Homeodomain-interacting protein kinase maintains neuronal homeostasis during normal Caenorhabditis elegans aging and systemically regulates longevity from serotonergic and GABAergic neurons.

Aging and the age-associated decline of the proteome is determined in part through neuronal control of evolutionarily conserved transcriptional effectors, which safeguard homeostasis under fluctuating metabolic and stress conditions by regulating an expansive proteostatic network. We have discovered the Caenorhabditis elegans homeodomain-interacting protein kinase (HPK-1) acts as a key transcriptional effector to preserve neuronal integrity, function, and proteostasis during aging. Loss of hpk-1 results in drastic dysregulation in expression of neuronal genes, including genes associated with neuronal aging. During normal aging hpk-1 expression increases throughout the nervous system more broadly than any other kinase. Within the aging nervous system, hpk-1 induction overlaps with key longevity transcription factors, which suggests that hpk-1 expression mitigates natural age-associated physiological decline. Consistently, pan-neuronal overexpression of hpk-1 extends longevity, preserves proteostasis both within and outside of the nervous system, and improves stress resistance. Neuronal HPK-1 improves proteostasis through kinase activity. HPK-1 functions cell non-autonomously within serotonergic and γ-aminobutyric acid (GABA)ergic neurons to improve proteostasis in distal tissues by specifically regulating distinct components of the proteostatic network. Increased serotonergic HPK-1 enhances the heat shock response and survival to acute stress. In contrast, GABAergic HPK-1 induces basal autophagy and extends longevity, which requires mxl-2 (MLX), hlh-30 (TFEB), and daf-16 (FOXO). Our work establishes hpk-1 as a key neuronal transcriptional regulator critical for preservation of neuronal function during aging. Further, these data provide novel insight as to how the nervous system partitions acute and chronic adaptive response pathways to delay aging by maintaining organismal homeostasis.

Proteins are essential for nearly every cellular process to sustain a healthy organism. A complex network of pathways and signalling molecules regulates the proteins so that they work correctly in a process known as proteostasis. As the body ages, this network can become damaged, which leads to the production of faulty proteins. Many proteins end up being misfolded ­ in other words, they are misshapen on the molecular level, which can be toxic for the cell. A build-up of such misfolded proteins is implicated in several neurological conditions, including Alzheimer's, Parkinson's and Huntington's disease. Cells have various ways to detect and respond to internal stressors, such as tissue or organ damage. For example, specific proteins in the nervous system can raise a 'central' alert when damage is detected, which then primes and coordinates the body's systems to respond in the peripheral cells and tissues. But exactly how this happens is still unclear. To find out more about the central coordination of stress responses, Lazaro-Pena et al. studied one such sensor protein, called HPK-1, in the roundworm C. elegans. They first overexpressed the protein in various tissues. This revealed that only when HPK-1 was overactive in nerve tissue, it protected proteins and prolonged the lifespan of the worms. An increased amount of HPK-1 improved the health span of the worms and older worms also moved better. However, genetically manipulated worms lacking HPK-1 in their nerve cells showed a faster decline in nervous system health as they aged, which could be reversed once HPK-1 was activated again. Lazaro-Pena et al. then measured the amount of HPK-1 in worms at different stages of their life. This showed that as the worms aged, the amount of HPK-1 increased in the nerve cells. The nerve cells in which HPK-1 levels increased overlapped with an increased expression of proteins associated with longevity. Moreover, when HPK-1 was overexpressed, it stimulated the release of other cell signals, which then triggered protective responses to prevent the misfolding and aggregation of proteins and to help degrade damaged proteins. This study shows for the first time that HPK-1 appears to play a protective role during normal ageing and that it may act as a key switch to stimulate other protective mechanisms. These findings may give rise to new insights into how the nervous system can coordinate many different stress responses, and ultimately delay ageing throughout the whole body.

Homeodomain-interacting protein kinase maintains neuronal homeostasis during normal Caenorhabditis elegans aging and systemically regulates longevity from serotonergic and GABAergic neurons.

Aging and the age-associated decline of the proteome is determined in part through neuronal control of evolutionarily conserved transcriptional effectors, which safeguard homeostasis under fluctuating metabolic and stress conditions by regulating an expansive proteostatic network. We have discovered the Caenorhabditis elegans h omeodomain-interacting p rotein k inase (HPK-1) acts as a key transcriptional effector to preserve neuronal integrity, function, and proteostasis during aging. Loss of hpk-1 results in drastic dysregulation in expression of neuronal genes, including genes associated with neuronal aging. During normal aging hpk-1 expression increases throughout the nervous system more broadly than any other kinase. Within the aging nervous system, hpk-1 induction overlaps with key longevity transcription factors, which suggests hpk-1 expression mitigates natural age-associated physiological decline. Consistently, pan-neuronal overexpression of hpk-1 extends longevity, preserves proteostasis both within and outside of the nervous system, and improves stress resistance. Neuronal HPK-1 improves proteostasis through kinase activity. HPK-1 functions cell non-autonomously within serotonergic and GABAergic neurons to improve proteostasis in distal tissues by specifically regulating distinct components of the proteostatic network. Increased serotonergic HPK-1 enhances the heat shock response and survival to acute stress. In contrast, GABAergic HPK-1 induces basal autophagy and extends longevity, which requires mxl-2 (MLX), hlh-30 (TFEB), and daf-16 (FOXO). Our work establishes hpk-1 as a key neuronal transcriptional regulator critical for preservation of neuronal function during aging. Further, these data provide novel insight as to how the nervous system partitions acute and chronic adaptive response pathways to delay aging by maintaining organismal homeostasis.

HSF-1: Guardian of the Proteome Through Integration of Longevity Signals to the Proteostatic Network.

Discoveries made in the nematode Caenorhabditis elegans revealed that aging is under genetic control. Since these transformative initial studies, C. elegans has become a premier model system for aging research. Critically, the genes, pathways, and processes that have fundamental roles in organismal aging are deeply conserved throughout evolution. This conservation has led to a wealth of knowledge regarding both the processes that influence aging and the identification of molecular and cellular hallmarks that play a causative role in the physiological decline of organisms. One key feature of age-associated decline is the failure of mechanisms that maintain proper function of the proteome (proteostasis). Here we highlight components of the proteostatic network that act to maintain the proteome and how this network integrates into major longevity signaling pathways. We focus in depth on the heat shock transcription factor 1 (HSF1), the central regulator of gene expression for proteins that maintain the cytosolic and nuclear proteomes, and a key effector of longevity signals.

Quantifying Tissue-Specific Proteostatic Decline in Caenorhabditis elegans.

The ability to maintain proper function and folding of the proteome (protein homeostasis) declines during normal aging, facilitating the onset of a growing number of age-associated diseases. For instance, proteins with polyglutamine expansions are prone to aggregation, as exemplified with the huntingtin protein and concomitant onset of Huntington's disease. The age-associated deterioration of the proteome has been widely studied through the use of transgenic Caenorhabditis elegans expressing polyQ repeats fused to a yellow fluorescent protein (YFP). This polyQ::YFP transgenic animal model facilitates the direct quantification of the age-associated decline of the proteome through imaging the progressive formation of fluorescent foci (i.e., protein aggregates) and subsequent onset of locomotion defects that develop as a result of the collapse of the proteome. Further, the expression of the polyQ::YFP transgene can be driven by tissue-specific promoters, allowing the assessment of proteostasis across tissues in the context of an intact multicellular organism. This model is highly amenable to genetic analysis, thus providing an approach to quantify aging that is complementary to lifespan assays. We describe how to accurately measure polyQ::YFP foci formation within either neurons or body wall muscle during aging, and the subsequent onset of behavioral defects. Next, we highlight how these approaches can be adapted for higher throughput, and potential future applications using other emerging strategies for C. elegans genetic analysis.

TIAM-1/GEF can shape somatosensory dendrites independently of its GEF activity by regulating F-actin localization.

Dendritic arbors are crucial for nervous system assembly, but the intracellular mechanisms that govern their assembly remain incompletely understood. Here, we show that the dendrites of PVD neurons in Caenorhabditis elegans are patterned by distinct pathways downstream of the DMA-1 leucine-rich transmembrane (LRR-TM) receptor. DMA-1/LRR-TM interacts through a PDZ ligand motif with the guanine nucleotide exchange factor TIAM-1/GEF in a complex with act-4/Actin to pattern higher order 4° dendrite branches by localizing F-actin to the distal ends of developing dendrites. Surprisingly, TIAM-1/GEF appears to function independently of Rac1 guanine nucleotide exchange factor activity. A partially redundant pathway, dependent on HPO-30/Claudin, regulates formation of 2° and 3° branches, possibly by regulating membrane localization and trafficking of DMA-1/LRR-TM. Collectively, our experiments suggest that HPO-30/Claudin localizes the DMA-1/LRR-TM receptor on PVD dendrites, which in turn can control dendrite patterning by directly modulating F-actin dynamics through TIAM-1/GEF.

Holothurians as a Model System to Study Regeneration.

Echinoderms possess an incredible regenerative capacity. Within this phylum, holothurians, better known as sea cucumbers, can regenerate most of their internal and external organs. While regeneration has been studied in several species, the most recent and extensive studies have been done in the species Holothuria glaberrima, the focus of most of our discussion. This chapter presents the model system and integrates the work that has been done to determine the major steps that take place, during regeneration of the intestinal and nervous system, from wound healing to the reestablishment of original function. We describe the cellular and molecular events associated with the regeneration processes and also describe the techniques that have been used, discuss the results, and explain the gaps in our knowledge that remain. We expect that the information provided here paves the road for new and young investigators to continue the study of the amazing potential of regeneration in members of the Echinodermata and how these studies will shed some light into the mechanisms that are common to many regenerative processes.

Synaptogenesis Is Modulated by Heparan Sulfate in Caenorhabditis elegans.

The nervous system regulates complex behaviors through a network of neurons interconnected by synapses. How specific synaptic connections are genetically determined is still unclear. Male mating is the most complex behavior in Caenorhabditis elegans It is composed of sequential steps that are governed by > 3000 chemical connections. Here, we show that heparan sulfates (HS) play a role in the formation and function of the male neural network. HS, sulfated in position 3 by the HS modification enzyme HST-3.1/HS 3-O-sulfotransferase and attached to the HS proteoglycan glypicans LON-2/glypican and GPN-1/glypican, functions cell-autonomously and nonautonomously for response to hermaphrodite contact during mating. Loss of 3-O sulfation resulted in the presynaptic accumulation of RAB-3, a molecule that localizes to synaptic vesicles, and disrupted the formation of synapses in a component of the mating circuits. We also show that the neural cell adhesion protein NRX-1/neurexin promotes and the neural cell adhesion protein NLG-1/neuroligin inhibits the formation of the same set of synapses in a parallel pathway. Thus, neural cell adhesion proteins and extracellular matrix components act together in the formation of synaptic connections.

Muscle- and Skin-Derived Cues Jointly Orchestrate Patterning of Somatosensory Dendrites.

Sensory dendrite arbors are patterned through cell-autonomously and non-cell-autonomously functioning factors [1-3]. Yet, only a few non-cell-autonomously acting proteins have been identified, including semaphorins [4, 5], brain-derived neurotrophic factors (BDNFs) [6], UNC-6/Netrin [7], and the conserved MNR-1/Menorin-SAX-7/L1CAM cell adhesion complex [8, 9]. This complex acts from the skin to pattern the stereotypic dendritic arbors of PVD and FLP somatosensory neurons in Caenorhabditis elegans through the leucine-rich transmembrane receptor DMA-1/LRR-TM expressed on PVD neurons [8, 9]. Here we describe a role for the diffusible C. elegans protein LECT-2, which is homologous to vertebrate leukocyte cell-derived chemotaxin 2 (LECT2)/Chondromodulin II. LECT2/Chondromodulin II has been implicated in a variety of pathological conditions [10-13], but the developmental functions of LECT2 have remained elusive. We find that LECT-2/Chondromodulin II is required for development of PVD and FLP dendritic arbors and can act as a diffusible cue from a distance to shape dendritic arbors. Expressed in body-wall muscles, LECT-2 decorates neuronal processes and hypodermal cells in a pattern similar to the cell adhesion molecule SAX-7/L1CAM. LECT-2 functions genetically downstream of the MNR-1/Menorin-SAX-7/L1CAM adhesion complex and upstream of the DMA-1 receptor. LECT-2 localization is dependent on SAX-7/L1CAM, but not on MNR-1/Menorin or DMA-1/LRR-TM, suggesting that LECT-2 functions as part of the skin-derived MNR-1/Menorin-SAX-7/L1CAM adhesion complex. Collectively, our findings suggest that LECT-2/Chondromodulin II acts as a muscle-derived, diffusible cofactor together with a skin-derived cell adhesion complex to orchestrate the molecular interactions of three tissues during patterning of somatosensory dendrites.

Holothurian Nervous System Diversity Revealed by Neuroanatomical Analysis.

The Echinodermata comprise an interesting branch in the phylogenetic tree of deuterostomes. Their radial symmetry which is reflected in their nervous system anatomy makes them a target of interest in the study of nervous system evolution. Until recently, the study of the echinoderm nervous system has been hindered by a shortage of neuronal markers. However, in recent years several markers of neuronal and fiber subpopulations have been described. These have been used to identify subpopulations of neurons and fibers, but an integrative study of the anatomical relationship of these subpopulations is wanting. We have now used eight commercial antibodies, together with three antibodies produced by our group to provide a comprehensive and integrated description and new details of the echinoderm neuroanatomy using the holothurian Holothuria glaberrima (Selenka, 1867) as our model system. Immunoreactivity of the markers used showed: (1) specific labeling patterns by markers in the radial nerve cords, which suggest the presence of specific nerve tracts in holothurians. (2) Nerves directly innervate most muscle fibers in the longitudinal muscles. (3) Similar to other deuterostomes (mainly vertebrates), their enteric nervous system is composed of a large and diverse repertoire of neurons and fiber phenotypes. Our results provide a first blueprint of the anatomical organization of cells and fibers that form the holothurian neural circuitry, and highlight the fact that the echinoderm nervous system shows unexpected diversity in cell and fiber types and their distribution in both central and peripheral nervous components.

The Adhesion Molecule KAL-1/anosmin-1 Regulates Neurite Branching through a SAX-7/L1CAM-EGL-15/FGFR Receptor Complex.

Neurite branching is essential for correct assembly of neural circuits, yet it remains a poorly understood process. For example, the neural cell adhesion molecule KAL-1/anosmin-1, which is mutated in Kallmann syndrome, regulates neurite branching through mechanisms largely unknown. Here, we show that KAL-1/anosmin-1 mediates neurite branching as an autocrine co-factor with EGL-17/FGF through a receptor complex consisting of the conserved cell adhesion molecule SAX-7/L1CAM and the fibroblast growth factor receptor EGL-15/FGFR. This protein complex, which appears conserved in humans, requires the immunoglobulin (Ig) domains of SAX-7/L1CAM and the FN(III) domains of KAL-1/anosmin-1 for formation in vitro as well as function in vivo. The kinase domain of the EGL-15/FGFR is required for branching, and genetic evidence suggests that ras-mediated signaling downstream of EGL-15/FGFR is necessary to effect branching. Our studies establish a molecular pathway that regulates neurite branching during development of the nervous system.

Complex cooperative functions of heparan sulfate proteoglycans shape nervous system development in Caenorhabditis elegans.

The development of the nervous system is a complex process requiring the integration of numerous molecular cues to form functional circuits. Many cues are regulated by heparan sulfates, a class of linear glycosaminoglycan polysaccharides. These sugars contain distinct modification patterns that regulate protein-protein interactions. Misexpressing the homolog of KAL-1/anosmin-1, a neural cell adhesion molecule mutant in Kallmann syndrome, in Caenorhabditis elegans causes a highly penetrant, heparan sulfate-dependent axonal branching phenotype in AIY interneurons. In an extended forward genetic screen for modifiers of this phenotype, we identified alleles in new as well as previously identified genes involved in HS biosynthesis and modification, namely the xylosyltransferase sqv-6, the HS-6-O-sulfotransferase hst-6, and the HS-3-O-sulfotransferase hst-3.2. Cell-specific rescue experiments showed that different HS biosynthetic and modification enzymes can be provided cell-nonautonomously by different tissues to allow kal-1-dependent branching of AIY. In addition, we show that heparan sulfate proteoglycan core proteins that carry the heparan sulfate chains act genetically in a highly redundant fashion to mediate kal-1-dependent branching in AIY neurons. Specifically, lon-2/glypican and unc-52/perlecan act in parallel genetic pathways and display synergistic interactions with sdn-1/syndecan to mediate kal-1 function. Because all of these heparan sulfate core proteins have been shown to act in different tissues, these studies indicate that KAL-1/anosmin-1 requires heparan sulfate with distinct modification patterns of different cellular origin for function. Our results support a model in which a three-dimensional scaffold of heparan sulfate mediates KAL-1/anosmin-1 and intercellular communication through complex and cooperative interactions. In addition, the genes we have identified could contribute to the etiology of Kallmann syndrome in humans.

Calbindin-D32k is localized to a subpopulation of neurons in the nervous system of the sea cucumber Holothuria glaberrima (Echinodermata).

Members of the calbindin subfamily serve as markers of subpopulations of neurons within the vertebrate nervous system. Although markers of these proteins are widely available and used, their application to invertebrate nervous systems has been very limited. In this study we investigated the presence and distribution of members of the calbindin subfamily in the sea cucumber Holothuria glaberrima (Selenka, 1867). Immunohistological experiments with antibodies made against rat calbindin 1, parvalbumin, and calbindin 2, showed that these antibodies labeled cells and fibers within the nervous system of H. glaberrima. Most of the cells and fibers were co-labeled with the neural-specific marker RN1, showing their neural specificity. These were distributed throughout all of the nervous structures, including the connective tissue plexi of the body wall and podia. Bioinformatics analyses of the possible antigen recognized by these markers showed that a calbindin 2-like protein present in the sea urchin Strongylocentrotus purpuratus, corresponded to the calbindin-D32k previously identified in other invertebrates. Western blots with anti-calbindin 1 and anti-parvalbumin showed that these markers recognized an antigen of approximately 32 kDa in homogenates of radial nerve cords of H. glaberrima and Lytechinus variegatus. Furthermore, immunoreactivity with anti-calbindin 1 and anti-parvalbumin was obtained to a fragment of calbindin-D32k of H. glaberrima. Our findings suggest that calbindin-D32k is present in invertebrates and its sequence is more similar to the vertebrate calbindin 2 than to calbindin 1. Thus, characterization of calbindin-D32k in echinoderms provides an important view of the evolution of this protein family and represents a valuable marker to study the nervous system of invertebrates.