A Novel Method to Model Chronic Traumatic Encephalopathy in <em>Drosophila</em>

Chronic Traumatic Encephalopathy (CTE) is an established neurodegenerative disease that is closely associated with exposure to repetitive mild Traumatic ...

Johns Hopkins University School of Medicine

Dxl6 is a member of the Drosophila melanogaster SR protein family, a group of nuclear proteins that are both essential splicing factors and specific splicing regulators. To get more insight of Dx16 function, we generated the monoclonal antibody against Dx16 and determined its expression pattern and subcellular location. It is mainly expressed in the nucleus of CNS in Drosophila embryos. In order to investigate the RNA-binding specificity of Dxl6, Dxl6-binding RNAs were identified by SELEX screen by using recombinant Dxl6 N-terminus protein as the target. These RNAs contained a consensus motif. Some pre-mRNAs from the corresponding genes showed splicing defects in the Dxl6-P-element insertional mutant fly. These results indicate that Dxl6 has unique functions in the removal of some introns during development.

Identification of RNA binding sequences of Drosophila SR protein DX16.

Neurexins are highly polymorphic cell-surface adhesive molecules in neurons. In cultured mammalian cell system, they were found to be involved in synaptogenesis. Here, we report for the first time that Drosophila neurexin is required for synapse formation and associative learning in larvae. Drosophila genome encodes a single functional neurexin (CG7050; Neurexin-1 or Nrx-1), which is a homolog of vertebrate alpha-neurexin. Neurexin-1 is expressed in central nervous system and highly enriched in synaptic regions of the ventral ganglion and brain. Neurexin-1 null mutants are viable and fertile, but have shortened lifespan. The synapse number is decreased in central nervous system in Neurexin-1 null mutants. In addition, Neurexin-1 null mutants exhibit associative learning defect in larvae.

Neurexin-1 is required for synapse formation and larvae associative learning in Drosophila.

The serine-arginine-rich (SR) proteins belong to a conserved splicing factor family that not only is essential for constitutive pre-mRNA splicing, but also plays important roles in regulation of alternative splicing. Dx16 is a member of SR protein family in Drosophila. In order to get more insight of dx16 function, we identified the proteins interacting with DX16 through yeast two-hybrid and GST-pull down assays. DX16 interacts with the U1 snRNP subunit CG7564, the SR protein RBP1 and the SR protein kinase DOA. The first and second serine-and arginine-rich regions of DOA are required for the interaction between DOA and DX16. DX16 could be phosphorylated by DOA in vitro and DX16 is highly phosphorylated in vivo. Immunofluorescence microscopy results reveal that doa and dx16 are both highly expressed in embryonic central nervous system. These results suggest that DX16 could be a novel SR protein phosphorylated by DOA and it may participate in the formation of splicing complex through its interactions with other splicing related proteins.

DX16 is a novel SR protein phosphorylated by DOA.

Chromosome movements in mitosis are orchestrated by dynamic interactions between spindle microtubules and the kinetochore, a multiprotein complex assembled onto centromeric DNA of the chromosome. Here we show that phosphorylation of human HsMis13 by Aurora B kinase is required for functional kinetochore assembly in HeLa cells. Aurora B interacts with HsMis13 in vitro and in vivo. HsMis13 is a cognate substrate of Aurora B, and the phosphorylation sites were mapped to Ser-100 and Ser-109. Suppression of Aurora B kinase by either small interfering RNA or chemical inhibitors abrogates the localization of HsMis13 but not HsMis12 to the kinetochore. In addition, non-phosphorylatable but not wild type and phospho-mimicking HsMis13 failed to localize to the kinetochore, demonstrating the requirement of phosphorylation by Aurora B for the assembly of HsMis13 to kinetochore. In fact, localization of HsMis13 to the kinetochore is spatiotemporally regulated by Aurora B kinase, which is essential for recruiting outer kinetochore components such as Ndc80 components and CENP-E for functional kinetochore assembly. Importantly, phospho-mimicking mutant HsMis13 restores the assembly of CENP-E to the kinetochore, and tension developed across the sister kinetochores in Aurora B-inhibited cells. Thus, we reason that HsMis13 phosphorylation by Aurora B is required for organizing a stable bi-oriented microtubule kinetochore attachment that is essential for faithful chromosome segregation in mitosis.

Phosphorylation of HsMis13 by Aurora B kinase is essential for assembly of functional kinetochore.

Neurexins &#x662F;&#x601D;&#x60F3;&#x8981;&#x672C;&#x5730;&#x5316;&#x7684;&#x7A81;&#x89E6;&#x524D;&#x819C;&#x7684;&#x795E;&#x7ECF;&#x5143;&#x7279;&#x5F02;&#x6027;&#x7EC6;&#x80DE;&#x8868;&#x9762;&#x5206;&#x5B50;&#x3002;&#x6700;&#x8FD1;&#x4F7F;&#x7528;&#x679C;&#x8747;&#x7684;&#x9057;&#x4F20;&#x5B66;&#x7814;&#x7A76;&#x6709;&#x7275;&#x8FDE;&#x5230;&#x5355;&#x4E2A;&#x679C;&#x8747; neurexin (dnrx) &#x5728;&#x9002;&#x5F53;&#x7684;&#x4F53;&#x7CFB;&#x7ED3;&#x6784;&#x3001; &#x53D1;&#x5C55;&#x548C;&#x529F;&#x80FD;&#x7684;&#x795E;&#x7ECF;&#x7A81;&#x89E6;&#x4F53;&#x5185;&#x7684;&#x81F3;&#x5173;&#x91CD;&#x8981;&#x7684;&#x4F5C;&#x7528;&#x3002;&#x7136;&#x800C;&#xFF0C;&#x8FD9;&#x4E9B;&#x884C;&#x52A8;&#x6240;&#x4F9D;&#x636E;&#x7684;&#x786E;&#x5207;&#x673A;&#x5236;&#x4E0D;&#x5145;&#x5206;&#x7684;&#x7406;&#x89E3;&#x3002;&#x6F84;&#x6E05; Neurexin &#x4F53;&#x5185;&#x7684;&#x5206;&#x5B50;&#x673A;&#x5236;&#xFF0C;&#x6211;&#x4EEC;&#x53EF;&#x4EE5;&#x91C7;&#x7528; dnrx &#x548C; caki &#x7684;&#x7A81;&#x53D8;&#x82CD;&#x8747;&#xFF0C;&#x52A0;&#x4E0A;&#x5404;&#x79CD;&#x65B9;&#x6CD5;&#xFF0C;&#x5E76;&#x5206;&#x6790;&#x52A8;&#x7269;&#x7684;&#x8FD0;&#x52A8;&#xFF0C;&#x7A81;&#x89E6;&#x56CA;&#x6CE1;&#x5FAA;&#x73AF;&#x548C;&#x795E;&#x7ECF;&#x808C;&#x8089;&#x63A5;&#x5934;&#x7684;&#x611F;&#x53D7;&#x5668;&#x3002;&#x6211;&#x4EEC;&#x53D1;&#x73B0; Dneurexin (DNRX) &#x662F;&#x91CD;&#x8981;&#x7684;&#x9057;&#x4F20;&#x76F8;&#x4E92;&#x4F5C;&#x7528;&#x4E0E;&#x652F;&#x67B6;&#x86CB;&#x767D;&#xFF0C;&#x901A;&#x8FC7;&#x8FD0;&#x52A8; CAKI/CMG&#xFF0C;&#x679C;&#x8747;&#x540C;&#x7CFB;&#x7269;&#x7684;&#x810A;&#x690E;&#x52A8;&#x7269;&#x7684;&#x6728;&#x6876;&#x3002;&#x7C7B;&#x4F3C;&#x4E8E;&#x54FA;&#x4E73;&#x52A8;&#x7269;&#x7684;&#x5BF9;&#x53E3;&#xFF0C;DNRX &#x662F;&#x7A81;&#x89E6;&#x56CA;&#x6CE1;&#x9A91;&#x81EA;&#x884C;&#x8F66;&#xFF0C;&#x8FD9;&#x5728;&#x795E;&#x7ECF;&#x808C;&#x8089;&#x63A5;&#x5934; &#xFF08;NMJ&#xFF09; &#x611F;&#x53D7;&#x5668;&#x4E2D;&#x8D77;&#x5173;&#x952E;&#x4F5C;&#x7528;&#x7684;&#x5FC5;&#x8981;&#x6761;&#x4EF6;&#x3002;&#x7136;&#x800C;&#xFF0C;&#x8FD9;&#x79CD;&#x76F8;&#x4E92;&#x4F5C;&#x7528;&#x4F3C;&#x4E4E;&#x4E0D;&#x9700;&#x8981;&#x9488;&#x5BF9;&#x7A81;&#x89E6;&#x7684; DNRX&#xFF0C;&#x4F46;&#x53EF;&#x80FD;&#x8F6C;&#x800C;&#x9700;&#x8981;&#x7684;&#x9002;&#x5F53;&#x7684;&#x7A81;&#x89E6;&#x529F;&#x80FD;&#xFF0C;&#x53EF;&#x80FD;&#x901A;&#x8FC7;&#x8C03;&#x8282;&#x7A81;&#x89E6;&#x56CA;&#x6CE1;&#x5FAA;&#x73AF;&#x8FC7;&#x7A0B;&#x3002;

Neurexin &#x4E0E; CAKI/CMG &#x9057;&#x4F20;&#x76F8;&#x4E92;&#x4F5C;&#x7528;&#x662F;&#x91CD;&#x8981;&#x7684;&#x679C;&#x8747;&#x795E;&#x7ECF;&#x808C;&#x8089;&#x63A5;&#x5934;&#x7A81;&#x89E6;&#x529F;&#x80FD;&#x3002;

Neuroligins belong to a highly conserved family of cell adhesion molecules that have been implicated in synapse formation and function. However, the precise in vivo roles of Neuroligins remain unclear. In the present study, we have analyzed the function of Drosophila neuroligin 2 (dnl2) in synaptic development and function. We show that dnl2 is strongly expressed in the embryonic and larval CNS and at the larval neuromuscular junction (NMJ). dnl2 null mutants are viable but display numerous structural defects at the NMJ, including reduced axonal branching and fewer synaptic boutons. dnl2 mutants also show an increase in the number of active zones per bouton but a decrease in the thickness of the subsynaptic reticulum and length of postsynaptic densities. dnl2 mutants also exhibit a decrease in the total glutamate receptor density and a shift in the subunit composition of glutamate receptors in favor of GluRIIA complexes. In addition to the observed defects in synaptic morphology, we also find that dnl2 mutants show increased transmitter release and altered kinetics of stimulus-evoked transmitter release. Importantly, the defects in presynaptic structure, receptor density, and synaptic transmission can be rescued by postsynaptic expression of dnl2. Finally, we show that dnl2 colocalizes and binds to Drosophila neurexin (dnrx) in vivo. However, whereas homozygous mutants for either dnl2 or dnrx are viable, double mutants are lethal and display more severe defects in synaptic morphology. Altogether, our data show that, although dnl2 is not absolutely required for synaptogenesis, it is required postsynaptically for synapse maturation and function.

Neuroligin 2 is required for synapse development and function at the Drosophila neuromuscular junction.

Synapse is a highly specialized inter-cellular structure between neurons or between a neuron and its target cell that mediates cell-cell communications. Ample results indicate that synaptic adhesion molecules are critically important in modulating the complexity and specificity of the synapse. And disruption of adhesive properties of synapses may lead to neurodevelopmental or neurodegenerative diseases. In this review, we will use the Drosophila NMJ as a model system for glutamatergic synapses to discuss the structure and function of homophilic and heterophilic synaptic adhesion molecules with special focus on recent findings in neurexins and neuroligins in Drosophila.

Cell adhesion molecules in Drosophila synapse development and function.

Neurexins are cell adhesion molecules involved in synapse formation and synaptic regulation. Mutations in the neurexin genes are linked to a number of neurodevelopmental disorders such as autism. Here, we show that the Drosophila homolog of α-Neurexin is critical for fly visual function. Lack of Neurexin leads to significantly impaired visual function due to reduced rhodopsin levels. We show that the decreased chromophore levels cause deficits in rhodopsin maturation and that Neurexin is required for retinoid transport. Using yeast two-hybrid screening, we identify that Neurexin interacts with apolipoprotein I (ApoL I), a product generated by cleavage of retinoid- and fatty acid-binding glycoprotein (RFABG) that functions in retinoid transport. Finally, we demonstrate that Neurexin is essential for the apolipoproteins level. Our results reveal a role for Neurexin in mediating retinoid transport and subsequent rhodopsin maturation and suggest that Neurexin regulates lipoprotein function.

Neurexin regulates visual function via mediating retinoid transport to promote rhodopsin maturation.

Neuroligins (Nlgs) are a family of cell adhesion molecules thought to be important for synapse maturation and function. Mammalian studies have shown that different Nlgs have different roles in synaptic maturation and function. In Drosophila melanogaster, the roles of Drosophila neuroligin1 (DNlg1), neuroligin2, and neuroligin4 have been examined. However, the roles of neuroligin3 (dnlg3) in synaptic development and function have not been determined. In this study, we used the Drosophila neuromuscular junctions (NMJs) as a model system to investigate the in vivo role of dnlg3. We showed that DNlg3 was expressed in both the CNS and NMJs where it was largely restricted to the postsynaptic site. We generated dnlg3 mutants and showed that these mutants exhibited an increased bouton number and reduced bouton size compared with the wild-type (WT) controls. Consistent with alterations in bouton properties, pre- and postsynaptic differentiations were affected in dnlg3 mutants. This included abnormal synaptic vesicle endocytosis, increased postsynaptic density length, and reduced GluRIIA recruitment. In addition to impaired synaptic development and differentiation, we found that synaptic transmission was reduced in dnlg3 mutants. Altogether, our data showed that DNlg3 was required for NMJ development, synaptic differentiation, and function.

Drosophila neuroligin3 regulates neuromuscular junction development and synaptic differentiation.

Tauopathies are a group of neurodegenerative disorders that include hereditary frontotemporal dementias (FTDs) such as FTD with parkinsonism linked to chromosome 17 (FTDP-17), as well as sporadic variants of FTDs like progressive supranuclear palsy (PSP), corticobasal degeneration (CBD), and Pick's disease. These diverse diseases all have in common the presence of abnormally phosphorylated tau aggregates. In this review, we will summarize key features of transgenic Drosophila models of tauopathies and a number of insights into disease mechanisms as well as therapeutic implications gained from the fruit fly models.

Studying tauopathies in Drosophila: A fruitful model.

Drosophila neurexin (DNRX) plays a critical role in proper architecture development and synaptic function in vivo. However, the temporal and spatial expression pattern of DNRX still remains unclear. For this study, we generated a novel Drosophila transgenic strain termed the DNRX-Gal4 transgenic line, with characteristic features in agreement with the endogenous DNRX expression pattern. DNRX expression was examined by driving the expression of a GFP reporter (nuclear-localized and membrane- localized GFP) using the DNRX-Gal4 promoter. We found that DNRX was expressed preferentially in central and motor neurons in embryos, larvae and adults, but not in glial cells. DNRX was expressed in pre- and post-synaptic areas in third instar larvae neuromuscular junctions (NMJs). Reporter expression was also observed in the salivary glands, guts, wings and legs of adult flies. In the adult brain, reporter expression was observed throughout several brain regions, including the mushroom body (MBs), antennal lobe (AL) and optic lobe neurons, which is consistent with endogenous DNRX expression via antibody staining. Interestingly, DNRX was also expressed in clock neurons. Meanwhile, we found that DNRX expression in the MBs was required for olfactory learning and memory.

Temporal and spatial expression of Drosophila Neurexin during the life cycle visualized using a DNRX-Gal4/UAS-reporter.

A rapidly progressive dementia case with pathological diagnosis of FTLD-UPS.

Abnormal accumulation of TDP-43 into cytoplasmic or nuclear inclusions with accompanying nuclear clearance, a common pathology initially identified in amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD), has also been found in Alzheimer' disease (AD). TDP-43 serves as a splicing repressor of nonconserved cryptic exons and that such function is compromised in brains of ALS and FTD patients, suggesting that nuclear clearance of TDP-43 underlies its inability to repress cryptic exons. However, whether TDP-43 cytoplasmic aggregates are a prerequisite for the incorporation of cryptic exons is not known. Here, we assessed hippocampal tissues from 34 human postmortem brains including cases with confirmed diagnosis of AD neuropathologic changes along with age-matched controls. We found that cryptic exon incorporation occurred in all AD cases exhibiting TDP-43 pathology. Furthermore, incorporation of cryptic exons was observed in the hippocampus when TDP-43 inclusions was restricted only to the amygdala, the earliest stage of TDP-43 progression. Importantly, cryptic exon incorporation could be detected in AD brains lacking TDP-43 inclusion but exhibiting nuclear clearance of TDP-43. These data supports the notion that the functional consequence of nuclear depletion of TDP-43 as determined by cryptic exon incorporation likely occurs as an early event of TDP-43 proteinopathy and may have greater contribution to the pathogenesis of AD than currently appreciated. Early detection and effective repression of cryptic exons in AD patients may offer important diagnostic and therapeutic implications for this devastating illness of the elderly.

Cryptic exon incorporation occurs in Alzheimer's brain lacking TDP-43 inclusion but exhibiting nuclear clearance of TDP-43.

Neuroligins (Nlgs) are transmembrane cell adhesion molecules playing essential roles in synapse development and function. Genetic mutations in neuroligin genes have been linked with some neurodevelopmental disorders such as autism. These mutated Nlgs are mostly retained in the endoplasmic reticulum (ER). However, the mechanisms underlying normal Nlg maturation and trafficking have remained largely unknown. Here, we found that Drosophila neuroligin 2 (DNlg2) undergoes proteolytic cleavage in the ER in a variety of Drosophila tissues throughout developmental stages. A region encompassing Y642-T698 is required for this process. The immature non-cleavable DNlg2 is retained in the ER and non-functional. The C-terminal fragment of DNlg2 instead of the full-length or non-cleavable DNlg2 is able to rescue neuromuscular junction defects and GluRIIB reduction induced by dnlg2 deletion. Intriguingly, the autism-associated R598C mutation in DNlg2 leads to similar marked defects in DNlg2 proteolytic process and ER export, revealing a potential role of the improper Nlg cleavage in autism pathogenesis. Collectively, our findings uncover a specific mechanism that controls DNlg2 maturation and trafficking via proteolytic cleavage in the ER, suggesting that the perturbed proteolytic cleavage of Nlgs likely contributes to autism disorder.

Mingkuan Sun

Division of Neuropathology,
Department of Pathology,
Division of Neuropathology, Department of Pathology

Johns Hopkins University School of Medicine

A Novel Method to Model Chronic Traumatic Encephalopathy in Drosophila

Mingkuan Sun

.css-o250i3{font-size:18px;font-family:Open Sans,sans-serif;line-height:21.6px;}Division of Neuropathology,Department of Pathology,Division of Neuropathology, Department of Pathology

Johns Hopkins University School of Medicine

A Novel Method to Model Chronic Traumatic Encephalopathy in Drosophila

Division of Neuropathology,
Department of Pathology,
Division of Neuropathology, Department of Pathology