From human to fly and back

During the screening of Pitt-Hopkins syndrome-like patients without TCF4 mutation, recessive defects in CNTNAP2 in a pair of siblings and an isolated patient as well as recessive defects in NRXN1 in an isolated patient were detected (chapter 4). This was quite exciting as NRXN1 and CNTNAP2 belong to the same superfamily of neurexins, though no common molecular functions were known so far (see below). A potential common molecular basis and maybe even a link to TCF4 could therefore provide an explanation for the similar phenotype in the patients. However, neither NRXN1 nor CNTNAP2 is expressed in appreciable levels in blood, and other tissues are difficult to obtain from living patients.

Therefore Drosophila melanogaster was utilized as a model organism for further functional studies as all three genes do have orthologs in the fly: daughterless (TCF4), Nrx-I (NRXN1), and Nrx-IV (CNTNP2).

Our first straightforward hypothesis was that TCF4 as a transcription factor could regulate the expression levels of NRXN1 and/or CNTNAP2. Ubiquitous knockdown of the TCF4 ortholog daughterless in the fly did not reveal conclusive data on altered expression levels of Nrx-I or Nrx-IV, the NRXN1 and CNTNAP2 orthologs, respectively, in the used system (chapter 4). However, recently another group reported a possible regulation of NRXN1 and CNTNAP2 through TCF4 in terms of a possible transactivation effect in cell system based promoter studies.316

NRXN1/Nrx-I has been known to be one of the key synapse organizing molecules by forming bridges over the synaptic cleft with its postsynaptic binding partners, the neuroligins.287 _{Vertebrate CNTNAP2, also termed CASPR2 has been mainly known for its} role in regulating neuron-glia contact and for colocalizing with K+- channels in the juxtaparanodal areas of Ranvier nodes in myelinated axons of both the central and peripheral nervous system.294,295 _{When this study was initiated, fly Nrx-IV was reported to be} almost exclusively expressed in glia-cells and to regulate glia-glia contact.293,296

The executed Drosophila experiments in this study brought several new insights (chapter 4) regarding NRXN1/Nrx-I and CNTNAP2/Nrx-IV and their possible interaction.

1) Nrx-IV was shown to play a role in neurons as neuronal knockdown lead to embryonic lethality. Previously, Nrx-IV was only considered to be expressed in glia cells in the fly.296 _{In parallel, two groups reported on neuronal Nrx-IV isoforms,}264,268 _{thus supporting} our conclusion that Nrx-IV plays a crucial role in neurons.

2) Nrx-IV is present at synapses. Previously, only a report on detection of Caspr2 in fractionated rat synaptic plasma had pointed to a possible synaptic presence.266

3) Nrx-I and Nrx-IV converge on synaptic active zone protein bruchpilot (brp) as a common target. Adding to the observation of decreased brp levels in Nrx-I mutants,270 _our work showed that both Nrx-I or Nrx-IV levels determine brp levels bidirectionally and that either Nrx-I or Nrx-IV overexpression induces identical changes in synaptic morphology.

Bruchpilot is a peripheral membrane protein being located at the presynaptic active zones, the location of neurotransmitter release into the synaptic cleft. Through its PDZ domain binding site it is involved in a large complex with other active zone proteins.298 _Of note, also Nrx-I and Nrx-IV contain C-terminal PDZ-domain binding sites, therefore raising the possibilty that all three proteins are assembled into one synaptic complex or macromolecular network. Brp shows high sequence and functional homology to the vertebrate family of ELKS/CAST proteins,298 _{corresponding to the human synaptic proteins} ERC1 and ERC2.

Summarizing, these findings in Drosophila helped to gain more insight into function of Nrx-I and Nrx-IV in the nervous system and particularly implicated Nrx-IV in synapse biology. It also helped to establish a functional link between Nrx-I and Nrx-IV as a possible explanation for the similar human phenotypes. Furthermore, with brp orthologs ERC1 and ERC2 it produced promising candidate genes to go back to humans and screen these genes for mutations in patients with a PTHS-like phenotype.

Therefore, in this study Drosophila proved to be a valuable model organism for functional testing in ID. However, it is important to know what to expect from an animal model like Drosophila. It is no equivalent to human patients, but the same is applicable for all other animal models or in vitro assays. It cannot provide final evidence for the pathogenicity of particular mutations, but it can provide functional support for the role of particular genes in cognitive and behavioral function and dysfunction. As successfully demonstrated in this thesis, it can help to establish molecular networks. This is in particularly interesting as the theme of overlapping phenotypes being caused by genes that are linked to each other in molecular networks178 _{gets more and more evident and attractive.}

Drosophila is not the answer to all our questions but it is an extremely suitable and valuable tool to efficiently add pieces to the puzzle that has been keeping and will keep scientists with an interest in ID - geneticists, and neurobiologists - busy for a long time.