DEC-7/SUSD2, a sushi domain-containing protein, regulates an ultradian behavior mediated by intestinal epithelial Ca2+ oscillations in Caenorhabditis elegans.

In Caenorhabditis elegans, rhythmic posterior body wall muscle contractions mediate the highly regular defecation cycle. These contractions are regulated by inositol-1,4,5-trisphosphate (InsP3) receptor-dependent Ca2+ oscillations in intestinal epithelial cells. Here, we find that mutations in dec-7, which encodes the nematode ortholog of the human Sushi domain-containing 2 protein (SUSD2), lead to an increase in InsP3 receptor-dependent rhythmic posterior body wall muscle contractions. DEC-7 is highly expressed in the intestinal epithelia and localizes to the cell-cell junction. The increase in rhythmic activity caused by the loss of dec-7 is dependent on the innexin gap junction protein INX-16. Moreover, DEC-7 is required for the clustering of INX-16 to the cell-cell junction of the intestinal epithelia. We hypothesize that DEC-7/SUSD2 regulates INX-16 activity to mediate the rhythmic frequency of the defecation motor program. Thus, our data indicate a critical role of a phylogenetically conserved cell-cell junction protein in mediating an ultradian rhythm in the intestinal epithelia of C. elegans.NEW & NOTEWORTHY The conserved complement group protein DEC-7/SUSD2 acts at the apical cell-cell junction of C. elegans intestinal epithelia to mediate gap junction protein organization and function to facilitate a Ca2+ wave-regulated ultradian behavior.

The Role of Mitochondrial Calcium Homeostasis in Alzheimer's and Related Diseases.

Calcium signaling is essential for neuronal function, and its dysregulation has been implicated across neurodegenerative diseases, including Alzheimer's disease (AD). A close reciprocal relationship exists between calcium signaling and mitochondrial function. Growing evidence in a variety of AD models indicates that calcium dyshomeostasis drastically alters mitochondrial activity which, in turn, drives neurodegeneration. This review discusses the potential pathogenic mechanisms by which calcium impairs mitochondrial function in AD, focusing on the impact of calcium in endoplasmic reticulum (ER)-mitochondrial communication, mitochondrial transport, oxidative stress, and protein homeostasis. This review also summarizes recent data that highlight the need for exploring the mechanisms underlying calcium-mediated mitochondrial dysfunction while suggesting potential targets for modulating mitochondrial calcium levels to treat neurodegenerative diseases such as AD.

Metabolic constraints of swelling-activated glutamate release in astrocytes and their implication for ischemic tissue damage.

Volume-regulated anion channel (VRAC) is a glutamate-permeable channel that is activated by physiological and pathological cell swelling and promotes ischemic brain damage. However, because VRAC opening requires cytosolic ATP, it is not clear if and how its activity is sustained in the metabolically compromised CNS. In the present study, we used cultured astrocytes - the cell type which shows prominent swelling in stroke - to model how metabolic stress and changes in gene expression may impact VRAC function in the ischemic and post-ischemic brain. The metabolic state of primary rat astrocytes was modified with chemical inhibitors and examined using luciferin-luciferase ATP assays and a Seahorse analyzer. Swelling-activated glutamate release was quantified with the radiotracer D-[3 H]aspartate. The specific contribution of VRAC to swelling-activated glutamate efflux was validated by RNAi knockdown of the essential subunit, leucine-rich repeat-containing 8A (LRRC8A); expression levels of VRAC components were measured with qRT-PCR. Using this methodology, we found that complete metabolic inhibition with the glycolysis blocker 2-deoxy-D-glucose and the mitochondrial poison sodium cyanide reduced astrocytic ATP levels by > 90% and abolished glutamate release from swollen cells (via VRAC). When only mitochondrial respiration was inhibited by cyanide or rotenone, the intracellular ATP levels and VRAC activity were largely preserved. Bypassing glycolysis by providing the mitochondrial substrates pyruvate and/or glutamine led to partial recovery of ATP levels and VRAC activity. Unexpectedly, the metabolic block of VRAC was overridden when ATP-depleted cells were exposed to extreme cell swelling (≥ 50% reduction in medium osmolarity). Twenty-four hour anoxic adaptation caused a moderate reduction in the expression levels of the VRAC component LRRC8A, but no significant changes in VRAC activity. Overall, our findings suggest that (i) astrocytic VRAC activity and metabolism can be sustained by low levels of glucose and (ii) the inhibitory influence of diminishing ATP levels and the stimulatory effect of cellular swelling are the two major factors that govern VRAC activity in the ischemic brain.

C. elegans Presenilin Mediates Inter-Organelle Contacts and Communication that Is Required for Lysosome Activity.

Compromised lysosome function is implicated in the pathology of many neurodegenerative diseases, including Alzheimer's disease (AD). Familial Alzheimer's disease (fAD) is caused primarily by mutations in the presenilin encoding genes, but the underlying mechanism remains obscure. Loss of the conserved C. elegans presenilin orthologue SEL-12 results in increased mitochondrial calcium, which promotes neurodegeneration. Here, we find that sel-12 mutant lysosomes, independent of SEL-12 proteolytic activity, are significantly enlarged and more alkaline due to increased ER-to-mitochondrial calcium signaling and concomitant mitochondrial oxidative stress. These defects and their dependence on mitochondrial calcium are recapitulated in human fAD fibroblasts, demonstrating a conserved role for mitochondrial calcium in presenilin-mediated lysosome dysfunction. sel-12 mutants also have increased contact surface area between the ER, mitochondria, and lysosomes, suggesting sel-12 has an additional role in modulating organelle contact and communication. Overall, we demonstrate that SEL-12 maintains lysosome acidity and lysosome health by controlling ER-to-mitochondrial calcium signaling.

Deregulation of Mitochondrial Calcium Handling Due to Presenilin Loss Disrupts Redox Homeostasis and Promotes Neuronal Dysfunction.

Mitochondrial dysfunction and oxidative stress are major contributors to the pathophysiology of neurodegenerative diseases, including Alzheimer's disease (AD). However, the mechanisms driving mitochondrial dysfunction and oxidative stress are unclear. Familial AD (fAD) is an early onset form of AD caused primarily by mutations in the presenilin-encoding genes. Previously, using Caenorhabditis elegans as a model system to study presenilin function, we found that loss of C. elegans presenilin orthologue SEL-12 results in elevated mitochondrial and cytosolic calcium levels. Here, we provide evidence that elevated neuronal mitochondrial generated reactive oxygen species (ROS) and subsequent neurodegeneration in sel-12 mutants are a consequence of the increase of mitochondrial calcium levels and not cytosolic calcium levels. We also identify mTORC1 signaling as a critical factor in sustaining high ROS in sel-12 mutants in part through its repression of the ROS scavenging system SKN-1/Nrf. Our study reveals that SEL-12/presenilin loss disrupts neuronal ROS homeostasis by increasing mitochondrial ROS generation and elevating mTORC1 signaling, which exacerbates this imbalance by suppressing SKN-1/Nrf antioxidant activity.

Increased mitochondrial calcium uptake and concomitant mitochondrial activity by presenilin loss promotes mTORC1 signaling to drive neurodegeneration.

Metabolic dysfunction and protein aggregation are common characteristics that occur in age-related neurodegenerative disease. However, the mechanisms underlying these abnormalities remain poorly understood. We have found that mutations in the gene encoding presenilin in Caenorhabditis elegans, sel-12, results in elevated mitochondrial activity that drives oxidative stress and neuronal dysfunction. Mutations in the human presenilin genes are the primary cause of familial Alzheimer's disease. Here, we demonstrate that loss of SEL-12/presenilin results in the hyperactivation of the mTORC1 pathway. This hyperactivation is caused by elevated mitochondrial calcium influx and, likely, the associated increase in mitochondrial activity. Reducing mTORC1 activity improves proteostasis defects and neurodegenerative phenotypes associated with loss of SEL-12 function. Consistent with high mTORC1 activity, we find that SEL-12 loss reduces autophagosome formation, and this reduction is prevented by limiting mitochondrial calcium uptake. Moreover, the improvements of proteostasis and neuronal defects in sel-12 mutants due to mTORC1 inhibition require the induction of autophagy. These results indicate that mTORC1 hyperactivation exacerbates the defects in proteostasis and neuronal function in sel-12 mutants and demonstrate a critical role of presenilin in promoting neuronal health.

Innexin function: minding the gap junction.

Gap junctions mediate intercellular communication and are critical for development and nervous system function. Initially thought to function solely as stand-alone molecules, it has now been shown that a stomatin-like protein regulates a gap junction channel in Caenorhabditis elegans.

Corrupted ER-mitochondrial calcium homeostasis promotes the collapse of proteostasis.

Aging and age-related diseases are associated with a decline of protein homeostasis (proteostasis), but the mechanisms underlying this decline are not clear. In particular, decreased proteostasis is a widespread molecular feature of neurodegenerative diseases, such as Alzheimer's disease (AD). Familial AD is largely caused by mutations in the presenilin encoding genes; however, their role in AD is not understood. In this study, we investigate the role of presenilins in proteostasis using the model system Caenorhabditis elegans. Previously, we found that mutations in C. elegans presenilin cause elevated ER to mitochondria calcium signaling, which leads to an increase in mitochondrial generated oxidative stress. This, in turn, promotes neurodegeneration. To understand the cellular mechanisms driving neurodegeneration, using several molecular readouts of protein stability in C. elegans, we find that presenilin mutants have widespread defects in proteostasis. Markedly, we demonstrate that these defects are independent of the protease activity of presenilin and that reduction in ER to mitochondrial calcium signaling can significantly prevent the proteostasis defects observed in presenilin mutants. Furthermore, we show that supplementing presenilin mutants with antioxidants suppresses the proteostasis defects. Our findings indicate that defective ER to mitochondria calcium signaling promotes proteostatic collapse in presenilin mutants by increasing oxidative stress.

The Ror receptor tyrosine kinase CAM-1 is required for ACR-16-mediated synaptic transmission at the C. elegans neuromuscular junction.

Nicotinic (cholinergic) neurotransmission plays a critical role in the vertebrate nervous system, underlies nicotine addiction, and nicotinic receptor dysfunction leads to neurological disorders. The C. elegans neuromuscular junction (NMJ) shares many characteristics with neuronal synapses, including multiple classes of postsynaptic currents. Here, we identify two genes required for the major excitatory current found at the C. elegans NMJ: acr-16, which encodes a nicotinic AChR subunit homologous to the vertebrate alpha7 subunit, and cam-1, which encodes a Ror receptor tyrosine kinase. acr-16 mutants lack fast cholinergic current at the NMJ and exhibit synthetic behavioral deficits with other known AChR mutants. In cam-1 mutants, ACR-16 is mislocalized and ACR-16-dependent currents are disrupted. The postsynaptic deficit in cam-1 mutants is accompanied by alterations in the distribution of cholinergic vesicles and associated synaptic proteins. We hypothesize that CAM-1 contributes to the localization or stabilization of postsynaptic ACR-16 receptors and presynaptic release sites.

Alpha spectrin is essential for morphogenesis and body wall muscle formation in Caenorhabditis elegans.

A common feature of multicellular animals is the ubiquitous presence of the spectrin cytoskeleton. Although discovered over 30 yr ago, the function of spectrin in non-erythrocytes has remained elusive. We have found that the spc-1 gene encodes the only alpha spectrin gene in the Caenorhabditis elegans genome. During embryogenesis, alpha spectrin localizes to the cell membrane in most if not all cells, starting at the first cell stage. Interestingly, this localization is dependent on beta spectrin but not beta(Heavy) spectrin. Furthermore, analysis of spc-1 mutants indicates that beta spectrin requires alpha spectrin to be stably recruited to the cell membrane. Animals lacking functional alpha spectrin fail to complete embryonic elongation and die just after hatching. These mutant animals have defects in the organization of the hypodermal apical actin cytoskeleton that is required for elongation. In addition, we find that the process of elongation is required for the proper differentiation of the body wall muscle. Specifically, when compared with myofilaments in wild-type animals the myofilaments of the body wall muscle in mutant animals are abnormally oriented relative to the longitudinal axis of the embryo, and the body wall muscle cells do not undergo normal cell shape changes.

Measurement of Oxygen Consumption Rates in Intact Caenorhabditis elegans.

Optimal mitochondrial function is critical for healthy cellular activity, particularly in cells that have high energy demands like those in the nervous system and muscle. Consistent with this, mitochondrial dysfunction has been associated with a myriad of neurodegenerative diseases and aging in general. Caenorhabditis elegans have been a powerful model system for elucidating the many intricacies of mitochondrial function. Mitochondrial respiration is a strong indicator of mitochondrial function and recently developed respirometers offer a state-of-the-art platform to measure respiration in cells. In this protocol, we provide a technique to analyze live, intact C. elegans. This protocol spans a period of ~7 days and includes steps for (1) growing and synchronization of C. elegans, (2) preparation of compounds to be injected and hydration of probes, (3) drug loading and cartridge equilibration, (4) preparation of worm assay plate and assay run, and (5) post-experiment data analysis.

Role of Presenilin in Mitochondrial Oxidative Stress and Neurodegeneration in Caenorhabditis elegans.

Neurodegenerative diseases like Alzheimer's disease (AD) are poised to become a global health crisis, and therefore understanding the mechanisms underlying the pathogenesis is critical for the development of therapeutic strategies. Mutations in genes encoding presenilin (PSEN) occur in most familial Alzheimer's disease but the role of PSEN in AD is not fully understood. In this review, the potential modes of pathogenesis of AD are discussed, focusing on calcium homeostasis and mitochondrial function. Moreover, research using Caenorhabditis elegans to explore the effects of calcium dysregulation due to presenilin mutations on mitochondrial function, oxidative stress and neurodegeneration is explored.

Measurement of ROS in Caenorhabditis elegans Using a Reduced Form of Fluorescein.

Oxidative stress is implicated in the pathogenesis of various neurodegenerative diseases, including Alzheimer's disease. Oxidative stress is a result of a disruption of the equilibrium between antioxidants and oxidants, in favor of oxidants. Since mitochondria are major sites of production and reduction of reactive oxygen species (ROS), measurement of ROS levels can help us determine if mitochondrial functional integrity has been compromised. In this protocol, we describe a method to measure the level of ROS in the nematode Caenorhabditis elegans, using chloromethyl-2,7'-dichlorodihydrofluorescein diacetate (CM-H2DCFDA).

Analysis of Mitochondrial Structure in the Body Wall Muscle of Caenorhabditis elegans.

Mitochondrial function is altered in various pathologies, highlighting the crucial role mitochondria plays in maintaining cellular homeostasis. Mitochondrial structure undergoes constant fission and fusion in response to changing cellular environment. Due to this, analyzing mitochondrial structure could provide insight into the physiological state of the cell. In this protocol, we describe a method to analyze mitochondrial structure in body wall muscles in the nematode Caenorhabditis elegans, using both transgenic and dye-based approaches.

Presenilin mutations deregulate mitochondrial Ca2+ homeostasis and metabolic activity causing neurodegeneration in Caenorhabditis elegans.

Mitochondrial dysfunction and subsequent metabolic deregulation is observed in neurodegenerative diseases and aging. Mutations in the presenilin (PSEN) encoding genes (PSEN1 and PSEN2) cause most cases of familial Alzheimer's disease (AD); however, the underlying mechanism of pathogenesis remains unclear. Here, we show that mutations in the C. elegans gene encoding a PSEN homolog, sel-12 result in mitochondrial metabolic defects that promote neurodegeneration as a result of oxidative stress. In sel-12 mutants, elevated endoplasmic reticulum (ER)-mitochondrial Ca2+ signaling leads to an increase in mitochondrial Ca2+ content which stimulates mitochondrial respiration resulting in an increase in mitochondrial superoxide production. By reducing ER Ca2+ release, mitochondrial Ca2+ uptake or mitochondrial superoxides in sel-12 mutants, we demonstrate rescue of the mitochondrial metabolic defects and prevent neurodegeneration. These data suggest that mutations in PSEN alter mitochondrial metabolic function via ER to mitochondrial Ca2+ signaling and provide insight for alternative targets for treating neurodegenerative diseases.

C. elegans PAT-4/ILK functions as an adaptor protein within integrin adhesion complexes.

BACKGROUND: Mammalian integrin-linked kinase (ILK) was identified in a yeast two-hybrid screen for proteins binding the integrin beta(1) subunit cytoplasmic domain. ILK has been implicated in integrin-mediated signaling and is also an adaptor within integrin-associated cytoskeletal complexes. RESULTS: We identified the C. elegans pat-4 gene in previous genetic screens for mutants unable to assemble integrin-mediated muscle cell attachments. Here, we report that pat-4 encodes the sole C. elegans homolog of ILK. In pat-4 null mutants, embryonic muscle cells form integrin foci, but the subsequent recruitment of vinculin and UNC-89 as well as actin and myosin filaments to these in vivo focal adhesion analogs is blocked. Conversely, PAT-4/ILK requires the ECM component UNC-52/perlecan, the transmembrane protein integrin, and the novel cytoplasmic attachment protein UNC-112 to be properly recruited to nascent attachments. Transgenically expressed "kinase-dead" ILK fully rescues pat-4 loss-of-function mutants. We also identify UNC-112 as a new binding partner for ILK. CONCLUSIONS: Our data strengthens the emerging view that ILK functions primarily as an adaptor protein within integrin adhesion complexes and identifies UNC-112 as a new ILK binding partner.

Decreased sensory stimulation reduces behavioral responding, retards development, and alters neuronal connectivity in Caenorhabditis elegans.

Activity-dependent plasticity is a critical component of nervous systems. We show that in Caenorhabditis elegans, worms raised in isolation made smaller responses to mechanosensory stimulation and were smaller and slower to begin laying eggs than age-matched group-raised worms. The glutamate receptor gene GLR-1 was critical for the observed alterations in behavior but not in size, whereas the cGMP-dependent protein kinase gene EGL-4 was critical for the observed changes in size but not the changes in behavior. Mechanosensory stimulation during development reversed the effects of isolation on behavior and began to reduce the effects of isolation on size. In C. elegans, the six mechanosensory touch neurons synapse onto the four pair of command interneurons for forward and backward movement. Touch (mechanosensory) neurons of worms raised in isolation expressed lower levels of green fluorescent protein (GFP)-tagged synaptobrevin than touch neurons of worms raised in colonies. Command interneurons of worms raised in isolation expressed lower levels of GFP-tagged glutamate receptors than command interneurons of worms raised in groups. Brief mechanical stimulation during larval development rescued the expression of GFP-tagged glutamate receptors but not GFP-tagged synaptobrevin. Together, these results indicate that the level of stimulation experienced by C. elegans during development profoundly affects the development of neuronal connectivity and has widespread cellular and behavioral consequences.

A Conserved GEF for Rho-Family GTPases Acts in an EGF Signaling Pathway to Promote Sleep-like Quiescence in Caenorhabditis elegans.

Sleep is evolutionarily conserved and required for organism homeostasis and survival. Despite this importance, the molecular and cellular mechanisms underlying sleep are not well understood. Caenorhabditis elegans exhibits sleep-like behavioral quiescence and thus provides a valuable, simple model system for the study of cellular and molecular regulators of this process. In C. elegans, epidermal growth factor receptor (EGFR) signaling is required in the neurosecretory neuron ALA to promote sleep-like behavioral quiescence after cellular stress. We describe a novel role for VAV-1, a conserved guanine nucleotide exchange factor (GEF) for Rho-family GTPases, in regulation of sleep-like behavioral quiescence. VAV-1, in a GEF-dependent manner, acts in ALA to suppress locomotion and feeding during sleep-like behavioral quiescence in response to cellular stress. Additionally, VAV-1 activity is required for EGF-induced sleep-like quiescence and normal levels of EGFR and secretory dense core vesicles in ALA. Importantly, the role of VAV-1 in promoting cellular stress-induced behavioral quiescence is vital for organism health because VAV-1 is required for normal survival after cellular stress.

DIM-1, a novel immunoglobulin superfamily protein in Caenorhabditis elegans, is necessary for maintaining bodywall muscle integrity.

The UNC-112 protein is required during initial muscle assembly in C. elegans to form dense bodies and M-lines. Loss of this protein results in arrest at the twofold stage of embryogenesis. In contrast, a missense mutation in unc-112 results in viable animals that have disorganized bodywall muscle and are paralyzed as adults. Loss or reduction of dim-1 gene function can suppress the severe muscle disruption and paralysis exhibited by these mutant hermaphrodites. The overall muscle structure in hermaphrodites lacking a functional dim-1 gene is slightly disorganized, and the myofilament lattice is not as strongly anchored to the muscle cell membrane as it is in wild-type muscle. The dim-1 gene encodes two polypeptides that contain three Ig-like repeats. The short DIM-1 protein isoform consists entirely of three Ig repeats and is sufficient for wild-type bodywall muscle structure and stability. DIM-1(S) localizes to the region of the muscle cell membrane around and between the dense bodies, which are the structures that anchor the actin filaments and may play a role in stabilizing the thin rather than the thick filament components of the sarcomere.

A γ-Secretase Independent Role for Presenilin in Calcium Homeostasis Impacts Mitochondrial Function and Morphology in Caenorhabditis elegans.

Mutations in the presenilin (PSEN) encoding genes (PSEN1 and PSEN2) occur in most early onset familial Alzheimer's Disease. Despite the identification of the involvement of PSEN in Alzheimer's Disease (AD) ∼20 years ago, the underlying role of PSEN in AD is not fully understood. To gain insight into the biological function of PSEN, we investigated the role of the PSEN homolog SEL-12 in Caenorhabditis elegans. Using genetic, cell biological, and pharmacological approaches, we demonstrate that mutations in sel-12 result in defects in calcium homeostasis, leading to mitochondrial dysfunction. Moreover, consistent with mammalian PSEN, we provide evidence that SEL-12 has a critical role in mediating endoplasmic reticulum (ER) calcium release. Furthermore, we found that in SEL-12-deficient animals, calcium transfer from the ER to the mitochondria leads to fragmentation of the mitochondria and mitochondrial dysfunction. Additionally, we show that the impact that SEL-12 has on mitochondrial function is independent of its role in Notch signaling, γ-secretase proteolytic activity, and amyloid plaques. Our results reveal a critical role for PSEN in mediating mitochondrial function by regulating calcium transfer from the ER to the mitochondria.