Martina Bremer

A conserved juxtacrine signal regulates synaptic partner recognition in Caenorhabditis elegans

BackgroundAn essential stage of neural development involves the assembly of neural circuits via formation of inter-neuronal connections. Early steps in neural circuit formation, including cell migration, axon guidance, and the localization of synaptic components, are well described. However, upon reaching their target region, most neurites still contact many potential partners. In order to assemble functional circuits, it is critical that within this group of cells, neurons identify and form connections only with their appropriate partners, a process we call synaptic partner recognition (SPR). To understand how SPR is mediated, we previously developed a genetically encoded fluorescent trans-synaptic marker called NLG-1 GRASP, which labels synaptic contacts between individual neurons of interest in dense cellular environments in the genetic model organism Caenorhabditis elegans.ResultsHere, we describe the first use of NLG-1 GRASP technology, to identify SPR genes that function in this critical process. The NLG-1 GRASP system allows us to assess synaptogenesis between PHB sensory neurons and AVA interneurons instantly in live animals, making genetic analysis feasible. Additionally, we employ a behavioral assay to specifically test PHB sensory circuit function. Utilizing this approach, we reveal a new role for the secreted UNC-6/Netrin ligand and its transmembrane receptor UNC-40/Deleted in colorectal cancer (DCC) in SPR. Synapses between PHB and AVA are severely reduced in unc-6 and unc-40 animals despite normal axon guidance and subcellular localization of synaptic components. Additionally, behavioral defects indicate a complete disruption of PHB circuit function in unc-40 mutants. Our data indicate that UNC-40 and UNC-6 function in PHB and AVA, respectively, to specify SPR. Strikingly, overexpression of UNC-6 in postsynaptic neurons is sufficient to promote increased PHB-AVA synaptogenesis and to potentiate the behavioral response beyond wild-type levels. Furthermore, an artificially membrane-tethered UNC-6 expressed in the postsynaptic neurons promotes SPR, consistent with a short-range signal between adjacent synaptic partners.ConclusionsThese results indicate that the conserved UNC-6/Netrin-UNC-40/DCC ligand-receptor pair has a previously unknown function, acting in a juxtacrine manner to specify recognition of individual postsynaptic neurons. Furthermore, they illustrate the potential of this new approach, combining NLG-1 GRASP and behavioral analysis, in gene discovery and characterization.

A Study Assessing the Potential of Negative Effects in Interdisciplinary Math–Biology Instruction

There is increasing enthusiasm for teaching approaches that combine mathematics and biology. The call for integrating more quantitative work in biology education has led to new teaching tools that improve quantitative skills. Little is known, however, about whether increasing interdisciplinary work can lead to adverse effects, such as the development of broader but shallower skills or the possibility that math anxiety causes some students to disengage in the classroom, or, paradoxically, to focus so much on the mathematics that they lose sight of its application for the biological concepts in the center of the unit at hand. We have developed and assessed an integrative learning module and found disciplinary learning gains to be equally strong in first-year students who actively engaged in embedded quantitative calculations as in those students who were merely presented with quantitative data in the context of interpreting biological and biostatistical results. When presented to advanced biology students, our quantitative learning tool increased test performance significantly. We conclude from our study that the addition of mathematical calculations to the first year and advanced biology curricula did not hinder overall student learning, and may increase disciplinary learning and data interpretation skills in advanced students.

Plasmablast frequency and trafficking receptor expression are altered in pediatric ulcerative colitis

Background: The incidence of pediatric ulcerative colitis (UC), a chronic autoinflammatory disease of the colon, is on the rise. Although an increased infiltration of B cells from the peripheral blood into the colon occurs in UC, B‐cell trafficking is understudied. We hypothesized that the frequency of circulating plasmablasts (PBs) and their trafficking receptor (TR) expression may be indicative of the location and degree of pathology in pediatric UC. Methods: We conducted multicolor flow cytometry analyses of circulating IgA+/− PBs and IgA+ memory B cells (MBCs) in pediatric UC patients with remission, mild, moderate, and severe state of disease (n = 12), and healthy pediatric (n = 2) and adult donors (n = 11). Results: Compared to healthy donors the average frequency of PBs among total peripheral blood lymphocytes is increased 30‐fold during severe UC activity, and positively correlates with Pediatric Ulcerative Colitis Activity Index score, C‐reactive protein level, and erythrocyte sedimentation rate. A greater percent of PBs in severe patients express the gut‐homing receptors &agr;4&bgr;7 and CCR10, and the inflammatory homing molecule P‐selectin ligand (P‐sel lig). The percent of IgA+ MBCs expressing &agr;4&bgr;7, however, is reduced. Furthermore, expression of the small intestine TR CCR9 is decreased on &agr;4&bgr;7high PBs, and on &agr;4&bgr;7high/CCR10high PBs and MBCs in these patients, consistent with preferential cell targeting to the colon. Conclusions: Peripheral blood PBs with a colon‐homing phenotype (&agr;4&bgr;7/CCR10/P‐sel lig) are elevated in children with severe UC. Screening this B‐cell subset may provide a complementary approach in monitoring disease activity or therapeutic efficacy in pediatric UC. (Inflamm Bowel Dis 2012;)

Differential expression of alpha 4 integrins on effector memory T helper cells during Bordetella infections. Delayed responses in Bordetella pertussis.

Bordetella pertussis (B. pertussis) is the causative agent of whooping cough, a respiratory disease that is reemerging worldwide. Mechanisms of selective lymphocyte trafficking to the airways are likely to be critical in the immune response to this pathogen. We compared murine infection by B. pertussis, B. parapertussis, and a pertussis toxin-deleted B. pertussis mutant (BpΔPTX) to test the hypothesis that effector memory T-helper cells (emTh) display an altered pattern of trafficking receptor expression in B. pertussis infection due to a defect in imprinting. Increased cell recruitment to the lungs at 5 days post infection (p.i.) with B. parapertussis, and to a lesser extent with BpΔPTX, coincided with an increased frequency of circulating emTh cells expressing the mucosal-associated trafficking receptors α4β7 and α4β1 while a reduced population of these cells was observed in B. pertussis infection. These cells were highly evident in the blood and lungs in B. pertussis infection only at 25 days p.i. when B. parapertussis and BpΔPTX infections were resolved. Although at 5 days p.i., an equally high percentage of lung dendritic cells (DCs) from all infections expressed maturation markers, this expression persisted only in B. pertussis infection at 25 days p.i. Furthermore, at 5 days p.i with B. pertussis, lung DCs migration to draining lymph nodes may be compromised as evidenced by decreased frequency of CCR7+ DCs, inhibited CCR7-mediated in vitro migration, and fewer DCs in lung draining lymph nodes. Lastly, a reduced frequency of allogeneic CD4+ cells expressing α4β1 was detected following co-culture with lung DCs from B. pertussis-infected mice, suggesting a defect in DC imprinting in comparison to the other infection groups. The findings in this study suggest that B. pertussis may interfere with imprinting of lung-associated trafficking receptors on T lymphocytes leading to extended survival in the host and a prolonged course of disease.

The KM-Algorithm Identifies Regulated Genes in Time Series Expression Data.

We present a statistical method to rank observed genes in gene expression time series experiments according to their degree of regulation in a biological process. The ranking may be used to focus on specific genes or to select meaningful subsets of genes from which gene regulatory networks can be built. Our approach is based on a state space model that incorporates hidden regulators of gene expression. Kalman (K) smoothing and maximum (M) likelihood estimation techniques are used to derive optimal estimates of the model parameters upon which a proposed regulation criterion is based. The statistical power of the proposed algorithm is investigated, and a real data set is analyzed for the purpose of identifying regulated genes in time dependent gene expression data. This statistical approach supports the concept that meaningful biological conclusions can be drawn from gene expression time series experiments by focusing on strong regulation rather than large expression values.

C. elegans avoids toxin-producing Streptomyces using a seven transmembrane domain chemosensory receptor

Predators and prey co-evolve, each maximizing their own fitness, but the effects of predator–prey interactions on cellular and molecular machinery are poorly understood. Here, we study this process using the predator Caenorhabditis elegans and the bacterial prey Streptomyces, which have evolved a powerful defense: the production of nematicides. We demonstrate that upon exposure to Streptomyces at their head or tail, nematodes display an escape response that is mediated by bacterially produced cues. Avoidance requires a predicted G-protein-coupled receptor, SRB-6, which is expressed in five types of amphid and phasmid chemosensory neurons. We establish that species of Streptomyces secrete dodecanoic acid, which is sensed by SRB-6. This behavioral adaptation represents an important strategy for the nematode, which utilizes specialized sensory organs and a chemoreceptor that is tuned to recognize the bacteria. These findings provide a window into the molecules and organs used in the coevolutionary arms race between predator and potential prey.

Kinesin-1 Acts with Netrin and DCC to Maintain Sensory Neuron Position in Caenorhabditis elegans

The organization of neurons and the maintenance of that arrangement are critical to brain function. Failure of these processes in humans can lead to severe birth defects, mental retardation, and epilepsy. Several kinesins have been shown to play important roles in cell migration in vertebrate systems, but few upstream and downstream pathway members have been identified. Here, we utilize the genetic model organism Caenorhabditis elegans to elucidate the pathway by which the C. elegans Kinesin-1 Heavy Chain (KHC)/KIF5 ortholog UNC-116 functions to maintain neuronal cell body position in the PHB sensory neurons. We find that UNC-116/KHC acts in part with the cell and axon migration molecules UNC-6/Netrin and UNC-40/DCC in this process, but in parallel to SAX-3/Robo. We have also identified several potential adaptor, cargo, and regulatory proteins that may provide insight into the mechanism of UNC-116/KHC’s function in this process. These include the cargo receptor UNC-33/CRMP2, the cargo adaptor protein UNC-76/FEZ and its regulator UNC-51/ULK, the cargo molecule UNC-69/SCOCO, and the actin regulators UNC-44/Ankyrin and UNC-34/Enabled. These genes also act in cell migration and axon outgrowth; however, many proteins that function in these processes do not affect PHB position. Our findings suggest an active posterior cell migration mediated by UNC-116/KHC occurs throughout development to maintain proper PHB cell body position and define a new pathway that mediates maintenance of neuronal cell body position.

Lipopolysaccharide‐induced inflammation does not alter muscle spindle afferent mechanosensation or sensory integration in the spinal cord of adult mice

Inflammation is known to alter nervous system function, but its effect on muscle spindle afferent mechanosensation and sensory integration in the spinal cord has not been well studied. We tested the hypothesis that systemic inflammation induced by an intraperitoneal injection of the endotoxin lipopolysaccharide (LPS; 7.5 × 105 endotoxin units/kg 18 h before experiment) would alter muscle spindle afferent mechanosensation and spinal cord excitability to Group Ia input in male and female adult C57Bl/6 mice. LPS injection caused a systemic immune response, evidenced by decreased white blood cell, monocyte, and lymphocyte concentrations in the blood, increased blood granulocyte concentration, and body weight loss. The immune response in both sexes was qualitatively similar. We used an in vitro muscle‐nerve preparation to assay muscle spindle afferent response to stretch and vibration. LPS injection did not significantly change the response to stretch or vibration, with the exception of small decreases in the ability to entrain to high‐frequency vibration in male mice. Similarly, LPS injection did not alter spinal cord excitability to Group Ia muscle spindle afferent input as measured by the Hoffmans reflex test in anesthetized mice (100 mg/kg ketamine, 10 mg/kg xylazine). Specifically, there were no changes in M or H wave latencies nor in the percentage of motor neurons excited by electrical afferent stimulation (Hmax/Mmax). Overall, we found no major alterations in muscle proprioceptor function or sensory integration following exposure to LPS at a dose and time course that causes changes in nociceptor function and central processing.

Diet induced obesity alters muscle spindle afferent function in adult mice

Populations with obesity are more likely to fall and exhibit balance instability. The reason for this is likely multifactorial, but there is some evidence that sensory function is impaired during obesity. We tested the hypothesis that muscle proprioceptor function is compromised in a mouse model of diet induced obesity. An in vitro muscle-nerve preparation was used to record muscle spindle afferent responses to physiological stretch and sinusoidal vibration. We compared the responses of C57/Bl6 male and female mice on a control diet (10% kcal fat) with those eating a high fat diet (HFD; 60% kcal fat) for 10 weeks (final age 14–15 weeks old). Following HFD feeding, adult mice of both sexes exhibited decreased muscle spindle afferent responses to muscle movement. Muscle spindle afferent firing rates during the plateau phase of stretch were significantly lower in both male and female HFD animals as were two measures of dynamic sensitivity (dynamic peak and dynamic index). Muscle spindle afferents in male mice on a HFD were also significantly less likely to entrain to vibration. Due to the importance of muscle spindle afferents to proprioception and motor control, decreased muscle spindle afferent responsiveness may contribute to balance instability during obesity.

The receptor protein tyrosine phosphatase CLR-1 is required for synaptic partner recognition

During neural circuit formation, most axons are guided to complex environments, coming into contact with multiple potential synaptic partners. However, it is critical that they recognize specific neurons with which to form synapses. Here, we utilize the split GFP-based marker Neuroligin-1 GFP Reconstitution Across Synaptic Partners (NLG-1 GRASP) to visualize specific synapses in live animals, and a circuit-specific behavioral assay to probe circuit function. We demonstrate that the receptor protein tyrosine phosphatase (RPTP) clr-1 is necessary for synaptic partner recognition (SPR) between the PHB sensory neurons and the AVA interneurons in C. elegans. Mutations in clr-1/RPTP result in reduced NLG-1 GRASP fluorescence and impaired behavioral output of the PHB circuit. Temperature-shift experiments demonstrate that clr-1/RPTP acts early in development, consistent with a role in SPR. Expression and cell-specific rescue experiments indicate that clr-1/RPTP functions in postsynaptic AVA neurons, and overexpression of clr-1/RPTP in AVA neurons is sufficient to direct additional PHB-AVA synaptogenesis. Genetic analysis reveals that clr-1/RPTP acts in the same pathway as the unc-6/Netrin ligand and the unc-40/DCC receptor, which act in AVA and PHB neurons, respectively. This study defines a new mechanism by which SPR is governed, and demonstrates that these three conserved families of molecules, with roles in neurological disorders and cancer, can act together to regulate communication between cells.