Sarasija, Shaarika et al. (2013) International Worm Meeting "Using C. elegans to explore the role of presenilin in the pathogenesis of Alzheimer's Disease."

Sarasija, Shaarika, Norman, Kenneth

[International Worm Meeting, 2013]

Mutations in the genes encoding Presenilin-1 and Presenilin-2 occur in early onset Familial Alzheimer's Disease (FAD), a rare form of Alzheimer's Disease (AD). However, the role of presenilins in AD has remained elusive. The C. elegans genes sel-12 and hop-1 encode transmembrane domain proteins orthologous to human presenilins. We are interested in investigating whether mutations in sel-12 and/or hop-1 can alter calcium homeostasis in C. elegans. We have found that the sel-12 null mutant is hypersensitive to the muscle cell acetylcholine receptor agonist, levamisole, and the acetylcholine esterase inhibitor, aldicarb, suggesting that the muscle of the sel-12 mutant is hyper-excitable. Introduction of a ryanodine receptor (RyR) null background into the sel-12 mutant rescues this hypersensitivity. Additionally, we have observed that sel-12 mutants are shorter in length, possibly caused by increased muscle tone, a condition commonly observed in FAD patients. To test this possibility, we stimulated GABAergic inhibitory motor neurons and found that sel-12 mutants have a reduced ability to relax their muscles. Together, these data suggest that sel-12 mutants have elevated levels of sarcoplasmic calcium, which leads to hyper-excitable muscle. Mitochondria are dynamic organelles and act as a significant cytosolic calcium buffer in cells. We discovered that the organization of muscle mitochondria are structurally disrupted and that reactive oxygen species are poorly metabolized in sel-12 mutants upon paraquat exposure. Furthermore, we found that reducing sarcoplasmic calcium levels by introducing the RyR null mutation into the sel-12 mutant background did not restore mitochondria organization. These results suggest that the mitochondria dysfunction observed in sel-12 mutants is not a result of calcium overloading, but rather the primary defect. Therefore, we hypothesize that SEL-12 is required for normal mitochondria function and that loss of mitochondria function in sel-12 mutants leads to elevated calcium levels and improper ROS metabolism. Since signaling mechanisms are well conserved, we believe using C. elegans as a model system will provide insight into the role presenilins play in the pathogenesis of AD.

Spooner, Patrick et al. (2009) International Worm Meeting "Does UNC-89 mediate excitation-contraction coupling in C. elegans?"

Norman, Kenneth, Spooner, Patrick

[International Worm Meeting, 2009]

Ca2+ is a ubiquitous signaling molecule that is either directly or indirectly involved in most if not all cell physiological functions. Previously, we have found that the Rho GTPase family guanine nucleotide exchange factor, VAV-1 is a crucial component involved in mediating rhythmic activities by regulating Ca2+ oscillations. To understand the role VAV-1 plays in Ca2+ regulation, we have generated transgenic strains that overexpress constitutively active VAV-1. These animals display abnormal rhythmic activities, including altered defecation cycle, ovulation and uncoordinated locomotion. To gain insight into the mechanism(s) underlying VAV-1''s role in regulating Ca2+, we have conducted a suppressor screen to identify mutations that can suppress the Unc phenotype associated with expression of constitutively active VAV-1 (see A. Fry''s poster). From the suppressors isolated, two suppressors are alleles of unc-89. unc-89 encodes several isoforms that are part of the titin immunoglobulin (Ig) superfamily, which includes several large Ig rich isoforms (>750 kDa) and two small isoforms (~160 kDa) (Benian et al., 1996; Small et al., 2004). UNC-89 and its mammalian homolog, obscurin, have been implicated in myofilament assembly and maintenance. To understand how unc-89 mutants can suppress constitutively active VAV-1, we are addressing the following questions: 1) Does the disorganization of the myofilaments in unc-89 mutations lead to disrupted actomyosin contraction, 2) Is the sarco-endoplasmic reticulum (SR) disorganized in unc-89 mutants, 3) Do unc-89 mutations affect Ca2+ signaling events required for excitation-contraction coupling? Thus far, we have found that the alleles of unc-89 that suppress constitutively active VAV-1 are slightly slow moving but show normal sinusoidal locomotion. This argues that contraction is not disrupted but may be reduced since these animals are slow moving. Additionally, we have found that in the late embryo and early larvae the myofilaments are normally arranged in unc-89 mutants; however, in late larval/adult stages the myofilaments are disorganized. This suggests that UNC-89 is important for growth and/or maintenance of the myofilaments. Additionally, we have found that the SR as well as UNC-68/Ryanodine receptors are also disorganized in unc-89 adults, which could lead to abnormal Ca2+ influx from the SR. Thus far, our data suggests that UNC-89 is important for myofilament/SR organization in adults and that loss of UNC-89 activity leads to reduced muscle activity either by reducing the amount of Ca2+ inducing myofilament contraction or altering myofilament sensitivity to Ca2+. Benian et al., 1996 JCB 132: 835-848. Small et al., 2004 J Mol Biol 342: 91-108.

Senthil, Mano et al. (2021) International Worm Meeting "Investigating the role of miro-1 in neurodegeneration using a C. elegans Alzheimer's disease model"

Norman, Kenneth, Senthil, Mano

[International Worm Meeting, 2021]

Alzheimer's Disease (AD) is a debilitating neurodegenerative condition affecting many elderly individuals. Although AD is generally considered a progressive, age-related condition, a subset of the population is genetically predisposed to developing AD early in life, a condition known as familial Alzheimer's Disease (FAD). FAD is primarily caused by mutations in one of three genes, Amyloid Precursor Protein (APP), Presenilin 1 (PSEN1), and Presenilin 2 (PSEN2), with most mutations occurring in PSEN1. PSEN1 functions the catalytic core of gamma-secretase and is involved in the processing of APP to amyloid beta (Abeta) peptides. and the regulation intracellular Ca2+ signaling. Abeta plaque accumulation has long been regarded as the disease promoting agent controlling AD like neurodegeneration. However, recent studies are showing a lack of correlation between Abeta plaques and AD symptom severity, pointing to the possibility of other mechanisms controlling neurodegeneration. PSEN1 is also known to regulate intracellular Ca2+ signaling, and mutations in PSEN1 have been shown to result in increased reactive oxygen species (ROS) production, oxidative stress, and neurodegeneration as a product of intracellular Ca2+ dysregulation. The mechanisms underlying these intracellular changes however remain unclear. We are looking to investigate the relationship between endoplasmic reticulum (ER)-mitochondrial Ca2+ signaling, mitochondrial ROS production, and neurodegeneration using C. elegans strains harboring mutations in the gene encoding the C. elegans PSEN1 homologue, sel-12. sel-12 mutants have been shown to display elevated intracellular Ca2+ levels and increased ER-mitochondrial contacts which are associated with increased mitochondrial Ca2+ uptake, mitochondrial ROS production, and neuronal dysfunction. To understand the mechanism underlying the sel-12 mitochondrial phenotype, we are investigating the role a specific mitochondrial outer membrane protein, MIRO-1, which has been shown to have roles in mitochondrial trafficking, mitochondrial Ca2+ regulation, and mitochondrial metabolism. By introducing a miro-1 null mutation into the sel-12 mutant background, we are investigating whether miro-1 has a role in the ER- mitochondrial Ca2+ homeostasis and if loss of miro-1 improves sel-12 mutant fitness. Thus far, our results indicate that disrupting miro-1 in the sel-12 mutant background restores the neurodegenerative phenotype observed in the FAD mutant model system.

Fry, Amanda et al. (2011) International Worm Meeting "Function of VAV-1 in nervous system control of locomotion."

Fry, Amanda, Norman, Kenneth

[International Worm Meeting, 2011]

C. elegans VAV-1 is a guanine nucleotide exchange factor (GEF) for Rho/Rac family GTPases and is homologous to the mammalian Vav proto-oncogenes. We found previously that VAV-1 regulates a variety of rhythmic activities in C. elegans, including pharyngeal pumping, ovulation and fertilization, and the defecation cycle, but its role in locomotion has remained unexplored. Interestingly, we have found that vav-1 null animals display an elevated rate of locomotion and are hypersensitive to aldicarb (an acetylcholine esterase inhibitor), indicating heightened nervous system activity. The aldicarb hypersensitivity observed in vav-1 mutants is rescued by pan-neural expression of VAV-1, and using further pharmacological assays, we have shown that this hypersensitivity is not due to altered response of vav-1 mutant muscle cells to acetylcholine. Together, these data indicate that VAV-1 inhibits locomotion through a nervous system mechanism. Remarkably, Vav3 knockout mice are known to exhibit sympathetic nervous system hyperactivity, suggesting a conserved role of Vav proteins in negatively regulating nervous system activity, but the mechanism by which this regulation occurs is not fully understood (1-3). Since VAV-1 is known to be upstream of regulators of nervous system development (e.g. Rho/Rac GTPases), and impaired GABAergic neuron function can lead to aldicarb hypersensitivity, we examined the structure and function of GABA neurons by a combination of cell biological and pharmacological approaches. These analyses showed that both the development and function of GABA-releasing neurons is normal. To identify the cells that express vav-1 and gain insight into the neural circuit that regulates locomotion, we have analyzed vav-1 reporter expression in vivo and found that VAV-1 is expressed in a subset of neurons in the head ganglia of the worm, and highly expressed in the ALA neuron. Interestingly, the only known function of the ALA neuron is to regulate behavioral quiescence, the quantification of which is the rate of locomotion (4). We intend to elucidate the mechanism responsible for the control of locomotion by VAV-1 by determining the requirement of VAV-1 in the ALA neuron, as well as investigating the participation of VAV-1 in signaling pathways active in this cell. 1. Sauzeau et al. (2006). Nat. Med. 12, 841-845 2. Sauzeau et al. (2010). Mol Biol Cell. 21, 4251-63 3. Quevedo C. (2010). 15, 1125-39 4. Van Buskirk, C. and Sternberg, P.W. (2007) Nat. Neurosci. 10, 1300-07.

Sarasija, Shaarika et al. (2015) International Worm Meeting "A novel gamma-secretase independent role for presenilin in mitochondrial function and morphology."

Sarasija, Shaarika, Norman, Kenneth

[International Worm Meeting, 2015]

Mutations in the presenilin encoding genes (PSEN1 and PSEN2) occur in most early onset familial Alzheimer's Disease (FAD). Despite the identification of the involvement of PSEN in AD about 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 have found that mutations in sel-12 result in defects in calcium homeostasis and mitochondrial function. Moreover, we provide evidence that SEL-12 has a critical role in mediating endoplasmic reticulum (ER) calcium release. In SEL-12 deficient animals, calcium release from the ER leads to fragmentation of the mitochondria and mitochondrial dysfunction. Additionally, we show that the role of SEL-12 is independent of its role in Notch signaling and the gamma-secretase proteolytic complex. Our results provide evidence for a novel role of PSEN in mediating mitochondrial function by regulating calcium release from the ER to the mitochondria.

Fry, Amanda et al. (2009) International Worm Meeting "Investigation of the signaling pathways of VAV-1, a Rho family guanine nucleotide exchange factor, in C. elegans."

Norman, Kenneth, Spooner, Patrick, Fry, Amanda

[International Worm Meeting, 2009]

C. elegans VAV-1 is a guanine nucleotide exchange factor (GEF) for Rho/Rac GTPases and is homologous to the mammalian Vav proto-oncogenes. VAV-1 is widely expressed and has been shown to regulate rhythmic activities such as ovulation and defecation in C. elegans. VAV-1 regulates these rhythmic activities by modulating intracellular calcium signaling in part through inositol 1,4,5-triphospate receptors, but other signaling mechanisms must be at work downstream of VAV-1. Transgenic animals expressing constitutively active VAV-1 have uncoordinated rhythmic activities and locomotion. Consistent with VAV-1 modulating calcium, calcium oscillations are also disorganized in these transgenic animals. By screening a mutagenized strain of C. elegans that expresses constitutively active VAV-1 for suppression of their uncoordinated locomotion phenotype, we aim to identify other components of VAV-1 signaling pathways. Several putative suppressors of constitutively active VAV-1 have been identified (also see P. Spooner''s poster). In order to determine the physical locations of the unidentified suppressor mutations in the C. elegans genome, single nucleotide polymorphism (SNP) mapping is currently being used. The location of one such suppressor mutation has been narrowed to genomic interval containing about 60 genes, and we are in the process of testing candidates for their ability to suppress constitutively active VAV-1. Additionally, recent evidence suggests that mammalian Vav2 and Vav3 are important for sympathetic nervous system function and hence control of normal heart function. Interestingly, we have found that vav-1 mutants display abnormal pharyngeal contraction and are hypersensitive to the acetylcholine esterase inhibitor, aldicarb. Worm strains that have impaired synaptic transmission are resistant to aldicarb whereas mutants with hyperactive synaptic transmission are hypersensitive to aldicarb. Therefore, these data suggest that VAV-1 likely functions to negatively regulate synaptic transmission. Our future studies will focus on identifying the mechanism underlying the synaptic transmission defect and cloning the suppressor mutations of constitutive VAV-1 activation.

Sarasija, Shaarika et al. (2017) International Worm Meeting "Loss of SEL-12 function leads to aberrant Ca2+ homeostasis and mitochondrial metabolism resulting in neurodegeneration."

Sarasija, Shaarika, Laboy, Jocelyn, Norman, Kenneth R.

[International Worm Meeting, 2017]

Maintenance of mitochondrial structure and activity is integral to cellular function and survival. 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. Using genetic, cell biological and pharmacological approaches, we have found that the Caenorhabditis elegans PSEN homolog, SEL-12, is required for Ca2+ homeostasis, mitochondrial function and neuronal health. In SEL-12 deficient animals, aberrant Ca2+ transfer from the ER to the mitochondria alters mitochondrial morphology and function. Furthermore, sel-12 mutations lead to neuronal structural defects in the form of ectopic sprouting, axonal lesions and breaks in the mechanosensory neurons and concomitant functional loss. Interestingly, these mitochondrial and neuronal defects are independent of SEL-12's role in the ?-secretase proteolytic complex and Notch signaling. Conversely, we find that reduction of ER-Ca2+ release or mitochondrial Ca2+ uptake rescues the mitochondrial changes and the neuronal structural and functional defects observed in sel-12 mutants. Moreover, we demonstrate that the treatment of sel-12 mutants with a mitochondrial targeted antioxidant also rescues these neurodegenerative phenotypes. Thus, we hypothesize that loss of SEL-12/presenilin function leads to aberrant ER-Ca2+ release and mitochondrial Ca2+ uptake, which increases mitochondrial oxidative phosphorylation leading to excessive superoxide production. Consistent with this hypothesis, we detect an increase in ATP levels, oxygen consumption and ROS levels in sel-12 mutants. Additionally, we find that reducing ER Ca2+ release or reducing mitochondrial Ca2+ uptake normalizes oxygen consumption and ROS levels in sel-12 mutants. Thus our data argues that loss of SEL-12/presenilin function leads to neuronal abnormalities by altering mitochondrial metabolic activity, which leads to toxic levels of superoxide production. Our results provide evidence for a novel gamma-secretase independent role of PSEN in mediating neuronal structural and functional integrity via regulation of ER-mitochondrial Ca2+ transfer.

Spooner, Patrick et al. (2011) International Worm Meeting "Immunoglobulin domain containing isoforms of UNC-89 (obscurin) are required for myofilament organization and calcium signaling in Caenorhabditis elegans."

Duran, M. Berenice, Spooner, Patrick, Benian, Guy, Bonner, Jennifer, Norman, Kenneth

[International Worm Meeting, 2011]

The force generating machinery in muscle is regulated by the influx of calcium ions into the muscle cytoplasm. Initially, depolarization of the sarcolemma leads to calcium entry via voltage gated calcium channels, which rapidly triggers the release of calcium from the sarcoplasmic reticulum via ryanodine receptors by calcium induced calcium release. Thus, calcium needs to be rapidly, accurately and reliably regulated and the calcium channels and contractile machinery must be maintained in a highly ordered arrangement for efficient and effective muscle contraction to occur. The mechanism underlying this highly organized configuration is not fully understood. Using a combination of genetic and cell biological techniques, we have found that the C. elegans ortholog of the giant muscle protein obscurin, UNC-89, is required for muscle cell organization. The unc-89 locus encodes at least 6 isoforms as large as 900 kDa, 4 large Immunoglobin (Ig) domain rich isoforms and two smaller tandem kinase containing isoforms (Small et al. 2004; Ferrara et al. 2005). From our analyses, we have found that large Ig domain rich isoforms of UNC-89 are critical for sarcomere and SR organization. Furthermore, we have found evidence that unc-89 mutants lacking the large Ig rich isoforms have defects in excitation-contraction coupling in the pharyngeal and body wall muscle, through the coordination of calcium influx. Thus, our data implicates the large Ig domain rich isoforms of UNC-89 in maintaining muscle cell architecture that is critical for efficient and effective muscle contraction.

Ryan, Kerry et al. (2021) International Worm Meeting "Disruption of mitochondrial calcium homeostasis by loss of presenilin promotes mTORC1 signaling to drive neurodegeneration"

Ryan, Kerry, Sarasija, Shaarika, Ashkavand, Zahra, Samarakoon, Rohan, Norman, Kenneth, Laboy, Jocelyn

[International Worm Meeting, 2021]

Metabolic dysfunction and protein aggregation are associated with neurodegenerative disorders such as Alzheimer's disease (AD). However, the mechanisms underlying these abnormalities remain poorly understood. Mutations in the presenilin encoding genes are the primary cause of early onset familial AD, but their role in the disease is unclear. The presenilins are primarily known to function as the catalytic component of the gamma-secretase complex, which is involved in the cleavage of the amyloid precursor protein (APP) to produce amyloid beta (Abeta) peptides, whose aggregation into Abeta plaques is considered the hallmark of AD. However, many studies have demonstrated a gamma-secretase independent role for presenilins in calcium homeostasis that is critical for neuronal health. Previously, we showed that loss of the Caenorhabditis elegans presenilin ortholog SEL-12 elevates mitochondrial calcium signaling, which increases mitochondrial metabolism to drive neurodegeneration and loss of protein homeostasis. Here, we demonstrate that this elevated mitochondrial calcium and concomitant mitochondrial hyperactivity promotes activation of the mechanistic Target of Rapamycin Complex 1 (mTORC1) pathway. We utilize several models of protein homeostasis to show mTORC1 hyperactivity contributes to protein homeostasis defects. Reducing mTORC1 activity improves neurodegenerative phenotypes associated with loss of SEL-12/presenilin function. We also show mTORC1 plays a cell-autonomous role in neurodegeneration and that rescue of neuronal mTORC1 signaling is sufficient to abrogate improvements to neuronal function caused by global mTORC1 inhibition. Consistent with high mTORC1 activity, we find that SEL-12/presenilin loss reduces autophagy, and this reduction is prevented by limiting mitochondrial calcium uptake. Furthermore, the improvements to protein homeostasis and neuronal function in sel-12 mutants due to mTORC1 inhibition require the induction of autophagy. Collectively, these results indicate that mTORC1 hyperactivation exacerbates the defects in protein homeostasis, autophagy, and neuronal function in sel-12 mutants and suggest a potential therapeutic target for treating AD.

Ken Norman et al. (2006) Neuronal Development, Synaptic Function, and Behavior Meeting "Does the Rho family guanine nucleotide exchange factor VAV-1 function to regulate muscle excitability in C. elegans?"

Ken Norman, Villu Maricq

[Neuronal Development, Synaptic Function, and Behavior Meeting, 2006]

Small G proteins of the Rho family are involved in many cellular functions, including cell cycle progression, cytoskeletal rearrangement, gene transcription, membrane transport and other signaling events. Due to their involvement in such a diverse array of cellular behaviors Rho proteins are highly regulated by several classes of proteins. One class that promotes Rho activity is the guanine nucleotide exchange factors (Rho GEFs). We have found that the Rho GEF VAV-1 is involved in regulating several rhythmic behaviors in C. elegans (Norman et al., 2005), including pharyngeal pumping, ovulation and defecation. In part, VAV-1 regulates these behaviors by modulating intracellular calcium levels via the signaling molecule IP3. vav-1 mutants have slow and asymmetric pharyngeal muscle contractions and abnormal calcium transients. These defects are rescued by specific expression of vav-1 in the pharyngeal muscle. Since mutations in the IP3 receptor, ITR-1, do not completely phenocopy vav-1 pharyngeal defects, vav-1 signaling may involve additional pathways. We have found that a gain of function mutation in the voltage gated potassium channel, UNC-103, is able to restore some normal pharyngeal activity in vav-1 mutants. The unc-103 gain of function mutation is thought to lead to hyperpolarized muscle cells resulting in less excitable muscle cells (Petersen et al., 2004). In another approach, we have found that vav-1 mutants are hypersensitive to the acetylcholine esterase inhibitor aldicarb. These data suggest that vav-1 mutants may have hyperexcitable muscle. To investigate this further, we generated egl-8; vav-1 double mutants. egl-8 encodes a phospholipase C beta; that promotes neurotransmitter release and when mutated results in aldicarb resistance. Introduction of the vav-1 mutation into an egl-8 mutant, restored normal aldicarb sensitivity. These results are consistent with VAV-1 regulating muscle excitability. We are now screening for additional genes that modify VAV-1 signaling.