measurement-oxygen-consumption-rates-intact-caenorhabditis

Measurement of Oxygen Consumption Rates in Intact <em>Caenorhabditis elegans</em>

Optimal mitochondrial function is critical for healthy cellular activity, particularly in cells that have high energy demands like those in the nervous ...

Albany Medical College

Calcium, a ubiquitous intracellular signaling molecule, controls a diverse array of cellular processes. Consequently, cells have developed strategies to modulate the shape of calcium signals in space and time. The force generating machinery in muscle is regulated by the influx and efflux of calcium ions into the muscle cytoplasm. In order for efficient and effective muscle contraction to occur, calcium needs to be rapidly, accurately and reliably regulated. The mechanisms underlying this highly regulated process are not fully understood. Here, we show that the Caenorhabditis elegans homolog of the giant muscle protein obscurin, UNC-89, is required for normal muscle cell architecture. The large immunoglobulin domain-rich isoforms of UNC-89 are critical for sarcomere and sarcoplasmic reticulum organization. Furthermore, we have found evidence that this structural organization is crucial for excitation-contraction coupling in the body wall muscle, through the coordination of calcium signaling. Thus, our data implicates UNC-89 in maintaining muscle cell architecture and that this precise organization is essential for optimal calcium mobilization and efficient and effective muscle contraction.

Large isoforms of UNC-89 (obscurin) are required for muscle cell architecture and optimal calcium release in Caenorhabditis elegans.

The complex molecular and cellular mechanisms underlying neuronal control of animal movement are not well understood. Locomotion of Caenorhabditis elegans is mediated by a neuronal circuit that produces coordinated sinusoidal movement. Here we utilize this simple, yet elegant, behaviour to show that VAV-1, a conserved guanine nucleotide exchange factor for Rho-family GTPases, negatively regulates motor circuit activity and the rate of locomotion. While vav-1 is expressed in a small subset of neurons, we find that VAV-1 function is required in a single interneuron, ALA, to regulate motor neuron circuit activity. Furthermore, we show by genetic and optogenetic manipulation of ALA that VAV-1 is required for the excitation and activation of this neuron. We find that ALA signalling inhibits command interneuron activity by abrogating excitatory signalling in the command interneurons, which is responsible for promoting motor neuron circuit activity. Together, our data describe a novel neuromodulatory role for VAV-1-dependent signalling in the regulation of motor circuit activity and locomotion. 

VAV-1 acts in a single interneuron to inhibit motor circuit activity 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. 

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

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

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  using chloromethyl-2,7'-dichlorodihydrofluorescein diacetate (CM-HDCFDA).

Measurement of ROS in  Using a Reduced Form of Fluorescein.

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  using both transgenic and dye-based approaches.

Analysis of Mitochondrial Structure in the Body Wall Muscle of .

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

Presenilin mutations deregulate mitochondrial Ca homeostasis and metabolic activity causing neurodegeneration in .

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  to explore the effects of calcium dysregulation due to presenilin mutations on mitochondrial function, oxidative stress and neurodegeneration is explored.

Article	Year
Large isoforms of UNC-89 (obscurin) are required for muscle cell architecture and optimal calcium release in Caenorhabditis elegans. PloS one\| PubMed ID: 22768340	2012
VAV-1 acts in a single interneuron to inhibit motor circuit activity in Caenorhabditis elegans. Nature communications\| PubMed ID: 25412913	2014
A γ-Secretase Independent Role for Presenilin in Calcium Homeostasis Impacts Mitochondrial Function and Morphology in Caenorhabditis elegans. Genetics\| PubMed ID: 26500256	2015
A Conserved GEF for Rho-Family GTPases Acts in an EGF Signaling Pathway to Promote Sleep-like Quiescence in Caenorhabditis elegans. Genetics\| PubMed ID: 26801183	2016
Measurement of ROS in Using a Reduced Form of Fluorescein. Bio-protocol\| PubMed ID: 29707606	2018
Analysis of Mitochondrial Structure in the Body Wall Muscle of . Bio-protocol\| PubMed ID: 29707607	2018
Presenilin mutations deregulate mitochondrial Ca homeostasis and metabolic activity causing neurodegeneration in . eLife\| PubMed ID: 29989545	2018
Role of Presenilin in Mitochondrial Oxidative Stress and Neurodegeneration in . Antioxidants (Basel, Switzerland)\| PubMed ID: 30149498	2018

Kenneth R. Norman

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