas remarkable advances have uncovered mechanisms that drive nervous system assembly, the processes responsible for the lifelong maintenance of nervous system architecture remain poorly understood. Subsequent to its establishment during embryogenesis, neuronal architecture is maintained throughout life in the face of the animal’s growth, maturation processes, the addition of new neurons, body movements, and aging. The C. elegans protein SAX-7, homologous to the vertebrate L1 protein family, is required for maintaining the organization of neuronal ganglia and fascicles after their successful initial embryonic development. To dissect the function of sax-7 in neuronal maintenance, we generated a null allele and sax-7S-isoform-specific alleles. We find that the null sax-7(qv30) is, in some contexts, more severe than previously described mutant alleles, and that the loss of sax-7S largely phenocopies the null, consistent with sax-7S being the key isoform in neuronal maintenance. Using a sfGFP::SAX-7S knock-in, we observe sax-7S to be predominantly expressed across the nervous system, from embryogenesis to adulthood. Yet, its role in maintaining neuronal organization is ensured by post-developmentally acting SAX-7S, as larval transgenic sax-7S(+) expression alone is sufficient to profoundly rescue the null mutants’ neuronal maintenance defects. Moreover, the majority of the protein SAX-7 appears to be cleaved, and we show that these cleaved SAX-7S fragments together, not individually, can fully support neuronal maintenance. These findings contribute to our understanding of the role of the conserved protein SAX-7/L1CAM in long-term neuronal maintenance, and may help decipher processes that go awry in some neurodegenerative conditions

Functional Requirements for Heparan Sulfate Biosynthesis in Morphogenesis and Nervous System Development in C. elegans

The regulation of cell migration is essential to animal development and physiology. Heparan sulfate proteoglycans shape the interactions of morphogens and guidance cues with their respective receptors to elicit appropriate cellular responses. Heparan sulfate proteoglycans consist of a protein core with attached heparan sulfate glycosaminoglycan chains, which are synthesized by glycosyltransferases of the exostosin (EXT) family. Abnormal HS chain synthesis results in pleiotropic consequences, including abnormal development and tumor formation. In humans, mutations in either of the exostosin genes EXT1 and EXT2 lead to osteosarcomas or multiple exostoses. Complete loss of any of the exostosin glycosyltransferases in mouse, fish, flies and worms leads to drastic morphogenetic defects and embryonic lethality. Here we identify and study previously unavailable viable hypomorphic mutations in the two C. elegans exostosin glycosyltransferases genes, rib-1 and rib-2. These partial loss-of-function mutations lead to a severe reduction of HS levels and result in profound but specific developmental defects, including abnormal cell and axonal migrations. We find that the expression pattern of the HS copolymerase is dynamic during embryonic and larval morphogenesis, and is sustained throughout life in specific cell types, consistent with HSPGs playing both developmental and post-developmental roles. Cell-type specific expression of the HS copolymerase shows that HS elongation is required in both the migrating neuron and neighboring cells to coordinate migration guidance. Our findings provide insights into general principles underlying HSPG function in development

The Batten Disease Palmitoyl Protein Thioesterase 1 Gene Regulates Neural Specification and Axon Connectivity during Drosophila Embryonic Development

Palmitoyl Protein Thioesterase 1 (PPT1) is an essential lysosomal protein in the mammalian nervous system whereby defects result in a fatal pediatric disease called Infantile Neuronal Ceroids Lipofuscinosis (INCL). Flies bearing mutations in the Drosophila ortholog Ppt1 exhibit phenotypes similar to the human disease: accumulation of autofluorescence deposits and shortened adult lifespan. Since INCL patients die as young children, early developmental neural defects due to the loss of PPT1 are postulated but have yet to be elucidated. Here we show that Drosophila Ppt1 is required during embryonic neural development. Ppt1 embryos display numerous neural defects ranging from abnormal cell fate specification in a number of identified precursor lineages in the CNS, missing and disorganized neurons, faulty motoneuronal axon trajectory, and discontinuous, misaligned, and incorrect midline crossings of the longitudinal axon bundles of the ventral nerve cord. Defects in the PNS include a decreased number of sensory neurons, disorganized chordotonal neural clusters, and abnormally shaped neurons with aberrant dendritic projections. These results indicate that Ppt1 is essential for proper neuronal cell fates and organization; and to establish the local environment for proper axon guidance and fasciculation. Ppt1 function is well conserved from humans to flies; thus the INCL pathologies may be due, in part, to the accumulation of various embryonic neural defects similar to that of Drosophila. These findings may be relevant for understanding the developmental origin of neural deficiencies in INCL

Investigation of Ppt1 and other lysosomal storage disease genes during drosophila neurogenesis

Infantile Neuronal Ceroid Lipofuscinoses (INCL) is a lysosomal storage disease caused by a defect in the Palmitoyl Protein Thioesterase 1 (PPT1) protein. Studies have shown that the Drosophila ortholog, Ppt1, is essential for proper embryonic neurogenesis. Without Ppt1, flies exhibit defects in neuronal precursor cell fates, axon misrouting, and fasciculation. This study extends this analysis by examining the role of other lysosomal storage disease genes during Drosophila neurogenesis, namely SAK, palmitoyl protein thioesterase 2, and spinster. Flies carrying mutations in these genes were individually crossed into the Ppt1 knockout background in order to investigate whether they genetically interact with Ppt1. Embryos from the mutant strains and crosses were stained with αBP102 using a 2-day immunohistochemistry protocol and digital images were taken of well-stained stage 16 embryos. Embryonic neurogenesis was analyzed in each embryo to determine whether mutations in SAK, Ppt2, or spinster disrupted nervous system development and/or interacted genetically with Ppt1. Fly strains mutant for SAK, Ppt2, and spinster exhibited abnormalities in nervous system development. When SAK was crossed into the Ppt1-mutant background, abnormalities increased; when spinster was crossed in the Ppt1-mutant background, abnormalities significantly decreased

Glypican Is a Modulator of Netrin-Mediated Axon Guidance.

Netrin is a key axon guidance cue that orients axon growth during neural circuit formation. However, the mechanisms regulating netrin and its receptors in the extracellular milieu are largely unknown. Here we demonstrate that in Caenorhabditis elegans, LON-2/glypican, a heparan sulfate proteoglycan, modulates UNC-6/netrin signaling and may do this through interactions with the UNC-40/DCC receptor. We show that developing axons misorient in the absence of LON-2/glypican when the SLT-1/slit guidance pathway is compromised and that LON-2/glypican functions in both the attractive and repulsive UNC-6/netrin pathways. We find that the core LON-2/glypican protein, lacking its heparan sulfate chains, and secreted forms of LON-2/glypican are functional in axon guidance. We also find that LON-2/glypican functions from the epidermal substrate cells to guide axons, and we provide evidence that LON-2/glypican associates with UNC-40/DCC receptor-expressing cells. We propose that LON-2/glypican acts as a modulator of UNC-40/DCC-mediated guidance to fine-tune axonal responses to UNC-6/netrin signals during migration

Genetic interactions between <i>zig-5, zig-8</i> and <i>sax-7</i>.

<p>(A) ASI and ASH neuronal displacements in mutant adult animals scored with the <i>oyIs14</i> reporter transgene. Wild-type and <i>zig-5 zig-8</i> double mutant pictures are the same as shown in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002819#pgen-1002819-g001" target="_blank">Figure 1C</a> and shown for comparison only. See legend to <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002819#pgen-1002819-g001" target="_blank">Figure <i>1C</i></a> for explanation of symbols. <i>Scale</i> bar is 5 µm. (B) Quantification of ASI and ASH neuronal position in genetic different backgrounds. “punc-14” is a panneuronal driver. <i>Punc-14::sax-7L</i>: DNA was injected at 75 ng/uL (line 1) or 50 ng/uL (lines 2 and 3). <i>Punc-14::sax-7S and Punc-14::sax7Δ11</i>: DNA was injected at 50 ng/uL. Proportions of different animal populations were compared using the z-test. “*” indicates p<0.001. (C) Genetic interactions of <i>zig-5, zig-8</i> and <i>sax-7</i> in controlling axon positioning in the ventral nerve cord. Quantification of PVQ axon flip-overs with transgene <i>oyIs14</i>. Error bars indicate s.e.p. Proportions of different animal populations were compared using the z-test. “*” indicates p<0.01. (D) Effect of ectopic expression of various forms of <i>sax-7</i> in a wild-type background. ASI and ASH neuronal position are quantified. Proportions of different animal populations were compared using the z-test. “*” indicates p<0.001. (E) Quantification of ASI and ASH neuronal position in <i>dig-1(ky188)</i> mutant animals. “<i>Punc-14</i>” is a panneuronal driver for expression of SAX-7S. There are no statistically significant differences between <i>dig-1</i> animals and any of the transgenic <i>dig-1</i> animals expressing <i>sax-7S.</i></p

Neuronal maintenance factors and the defects caused by their removal.

<p>(A) Schematic protein structures and alleles used in this study. (B) Summary of previous <i>in vitro</i> and <i>in vivo</i> adhesion studies <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002819#pgen.1002819-Sasakura1" target="_blank">[6]</a>, <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002819#pgen.1002819-Pocock1" target="_blank">[7]</a>. Star indicates a shortened hinge region which prevents formation of the horseshoe configuration <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002819#pgen.1002819-Pocock1" target="_blank">[7]</a>. (C) ASI and ASH neuronal displacements observed in <i>zig-5(ok1065)</i> and <i>zig-8(ok561)</i> single and double mutant adult animals with the <i>oyIs14</i> reporter transgene. Blue arrowheads indicate position of the nerve ring and red arrowheads position of neuronal soma, which is scored relative to position of the nerve ring (wild type: behind nerve ring; mutant: on top of to nerve ring). Anterior to left, dorsal on top. Scale bar is 5 µm. (D) Quantification of ASI and ASH neuronal displacement in single and double mutants of the <i>zig</i> gene family. Alleles are described in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002819#pgen.1002819-Benard3" target="_blank">[11]</a>. Error bars indicate s.e.p.. Proportions of different animal populations were compared using the z-test. “*” indicates p<0.001.</p

Rescue of <i>zig-5 zig-8</i> mutant phenotypes.

1<p>On top of or anterior to nerve ring. Scored with <i>oyIs14 (sra-6::gfp)</i>.</p>2<p>Repeated from <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002819#pgen-1002819-g001" target="_blank">Figure 1</a> for comparison.</p>3<p>Compared to non-transgenic control (74% defective). Injection of <i>zig-5</i> and <i>zig-8</i> expressed under the control of a number of neuronal or pharyngeal promoters, at a range of concentrations, did not produce better rescue of the mutant phenotypes than that obtained with the fosmid and YAC.</p