Godenschwege, Tanja A.

Nicotinic acetylcholine receptors (nAChRs) are pentameric ligand-gated ion channels. The a7 subtype of nAChRs is involved
in neurological pathologies such as Parkinson’s disease, Alzheimer’s disease, addiction, epilepsy and autism spectrum
disorders. The Drosophila melanogaster a7 (Da7) has the closest sequence homology to the vertebrate a7 subunit and it can
form homopentameric receptors just as the vertebrate counterpart. The Da7 subunits are essential for the function of the
Giant Fiber circuit, which mediates the escape response of the fly. To further characterize the receptor function, we
generated different missense mutations in the Da7 nAChR’s ligand binding domain. We characterized the effects of
targeted expression of two UAS-constructs carrying a single mutation, D197A and Y195T, as well as a UAS-construct carrying
a triple D77T, L117Q, I196P mutation in a Da7 null mutant and in a wild type background. Expression of the triple mutation
was able to restore the function of the circuit in Da7 null mutants and had no disruptive effects when expressed in wild
type. In contrast, both single mutations severely disrupted the synaptic transmission of Da7-dependent but not
glutamatergic or gap junction dependent synapses in wild type background, and did not or only partially rescued the
synaptic defects of the null mutant. These observations are consistent with the formation of hybrid receptors, consisting of
D197A or Y195T subunits and wild type Da7 subunits, in which the binding of acetylcholine or acetylcholine-induced
conformational changes of the Da7 receptor are altered and causes inhibition of cholinergic responses. Thus targeted
expression of D197A or Y195T can be used to selectively disrupt synaptic transmission of Da7-dependent synapses in
neuronal circuits. Hence, these constructs can be used as tools to study learning and memory or addiction associated
behaviors by allowing the manipulation of neuronal processing in the circuits without affecting other cellular signaling.

A large number of different pathological L1CAM mutations have been identified that result in a broad spectrum of
neurological and non-neurological phenotypes. While many of these mutations have been characterized for their effects on
homophilic and heterophilic interactions, as well as expression levels in vitro, there are only few studies on their biological
consequences in vivo. The single L1-type CAM gene in Drosophila, neuroglian (nrg), has distinct functions during axon
guidance and synapse formation and the phenotypes of nrg mutants can be rescued by the expression of human L1CAM.
We previously showed that the highly conserved intracellular FIGQY Ankyrin-binding motif is required for L1CAM-mediated
synapse formation, but not for neurite outgrowth or axon guidance of the Drosophila giant fiber (GF) neuron. Here, we use
the GF as a model neuron to characterize the pathogenic L120V, Y1070C, C264Y, H210Q, E309K and R184Q extracellular
L1CAM missense mutations and a L1CAM protein with a disrupted ezrin–moesin–radixin (ERM) binding site to investigate
the signaling requirements for neuronal development. We report that different L1CAM mutations have distinct effects on
axon guidance and synapse formation. Furthermore, L1CAM homophilic binding and signaling via the ERM motif is essential
for axon guidance in Drosophila. In addition, 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. Thus, the pathological phenotypes observed in humans are likely to be caused by the disruption of signaling
required for both, guidance and synaptogenesis.