Functional Study of Mammalian Neph Proteins in <em>Drosophila melanogaster</em>

<div><p>Neph molecules are highly conserved immunoglobulin superfamily proteins (IgSF) which are essential for multiple morphogenetic processes, including glomerular development in mammals and neuronal as well as nephrocyte development in <em>D. melanogaster</em>. While <em>D. melanogaster</em> expresses two Neph-like proteins (Kirre and IrreC/Rst), three Neph proteins (Neph1–3) are expressed in the mammalian system. However, although these molecules are highly abundant, their molecular functions are still poorly understood. Here we report on a fly system in which we overexpress and replace endogenous Neph homologs with mammalian Neph1–3 proteins to identify functional Neph protein networks required for neuronal and nephrocyte development. Misexpression of Neph1, but neither Neph2 nor Neph3, phenocopies the overexpression of endogenous Neph molecules suggesting a functional diversity of mammalian Neph family proteins. Moreover, structure-function analysis identified a conserved and specific Neph1 protein motif that appears to be required for the functional replacement of Kirre. Hereby, we establish <em>D. melanogaster</em> as a genetic system to specifically model molecular Neph1 functions <em>in vivo</em> and identify a conserved amino acid motif linking Neph1 to <em>Drosophila</em> Kirre function.</p> </div

GCN fusion requires the cytoplasmic part of Kirre containing the KIN1 motif.

<p><b>A.</b> Scanning electron micrographs of GCNs at third larval stage. Scale bar: 20 µm. <b>B.</b> Quantitation of GCN circularity revealing that Kirre versions CT1– CT3 which still contain the KIN1 motif are able to partially restore the wildtype situation. However, Kirre-CT4 missing the conserved KIN1 motif is not able to rescue the GCN fusion phenotype. *P value <0,0001 (unpaired t-test with Welchs correction). Genotypes: Control: <i>+/sns-GAL4</i>; <i>kirre^-</i>: <i>Df(1)duf</i> <i>^sps-1</i>; CT1 rescue: <i>Df(1)duf</i> <i>^sps-1/y; UAS-CT1/sns-GAL4</i>; CT2 rescue: <i>Df(1)duf</i> <i>^sps-1/y; UAS-CT2/sns-GAL4</i>; CT3 rescue: <i>Df(1)duf</i> <i>^sps-1/y; UAS-CT3/sns-GAL4,</i> CT4 rescue: <i>Df(1)duf</i> <i>^sps-1/y; UAS-CT4/sns-GAL4.</i></p

Neph1 can mimic neuronal and eye phenotypes of overexpressed Kirre or IrreC/Rst.

<p>Scanning electron micrograph of adult <i>Drosophila</i> eyes (<b>A,B,F,G,K,L,P,Q</b>), close-up of the eye (<b>B,G,L,Q</b>) and light micrographs of semithin sections (<b>C,H,M,R</b>). The control shows the regular crystal like arrangement of an <i>Drosophila</i> eye (<b>A,B</b>). <i>Sev-GAL4</i> induced misexpression of IrreC/Rst (<b>F,G</b>) or Kirre (<b>K,L</b>) results in a rough eye phenotype. Misexpression of Neph1 with <i>sev-GAL4</i> also causes a rough eye phenotype (<b>P,Q</b>). All three genotypes exhibit fusion of ommatidia (<b>H,M,R</b>). Genotypes: <i>sev-GAL4/+</i> (<b>A,B,C</b>); <i>sev-GAL4/UAS-irreC/rst</i> (<b>F,G,H</b>); <i>sev-GAL4/+,UAS-kirre/+</i> (<b>K,L</b>); <i>sev-GAL4/UAS-neph1_V5</i> (<b>P,Q,R</b>). Auto fluorescence micrographs of adult <i>Drosophila</i> optic lobes (<b>D,I,N,S</b>). The control fly shows the typical wildtype-like arrangement of the neuropils of the <i>Drosophila</i> optic lobe (<b>D,E</b>). Overexpression of Rst (<b>I</b>) or Kirre (<b>N</b>) causes severe misrouting of fibers in medulla and lobula complex and a disorganization of these neuropils (<b>J,O</b>). Misexpression of Neph1 leads to a similar phenotype (<b>S,T</b>). Optic lobe drawing: la: lamina, me: medulla, lo: lobula, lp: lobula plate. Genotypes: <i>Mz1369-GAL4/+</i> (<b>D</b>); <i>Mz1369-GAL4/UAS-rst</i> (<b>I</b>); <i>Mz1369-GAL4/+,UAS-kirre/+</i> (<b>N</b>); <i>Mz1369-GAL4/UAS-neph1_V5</i> (<b>S</b>).</p

Schematic drawing of the identified KIN1 motif and its position in the protein sequences.

<p><b>A.</b> MEME generated Bitlogo of the KIN1 motif. <b>B.</b> Structural comparison of Neph and Neph-like proteins. Blue: Ig domain. Grey: transmembrane domain. Yellow: KIN1 motif. Scale: number of amino acids. <b>C.</b> Surface representation of the structural model of the cytoplasmic domain of Neph1 is shown in green with the KIN1 motif highlighted in yellow.</p

Third larval stage <i>kirre^-</i> GCNs are hyperfused. Neph1 can rescue the <i>kirre^-</i> phenotype.

<p><b>A.</b> Scanning electron micrographs of GCNs at third larval stage. Scale bar: 20 µm. <b>B.</b> The fact that hyperfused GCNs lose their spherical shape was used to quantify the rescue efficiency. Kirre or Neph1 expression is sufficient to significantly rescue the <i>kirre^-</i> phenotype. *P value <0,0001 (unpaired t-test with Welchs correction). Genotypes: Control: <i>+/sns-GAL4. kirre^-</i>: <i>Df(1)duf</i> <i>^sps-1/y</i>. Kirre rescue: <i>Df(1)duf</i> <i>^sps-1/y; UAS-Kirre/sns-GAL4.</i> Neph1 rescue: <i>Df(1)duf</i> <i>^sps-1/y; UAS-neph1/sns-GAL4</i>. Neph2 rescue: <i>Df(1)duf</i> <i>^sps-1/y; UAS-neph2/sns-GAL4</i>. Neph3 rescue: <i>Df(1)duf</i> <i>^sps-1/y; UAS-neph3/sns-GAL4.</i></p

IRM protein misexpression in GCNs can induce clustering and/or hyperfusion.

<p><b>A-F.</b> Scanning electron micrograph of <i>Drosophila</i> GCNs at third larval stage and immunoreactivity of the corresponding genotypes. The control shows the distribution of misexpressed membrane associated mCD8::GFP. The misexpressed protein is endocytosed if it is not stabilized in the nephrocyte diaphragm. The binucleate GCNs are separated (<b>A</b>). IrreC/Rst misexpression leads to clustering and hyperfusion of GCNs (<b>B</b>). Kirre overexpression leads to a similar phenotype. (<b>C</b>). Neph1 misexpression also leads to clustering and fusion of GCNs (<b>D</b>)<b>.</b> Neph2 misexpression does not interfere with the fusion of GCNs (<b>E</b>). The arrow head marks the enriched Neph2 immunoreactivity at cell-cell contacts. Misexpression of Neph3 does not interfere with the GCN fusion. Immunoreactivity shows that the Neph3 expression pattern is similar to the GFP control (<b>F</b>).</p

TBC1D24 genotype-phenotype correlation: Epilepsies and other neurologic features.

OBJECTIVE: To evaluate the phenotypic spectrum associated with mutations in TBC1D24. METHODS: We acquired new clinical, EEG, and neuroimaging data of 11 previously unreported and 37 published patients. TBC1D24 mutations, identified through various sequencing methods, can be found online (http://lovd.nl/TBC1D24). RESULTS: Forty-eight patients were included (28 men, 20 women, average age 21 years) from 30 independent families. Eighteen patients (38%) had myoclonic epilepsies. The other patients carried diagnoses of focal (25%), multifocal (2%), generalized (4%), and unclassified epilepsy (6%), and early-onset epileptic encephalopathy (25%). Most patients had drug-resistant epilepsy. We detail EEG, neuroimaging, developmental, and cognitive features, treatment responsiveness, and physical examination. In silico evaluation revealed 7 different highly conserved motifs, with the most common pathogenic mutation located in the first. Neuronal outgrowth assays showed that some TBC1D24 mutations, associated with the most severe TBC1D24-associated disorders, are not necessarily the most disruptive to this gene function. CONCLUSIONS: TBC1D24-related epilepsy syndromes show marked phenotypic pleiotropy, with multisystem involvement and severity spectrum ranging from isolated deafness (not studied here), benign myoclonic epilepsy restricted to childhood with complete seizure control and normal intellect, to early-onset epileptic encephalopathy with severe developmental delay and early death. There is no distinct correlation with mutation type or location yet, but patterns are emerging. Given the phenotypic breadth observed, TBC1D24 mutation screening is indicated in a wide variety of epilepsies. A TBC1D24 consortium was formed to develop further research on this gene and its associated phenotypes.peerReviewe