Genetics最新文献

Neurodegenerative diseases such as Alzheimer's and Parkinson's currently affect ∼25 million people worldwide. The global incidence of traumatic brain injury (TBI) is estimated at ∼70 million/year. Both neurodegenerative diseases and TBI remain without effective treatments. We are utilizing adult Drosophila melanogaster to investigate the mechanisms of brain regeneration with the long-term goal of identifying targets for neural regenerative therapies. We specifically focused on neurogenesis, i.e., the generation of new cells, as opposed to the regrowth of specific subcellular structures such as axons. Like mammals, Drosophila have few proliferating cells in the adult brain. Nonetheless, within 24 hours of a penetrating traumatic brain injury (PTBI) to the central brain, there is a significant increase in the number of proliferating cells. We subsequently detect both new glia and new neurons and the formation of new axon tracts that target appropriate brain regions. Glial cells divide rapidly upon injury to give rise to new glial cells. Other cells near the injury site upregulate neural progenitor genes including asense and deadpan and later give rise to the new neurons. Locomotor abnormalities observed after PTBI are reversed within 2 weeks of injury, supporting the idea that there is functional recovery. Together, these data indicate that adult Drosophila brains are capable of neuronal repair. We anticipate that this paradigm will facilitate the dissection of the mechanisms of neural regeneration and that these processes will be relevant to human brain repair.

In our group, we aim to understand metabolism in the nematode Caenorhabditis elegans and its relationships with gene expression, physiology, and the response to therapeutic drugs. Visualization of the metabolic pathways that comprise the metabolic network is extremely useful for interpreting a wide variety of experiments. Detailed annotated metabolic pathway maps for C. elegans are mostly limited to pan-organismal maps, many with incomplete or inaccurate pathway and enzyme annotations. Here, we present WormPaths, which is composed of two parts: (1) the careful manual annotation of metabolic genes into pathways, categories, and levels, and (2) 62 pathway maps that include metabolites, metabolite structures, genes, reactions, and pathway connections between maps. These maps are available on the WormFlux website. We show that WormPaths provides easy-to-navigate maps and that the different levels in WormPaths can be used for metabolic pathway enrichment analysis of transcriptomic data. In the future, we envision further developing these maps to be more interactive, analogous to road maps that are available on mobile devices.

Communicating editor: B. Grant The composition and biophysical properties of cellular membranes must be tightly regulated to maintain the proper functions of myriad processes within cells. To better understand the importance of membrane homeostasis, we assembled a panel of five Caenorhabditis elegans strains that show a wide span of membrane composition and properties, ranging from excessively rich in saturated fatty acids (SFAs) and rigid to excessively rich in polyunsaturated fatty acids (PUFAs) and fluid. The genotypes of the five strain are, from most rigid to most fluid: paqr-1(tm3262); paqr-2(tm3410), paqr-2(tm3410), N2 (wild-type), mdt-15(et14); nhr-49(et8), and mdt-15(et14); nhr-49(et8); acs-13(et54). We confirmed the excess SFA/rigidity-to-excess PUFA/fluidity gradient using the methods of fluorescence recovery after photobleaching (FRAP) and lipidomics analysis. The five strains were then studied for a variety of cellular and physiological traits and found to exhibit defects in: permeability, lipid peroxidation, growth at different temperatures, tolerance to SFA-rich diets, lifespan, brood size, vitellogenin trafficking, oogenesis, and autophagy during starvation. The excessively rigid strains often exhibited defects in opposite directions compared to the excessively fluid strains. We conclude that deviation from wild-type membrane homeostasis is pleiotropically deleterious for numerous cellular/physiological traits. The strains introduced here should prove useful to further study the cellular and physiological consequences of impaired membrane homeostasis.

In Caenorhabditis elegans, the cha-1 gene encodes choline acetyltransferase (ChAT), the enzyme that synthesizes the neurotransmitter acetylcholine. We have analyzed a large number of cha-1 hypomorphic mutants, most of which are missense alleles. Some homozygous cha-1 mutants have approximately normal ChAT immunoreactivity; many other alleles lead to consistent reductions in synaptic immunostaining, although the residual protein appears to be stable. Regardless of protein levels, neuromuscular function of almost all mutants is temperature-sensitive, i.e., neuromuscular function is worse at 25° than at 14°. We show that the temperature effects are not related to acetylcholine release, but specifically to alterations in acetylcholine synthesis. This is not a temperature-dependent developmental phenotype, because animals raised at 20° to young adulthood and then shifted for 2 h to either 14° or 25° had swimming and pharyngeal pumping rates similar to animals grown and assayed at either 14° or 25°, respectively. We also show that the temperature-sensitive phenotypes are not limited to missense alleles; rather, they are a property of most or all severe cha-1 hypomorphs. We suggest that our data are consistent with a model of ChAT protein physically, but not covalently, associated with synaptic vesicles; and there is a temperature-dependent equilibrium between vesicle-associated and cytoplasmic (i.e., soluble) ChAT. Presumably, in severe cha-1 hypomorphs, increasing the temperature would promote dissociation of some of the mutant ChAT protein from synaptic vesicles, thus removing the site of acetylcholine synthesis (ChAT) from the site of vesicular acetylcholine transport. This, in turn, would decrease the rate and extent of vesicle-filling, thus increasing the severity of the behavioral deficits.

The axis of the vertebrate neural tube is patterned, in part, by a ventral to dorsal gradient of Shh signaling. In the ventral spinal cord, Shh induces concentration-dependent expression of transcription factors, subdividing neural progenitors into distinct domains that subsequently produce distinct neuronal and glial subtypes. In particular, progenitors of the pMN domain express the bHLH transcription factor Olig2 and produce motor neurons followed by oligodendrocytes, the myelinating glial cell type of the central nervous system. In addition to its role in patterning ventral progenitors, Shh signaling must be maintained through development to specify pMN progenitors for oligodendrocyte fate. Using a forward genetic screen in zebrafish for mutations that disrupt the development of oligodendrocytes, we identified a new mutant allele of boc, which encodes a type I transmembrane protein that functions as a coreceptor for Shh. Embryos homozygous for the bocco25 allele, which creates a missense mutation in a Fibronectin type III domain that binds Shh, have normally patterned spinal cords but fail to maintain pMN progenitors, resulting in a deficit of oligodendrocytes. Using a sensitive fluorescent detection method for in situ RNA hybridization, we found that spinal cord cells express boc in a graded fashion that is inverse to the gradient of Shh signaling activity and that boc function is necessary to maintain pMN progenitors by shaping the Shh signaling gradient.

Parkinson's disease (PD) is primarily characterized by the loss of dopaminergic (DA) neurons in the brain. However, little is known about why DA neurons are selectively vulnerable to PD. To identify genes that are associated with DA neuron loss, we screened through 201 wild-caught populations of Drosophila melanogaster as part of the Drosophila Genetic Reference Panel. Here, we identify the top-associated genes containing single-nucleotide polymorphisms that render DA neurons vulnerable. These genes were further analyzed by using mutant analysis and tissue-specific knockdown for functional validation. We found that this loss of DA neurons caused progressive locomotor dysfunction in mutants and gene knockdown analysis. The identification of genes associated with the progressive loss of DA neurons should help to uncover factors that render these neurons vulnerable in PD, and possibly develop strategies to make these neurons more resilient.

A missense mutant, unc-17(e245), which affects the Caenorhabditis elegans vesicular acetylcholine transporter UNC-17, has a severe uncoordinated phenotype, allowing efficient selection of dominant suppressors that revert this phenotype to wild-type. Such selections permitted isolation of numerous suppressors after EMS (ethyl methanesulfonate) mutagenesis, leading to demonstration of delays in mutation fixation after initial EMS treatment, as has been shown in T4 bacteriophage but not previously in eukaryotes. Three strong dominant extragenic suppressor loci have been defined, all of which act specifically on allele e245, which causes a G347R mutation in UNC-17. Two of the suppressors (sup-1 and sup-8/snb-1) have previously been shown to encode synaptic proteins able to interact directly with UNC-17. We found that the remaining suppressor, sup-2, corresponds to a mutation in erd-2.1, which encodes an endoplasmic reticulum retention protein; sup-2 causes a V186E missense mutation in transmembrane helix 7 of ERD-2.1. The same missense change introduced into the redundant paralogous gene erd-2.2 also suppressed unc-17(e245). Suppression presumably occurred by compensatory charge interactions between transmembrane helices of UNC-17 and ERD-2.1 or ERD-2.2, as previously proposed in work on suppression by SUP-1(G84E) or SUP-8(I97D)/synaptobrevin. erd-2.1(V186E) homozygotes were fully viable, but erd-2.1(V186E); erd-2.2(RNAi) exhibited synthetic lethality [like erd-2.1(RNAi); erd-2.2(RNAi)], indicating that the missense change in ERD-2.1 impairs its normal function in the secretory pathway but may allow it to adopt a novel moonlighting function as an unc-17 suppressor.

Egg laying in the nematode worm Caenorhabditis elegans is a two-state behavior modulated by internal and external sensory input. We have previously shown that homeostatic feedback of embryo accumulation in the uterus regulates bursting activity of the serotonergic HSN command neurons that sustains the egg-laying active state. How sensory feedback of egg release signals to terminate the egg-laying active state is less understood. We find that Gαo, a conserved Pertussis Toxin-sensitive G protein, signals within HSN to inhibit egg-laying circuit activity and prevent entry into the active state. Gαo signaling hyperpolarizes HSN, reducing HSN Ca2+ activity and input onto the postsynaptic vulval muscles. Loss of inhibitory Gαo signaling uncouples presynaptic HSN activity from a postsynaptic, stretch-dependent homeostat, causing precocious entry into the egg-laying active state when only a few eggs are present in the uterus. Feedback of vulval opening and egg release activates the uv1 neuroendocrine cells which release NLP-7 neuropeptides which signal to inhibit egg laying through Gαo-independent mechanisms in the HSNs and Gαo-dependent mechanisms in cells other than the HSNs. Thus, neuropeptide and inhibitory Gαo signaling maintain a bi-stable state of electrical excitability that dynamically controls circuit activity in response to both external and internal sensory input to drive a two-state behavior output.