Godenschwege, Tanja A.

L1-type cell adhesion molecule (L1CAM) plays an essential role in the
development of nervous system and is also highly relevant for the progression of diseases
such as Alzheimer’s disease, stroke and cancers, some of the leading causes of human
mortality. In addition to its canonical role as a plasma membrane protein organizing the
cytoskeleton, recent in vitro studies have revealed that transmembrane as well as cytosolic
fragments of proteolytically cleaved vertebrate L1CAM translocate to the nucleus and
regulate expression of genes involved in DNA post-replication repair, cell cycle control,
migration and differentiation. However, little is known about the in vivo function of
L1CAM in the adult nervous system.
This dissertation research focuses on studying in vivo nuclear translocation and
function of L1CAM. Using the Drosophila model system, we first show that the sole
Drosophila L1CAM homolog, Neuroglian (Nrg), is proteolytically cleaved by Alzheimer’s
associated secretases, similar to L1CAM, and is also translocated to the nucleus in the adult nervous system. Subsequently, we have shown that the deletion of highly conserved
Ankyrin binding domain or FIGQY motif disrupts nuclear import. Further experiments
have revealed that the nuclear translocation of Nrg is in fact regulated by the
phosphorylation of the FIGQY motif. Importantly, our studies also show transgenic
expression of full-length Nrg or the intracellular domain of Nrg resulted in increased myc
expression, which is associated with increased sensitivity to oxidative stress and reduced
life span. On the other hand, deletion of the FIGQY motif or mutations preventing its
phosphorylation led to decrease in myc expression. In summary, we have identified a novel
role for the highly conserved Ankyrin binding domain in nuclear translocation and
transcriptional regulation of the Drosophila myc oncogene, which is of high relevance to
neurodegenerative diseases and cancer associated with oxidative stress.

PTP69D is a receptor protein tyrosine phosphatase RPTP with two intracellular catalytic
domains Cat1 and Cat2, which has been shown to play a role in axon outgrowth and guidance of
embryonic motoneurons as well as targeting of photoreceptor neurons in the visual system of
Drosophila melanogaster. Here, we characterized the developmental role of PTP69D in the giant
fiber GF neurons; two interneurons in the central nervous system CNS that control the escape
response of the fly. In addition to guidance and targeting functions, our studies reveal an
additional role for PTP69D in synaptic terminal growth in the CNS. We found that inhibition of
phosphatase activity in catalytic domain Cat1 proximal to the transmembrane domain did not
affect axon guidance or targeting but resulted in stunted terminal growth of the GFs. Cell
autonomous rescue and knockdown experiments demonstrated a function for PTP69D in the
GFs, but not its postsynaptic target neurons. In addition, complementation studies and structurefunction
analyses revealed that for GF terminal growth Cat1 function of PTP69D requires the
Immunoglobulin and the Cat2 domain, but not the Fibronectin III and the Membrane Proximal
Region domains. In contrast, the Fibronectin III, but not the Immunoglobulin domains, were
previously shown to be essential for axon targeting of photoreceptor neurons. Thus, our studies
uncover a novel role for PTP69D in synaptic terminal growth in the CNS that is mechanistically
distinct from its function during earlier developmental process.

L1-type cell adhesion molecule (L1-CAM) is a synaptic membrane protein that is associated with L1 syndrome, which exhibits spasticity, intellectual disability and hydrocephalus Neuroglian (Nrg) is the invertebrate homologue of L1-CAM in Drosophila melanogaster. In vitro studies have shown L1-CAM is proteolytically cleaved and the intracellular domain (ICD) translocates to the nucleus. There it is involved in the upregulation of genes that are involved in DNA damage response, cell cycle progression, apoptosis and cellular differentiation. In some forms of Alzheimer’s Disease (AD) proteolytic cleavage of L1-CAM are enhanced. We studied the effects of expression of NrgICD in vivo. Our results indicate that ubiquitous expression of NrgICD like its vertebrate homologue resulted in upregulation of NBS1 and c-myc in Drosophila. We found that the ubiquitous expression of NrgICD resulted in reduced viability in various models of oxidative stress. This suggests that enhanced proteolytic cleavage of Nrg/L1-CAM contributes to the pathology of AD. Our results may provide new insights into the cellular mechanisms of neurodegenerative diseases.

PTP69D is a receptor protein tyrosine phosphatase (RPTP) with two intracellular catalytic domains (Cat1 and Cat2), which has been shown to play a role in axon
outgrowth and guidance of embryonic motorneurons, as well as targeting of photoreceptor neurons in the visual system of Drosophila melanogaster. Here, we
characterized the developmental role of PTP69D in the giant fiber (GF) neurons; two
interneurons in the central nervous system (CNS) that control the escape response of the fly. In addition to guidance and targeting functions, our studies reveal an additional role for PTP69D in synaptic terminal growth in the CNS. We found that inhibition of
phosphatase activity in catalytic domain (Cat1) proximal to the transmembrane domain
did not affect axon guidance or targeting but resulted in stunted terminal growth of the
GFs. Cell autonomous rescue and knockdown experiments demonstrated a function for
PTP69D in the GFs, but not its postsynaptic target neurons. In addition,complementation studies and structure-function analyses revealed that for GF terminal growth, Cat1 function of PTP69D requires the immunoglobulin and the Cat2 domain but not the fibronectin type III repeats nor the membrane proximal region. In contrast, the fibronectin type III repeats, but not the immunoglobulin domains, were previously shown to be essential for axon targeting of photoreceptor neurons. Thus, our studies uncover a novel role for PTP69D in synaptic terminal growth in the CNS that is mechanistically distinct from its function during earlier developmental processes.

Neuronal cell adhesion molecules of L1 family play a critical role in proper nervous system development. Various mutations on human L1-CAM that lead to severe
neurodevelopmental disorders like retardation, spasticity etc. termed under L1 syndrome. The vertebrr their roles in axon pathfinding, neurite extension and cell migration, howeverate L1CAM and its homolog in Drosophila, neuroglian (nrg) have been well studied fo, much less is known about the mechanisms by which they fine tune synaptic connectivity to control the development and maintenance of synaptic connections within neuronal circuits. Here we characterized the essential role of nrg in regulating synaptic structure and function in vivo in a well characterized Drosophila central synapse model neuron, the Giant Fiber (GF) system. Previous studies from our lab revealed that the phosphorylation status of the tyrosine in the Ankyrin binding FIGQY motif in the intracellular domain of Nrg iscrucial for synapse formation of the GF to Tergo-Trochanteral Motor neuron (TTMn) synapse in the GF circuit.
The present work provided us with novel insights into the role of Nrg-Ank interaction in regulating Nrg function during synapse formation and maintenance. By
utilizing a sophisticated Pacman based genomic rescue strategy we have shown that
dynamic regulation of the Neuroglian–Ankyrin interaction is required to coordinate
transsynaptic development in the GF–TTMn synapse. In contrast, the strength of Ankyrin binding directly controls the balance between synapse formation and maintenance at the NMJ.
Human L1 pathological mutations affect different biological processes distinctively
and thus their proper characterization in vivo is essential to understand L1CAM function.
By utilizing nrg14;P[nrg180ΔFIGQY] mutants that have exclusive synaptic defects and the previously characterized nrg849 allele that affected both GF guidance and synaptic function, we were able to analyze pathological L1CAM missense mutations with respect to their effects on guidance and synapse formation in vivo. We found that the human pathological H210Q, R184Q and Y1070C, but not the E309K and L120V L1CAM mutations affect outside-in signaling via the FIGQY Ankyrin binding domain which is required for synapse formation and not for axon guidance while L1CAM homophilic binding and signaling via the ERM motif is essential for axon guidance in Drosophila.

Conopeptides are found in the venom of marine cone snails, aiding in the paralysis of their prey, and have been shown to have potential therapeutic uses in humans. Conopressins are conopeptides that target vasopressin/oxytocin receptors in vascular smooth muscle cells that are found within blood vessels. The crustacean cardioactive peptide (CCAP) is a homologous peptide found in crustaceans and has been shown to behave as a cardioaccelerator in a homologous system. This study describes the effects of CCAP in Drosophila larvae. We find that CCAP has an inotropic effect by causing a change in the contraction of blood vessels. We further investigate the effects of another possibly cardioactive conopeptide, γ-conopressin-vil, in Drosophila larvae. Elucidating the effects of conopetides in Drosophila larvae may translate to cardioactive therapeutic uses in mammalian systems.