Anhydrobiotic organisms have the remarkable ability to lose extensive amounts of body water and survive in an ametabolic, suspended animation state. Distributed to various taxa of life, these organisms have evolved strategies to efficiently protect their cell membranes and proteins against extreme water loss. At the molecular level, a variety of mutually non-exclusive mechanisms have been proposed to account particularly for preserving the integrity of the cell membranes in the desiccated state. Recently, it has been shown that the dauer larva of the nematode Caenorhabditis elegans is anhydrobiotic and accumulates high amounts of trehalose during preparation for harsh desiccation (preconditioning), thereby allowing for a reversible desiccation / rehydration cycle. Here, we have used this genetic model to study the biophysical manifestations of anhydrobiosis and show that, in addition to trehalose accumulation, the dauer larvae exhibit a systemic chemical response upon preconditioning by dramatically reducing their phosphatidylcholine (PC) content. The C. elegans strain daf-2 was chosen for these studies, because it forms a constitutive dauer state under appropriate growth conditions. Using complementary approaches such as chemical analysis, time-resolved FTIR-spectroscopy, Langmuir-Blodgett monolayers, and fluorescence spectroscopy, it is shown that this chemical adaptation of the phospholipid (PL) composition has key consequences for their interaction with trehalose. Infrared-spectroscopic experiments were designed and automated to particularly address structural changes during fast hydration transients. Importantly, the coupling of headgroup hydration to acyl chain order at low humidity was found to be altered on the environmentally relevant time scale of seconds. PLs from preconditioned larvae with reduced PC content exhibit a higher trehalose affinity, a stronger hydration-induced gain in acyl chain free volume, and a wider spread of structural relaxation rates during lyotropic transitions and sub- headgroup H-bond interactions as compared to PLs from non-preconditioned larvae. The effects are related to the intrinsically different hydration properties of PC and phosphatidylethanolamine (PE) headgroups, and lead to a larger hydration-dependent rearrangement of trehalose-mediated H-bond network in PLs from preconditioned larvae. This results in a lipid compressibility modulus of ∼0.5 mN/m and 1.2 mN/m for PLs derived from preconditioned and non-preconditioned larvae, respectively. The ensemble of these changes evidences a genetically controlled chemical tuning of the native lipid composition of a true anhydrobiote to functionally interact with a ubiquitous protective disaccharide. The biological relevance of this adaptation is the preservation of plasma membrane integrity by relieving mechanical strain from desiccated trehalose- containing cells during fast rehydration. Finally, the thermo-tropic lipid phase behavior was studied by temperature-dependent ATR-FTIR and fluorescence spectroscopy of LAURDAN-labeled PLs. The results show that the adaptation to drought, which is accomplished to a significant part by the reduction of the PC content, relies on reducing thermo-tropic and enhancing lyotropic phase transitions. The data are interpreted on a molecular level emphasizing the influence of trehalose on the lipid phase transition under biologically relevant conditions by a detailed analysis of the lipid C=O H-bond environment. The salient feature of the deduced model is a dynamic interaction of trehalose at the PL headgroup region. It is proposed here that the location of trehalose is changed from a more peripheral to a more sub-headgroup-associated position. This appears to be particularly pronounced in PLs from preconditioned worms. The sugar slides deeper into the inter-headgroup space during hydration and thereby supports a quick lateral expansion such that membranes can more readily adapt to the volume changes in the swelling biological material at reduced humidity. The data show that the nature of the headgroup is crucial for its interaction with trehalose and there is no general mechanism by which the sugar affects lipidic phase transitions. The intercalation into a phosphatidylethanolamine-rich membrane appears to be unique. In this case, neither the phase transition temperature nor its width is affected by the protective sugar, whereas strong effects on these parameters were observed with other model lipids. With respect to membrane preservation, desiccation tolerance may be largely dependent on reducing phosphatidylcholine and increasing the phsophatidylethanolamine content in order to optimize trehalose headgroup interactions. As a consequence, fast mechanical adaptation of cell membranes to hydration-induced strain can be realized.

Parkinson’s disease (PD) is a devastating neurodegenerative disease and the second most prevalent after Alzheimer’s disease. The most characteristic hallmark of Parkinson’s is the presence of Lewy Bodies, clumps of aggregated α-synuclein protein, in the Substantia Nigra. While much has been said and theorized about α-synuclein, mitochondrial dysregulation in neurons of Parkinson’s patients is an equally important consideration due to the role that the mitochondria plays in supplying neurons with their energy needs through ATP. C. elegans is a non-vertebrate animal often used to study aging and neurodegenerative disease due to its simple, well characterized genome. This literature review aims to outline the genetic and some environmental factors that cause mitochondrial dysregulation leading to the progressive neurodegeneration witnessed in Parkinson’s, as modeled in C. elegans. Through a select review of studies done on C. elegans homolog of genes associated with mitochondrial function, this review aims to elucidate the mechanism by which each mutation not only causes the deficits seen in PD on its own but also how it interacts with other genes to worsen or alleviate symptoms. Ultimately, understanding these pathways and mechanism will be crucial to discovering and creating new therapeutic treatments and targets.

In the nervous system, billions of neurons undergo a multistep process to establish functional circuits. This entails accurate extension of dendritic and axonal processes and coordinated efforts of pre- and postsynaptic neurons to form synaptic connections. Although many axon guidance molecules and synaptic organizers have been identified, the molecular redundancy and the vast number of synapses in the brain has complicated attempts to define their precise roles. In order to understand the molecular mechanisms that encompass these processes, my studies utilize the genetic strengths and cellular precision available in Caenorhabditis elegans for in vivo investigations of nervous system development. In this work, I unravel cell-specific requirements for the transmembrane receptor integrin in regulating developmental axon guidance of GABAergic motor neurons. Furthermore, I address important questions about mechanisms of synapse formation and maintenance using a novel dendritic spine model in C. elegans. Using high resolution microscopy, I find that the formation of immature presynaptic vesicles and postsynaptic receptors are established prior to the outgrowth of dendritic spines at nascent synapses. During this early period of synapse formation, the kinesin-3 family protein UNC-104/KIF1A transports a transsynaptic adhesion molecule neurexin/NRX-1 to developing active zones, in order to maintain postsynaptic receptors and dendritic spines in the mature circuit. In the absence of nrx-1, spines initially form normally but collapse following their extension. These findings demonstrate that presynaptic NRX-1 is required to maintain postsynaptic structures. Together my work provides new insights into molecular mechanisms that define spatiotemporal characteristics of nervous system development and the maintenance of connectivity.

The inability to repair axonal damage is a feature of neurological impairment after injury and in neurodegenerative diseases. Axonal repair after injury depends in part on intrinsic factors. Several genes cell-autonomously regulate both axon regeneration and degeneration in response to injury. Recently, Sarm1 has emerged as a key regulator of neurodegeneration. Whether Sarm1 plays a role in axon regeneration is unknown. In this thesis, I identified a role for the C. elegans homolog of Sarm1, tir-1, as a negative regulator of axon regeneration. Investigating the genes which regulate axon regeneration and degeneration has been hindered by technical difficulties in visualizing and manipulating both of these processes in vivo simultaneously. To circumvent this challenge, I developed a new model of axon injury, where both axon regeneration and degeneration can be monitored in vivo with single neuron resolution in C. elegans. I found that the C. elegans homolog of Sarm1, tir-1, strongly inhibits axon regeneration in response to injury. I found that TIR-1 functions cell-intrinsically and that its subcellular localization is dynamically regulated in response to injury. To regulate both axon regeneration and degeneration after injury, I found that TIR-1 function is determined by interaction with two distinct genetic pathways. Together, this work reveals a novel role for tir-1/Sarm1 in axon regeneration, increases our understanding of the injury response, provides new avenues of investigation for studies of TIR-1/SARM1, and inspires candidate approaches to repair the injured nervous system.

Host metabolism is profoundly altered during bacterial infection, both as a consequence of immune activation and secondary to virulence strategies of invading pathogens. As a result, the metabolic pathways that regulate nutrient acquisition, energy storage, and resource allocation in host cells must adapt to pathogen stress in order to meet the physiological demands of the host during infection. In this work, we uncover that the synthesis of the monounsaturated fatty acid (MUFA) oleate is necessary for the pathogen-mediated induction of immune defense genes. Accordingly, C. elegans deficient in oleate production are hypersusceptible to infection with diverse human pathogens, which can be rescued by the addition of exogenous oleate. However, oleate is not sufficient to drive protective immune activation. Oleate is also important for proper lipid storage and abundance. We found that exposure to pathogenic bacteria drives rapid somatic depletion of lipid stores in C. elegans. Activating the p38/MAPK immune signaling pathway in the absence of pathogens was also sufficient to drive loss of somatic fat. In addition, we found that transcriptional suppression of MUFA synthesis occurs during P. aeruginosa infection, in a manner dependent on pathogen virulence. Finally, we showed that the host compensates for the pathogen-induced depletion of fatty acids by promoting the redistribution of oleate from non-intestinal tissues to support immune function in the intestine. Together, these data add to the known health-promoting effects of MUFAs, and suggest an ancient link between nutrient stores, metabolism, and host responses to bacterial infection.

Neuropeptide signaling play critical roles in maintaining distinct behavioral states and orchestrating transitions between them. However, elucidating the mechanisms underlying neuropeptide modulation of neural circuits in vivo remains a major challenge. The nematode Caenorhabditis elegans serves as an excellent model organism to study neuropeptide signaling mechanisms encoded in relatively simple neural circuits. We have used the C. elegans egg-laying circuit as a model to understand how neuropeptide signaling modifies circuit activity to generate opposing behavioral outcomes. C. elegans egg-laying behavior is composed of alternating cycles of two states – short bursts of egg deposition (active phases) and prolonged periods of quiescence (inactive phases). We have identified two neuropeptides (NLP-7 and FLP-11) that are locally released from a group of neurosecretory cells (uv1) and coordinate the temporal organization of egglaying by prolonging the duration of inactive phases. These neuropeptides regulate activity within the core circuit by inhibiting serotonergic transmission between its individual components (HSN motorneurons and Vm2 vulval muscles). This inhibition is achieved at least in part, by reducing synaptic vesicle abundance in the HSN synaptic regions. To identify potential downstream signaling components that mediate the actions of these neuropeptides, we have performed a forward genetic screen and have identified a strong candidate. In addition, we are trying to identify the receptor(s) of these neuropeptides by using a candidate gene approach. Together, we demonstrate that local neuropeptide signaling maintains the periodicity of distinct behavioral states by regulating serotonergic transmission in the core neural circuit.

How do organisms attain the capacity to regenerate a structure, entire body, or not to regenerate? These are fundamental questions in biology for understanding how replicative systems are evolved to renew, age, and/or die. One outstanding question in regenerative biology that attracts attention is how and why the human central nervous system fails to regenerate after injury. Nervous system injuries are characterized by axonal damage and loss of synaptic function that contribute to debilitating neuronal dysfunctions. Although the molecular underpinnings of axon regeneration are well characterized, very little is known about how and what molecular pathways modulate reformation of synapses within regenerating axons to restore function. Thus, understanding the fundamental molecular and genetic mechanisms of functional axon regeneration (FAR), restoration of both axon and synapse, for the functional recovery of the nervous system remains elusive. In Chapter I, I outline the biology of regeneration and provide evolutionary perspectives of this phenomenon. Then, I provide clinical perspectives of central nervous system regeneration and therapeutic innovations. I next introduce the regulators of axon regeneration and how C. elegans as a genetic system allows detailed characterization of axon regeneration. In Chapter II, using C. elegans as a platform, I show how axon regeneration and synaptic reformation are controlled by distinct genetic pathways. I show how Poly-ADP ribose polymerase (PARP) pathway modulates functional restoration by regulating divergent genetic pathways leading to axon regeneration and synapse restoration. Finally, in Chapter III, I summarize the model of axon regeneration, evolutionary perspectives, and epistemic limitations of C. elegans axon regeneration.

The gene lin-42 is an ortholog of the mammalian period gene, a component of the circadian pathway that converts environmental stimuli into behavioral and physiological outputs over 24 hours. Mammalian period also regulates adult stem cell differentiation, although this function is poorly understood. The structure, function and expression of lin-42 are all similar to period. Therefore, we are studying lin-42 regulation and function during C. elegans larval development as a model for understanding period control of mammalian stem/progenitor cell development. Previous work has shown that ZTF-16 is a regulator of lin-42 transcription. The lin-42 locus encodes three isoforms, and we have characterized lin-42 isoform specific regulation by ZTF-16 through phenotypic assays and analysis of transcriptional reporter strains. Our data show that ZTF-16 regulates the cyclic expression of lin-42A and lin-42B during larval development. However, ztf-16 is not expressed during the adult stage and does not regulate lin-42C, which is expressed only in adults and may be responsible for the circadian functions of lin-42. We also show that ztf-16 reduction-of-function mutations phenocopy loss-of- function phenotypes of the lin-42A/B isoforms. Finally, we have found that deletion of a putative ZTF-16 transcription factor binding site within the lin-42BC promoter abolishes tissue-specific expression patterns. Together, these data indicate that ZTF-16 is required to regulate the expression of lin-42A/B during C. elegans development, and may do this by direct binding to the lin-42BC promoter. Our  findings pave the way for testing the possible regulation of period expression by HIL-family transcription factors in mammalian tissues. / Master of Science

京都大学 / 0048 / 新制・課程博士 / 博士(理学) / 甲第18110号 / 理博第3988号 / 新制||理||1575(附属図書館) / 30968 / 京都大学大学院理学研究科生物科学専攻 / (主査)准教授 秋山 秋梅, 教授 沼田 英治, 教授 疋田 努 / 学位規則第4条第1項該当 / Doctor of Science / Kyoto University / DGAM

京都大学 / 0048 / 新制・課程博士 / 博士(生命科学) / 甲第20523号 / 生博第365号 / 新制||生||48(附属図書館) / 京都大学大学院生命科学研究科統合生命科学専攻 / (主査)教授 西田 栄介, 教授 松本 智裕, 教授 豊島 文子 / 学位規則第4条第1項該当 / Doctor of Philosophy in Life Sciences / Kyoto University / DFAM