Glial loss of the metallo ß-lactamase domain containing protein, SWIP-10, induces age- and glutamate-signaling dependent, dopamine neuron degeneration.

Across phylogeny, glutamate (Glu) signaling plays a critical role in regulating neural excitability, thus supporting many complex behaviors. Perturbed synaptic and extrasynaptic Glu homeostasis in the human brain has been implicated in multiple neuropsychiatric and neurodegenerative disorders including Parkinson's disease, where theories suggest that excitotoxic insults may accelerate a naturally occurring process of dopamine (DA) neuron degeneration. In C. elegans, mutation of the glial expressed gene, swip-10, results in Glu-dependent DA neuron hyperexcitation that leads to elevated DA release, triggering DA signaling-dependent motor paralysis. Here, we demonstrate that swip-10 mutations induce premature and progressive DA neuron degeneration, with light and electron microscopy studies demonstrating the presence of dystrophic dendritic processes, as well as shrunken and/or missing cell soma. As with paralysis, DA neuron degeneration in swip-10 mutants is rescued by glial-specific, but not DA neuron-specific expression of wildtype swip-10, consistent with a cell non-autonomous mechanism. Genetic studies implicate the vesicular Glu transporter VGLU-3 and the cystine/Glu exchanger homolog AAT-1 as potential sources of Glu signaling supporting DA neuron degeneration. Degeneration can be significantly suppressed by mutations in the Ca2+ permeable Glu receptors, nmr-2 and glr-1, in genes that support intracellular Ca2+ signaling and Ca2+-dependent proteolysis, as well as genes involved in apoptotic cell death. Our studies suggest that Glu stimulation of nematode DA neurons in early larval stages, without the protective actions of SWIP-10, contributes to insults that ultimately drive DA neuron degeneration. The swip-10 model may provide an efficient platform for the identification of molecular mechanisms that enhance risk for Parkinson's disease and/or the identification of agents that can limit neurodegenerative disease progression.

Decreased function of survival motor neuron protein impairs endocytic pathways.

Spinal muscular atrophy (SMA) is caused by depletion of the ubiquitously expressed survival motor neuron (SMN) protein, with 1 in 40 Caucasians being heterozygous for a disease allele. SMN is critical for the assembly of numerous ribonucleoprotein complexes, yet it is still unclear how reduced SMN levels affect motor neuron function. Here, we examined the impact of SMN depletion in Caenorhabditis elegans and found that decreased function of the SMN ortholog SMN-1 perturbed endocytic pathways at motor neuron synapses and in other tissues. Diminished SMN-1 levels caused defects in C. elegans neuromuscular function, and smn-1 genetic interactions were consistent with an endocytic defect. Changes were observed in synaptic endocytic proteins when SMN-1 levels decreased. At the ultrastructural level, defects were observed in endosomal compartments, including significantly fewer docked synaptic vesicles. Finally, endocytosis-dependent infection by JC polyomavirus (JCPyV) was reduced in human cells with decreased SMN levels. Collectively, these results demonstrate for the first time, to our knowledge, that SMN depletion causes defects in endosomal trafficking that impair synaptic function, even in the absence of motor neuron cell death.

A novel function for the Caenorhabditis elegans torsin OOC-5 in nucleoporin localization and nuclear import.

Torsin proteins are AAA+ ATPases that localize to the endoplasmic reticular/nuclear envelope (ER/NE) lumen. A mutation that markedly impairs torsinA function causes the CNS disorder DYT1 dystonia. Abnormalities of NE membranes have been linked to torsinA loss of function and the pathogenesis of DYT1 dystonia, leading us to investigate the role of the Caenorhabditis elegans torsinA homologue OOC-5 at the NE. We report a novel role for torsin in nuclear pore biology. In ooc-5-mutant germ cell nuclei, nucleoporins (Nups) were mislocalized in large plaques beginning at meiotic entry and persisted throughout meiosis. Moreover, the KASH protein ZYG-12 was mislocalized in ooc-5 gonads. Nups were mislocalized in adult intestinal nuclei and in embryos from mutant mothers. EM analysis revealed vesicle-like structures in the perinuclear space of intestinal and germ cell nuclei, similar to defects reported in torsin-mutant flies and mice. Consistent with a functional disruption of Nups, ooc-5-mutant embryos displayed impaired nuclear import kinetics, although the nuclear pore-size exclusion barrier was maintained. Our data are the first to demonstrate a requirement for a torsin for normal Nup localization and function and suggest that these functions are likely conserved.

The nphp-2 and arl-13 genetic modules interact to regulate ciliogenesis and ciliary microtubule patterning in C. elegans.

Cilia are microtubule-based cellular organelles that mediate signal transduction. Cilia are organized into several structurally and functionally distinct compartments: the basal body, the transition zone (TZ), and the cilia shaft. In vertebrates, the cystoprotein Inversin localizes to a portion of the cilia shaft adjacent to the TZ, a region termed the "Inversin compartment" (InvC). The mechanisms that establish and maintain the InvC are unknown. In the roundworm C. elegans, the cilia shafts of amphid channel and phasmid sensory cilia are subdivided into two regions defined by different microtubule ultrastructure: a proximal doublet-based region adjacent to the TZ, and a distal singlet-based region. It has been suggested that C. elegans cilia also possess an InvC, similarly to mammalian primary cilia. Here we explored the biogenesis, structure, and composition of the C. elegans ciliary doublet region and InvC. We show that the InvC is conserved and distinct from the doublet region. nphp-2 (the C. elegans Inversin homolog) and the doublet region genes arl-13, klp-11, and unc-119 are redundantly required for ciliogenesis. InvC and doublet region genes can be sorted into two modules-nphp-2+klp-11 and arl-13+unc-119-which are both antagonized by the hdac-6 deacetylase. The genes of this network modulate the sizes of the NPHP-2 InvC and ARL-13 doublet region. Glutamylation, a tubulin post-translational modification, is not required for ciliary targeting of InvC and doublet region components; rather, glutamylation is modulated by nphp-2, arl-13, and unc-119. The ciliary targeting and restricted localization of NPHP-2, ARL-13, and UNC-119 does not require TZ-, doublet region, and InvC-associated genes. NPHP-2 does require its calcium binding EF hand domain for targeting to the InvC. We conclude that the C. elegans InvC is distinct from the doublet region, and that components in these two regions interact to regulate ciliogenesis via cilia placement, ciliary microtubule ultrastructure, and protein localization.

Extracellular leucine-rich repeat proteins are required to organize the apical extracellular matrix and maintain epithelial junction integrity in C. elegans.

Epithelial cells are linked by apicolateral junctions that are essential for tissue integrity. Epithelial cells also secrete a specialized apical extracellular matrix (ECM) that serves as a protective barrier. Some components of the apical ECM, such as mucins, can influence epithelial junction remodeling and disassembly during epithelial-to-mesenchymal transition (EMT). However, the molecular composition and biological roles of the apical ECM are not well understood. We identified a set of extracellular leucine-rich repeat only (eLRRon) proteins in C. elegans (LET-4 and EGG-6) that are expressed on the apical surfaces of epidermal cells and some tubular epithelia, including the excretory duct and pore. A previously characterized paralog, SYM-1, is also expressed in epidermal cells and secreted into the apical ECM. Related mammalian eLRRon proteins, such as decorin or LRRTM1-3, influence stromal ECM or synaptic junction organization, respectively. Mutants lacking one or more of the C. elegans epithelial eLRRon proteins show multiple defects in apical ECM organization, consistent with these proteins contributing to the embryonic sheath and cuticular ECM. Furthermore, epithelial junctions initially form in the correct locations, but then rupture at the time of cuticle secretion and remodeling of cell-matrix interactions. This work identifies epithelial eLRRon proteins as important components and organizers of the pre-cuticular and cuticular apical ECM, and adds to the small but growing body of evidence linking the apical ECM to epithelial junction stability. We propose that eLRRon-dependent apical ECM organization contributes to cell-cell adhesion and may modulate epithelial junction dynamics in both normal and disease situations.

A homolog of FHM2 is involved in modulation of excitatory neurotransmission by serotonin in C. elegans.

The C. elegans eat-6 gene encodes a Na(+), K(+)-ATPase alpha subunit and is a homolog of the familial hemiplegic migraine candidate gene FHM2. Migraine is the most common neurological disorder linked to serotonergic dysfunction. We sought to study the pathophysiological mechanisms of migraine and their relation to serotonin (5-HT) signaling using C. elegans as a genetic model. In C. elegans, exogenous 5-HT inhibits paralysis induced by the acetylcholinesterase inhibitor aldicarb. We found that the eat-6(ad467) mutation or RNAi of eat-6 increases aldicarb sensitivity and causes complete resistance to 5-HT treatment, indicating that EAT-6 is a component of the pathway that couples 5-HT signaling and ACh neurotransmission. While a postsynaptic role of EAT-6 at the bodywall NMJs has been well established, we found that EAT-6 may in addition regulate presynaptic ACh neurotransmission. We show that eat-6 is expressed in ventral cord ACh motor neurons, and that cell-specific RNAi of eat-6 in the ACh neurons leads to hypersensitivity to aldicarb. Electron microscopy showed an increased number of synaptic vesicles in the ACh neurons in the eat-6(ad467) mutant. Genetic analyses suggest that EAT-6 interacts with EGL-30 Galphaq, EGL-8 phospholipase C and SLO-1 BK channel signaling to modulate ACh neurotransmission and that either reduced or excessive EAT-6 function may lead to increased ACh neurotransmission. Study of the interaction between eat-6 and 5-HT receptors revealed both stimulatory and inhibitory 5-HT inputs to the NMJs. We show that the inhibitory and stimulatory 5-HT signals arise from distinct 5-HT neurons. The role of eat-6 in modulation of excitatory neurotransmission by 5-HT may provide a genetic explanation for the therapeutic effects of the drugs targeting 5-HT receptors in the treatment of migraine patients.

Oncogenic cooperation and coamplification of developmental transcription factor genes in lung cancer.

We used high-resolution array analysis to discover a recurrent lung cancer amplicon located at 14q13.3. Low-level gain of this region was detected in 15% of lung cancer samples, and high-level amplification was detected in an additional 4% of samples. High-level focal amplification appears to be specific to lung cancers, because it was not detected in >500 samples of other tumor types. Mapping of the commonly amplified region revealed there are three genes in the core region, all of which encode transcription factors with either established lung developmental function (TTF1/NKX2-1, NKX2-8) or potential lung developmental function (PAX9). All three genes were overexpressed to varying degrees in amplified samples, although TTF1/NKX2-1 was not expressed in the squamous cancer subtype, consistent with previous reports. Remarkably, overexpression of any pairwise combination of these genes showed pronounced synergy in promoting the proliferation of immortalized human lung epithelial cells. Analysis of human lung cancer cell lines by both RNAi and ectopic overexpression further substantiates an oncogenic role for these transcription factors. These results, taken together with previous reports of oncogenic alterations of transcription factors involved in lung development (p63, CEBPA), suggest genetic alterations that directly interfere with transcriptional networks normally regulating lung development may be a more common feature of lung cancer than previously realized.

Identification of alterations in DNA copy number in host stromal cells during tumor progression.

The interactions between cancer cells and the surrounding host stromal tissue play a critical role in tumor progression and metastasis, but the molecular nature of this relationship remains largely uncharacterized. Furthermore, although genetic changes of neoplastic cells in tumors contribute significantly to tumor progression, it is not known whether similar changes occur in the adjacent host stromal microenvironment and whether they contribute to or inhibit tumorigenesis. To address this question in an unbiased and genome-wide manner, we applied high-resolution DNA copy number analysis to murine stromal DNA isolated from human xenograft tumors that were formed in immunodeficient mice. We show that numerous amplifications and deletions are found within the host stromal microenvironment, suggesting that alterations in host DNA copy number can occur and may play a significant role in modifying tumor-stromal interactions.

Representational oligonucleotide microarray analysis: a high-resolution method to detect genome copy number variation.

We have developed a methodology we call ROMA (representational oligonucleotide microarray analysis), for the detection of the genomic aberrations in cancer and normal humans. By arraying oligonucleotide probes designed from the human genome sequence, and hybridizing with "representations" from cancer and normal cells, we detect regions of the genome with altered "copy number." We achieve an average resolution of 30 kb throughout the genome, and resolutions as high as a probe every 15 kb are practical. We illustrate the characteristics of probes on the array and accuracy of measurements obtained using ROMA. Using this methodology, we identify variation between cancer and normal genomes, as well as between normal human genomes. In cancer genomes, we readily detect amplifications and large and small homozygous and hemizygous deletions. Between normal human genomes, we frequently detect large (100 kb to 1 Mb) deletions or duplications. Many of these changes encompass known genes. ROMA will assist in the discovery of genes and markers important in cancer, and the discovery of loci that may be important in inherited predispositions to disease.

Genomic amplification and oncogenic properties of the KCNK9 potassium channel gene.

Representational difference analysis (RDA) of human breast cancer was used to discover a novel amplicon located at chromosomal region 8q24.3. We examined a series of breast cancer samples harboring amplification of this region and determined that KCNK9 is the sole overexpressed gene within the amplification epicenter. KCNK9 encodes a potassium channel that is amplified from 3-fold to 10-fold in 10% of breast tumors and overexpressed from 5-fold to over 100-fold in 44% of breast tumors. Overexpression of KCNK9 in cell lines promotes tumor formation and confers resistance to both hypoxia and serum deprivation, suggesting that its amplification and overexpression plays a physiologically important role in human breast cancer.

PRC17, a novel oncogene encoding a Rab GTPase-activating protein, is amplified in prostate cancer.

We used cDNA-based genomic microarrays to examine DNA copy number changes in a panel of prostate tumors and found a previously undescribed amplicon on chromosome 17 containing a novel overexpressed gene that we termed prostate cancer gene 17 (PRC17). When overexpressed in 3T3 mouse fibroblast cells, PRC17 induced growth in low serum, loss of contact inhibition, and tumor formation in nude mice. The PRC17 gene product contains a GTPase-activating protein (GAP) catalytic core motif found in various Rab/Ypt GAPs, including RN-Tre. Similar to RN-Tre, we found that PRC17 protein interacts directly with Rab5 and stimulates its GTP hydrolysis. Point mutations that alter conserved amino acid residues within the PRC17 GAP domain abolished its transforming abilities, suggesting that GAP activity is essential for its oncogenic function. Whereas PRC17 is amplified in 15% of prostate cancers, it is highly overexpressed in approximately one-half of metastatic prostate tumors. The potent oncogenic activity of PRC17 is likely to influence the tumorigenic phenotype of these prostate cancers.

Oncogenic properties of PPM1D located within a breast cancer amplification epicenter at 17q23.

We found that PPM1D, encoding a serine/threonine protein phosphatase, lies within an epicenter of the region at 17q23 that is amplified in breast cancer. We show that overexpression of this gene confers two oncogenic phenotypes on cells in culture: attenuation of apoptosis induced by serum starvation and transformation of primary cells in cooperation with RAS.