Protein-based, fluorescent biosensors power basic research on cell signaling in health and disease, but their use in automated laboratories is limited. We have now created two live-cell assays, one ...for diacyl glycerol and another for cAMP, that are robust (Z′ > 0.7) and easily deployed on standard fluorescence plate readers. We describe the development of these assays, focusing on the parameters that were critical for optimization, in the hopes that the lessons learned can be generalized to the development of new biosensor-based assays.
Many neurodegenerative diseases induce high levels of sustained cellular stress and alter a number of cellular processes. To examine how different mutations associated with neurodegenerative disease ...affect cell stress and signaling, we created live-cell assays for endoplasmic reticulum (ER)-mediated cell stress and second messenger signaling. We first examined neurodegenerative mutations associated with direct ER stress by exploring the effect of rhodopsin mutations on ER stress and Ca
signaling. The rhodopsin P23H mutation, the most common mutation in autosomal dominant Retinitis Pigmentosa (RP), produced increased ER stress levels compared to wild type (WT) rhodopsin. Moreover, this increase in cell stress correlated with blunted Ca
signaling in a stress-dependent manner. Analysis of single-cell Ca
signaling profiles revealed unique Ca
signaling responses exist in cells expressing WT or P23H rhodopsin, consistent with the idea that second messenger signaling is affected by cell stress. To explore the use of the ER-stress biosensor in neurodegenerative diseases that may not have a direct effect on ER-mediated cell stress, we examined how various mutants of α-synuclein and TDP-43 affected ER stress. Mutants of both α-synuclein and TDP-43 associated with Parkinson's disease (PD) and Amyotrophic lateral sclerosis (ALS) demonstrated increased ER stress compared to WT proteins. To examine the effect of α-synuclein and TDP-43 mutants on cellular signaling, we created a second live-cell assay to monitor changes in cAMP signaling during expression of various forms of α-synuclein and TDP-43. The increased cell stress caused by expression of the mutant proteins was accompanied by changes in phosphodiesterase activity. Both HEK293T and SH-SY5Y cells expressing these proteins displayed a shift towards increased cAMP degradation rates, likely due to increased phosphodiesterase activity. Together these data illustrate how biosensors for cellular stress and signaling can provide nuanced, new views of neurodegenerative disease processes.
This study investigated the role of prion infection of the olfactory mucosa in the shedding of prion infectivity into nasal secretions. Prion infection with the HY strain of the transmissible mink ...encephalopathy (TME) agent resulted in a prominent infection of the olfactory bulb and the olfactory sensory epithelium including the olfactory receptor neurons (ORNs) and vomeronasal receptor neurons (VRNs), whose axons comprise the two olfactory cranial nerves. A distinct glycoform of the disease-specific isoform of the prion protein, PrP(Sc), was found in the olfactory mucosa compared to the olfactory bulb, but the total amount of HY TME infectivity in the nasal turbinates was within 100-fold of the titer in the olfactory bulb. PrP(Sc) co-localized with olfactory marker protein in the soma and dendrites of ORNs and VRNs and also with adenylyl cyclase III, which is present in the sensory cilia of ORNs that project into the lumen of the nasal airway. Nasal lavages from HY TME-infected hamsters contained prion titers as high as 10(3.9) median lethal doses per ml, which would be up to 500-fold more infectious in undiluted nasal fluids. These findings were confirmed using the rapid PrP(Sc) amplification QuIC assay, indicating that nasal swabs have the potential to be used for prion diagnostics. These studies demonstrate that prion infection in the olfactory epithelium is likely due to retrograde spread from the olfactory bulb along the olfactory and vomeronasal axons to the soma, dendrites, and cilia of these peripheral neurons. Since prions can replicate to high levels in neurons, we propose that ORNs can release prion infectivity into nasal fluids. The continual turnover and replacement of mature ORNs throughout the adult lifespan may also contribute to prion shedding from the nasal passage and could play a role in transmission of natural prion diseases in domestic and free-ranging ruminants.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
In this study, we investigated the role of damage to the nasal mucosa in the shedding of prions into nasal samples as a pathway for prion transmission. Here, we demonstrate that prions can replicate ...to high levels in the olfactory sensory epithelium (OSE) in hamsters and that induction of apoptosis in olfactory receptor neurons (ORNs) in the OSE resulted in sloughing off of the OSE from nasal turbinates into the lumen of the nasal airway. In the absence of nasotoxic treatment, olfactory marker protein (OMP), which is specific for ORNs, was not detected in nasal lavage samples. However, after nasotoxic treatment that leads to apoptosis of ORNs, both OMP and prion proteins were present in nasal lavage samples. The cellular debris that was released from the OSE into the lumen of the nasal airway was positive for both OMP and the disease-specific isoform of the prion protein, PrPSc. By using the real-time quaking-induced conversion assay to quantify prions, a 100- to 1,000-fold increase in prion seeding activity was observed in nasal lavage samples following nasotoxic treatment. Since neurons replicate prions to higher levels than other cell types and ORNs are the most environmentally exposed neurons, we propose that an increase in ORN apoptosis or damage to the nasal mucosa in a host with a preexisting prion infection of the OSE could lead to a substantial increase in the release of prion infectivity into nasal samples. This mechanism of prion shedding from the olfactory mucosa could contribute to prion transmission.
Chronic wasting disease (CWD) is an emerging prion disease of free-ranging and captive cervids in North America. In this study we established a rodent model for CWD in Syrian golden hamsters that ...resemble key features of the disease in cervids including cachexia and infection of cardiac muscle. Following one to three serial passages of CWD from white-tailed deer into transgenic mice expressing the hamster prion protein gene, CWD was subsequently passaged into Syrian golden hamsters. In one passage line there were preclinical changes in locomotor activity and a loss of body mass prior to onset of subtle neurological symptoms around 340 days. The clinical symptoms included a prominent wasting disease, similar to cachexia, with a prolonged duration. Other features of CWD in hamsters that were similar to cervid CWD included the brain distribution of the disease-specific isoform of the prion protein, PrP(Sc), prion infection of the central and peripheral neuroendocrine system, and PrP(Sc) deposition in cardiac muscle. There was also prominent PrP(Sc) deposition in the nasal mucosa on the edge of the olfactory sensory epithelium with the lumen of the nasal airway that could have implications for CWD shedding into nasal secretions and disease transmission. Since the mechanism of wasting disease in prion diseases is unknown this hamster CWD model could provide a means to investigate the physiological basis of cachexia, which we propose is due to a prion-induced endocrinopathy. This prion disease phenotype has not been described in hamsters and we designate it as the 'wasting' or WST strain of hamster CWD.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
GENIe is a Genetically Encoded Nucleotide Indicator for monitoring cGMP, a second messenger in cell signaling that mediates many important physiological processes. Phosphodiesterase (PDE) inhibitors ...and soluble guanylate cyclase (sGC) activators modulate cGMP levels to produce beneficial effects on pulmonary and heart disease, erectile dysfunction, and other conditions. The timing, location and strength of cGMP signaling is tightly regulated by nearly 100 isoforms of cyclic nucleotide PDEs (Bender and Bravo 2006). Some isoforms are specific for cGMP, while others are specific for cAMP. Some are nonspecific and act on both cAMP and cGMP.
We have developed a new, very bright mNeonGreen fluorescent biosensor to monitor cGMP levels in any living cell type. The sensor produces robust changes in fluorescence intensity that are readily detected on fluorescence imaging systems or plate readers. Since different PDE isoforms have different substrate specificities, we did parallel assays of cGMP and cAMP in HEK293 cells expressing either the cGMP biosensor (GENIe) or the cAMP biosensor (cADDis). These sensors produced markedly different fluorescent profiles of cGMP and cAMP responses when treated with different PDE inhibitors. Our results indicate that the combination of these assays provide useful insights into the specific action of a particular PDE inhibitor in a living cell. Since these sensors can be genetically targeted, and since many biologically relevant cell types express more than one PDE, these tools provide a new approach to understanding the specific action of PDE inhibitors in health and disease.
We tested GENIe in HEK293T cells on a BioTek Synergy MX automated fluorescence plate reader. cGMP production was initiated by adding sodium nitroprusside (SNP), a nitric oxide donor, in both the presence and absence of the PDE5 selective inhibitor, Sildenafil. Fluorescence intensity changed dramatically in the presence of Sildenafil, as degradation of cGMP is inhibited by the drug. To optimize the response in HEK293T cells, we over‐expressed sGC, treated with SNP, and observed a significant improvement in the amplitude of the signal accompanied by faster kinetics and an increased sensitivity to the nitric oxide donor SNP. We expect that endogenous levels of sGC will vary in different cell types and these parameters should be varied accordingly.
To examine PDE inhibitor specificity, we monitored responses from both cADDis and GENIe in the presence of different PDE inhibitors. IBMX, a non‐specific inhibitor, produced similar responses from both sensors. Sildenafil and Rolipram indicated cGMP vs cAMP specificity. Sildenafil exhibited selective inhibition of the cGMP‐specific PDE5, while Rolipram exhibited selective inhibition of the cAMP specific PDE4.
Support or Funding Information
Funding from NSF SBIR Phase IIB 1430878
This is from the Experimental Biology 2018 Meeting. There is no full text article associated with this published in The FASEB Journal.
Abstract only
We present a set of robust, easy to use, genetically encoded sensors for the detection of cyclic AMP (cAMP), diacyl‐glycerol (DAG), and PIP
2
in a variety of living cell types. The ...sensors fluoresce in either the green or red part of the visible spectrum, and they can be combined with one another, or other colored sensors, for the simultaneous detection of multiple components of signal transduction pathways. The sensors are packaged in BacMam, a modified baculovirus, under the control of CMV, synapsin, EF1a, or CAG promoter systems for ease of use in a wide variety of cells.
We present examples of the simultaneous imaging of cAMP and DAG, cAMP and PIP
2,
and DAG and PIP
2
kinetics. We use the cAMP sensors to detect, quantify, and compare Gi and Gs mediated signaling across CHO, HEK293, and NIH 3T3 cells. The cAMP sensor is remarkably sensitive, and it can be used to detect either Gs activation of adenyl cyclase or Gi inhibition of adenyl cyclase in the same cells. Notably, our technology allows for Gi detection without the need for forskolin or IBMX pre‐treatment. These sensors are not only easy to use on standard microscopes, but they excel on automated plate readers, producing Z’ values > 0.8.
Cell-based assays to detect Gαi signaling are often indirect, frequently involve complex pharmacological interventions, and are usually blind to the kinetics of the signaling. Our goal was to develop ...a simple, direct measure of Gαi signaling in living cells. We previously reported our fluorescent cADDis assay and showed that it reliably detects Gαs-mediated increases in cAMP levels. Agonists that stimulate a Gs-coupled receptor produce changes in the intensity of bright green or red fluorescent protein sensors that can be followed over time using automated fluorescence plate readers or fluorescence imaging systems. Since the cADDis sensors can monitor Gαs-mediated increases in adenylyl cyclase activity, in theory they should also be capable of detecting Gαi-mediated decreases. Here we apply our green fluorescent cADDis sensor to the detection of Gαi-mediated inhibition of adenylyl cyclase activity. We validated and optimized the assay in living HEK 293T cells using several known Gαi-coupled receptors and agonists, and we report robust Z' statistics and consistent EC
responses.
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