Position
Chief,
Cellular Stress and Inflammation Section
Associate Director,
HIV Research
Director,
Transgenic Rat Project
Deputy Associate Director of Technology,
Technology Development Initiative
Contact
Biomedical Research Center251 Bayview Boulevard
Suite 200
Room 06A713
Baltimore, MD 21224
Phone: 667-312-5194
Email: bharvey@mail.nih.gov
Education
Ph.D. - University of Rochester, Neurobiology and Anatomy
Research Interests
Dr. Harvey received his Ph.D. in Neurobiology and Anatomy from the University of Rochester where he began his interest in the cellular mechanisms of neuronal dysfunction. He worked on gene delivery and gene therapy in models of central nervous system diseases. He joined NIDA in 2002 where he brought methods for viral vector production to the NIDA IRP. Dr. Harvey’s research uses viral vectors to study the molecular and cellular processes associated with substance abuse using rodent models of drug toxicity and neurodegeneration. His laboratory recently discovered a cellular phenomenon termed “exodosis” which occurs when resident proteins within a cell’s endoplasmic reticulum are secreted in response to calcium depletion. His research program studies pathophysiological consequences of exodosis and ER stress triggered by substances of abuse and other neurological diseases. Through a collaboration with the National Center for Advancing Translational Sciences, a high throughput screen has identified novel and FDA-approved drugs capable of alleviating ER stress and exodosis to potentially treat human diseases. Lastly, Dr. Harvey’s group is developing blood-based biomarkers to monitor exodosis and ER stress in human disease.
Publications
Selected Publications
2024
Dossat, Amanda M; Trychta, Kathleen A; Glotfelty, Elliot J; Hinkle, Joshua J; Fortuno, Lowella V; Gore, Lana N; Richie, Christopher T; Harvey, Brandon K
Excitotoxic glutamate levels cause the secretion of resident endoplasmic reticulum proteins Journal Article
In: J Neurochem, vol. 168, no. 9, pp. 2461–2478, 2024, ISSN: 1471-4159.
@article{pmid38491746,
title = {Excitotoxic glutamate levels cause the secretion of resident endoplasmic reticulum proteins},
author = {Amanda M Dossat and Kathleen A Trychta and Elliot J Glotfelty and Joshua J Hinkle and Lowella V Fortuno and Lana N Gore and Christopher T Richie and Brandon K Harvey},
url = {https://pubmed.ncbi.nlm.nih.gov/38491746/},
doi = {10.1111/jnc.16093},
issn = {1471-4159},
year = {2024},
date = {2024-09-01},
urldate = {2024-09-01},
journal = {J Neurochem},
volume = {168},
number = {9},
pages = {2461--2478},
abstract = {Dysregulation of synaptic glutamate levels can lead to excitotoxicity such as that observed in stroke, traumatic brain injury, and epilepsy. The role of increased intracellular calcium (Ca) in the development of excitotoxicity is well established. However, less is known regarding the impact of glutamate on endoplasmic reticulum (ER)-Ca-mediated processes such as proteostasis. To investigate this, we expressed a secreted ER Ca modulated protein (SERCaMP) in primary cortical neurons to monitor exodosis, a phenomenon whereby ER calcium depletion causes the secretion of ER-resident proteins that perform essential functions to the ER and the cell. Activation of glutamatergic receptors (GluRs) led to an increase in SERCaMP secretion indicating that normally ER-resident proteins are being secreted in a manner consistent with ER Ca depletion. Antagonism of ER Ca channels attenuated the effects of glutamate and GluR agonists on SERCaMP release. We also demonstrate that endogenous proteins containing an ER retention/retrieval sequence (ERS) are secreted in response to GluR activation supporting that neuronal activation by glutamate promotes ER exodosis. Ectopic expression of KDEL receptors attenuated the secretion of ERS-containing proteins caused by GluR agonists. Taken together, our data indicate that excessive GluR activation causes disruption of neuronal proteostasis by triggering the secretion of ER-resident proteins through ER Ca depletion and describes a new facet of excitotoxicity.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}
Dysregulation of synaptic glutamate levels can lead to excitotoxicity such as that observed in stroke, traumatic brain injury, and epilepsy. The role of increased intracellular calcium (Ca) in the development of excitotoxicity is well established. However, less is known regarding the impact of glutamate on endoplasmic reticulum (ER)-Ca-mediated processes such as proteostasis. To investigate this, we expressed a secreted ER Ca modulated protein (SERCaMP) in primary cortical neurons to monitor exodosis, a phenomenon whereby ER calcium depletion causes the secretion of ER-resident proteins that perform essential functions to the ER and the cell. Activation of glutamatergic receptors (GluRs) led to an increase in SERCaMP secretion indicating that normally ER-resident proteins are being secreted in a manner consistent with ER Ca depletion. Antagonism of ER Ca channels attenuated the effects of glutamate and GluR agonists on SERCaMP release. We also demonstrate that endogenous proteins containing an ER retention/retrieval sequence (ERS) are secreted in response to GluR activation supporting that neuronal activation by glutamate promotes ER exodosis. Ectopic expression of KDEL receptors attenuated the secretion of ERS-containing proteins caused by GluR agonists. Taken together, our data indicate that excessive GluR activation causes disruption of neuronal proteostasis by triggering the secretion of ER-resident proteins through ER Ca depletion and describes a new facet of excitotoxicity.
2023
Svarcbahs, Reinis; Blossom, Sarah M; Baffoe-Bonnie, Helena S; Trychta, Kathleen A; Greer, Lacey K; Pickel, James; Henderson, Mark J; Harvey, Brandon K
A transgenic mouse line for assaying tissue-specific changes in endoplasmic reticulum proteostasis Journal Article
In: Transgenic Res, vol. 32, no. 3, pp. 209–221, 2023, ISSN: 1573-9368.
@article{pmid37133648,
title = {A transgenic mouse line for assaying tissue-specific changes in endoplasmic reticulum proteostasis},
author = {Reinis Svarcbahs and Sarah M Blossom and Helena S Baffoe-Bonnie and Kathleen A Trychta and Lacey K Greer and James Pickel and Mark J Henderson and Brandon K Harvey},
url = {https://pubmed.ncbi.nlm.nih.gov/37133648/},
doi = {10.1007/s11248-023-00349-7},
issn = {1573-9368},
year = {2023},
date = {2023-06-01},
urldate = {2023-06-01},
journal = {Transgenic Res},
volume = {32},
number = {3},
pages = {209--221},
abstract = {Maintenance of calcium homeostasis is important for proper endoplasmic reticulum (ER) function. When cellular stress conditions deplete the high concentration of calcium in the ER, ER-resident proteins are secreted into the extracellular space in a process called exodosis. Monitoring exodosis provides insight into changes in ER homeostasis and proteostasis resulting from cellular stress associated with ER calcium dysregulation. To monitor cell-type specific exodosis in the intact animal, we created a transgenic mouse line with a Gaussia luciferase (GLuc)-based, secreted ER calcium-modulated protein, SERCaMP, preceded by a LoxP-STOP-LoxP (LSL) sequence. The Cre-dependent LSL-SERCaMP mice were crossed with albumin (Alb)-Cre and dopamine transporter (DAT)-Cre mouse lines. GLuc-SERCaMP expression was characterized in mouse organs and extracellular fluids, and the secretion of GLuc-SERCaMP in response to cellular stress was monitored following pharmacological depletion of ER calcium. In LSL-SERCaMP × Alb-Cre mice, robust GLuc activity was observed only in the liver and blood, whereas in LSL-SERCaMP × DAT-Cre mice, GLuc activity was seen in midbrain dopaminergic neurons and tissue samples innervated by dopaminergic projections. After calcium depletion, we saw increased GLuc signal in the plasma and cerebrospinal fluid collected from the Alb-Cre and DAT-Cre crosses, respectively. This mouse model can be used to investigate the secretion of ER-resident proteins from specific cell and tissue types during disease pathogenesis and may aid in the identification of therapeutics and biomarkers of disease.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}
Maintenance of calcium homeostasis is important for proper endoplasmic reticulum (ER) function. When cellular stress conditions deplete the high concentration of calcium in the ER, ER-resident proteins are secreted into the extracellular space in a process called exodosis. Monitoring exodosis provides insight into changes in ER homeostasis and proteostasis resulting from cellular stress associated with ER calcium dysregulation. To monitor cell-type specific exodosis in the intact animal, we created a transgenic mouse line with a Gaussia luciferase (GLuc)-based, secreted ER calcium-modulated protein, SERCaMP, preceded by a LoxP-STOP-LoxP (LSL) sequence. The Cre-dependent LSL-SERCaMP mice were crossed with albumin (Alb)-Cre and dopamine transporter (DAT)-Cre mouse lines. GLuc-SERCaMP expression was characterized in mouse organs and extracellular fluids, and the secretion of GLuc-SERCaMP in response to cellular stress was monitored following pharmacological depletion of ER calcium. In LSL-SERCaMP × Alb-Cre mice, robust GLuc activity was observed only in the liver and blood, whereas in LSL-SERCaMP × DAT-Cre mice, GLuc activity was seen in midbrain dopaminergic neurons and tissue samples innervated by dopaminergic projections. After calcium depletion, we saw increased GLuc signal in the plasma and cerebrospinal fluid collected from the Alb-Cre and DAT-Cre crosses, respectively. This mouse model can be used to investigate the secretion of ER-resident proteins from specific cell and tissue types during disease pathogenesis and may aid in the identification of therapeutics and biomarkers of disease.
2022
Jäntti, Maria H; Jackson, Shelley N; Kuhn, Jeffrey; Parkkinen, Ilmari; Sree, Sreesha; Hinkle, Joshua J; Jokitalo, Eija; Deterding, Leesa J; Harvey, Brandon K
Palmitate and thapsigargin have contrasting effects on ER membrane lipid composition and ER proteostasis in neuronal cells Journal Article
In: Biochim Biophys Acta Mol Cell Biol Lipids, vol. 1867, no. 11, pp. 159219, 2022, ISSN: 1879-2618.
@article{pmid35981704,
title = {Palmitate and thapsigargin have contrasting effects on ER membrane lipid composition and ER proteostasis in neuronal cells},
author = {Maria H Jäntti and Shelley N Jackson and Jeffrey Kuhn and Ilmari Parkkinen and Sreesha Sree and Joshua J Hinkle and Eija Jokitalo and Leesa J Deterding and Brandon K Harvey},
url = {https://pubmed.ncbi.nlm.nih.gov/35981704/},
doi = {10.1016/j.bbalip.2022.159219},
issn = {1879-2618},
year = {2022},
date = {2022-11-01},
urldate = {2022-11-01},
journal = {Biochim Biophys Acta Mol Cell Biol Lipids},
volume = {1867},
number = {11},
pages = {159219},
abstract = {The endoplasmic reticulum (ER) is an organelle that performs several key functions such as protein synthesis and folding, lipid metabolism and calcium homeostasis. When these functions are disrupted, such as upon protein misfolding, ER stress occurs. ER stress can trigger adaptive responses to restore proper functioning such as activation of the unfolded protein response (UPR). In certain cells, the free fatty acid palmitate has been shown to induce the UPR. Here, we examined the effects of palmitate on UPR gene expression in a human neuronal cell line and compared it with thapsigargin, a known depletor of ER calcium and trigger of the UPR. We used a Gaussia luciferase-based reporter to assess how palmitate treatment affects ER proteostasis and calcium homeostasis in the cells. We also investigated how ER calcium depletion by thapsigargin affects lipid membrane composition by performing mass spectrometry on subcellular fractions and compared this to palmitate. Surprisingly, palmitate treatment did not activate UPR despite prominent changes to membrane phospholipids. Conversely, thapsigargin induced a strong UPR, but did not significantly change the membrane lipid composition in subcellular fractions. In summary, our data demonstrate that changes in membrane lipid composition and disturbances in ER calcium homeostasis have a minimal influence on each other in neuronal cells. These data provide new insight into the adaptive interplay of lipid homeostasis and proteostasis in the cell.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}
The endoplasmic reticulum (ER) is an organelle that performs several key functions such as protein synthesis and folding, lipid metabolism and calcium homeostasis. When these functions are disrupted, such as upon protein misfolding, ER stress occurs. ER stress can trigger adaptive responses to restore proper functioning such as activation of the unfolded protein response (UPR). In certain cells, the free fatty acid palmitate has been shown to induce the UPR. Here, we examined the effects of palmitate on UPR gene expression in a human neuronal cell line and compared it with thapsigargin, a known depletor of ER calcium and trigger of the UPR. We used a Gaussia luciferase-based reporter to assess how palmitate treatment affects ER proteostasis and calcium homeostasis in the cells. We also investigated how ER calcium depletion by thapsigargin affects lipid membrane composition by performing mass spectrometry on subcellular fractions and compared this to palmitate. Surprisingly, palmitate treatment did not activate UPR despite prominent changes to membrane phospholipids. Conversely, thapsigargin induced a strong UPR, but did not significantly change the membrane lipid composition in subcellular fractions. In summary, our data demonstrate that changes in membrane lipid composition and disturbances in ER calcium homeostasis have a minimal influence on each other in neuronal cells. These data provide new insight into the adaptive interplay of lipid homeostasis and proteostasis in the cell.
Greer, Lacey K; Meilleur, Katherine G; Harvey, Brandon K; Wires, Emily S
Identification of ER/SR resident proteins as biomarkers for ER/SR calcium depletion in skeletal muscle cells Journal Article
In: Orphanet J Rare Dis, vol. 17, no. 1, pp. 225, 2022, ISSN: 1750-1172.
@article{pmid35698232,
title = {Identification of ER/SR resident proteins as biomarkers for ER/SR calcium depletion in skeletal muscle cells},
author = {Lacey K Greer and Katherine G Meilleur and Brandon K Harvey and Emily S Wires},
url = {https://pubmed.ncbi.nlm.nih.gov/35698232/},
doi = {10.1186/s13023-022-02368-9},
issn = {1750-1172},
year = {2022},
date = {2022-06-01},
urldate = {2022-06-01},
journal = {Orphanet J Rare Dis},
volume = {17},
number = {1},
pages = {225},
abstract = {BACKGROUND: Aberrations to endoplasmic/sarcoplasmic reticulum (ER/SR) calcium concentration can result in the departure of endogenous proteins in a phenomenon termed exodosis. Redistribution of the ER/SR proteome can have deleterious effects to cell function and cell viability, often contributing to disease pathogenesis. Many proteins prone to exodosis reside in the ER/SR via an ER retention/retrieval sequence (ERS) and are involved in protein folding, protein modification, and protein trafficking. While the consequences of their extracellular presence have yet to be fully delineated, the proteins that have undergone exodosis may be useful for biomarker development. Skeletal muscle cells rely upon tightly coordinated ER/SR calcium release for muscle contractions, and perturbations to calcium homeostasis can result in myopathies. Ryanodine receptor type-1 (RYR1) is a calcium release channel located in the SR. Mutations to the RYR1 gene can compromise calcium homeostasis leading to a vast range of clinical phenotypes encompassing hypotonia, myalgia, respiratory insufficiency, ophthalmoplegia, fatigue and malignant hyperthermia (MH). There are currently no FDA approved treatments for RYR1-related myopathies (RYR1-RM).nnRESULTS: Here we examine the exodosis profile of skeletal muscle cells following ER/SR calcium depletion. Proteomic analysis identified 4,465 extracellular proteins following ER/SR calcium depletion with 1,280 proteins significantly different than vehicle. A total of 54 ERS proteins were identified and 33 ERS proteins significantly increased following ER/SR calcium depletion. Specifically, ERS protein, mesencephalic astrocyte-derived neurotrophic factor (MANF), was elevated following calcium depletion, making it a potential biomarker candidate for human samples. Despite no significant elevation of MANF in plasma levels among healthy volunteers and RYR1-RM individuals, MANF plasma levels positively correlated with age in RYR1-RM individuals, presenting a potential biomarker of disease progression. Selenoprotein N (SEPN1) was also detected only in extracellular samples following ER/SR calcium depletion. This protein is integral to calcium handling and SEPN1 variants have a causal role in SEPN1-related myopathies (SEPN1-RM). Extracellular presence of ER/SR membrane proteins may provide new insight into proteomic alterations extending beyond ERS proteins. Pre-treatment of skeletal muscle cells with bromocriptine, an FDA approved drug recently found to have anti-exodosis effects, curbed exodosis of ER/SR resident proteins.nnCONCLUSION: Changes to the extracellular content caused by intracellular calcium dysregulation presents an opportunity for biomarker development and drug discovery.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}
BACKGROUND: Aberrations to endoplasmic/sarcoplasmic reticulum (ER/SR) calcium concentration can result in the departure of endogenous proteins in a phenomenon termed exodosis. Redistribution of the ER/SR proteome can have deleterious effects to cell function and cell viability, often contributing to disease pathogenesis. Many proteins prone to exodosis reside in the ER/SR via an ER retention/retrieval sequence (ERS) and are involved in protein folding, protein modification, and protein trafficking. While the consequences of their extracellular presence have yet to be fully delineated, the proteins that have undergone exodosis may be useful for biomarker development. Skeletal muscle cells rely upon tightly coordinated ER/SR calcium release for muscle contractions, and perturbations to calcium homeostasis can result in myopathies. Ryanodine receptor type-1 (RYR1) is a calcium release channel located in the SR. Mutations to the RYR1 gene can compromise calcium homeostasis leading to a vast range of clinical phenotypes encompassing hypotonia, myalgia, respiratory insufficiency, ophthalmoplegia, fatigue and malignant hyperthermia (MH). There are currently no FDA approved treatments for RYR1-related myopathies (RYR1-RM).nnRESULTS: Here we examine the exodosis profile of skeletal muscle cells following ER/SR calcium depletion. Proteomic analysis identified 4,465 extracellular proteins following ER/SR calcium depletion with 1,280 proteins significantly different than vehicle. A total of 54 ERS proteins were identified and 33 ERS proteins significantly increased following ER/SR calcium depletion. Specifically, ERS protein, mesencephalic astrocyte-derived neurotrophic factor (MANF), was elevated following calcium depletion, making it a potential biomarker candidate for human samples. Despite no significant elevation of MANF in plasma levels among healthy volunteers and RYR1-RM individuals, MANF plasma levels positively correlated with age in RYR1-RM individuals, presenting a potential biomarker of disease progression. Selenoprotein N (SEPN1) was also detected only in extracellular samples following ER/SR calcium depletion. This protein is integral to calcium handling and SEPN1 variants have a causal role in SEPN1-related myopathies (SEPN1-RM). Extracellular presence of ER/SR membrane proteins may provide new insight into proteomic alterations extending beyond ERS proteins. Pre-treatment of skeletal muscle cells with bromocriptine, an FDA approved drug recently found to have anti-exodosis effects, curbed exodosis of ER/SR resident proteins.nnCONCLUSION: Changes to the extracellular content caused by intracellular calcium dysregulation presents an opportunity for biomarker development and drug discovery.
Trychta, Kathleen A; Harvey, Brandon K
Caffeine and MDMA (Ecstasy) Exacerbate ER Stress Triggered by Hyperthermia Journal Article
In: Int J Mol Sci, vol. 23, no. 4, 2022, ISSN: 1422-0067.
@article{pmid35216090,
title = {Caffeine and MDMA (Ecstasy) Exacerbate ER Stress Triggered by Hyperthermia},
author = {Kathleen A Trychta and Brandon K Harvey},
url = {https://pubmed.ncbi.nlm.nih.gov/35216090/},
doi = {10.3390/ijms23041974},
issn = {1422-0067},
year = {2022},
date = {2022-02-01},
urldate = {2022-02-01},
journal = {Int J Mol Sci},
volume = {23},
number = {4},
abstract = {Drugs of abuse can cause local and systemic hyperthermia, a known trigger of endoplasmic reticulum (ER) stress and the unfolded protein response (UPR). Another trigger of ER stress and UPR is ER calcium depletion, which causes ER exodosis, the secretion of ER-resident proteins. In rodent models, club drugs such as 3,4-methylenedioxymethamphetamine (MDMA, 'ecstasy') can create hyperthermic conditions in the brain and cause toxicity that is affected by the environmental temperature and the presence of other drugs, such as caffeine. In human studies, MDMA stimulated an acute, dose-dependent increase in core body temperature, but an examination of caffeine and MDMA in combination remains a topic for clinical research. Here we examine the secretion of ER-resident proteins and activation of the UPR under combined exposure to MDMA and caffeine in a cellular model of hyperthermia. We show that hyperthermia triggers the secretion of normally ER-resident proteins, and that this aberrant protein secretion is potentiated by the presence of MDMA, caffeine, or a combination of the two drugs. Hyperthermia activates the UPR but the addition of MDMA or caffeine does not alter the canonical UPR gene expression despite the drug effects on ER exodosis of UPR-related proteins. One exception was increased mRNA levels in MDMA-treated cells exposed to hyperthermia. These findings suggest that club drug use under hyperthermic conditions exacerbates disruption of ER proteostasis, contributing to cellular toxicity.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}
Drugs of abuse can cause local and systemic hyperthermia, a known trigger of endoplasmic reticulum (ER) stress and the unfolded protein response (UPR). Another trigger of ER stress and UPR is ER calcium depletion, which causes ER exodosis, the secretion of ER-resident proteins. In rodent models, club drugs such as 3,4-methylenedioxymethamphetamine (MDMA, 'ecstasy') can create hyperthermic conditions in the brain and cause toxicity that is affected by the environmental temperature and the presence of other drugs, such as caffeine. In human studies, MDMA stimulated an acute, dose-dependent increase in core body temperature, but an examination of caffeine and MDMA in combination remains a topic for clinical research. Here we examine the secretion of ER-resident proteins and activation of the UPR under combined exposure to MDMA and caffeine in a cellular model of hyperthermia. We show that hyperthermia triggers the secretion of normally ER-resident proteins, and that this aberrant protein secretion is potentiated by the presence of MDMA, caffeine, or a combination of the two drugs. Hyperthermia activates the UPR but the addition of MDMA or caffeine does not alter the canonical UPR gene expression despite the drug effects on ER exodosis of UPR-related proteins. One exception was increased mRNA levels in MDMA-treated cells exposed to hyperthermia. These findings suggest that club drug use under hyperthermic conditions exacerbates disruption of ER proteostasis, contributing to cellular toxicity.
2021
Wires, Emily S; Trychta, Kathleen A; Kennedy, Lacey M; Harvey, Brandon K
The Function of KDEL Receptors as UPR Genes in Disease Journal Article
In: Int J Mol Sci, vol. 22, no. 11, 2021, ISSN: 1422-0067.
@article{pmid34063979,
title = {The Function of KDEL Receptors as UPR Genes in Disease},
author = {Emily S Wires and Kathleen A Trychta and Lacey M Kennedy and Brandon K Harvey},
url = {https://pubmed.ncbi.nlm.nih.gov/34063979/},
doi = {10.3390/ijms22115436},
issn = {1422-0067},
year = {2021},
date = {2021-05-01},
urldate = {2021-05-01},
journal = {Int J Mol Sci},
volume = {22},
number = {11},
abstract = {The KDEL receptor retrieval pathway is essential for maintaining resident proteins in the endoplasmic reticulum (ER) lumen. ER resident proteins serve a variety of functions, including protein folding and maturation. Perturbations to the lumenal ER microenvironment, such as calcium depletion, can cause protein misfolding and activation of the unfolded protein response (UPR). Additionally, ER resident proteins are secreted from the cell by overwhelming the KDEL receptor retrieval pathway. Recent data show that KDEL receptors are also activated during the UPR through the IRE1/XBP1 signaling pathway as an adaptive response to cellular stress set forth to reduce the loss of ER resident proteins. This review will discuss the emerging connection between UPR activation and KDEL receptors as it pertains to ER proteostasis and disease states.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}
The KDEL receptor retrieval pathway is essential for maintaining resident proteins in the endoplasmic reticulum (ER) lumen. ER resident proteins serve a variety of functions, including protein folding and maturation. Perturbations to the lumenal ER microenvironment, such as calcium depletion, can cause protein misfolding and activation of the unfolded protein response (UPR). Additionally, ER resident proteins are secreted from the cell by overwhelming the KDEL receptor retrieval pathway. Recent data show that KDEL receptors are also activated during the UPR through the IRE1/XBP1 signaling pathway as an adaptive response to cellular stress set forth to reduce the loss of ER resident proteins. This review will discuss the emerging connection between UPR activation and KDEL receptors as it pertains to ER proteostasis and disease states.
Henderson, Mark J; Trychta, Kathleen A; Yang, Shyh-Ming; Bäck, Susanne; Yasgar, Adam; Wires, Emily S; Danchik, Carina; Yan, Xiaokang; Yano, Hideaki; Shi, Lei; Wu, Kuo-Jen; Wang, Amy Q; Tao, Dingyin; ohalmi, Gergely Zahoránszky-KH; Hu, Xin; Xu, Xin; Maloney, David; Zakharov, Alexey V; Rai, Ganesha; Urano, Fumihiko; Airavaara, Mikko; Gavrilova, Oksana; Jadhav, Ajit; Wang, Yun; Simeonov, Anton; Harvey, Brandon K
A target-agnostic screen identifies approved drugs to stabilize the endoplasmic reticulum-resident proteome Journal Article
In: Cell Reports, vol. 35, no. 4, 2021, ISBN: 2211-1247.
@article{Henderson:2021aa,
title = {A target-agnostic screen identifies approved drugs to stabilize the endoplasmic reticulum-resident proteome},
author = {Mark J Henderson and Kathleen A Trychta and Shyh-Ming Yang and Susanne Bäck and Adam Yasgar and Emily S Wires and Carina Danchik and Xiaokang Yan and Hideaki Yano and Lei Shi and Kuo-Jen Wu and Amy Q Wang and Dingyin Tao and Gergely Zahoránszky-K{H o}halmi and Xin Hu and Xin Xu and David Maloney and Alexey V Zakharov and Ganesha Rai and Fumihiko Urano and Mikko Airavaara and Oksana Gavrilova and Ajit Jadhav and Yun Wang and Anton Simeonov and Brandon K Harvey},
url = {https://pubmed.ncbi.nlm.nih.gov/33910017/},
doi = {10.1016/j.celrep.2021.109040},
isbn = {2211-1247},
year = {2021},
date = {2021-04-27},
urldate = {2021-04-27},
booktitle = {Cell Reports},
journal = {Cell Reports},
volume = {35},
number = {4},
publisher = {Elsevier},
abstract = {Endoplasmic reticulum (ER) dysregulation is associated with pathologies including neurodegenerative, muscular, and diabetic conditions. Depletion of ER calcium can lead to the loss of resident proteins in a process termed exodosis. To identify compounds that attenuate the redistribution of ER proteins under pathological conditions, we performed a quantitative high-throughput screen using the Gaussia luciferase (GLuc)-secreted ER calcium modulated protein (SERCaMP) assay, which monitors secretion of ER-resident proteins triggered by calcium depletion. We identify several clinically used drugs, including bromocriptine, and further characterize them using assays to measure effects on ER calcium, ER stress, and ER exodosis. Bromocriptine elicits protective effects in cell-based models of exodosis as well as in vivo models of stroke and diabetes. Bromocriptine analogs with reduced dopamine receptor activity retain similar efficacy in stabilizing the ER proteome, indicating a non-canonical mechanism of action. This study describes a strategic approach to identify small-molecule drugs capable of improving ER proteostasis in human disease conditions.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}
Endoplasmic reticulum (ER) dysregulation is associated with pathologies including neurodegenerative, muscular, and diabetic conditions. Depletion of ER calcium can lead to the loss of resident proteins in a process termed exodosis. To identify compounds that attenuate the redistribution of ER proteins under pathological conditions, we performed a quantitative high-throughput screen using the Gaussia luciferase (GLuc)-secreted ER calcium modulated protein (SERCaMP) assay, which monitors secretion of ER-resident proteins triggered by calcium depletion. We identify several clinically used drugs, including bromocriptine, and further characterize them using assays to measure effects on ER calcium, ER stress, and ER exodosis. Bromocriptine elicits protective effects in cell-based models of exodosis as well as in vivo models of stroke and diabetes. Bromocriptine analogs with reduced dopamine receptor activity retain similar efficacy in stabilizing the ER proteome, indicating a non-canonical mechanism of action. This study describes a strategic approach to identify small-molecule drugs capable of improving ER proteostasis in human disease conditions.
Trychta, Kathleen A; Xie, Bing; Verma, Ravi Kumar; Xu, Min; Shi, Lei; Harvey, Brandon K
Computational Modeling of C-Terminal Tails to Predict the Calcium-Dependent Secretion of Endoplasmic Reticulum Resident Proteins Journal Article
In: Front Chem, vol. 9, pp. 689608, 2021, ISSN: 2296-2646.
@article{pmid34268295,
title = {Computational Modeling of C-Terminal Tails to Predict the Calcium-Dependent Secretion of Endoplasmic Reticulum Resident Proteins},
author = {Kathleen A Trychta and Bing Xie and Ravi Kumar Verma and Min Xu and Lei Shi and Brandon K Harvey},
url = {34268295},
doi = {10.3389/fchem.2021.689608},
issn = {2296-2646},
year = {2021},
date = {2021-01-01},
urldate = {2021-01-01},
journal = {Front Chem},
volume = {9},
pages = {689608},
abstract = {The lumen of the endoplasmic reticulum (ER) has resident proteins that are critical to perform the various tasks of the ER such as protein maturation and lipid metabolism. These ER resident proteins typically have a carboxy-terminal ER retention/retrieval sequence (ERS). The canonical ERS that promotes ER retrieval is Lys-Asp-Glu-Leu (KDEL) and when an ER resident protein moves from the ER to the Golgi, KDEL receptors (KDELRs) in the Golgi recognize the ERS and return the protein to the ER lumen. Depletion of ER calcium leads to the mass departure of ER resident proteins in a process termed exodosis, which is regulated by KDELRs. Here, by combining computational prediction with machine learning-based models and experimental validation, we identify carboxy tail sequences of ER resident proteins divergent from the canonical "KDEL" ERS. Using molecular modeling and simulations, we demonstrated that two representative non-canonical ERS can stably bind to the KDELR. Collectively, we developed a method to predict whether a carboxy-terminal sequence acts as a putative ERS that would undergo secretion in response to ER calcium depletion and interacts with the KDELRs. The interaction between the ERS and the KDELR extends beyond the final four carboxy terminal residues of the ERS. Identification of proteins that undergo exodosis will further our understanding of changes in ER proteostasis under physiological and pathological conditions where ER calcium is depleted.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}
The lumen of the endoplasmic reticulum (ER) has resident proteins that are critical to perform the various tasks of the ER such as protein maturation and lipid metabolism. These ER resident proteins typically have a carboxy-terminal ER retention/retrieval sequence (ERS). The canonical ERS that promotes ER retrieval is Lys-Asp-Glu-Leu (KDEL) and when an ER resident protein moves from the ER to the Golgi, KDEL receptors (KDELRs) in the Golgi recognize the ERS and return the protein to the ER lumen. Depletion of ER calcium leads to the mass departure of ER resident proteins in a process termed exodosis, which is regulated by KDELRs. Here, by combining computational prediction with machine learning-based models and experimental validation, we identify carboxy tail sequences of ER resident proteins divergent from the canonical "KDEL" ERS. Using molecular modeling and simulations, we demonstrated that two representative non-canonical ERS can stably bind to the KDELR. Collectively, we developed a method to predict whether a carboxy-terminal sequence acts as a putative ERS that would undergo secretion in response to ER calcium depletion and interacts with the KDELRs. The interaction between the ERS and the KDELR extends beyond the final four carboxy terminal residues of the ERS. Identification of proteins that undergo exodosis will further our understanding of changes in ER proteostasis under physiological and pathological conditions where ER calcium is depleted.
2020
Jӓntti, Maria; Harvey, Brandon K
Trophic activities of endoplasmic reticulum proteins CDNF and MANF Journal Article
In: Cell Tissue Res, vol. 382, no. 1, pp. 83–100, 2020, ISSN: 1432-0878.
@article{pmid32845431,
title = {Trophic activities of endoplasmic reticulum proteins CDNF and MANF},
author = {Maria Jӓntti and Brandon K Harvey},
url = {https://pubmed.ncbi.nlm.nih.gov/32845431/},
doi = {10.1007/s00441-020-03263-0},
issn = {1432-0878},
year = {2020},
date = {2020-10-01},
urldate = {2020-10-01},
journal = {Cell Tissue Res},
volume = {382},
number = {1},
pages = {83--100},
abstract = {Mesencephalic astrocyte-derived neurotrophic factor (MANF) and cerebral dopamine neurotrophic factor (CDNF) are endoplasmic reticulum (ER) luminal proteins that confer trophic activities in a wide range of tissues under diverse pathological conditions. Despite initially being classified as neurotrophic factors, neither protein structurally nor functionally resembles bona fide neurotrophic factors. Their highly homologous structures comprise a unique globular, saposin-like domain within the N-terminus joined by a flexible linker to a C-terminus containing a SAP-like domain, CXXC motif and an ER retention sequence. Neurotrophic factors exert effects by binding to cognate receptors in the plasma membrane; however, no cell surface receptors have been identified for MANF and CDNF. Both can act as unfolded protein response (UPR) genes that modulate the UPR and inflammatory processes. The trophic activity of MANF and CDNF extends beyond the central nervous system with MANF being crucial for the development of pancreatic β cells and both have trophic effects in a variety of diseases related to the liver, heart, skeletal tissue, kidney and peripheral nervous system. In this article, the unique features of MANF and CDNF, such as their structure and mechanisms of action related to ER stress and inflammation, will be reviewed. Recently identified interactions with lipids and membrane trafficking will also be described. Lastly, their function and therapeutic potential in different diseases including a recent clinical trial using CDNF to treat Parkinson's disease will be discussed. Collectively, this review will highlight MANF and CDNF as broad-acting trophic factors that regulate functions of the endoplasmic reticulum.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}
Mesencephalic astrocyte-derived neurotrophic factor (MANF) and cerebral dopamine neurotrophic factor (CDNF) are endoplasmic reticulum (ER) luminal proteins that confer trophic activities in a wide range of tissues under diverse pathological conditions. Despite initially being classified as neurotrophic factors, neither protein structurally nor functionally resembles bona fide neurotrophic factors. Their highly homologous structures comprise a unique globular, saposin-like domain within the N-terminus joined by a flexible linker to a C-terminus containing a SAP-like domain, CXXC motif and an ER retention sequence. Neurotrophic factors exert effects by binding to cognate receptors in the plasma membrane; however, no cell surface receptors have been identified for MANF and CDNF. Both can act as unfolded protein response (UPR) genes that modulate the UPR and inflammatory processes. The trophic activity of MANF and CDNF extends beyond the central nervous system with MANF being crucial for the development of pancreatic β cells and both have trophic effects in a variety of diseases related to the liver, heart, skeletal tissue, kidney and peripheral nervous system. In this article, the unique features of MANF and CDNF, such as their structure and mechanisms of action related to ER stress and inflammation, will be reviewed. Recently identified interactions with lipids and membrane trafficking will also be described. Lastly, their function and therapeutic potential in different diseases including a recent clinical trial using CDNF to treat Parkinson's disease will be discussed. Collectively, this review will highlight MANF and CDNF as broad-acting trophic factors that regulate functions of the endoplasmic reticulum.
2019
Campbell, Lee A; Richie, Christopher T; Maggirwar, Nishad S; Harvey, Brandon K
Cas9 Ribonucleoprotein Complex Delivery: Methods and Applications for Neuroinflammation. Journal Article
In: J Neuroimmune Pharmacol, vol. 14, no. 4, pp. 565–577, 2019, ISSN: 1557-1904 (Electronic); 1557-1890 (Linking).
@article{Campbell:2019aa,
title = {Cas9 Ribonucleoprotein Complex Delivery: Methods and Applications for Neuroinflammation.},
author = {Lee A Campbell and Christopher T Richie and Nishad S Maggirwar and Brandon K Harvey},
url = {https://www.ncbi.nlm.nih.gov/pubmed/31172397},
doi = {10.1007/s11481-019-09856-z},
issn = {1557-1904 (Electronic); 1557-1890 (Linking)},
year = {2019},
date = {2019-12-01},
urldate = {2019-12-01},
journal = {J Neuroimmune Pharmacol},
volume = {14},
number = {4},
pages = {565--577},
address = {Intramural Research Program, Biomedical Research Center, National Institute on Drug Abuse, Suite 200, 251 Bayview Blvd, Baltimore, MD, 21224, USA. lee.campbell@nih.gov.},
abstract = {The CRISPR/Cas9 system is a revolutionary gene editing technology that combines simplicity of use and efficiency of mutagenesis. As this technology progresses toward human therapies, valid concerns including off-target mutations and immunogenicity must be addressed. One approach to address these issues is to minimize the presence of the CRISPR/Cas9 components by maintaining a tighter temporal control of Cas9 endonuclease and reducing the time period of activity. This has been achieved to some degree by delivering the CRISPR/Cas9 system via pre-formed Cas9 + gRNA ribonucleoprotein (RNP) complexes. In this review, we first discuss the molecular modifications that can be made using CRISPR/Cas9 and provide an overview of current methods for delivering Cas9 RNP complexes both in vitro and in vivo. We conclude with examples of how Cas9 RNP delivery may be used to target neuroinflammatory processes, namely in regard to viral infections of the central nervous system and neurodegenerative diseases. We propose that Cas9 RNP delivery is a viable approach when considering the CRISPR/Cas9 system for both experimentation and the treatment of disease. Graphical Abstract.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}
The CRISPR/Cas9 system is a revolutionary gene editing technology that combines simplicity of use and efficiency of mutagenesis. As this technology progresses toward human therapies, valid concerns including off-target mutations and immunogenicity must be addressed. One approach to address these issues is to minimize the presence of the CRISPR/Cas9 components by maintaining a tighter temporal control of Cas9 endonuclease and reducing the time period of activity. This has been achieved to some degree by delivering the CRISPR/Cas9 system via pre-formed Cas9 + gRNA ribonucleoprotein (RNP) complexes. In this review, we first discuss the molecular modifications that can be made using CRISPR/Cas9 and provide an overview of current methods for delivering Cas9 RNP complexes both in vitro and in vivo. We conclude with examples of how Cas9 RNP delivery may be used to target neuroinflammatory processes, namely in regard to viral infections of the central nervous system and neurodegenerative diseases. We propose that Cas9 RNP delivery is a viable approach when considering the CRISPR/Cas9 system for both experimentation and the treatment of disease. Graphical Abstract.
Back, Susanne; Dossat, Amanda; Parkkinen, Ilmari; Koivula, Pyry; Airavaara, Mikko; Richie, Christopher T; Chen, Yun-Hsiang; Wang, Yun; Harvey, Brandon K
Neuronal Activation Stimulates Cytomegalovirus Promoter-Driven Transgene Expression. Journal Article
In: Mol Ther Methods Clin Dev, vol. 14, pp. 180–188, 2019, ISSN: 2329-0501 (Print); 2329-0501 (Linking).
@article{Back:2019aab,
title = {Neuronal Activation Stimulates Cytomegalovirus Promoter-Driven Transgene Expression.},
author = {Susanne Back and Amanda Dossat and Ilmari Parkkinen and Pyry Koivula and Mikko Airavaara and Christopher T Richie and Yun-Hsiang Chen and Yun Wang and Brandon K Harvey},
url = {https://www.ncbi.nlm.nih.gov/pubmed/31380464},
doi = {10.1016/j.omtm.2019.06.006},
issn = {2329-0501 (Print); 2329-0501 (Linking)},
year = {2019},
date = {2019-07-03},
urldate = {2019-07-03},
journal = {Mol Ther Methods Clin Dev},
volume = {14},
pages = {180--188},
address = {Molecular Mechanisms of Cellular Stress and Inflammation Section, National Institute on Drug Abuse, NIH, Baltimore, MD 21224, USA.},
abstract = {The cytomegalovirus (CMV) immediate early promoter has been extensively developed and exploited for transgene expression in vitro and in vivo, including human clinical trials. The CMV promoter has long been considered a stable, constitutive, and ubiquitous promoter for transgene expression. Using two different CMV-based promoters, we found an increase in CMV-driven transgene expression in the rodent brain and in primary neuronal cultures in response to methamphetamine, glutamate, kainic acid, and activation of G protein-coupled receptor signaling using designer receptors exclusively activated by designer drugs (DREADDs). In contrast, promoters derived from human synapsin 1 (hSYN1) gene or elongation factor 1alpha (EF1alpha) did not exhibit altered transgene expression in response to the same neuronal stimulations. Overall, our results suggest that the long-standing assertion that the CMV promoter confers constitutive expression in neurons should be reevaluated, and future studies should empirically determine the activity of the CMV promoter in a given application.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}
The cytomegalovirus (CMV) immediate early promoter has been extensively developed and exploited for transgene expression in vitro and in vivo, including human clinical trials. The CMV promoter has long been considered a stable, constitutive, and ubiquitous promoter for transgene expression. Using two different CMV-based promoters, we found an increase in CMV-driven transgene expression in the rodent brain and in primary neuronal cultures in response to methamphetamine, glutamate, kainic acid, and activation of G protein-coupled receptor signaling using designer receptors exclusively activated by designer drugs (DREADDs). In contrast, promoters derived from human synapsin 1 (hSYN1) gene or elongation factor 1alpha (EF1alpha) did not exhibit altered transgene expression in response to the same neuronal stimulations. Overall, our results suggest that the long-standing assertion that the CMV promoter confers constitutive expression in neurons should be reevaluated, and future studies should empirically determine the activity of the CMV promoter in a given application.
Back, Susanne; Necarsulmer, Julie; Whitaker, Leslie R; Coke, Lamarque M; Koivula, Pyry; Heathward, Emily J; Fortuno, Lowella V; Zhang, Yajun; Yeh, Grace C; Baldwin, Heather A; Spencer, Morgan D; Mejias-Aponte, Carlos A; Pickel, James; Hoffman, Alexander F; Spivak, Charles E; Lupica, Carl R; Underhill, Suzanne M; Amara, Susan G; Domanskyi, Andrii; Anttila, Jenni E; Airavaara, Mikko; Hope, Bruce T; Hamra, Kent F; Richie, Christopher T; Harvey, Brandon K
Neuron-Specific Genome Modification in the Adult Rat Brain Using CRISPR-Cas9 Transgenic Rats. Journal Article
In: Neuron, vol. 102, no. 1, pp. 105–119, 2019, ISSN: 1097-4199 (Electronic); 0896-6273 (Linking).
@article{Back:2019aac,
title = {Neuron-Specific Genome Modification in the Adult Rat Brain Using CRISPR-Cas9 Transgenic Rats.},
author = {Susanne Back and Julie Necarsulmer and Leslie R Whitaker and Lamarque M Coke and Pyry Koivula and Emily J Heathward and Lowella V Fortuno and Yajun Zhang and Grace C Yeh and Heather A Baldwin and Morgan D Spencer and Carlos A Mejias-Aponte and James Pickel and Alexander F Hoffman and Charles E Spivak and Carl R Lupica and Suzanne M Underhill and Susan G Amara and Andrii Domanskyi and Jenni E Anttila and Mikko Airavaara and Bruce T Hope and Kent F Hamra and Christopher T Richie and Brandon K Harvey},
url = {https://www.ncbi.nlm.nih.gov/pubmed/30792150},
doi = {10.1016/j.neuron.2019.01.035},
issn = {1097-4199 (Electronic); 0896-6273 (Linking)},
year = {2019},
date = {2019-04-03},
urldate = {2019-04-03},
journal = {Neuron},
volume = {102},
number = {1},
pages = {105--119},
address = {Molecular Mechanisms of Cellular Stress and Inflammation Section, Intramural Research Program, National Institute on Drug Abuse, National Institutes of Health, Baltimore, MD 21224, USA.},
abstract = {Historically, the rat has been the preferred animal model for behavioral studies. Limitations in genome modification have, however, caused a lag in their use compared to the bevy of available transgenic mice. Here, we have developed several transgenic tools, including viral vectors and transgenic rats, for targeted genome modification in specific adult rat neurons using CRISPR-Cas9 technology. Starting from wild-type rats, knockout of tyrosine hydroxylase was achieved with adeno-associated viral (AAV) vectors expressing Cas9 or guide RNAs (gRNAs). We subsequently created an AAV vector for Cre-dependent gRNA expression as well as three new transgenic rat lines to specifically target CRISPR-Cas9 components to dopaminergic neurons. One rat represents the first knockin rat model made by germline gene targeting in spermatogonial stem cells. The rats described herein serve as a versatile platform for making cell-specific and sequence-specific genome modifications in the adult brain and potentially other Cre-expressing tissues of the rat.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}
Historically, the rat has been the preferred animal model for behavioral studies. Limitations in genome modification have, however, caused a lag in their use compared to the bevy of available transgenic mice. Here, we have developed several transgenic tools, including viral vectors and transgenic rats, for targeted genome modification in specific adult rat neurons using CRISPR-Cas9 technology. Starting from wild-type rats, knockout of tyrosine hydroxylase was achieved with adeno-associated viral (AAV) vectors expressing Cas9 or guide RNAs (gRNAs). We subsequently created an AAV vector for Cre-dependent gRNA expression as well as three new transgenic rat lines to specifically target CRISPR-Cas9 components to dopaminergic neurons. One rat represents the first knockin rat model made by germline gene targeting in spermatogonial stem cells. The rats described herein serve as a versatile platform for making cell-specific and sequence-specific genome modifications in the adult brain and potentially other Cre-expressing tissues of the rat.
Campbell, Lee A; Coke, Lamarque M; Richie, Christopher T; Fortuno, Lowella V; Park, Aaron Y; Harvey, Brandon K
Gesicle-Mediated Delivery of CRISPR/Cas9 Ribonucleoprotein Complex for Inactivating the HIV Provirus. Journal Article
In: Mol Ther, vol. 27, no. 1, pp. 151–163, 2019, ISSN: 1525-0024 (Electronic); 1525-0016 (Linking).
@article{Campbell:2019ab,
title = {Gesicle-Mediated Delivery of CRISPR/Cas9 Ribonucleoprotein Complex for Inactivating the HIV Provirus.},
author = {Lee A Campbell and Lamarque M Coke and Christopher T Richie and Lowella V Fortuno and Aaron Y Park and Brandon K Harvey},
url = {https://www.ncbi.nlm.nih.gov/pubmed/30389355},
doi = {10.1016/j.ymthe.2018.10.002},
issn = {1525-0024 (Electronic); 1525-0016 (Linking)},
year = {2019},
date = {2019-01-02},
urldate = {2019-01-02},
journal = {Mol Ther},
volume = {27},
number = {1},
pages = {151--163},
address = {Intramural Research Program, National Institute on Drug Abuse, Biomedical Research Center, Suite 200, 251 Bayview Boulevard, Baltimore, MD 21224, USA. Electronic address: lee.campbell@nih.gov.},
abstract = {Investigators have utilized the CRISPR/Cas9 gene-editing system to specifically target well-conserved regions of HIV, leading to decreased infectivity and pathogenesis in vitro and ex vivo. We utilized a specialized extracellular vesicle termed a "gesicle" to efficiently, yet transiently, deliver Cas9 in a ribonucleoprotein form targeting the HIV long terminal repeat (LTR). Gesicles are produced through expression of vesicular stomatitis virus glycoprotein and package protein as their cargo, thus bypassing the need for transgene delivery, and allowing finer control of Cas9 expression. Using both NanoSight particle and western blot analysis, we verified production of Cas9-containing gesicles by HEK293FT cells. Application of gesicles to CHME-5 microglia resulted in rapid but transient transfer of Cas9 by western blot, which is present at 1 hr, but is undetectable by 24 hr post-treatment. Gesicle delivery of Cas9 protein preloaded with guide RNA targeting the HIV LTR to HIV-NanoLuc CHME-5 cells generated mutations within the LTR region and copy number loss. Finally, we demonstrated that this treatment resulted in reduced proviral activity under basal conditions and after stimulation with pro-inflammatory factors lipopolysaccharide (LPS) or tumor necrosis factor alpha (TNF-alpha). These data suggest that gesicles are a viable alternative approach to deliver CRISPR/Cas9 technology.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}
Investigators have utilized the CRISPR/Cas9 gene-editing system to specifically target well-conserved regions of HIV, leading to decreased infectivity and pathogenesis in vitro and ex vivo. We utilized a specialized extracellular vesicle termed a "gesicle" to efficiently, yet transiently, deliver Cas9 in a ribonucleoprotein form targeting the HIV long terminal repeat (LTR). Gesicles are produced through expression of vesicular stomatitis virus glycoprotein and package protein as their cargo, thus bypassing the need for transgene delivery, and allowing finer control of Cas9 expression. Using both NanoSight particle and western blot analysis, we verified production of Cas9-containing gesicles by HEK293FT cells. Application of gesicles to CHME-5 microglia resulted in rapid but transient transfer of Cas9 by western blot, which is present at 1 hr, but is undetectable by 24 hr post-treatment. Gesicle delivery of Cas9 protein preloaded with guide RNA targeting the HIV LTR to HIV-NanoLuc CHME-5 cells generated mutations within the LTR region and copy number loss. Finally, we demonstrated that this treatment resulted in reduced proviral activity under basal conditions and after stimulation with pro-inflammatory factors lipopolysaccharide (LPS) or tumor necrosis factor alpha (TNF-alpha). These data suggest that gesicles are a viable alternative approach to deliver CRISPR/Cas9 technology.
2018
Trychta, Kathleen A; Heathward, Emily J; Sulima, Agnieszka; Back, Susanne; Farokhnia, Mehdi; Richie, Christopher T; Leggio, Lorenzo; Rice, Kenner C; Harvey, Brandon K
Extracellular esterase activity as an indicator of endoplasmic reticulum calcium depletion. Journal Article
In: Biomarkers, pp. 1–10, 2018, ISSN: 1366-5804 (Electronic); 1354-750X (Linking).
@article{Trychta:2018aa,
title = {Extracellular esterase activity as an indicator of endoplasmic reticulum calcium depletion.},
author = {Kathleen A Trychta and Emily J Heathward and Agnieszka Sulima and Susanne Back and Mehdi Farokhnia and Christopher T Richie and Lorenzo Leggio and Kenner C Rice and Brandon K Harvey},
url = {https://www.ncbi.nlm.nih.gov/pubmed/30095301},
doi = {10.1080/1354750X.2018.1490968},
issn = {1366-5804 (Electronic); 1354-750X (Linking)},
year = {2018},
date = {2018-08-10},
urldate = {2018-08-10},
journal = {Biomarkers},
pages = {1--10},
address = {a Molecular Mechanisms of Cellular Stress and Inflammation , Intramural Research Program, National Institute on Drug Abuse , Baltimore , MD , USA.},
abstract = {CONTEXT: Endoplasmic reticulum (ER) calcium depletion is associated with diverse diseases, including cardiac, hepatic, and neurologic diseases. OBJECTIVE: The aim of the present study was to identify and characterize an endogenous protein that could be used to monitor ER calcium depletion comparably to a previously described exogenous reporter protein. MATERIALS AND METHODS: The use of a selective esterase-fluorescein diester pair allowed for carboxylesterase activity in extracellular fluid to be measured using a fluorescent readout. Cell culture media from three different cell lines, rat plasma, and human serum all possess quantifiable amounts of esterase activity. RESULTS: Fluorescence produced by the interaction of carboxylesterases with a fluorescein diester substrate tracks with pharmacological and physiological inducers of ER calcium depletion. The fluorescence measured for in vitro and in vivo samples were consistent with ER calcium depletion being the trigger for increased esterase activity. DISCUSSION: Decreased luminal ER calcium causes ER resident esterases to be released from the cell, and, when assessed concurrently with other disease biomarkers, these esterases may provide insight into the role of ER calcium homeostasis in human diseases. CONCLUSION: Our results indicate that carboxylesterases are putative markers of ER calcium dysfunction.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}
CONTEXT: Endoplasmic reticulum (ER) calcium depletion is associated with diverse diseases, including cardiac, hepatic, and neurologic diseases. OBJECTIVE: The aim of the present study was to identify and characterize an endogenous protein that could be used to monitor ER calcium depletion comparably to a previously described exogenous reporter protein. MATERIALS AND METHODS: The use of a selective esterase-fluorescein diester pair allowed for carboxylesterase activity in extracellular fluid to be measured using a fluorescent readout. Cell culture media from three different cell lines, rat plasma, and human serum all possess quantifiable amounts of esterase activity. RESULTS: Fluorescence produced by the interaction of carboxylesterases with a fluorescein diester substrate tracks with pharmacological and physiological inducers of ER calcium depletion. The fluorescence measured for in vitro and in vivo samples were consistent with ER calcium depletion being the trigger for increased esterase activity. DISCUSSION: Decreased luminal ER calcium causes ER resident esterases to be released from the cell, and, when assessed concurrently with other disease biomarkers, these esterases may provide insight into the role of ER calcium homeostasis in human diseases. CONCLUSION: Our results indicate that carboxylesterases are putative markers of ER calcium dysfunction.
2017
Campbell, Lee A; Richie, Christopher T; Zhang, Yajun; Heathward, Emily J; Coke, Lamarque M; Park, Emily Y; Harvey, Brandon K
In vitro modeling of HIV proviral activity in microglia. Journal Article
In: FEBS J, vol. 284, no. 23, pp. 4096–4114, 2017, ISSN: 1742-4658 (Electronic); 1742-464X (Linking).
@article{Campbell:2017aa,
title = {In vitro modeling of HIV proviral activity in microglia.},
author = {Lee A Campbell and Christopher T Richie and Yajun Zhang and Emily J Heathward and Lamarque M Coke and Emily Y Park and Brandon K Harvey},
url = {https://www.ncbi.nlm.nih.gov/pubmed/29114997},
doi = {10.1111/febs.14293},
issn = {1742-4658 (Electronic); 1742-464X (Linking)},
year = {2017},
date = {2017-12-01},
urldate = {2017-12-01},
journal = {FEBS J},
volume = {284},
number = {23},
pages = {4096--4114},
address = {Intramural Research Program, National Institute on Drug Abuse, Biomedical Research Center, Baltimore, MD, USA.},
abstract = {Microglia, the resident macrophages of the brain, play a key role in the pathogenesis of HIV-associated neurocognitive disorders (HAND) due to their productive infection by HIV. This results in the release of neurotoxic viral proteins and pro-inflammatory compounds which negatively affect the functionality of surrounding neurons. Because models of HIV infection within the brain are limited, we aimed to create a novel microglia cell line with an integrated HIV provirus capable of recreating several hallmarks of HIV infection. We utilized clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 gene editing technology and integrated a modified HIV provirus into CHME-5 immortalized microglia to create HIV-NanoLuc CHME-5. In the modified provirus, the Gag-Pol region is replaced with the coding region for NanoLuciferase (NanoLuc), which allows for the rapid assay of HIV long terminal repeat activity using a luminescent substrate, while still containing the necessary genetic material to produce established neurotoxic viral proteins (e.g. tat, nef, gp120). We confirmed that HIV-NanoLuc CHME-5 microglia express NanoLuc, along with the HIV viral protein Nef. We subsequently exposed these cells to a battery of experiments to modulate the activity of the provirus. Proviral activity was enhanced by treating the cells with pro-inflammatory factors lipopolysaccharide (LPS) and tumor necrosis factor alpha and by overexpressing the viral regulatory protein Tat. Conversely, genetic modification of the toll-like receptor-4 gene by CRISPR/Cas9 reduced LPS-mediated proviral activation, and pharmacological application of NF-kappaB inhibitor sulfasalazine similarly diminished proviral activity. Overall, these data suggest that HIV-NanoLuc CHME-5 may be a useful tool in the study of HIV-mediated neuropathology and proviral regulation.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}
Microglia, the resident macrophages of the brain, play a key role in the pathogenesis of HIV-associated neurocognitive disorders (HAND) due to their productive infection by HIV. This results in the release of neurotoxic viral proteins and pro-inflammatory compounds which negatively affect the functionality of surrounding neurons. Because models of HIV infection within the brain are limited, we aimed to create a novel microglia cell line with an integrated HIV provirus capable of recreating several hallmarks of HIV infection. We utilized clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 gene editing technology and integrated a modified HIV provirus into CHME-5 immortalized microglia to create HIV-NanoLuc CHME-5. In the modified provirus, the Gag-Pol region is replaced with the coding region for NanoLuciferase (NanoLuc), which allows for the rapid assay of HIV long terminal repeat activity using a luminescent substrate, while still containing the necessary genetic material to produce established neurotoxic viral proteins (e.g. tat, nef, gp120). We confirmed that HIV-NanoLuc CHME-5 microglia express NanoLuc, along with the HIV viral protein Nef. We subsequently exposed these cells to a battery of experiments to modulate the activity of the provirus. Proviral activity was enhanced by treating the cells with pro-inflammatory factors lipopolysaccharide (LPS) and tumor necrosis factor alpha and by overexpressing the viral regulatory protein Tat. Conversely, genetic modification of the toll-like receptor-4 gene by CRISPR/Cas9 reduced LPS-mediated proviral activation, and pharmacological application of NF-kappaB inhibitor sulfasalazine similarly diminished proviral activity. Overall, these data suggest that HIV-NanoLuc CHME-5 may be a useful tool in the study of HIV-mediated neuropathology and proviral regulation.
Wires, Emily S; Trychta, Kathleen A; Back, Susanne; Sulima, Agnieszka; Rice, Kenner C; Harvey, Brandon K
High fat diet disrupts endoplasmic reticulum calcium homeostasis in the rat liver. Journal Article
In: J Hepatol, vol. 67, no. 5, pp. 1009–1017, 2017, ISSN: 1600-0641 (Electronic); 0168-8278 (Linking).
@article{cite-key,
title = {High fat diet disrupts endoplasmic reticulum calcium homeostasis in the rat liver.},
author = {Emily S Wires and Kathleen A Trychta and Susanne Back and Agnieszka Sulima and Kenner C Rice and Brandon K Harvey},
url = {https://www.ncbi.nlm.nih.gov/pubmed/28596111},
doi = {10.1016/j.jhep.2017.05.023},
issn = {1600-0641 (Electronic); 0168-8278 (Linking)},
year = {2017},
date = {2017-07-17},
urldate = {2017-07-17},
journal = {J Hepatol},
volume = {67},
number = {5},
pages = {1009--1017},
address = {Molecular Mechanisms of Cellular Stress and Inflammation Unit, Intramural Research Program, National Institute on Drug Abuse, Baltimore, MD 21224, USA.},
abstract = {BACKGROUND & AIMS: Disruption to endoplasmic reticulum (ER) calcium homeostasis has been implicated in obesity, however, the ability to longitudinally monitor ER calcium fluctuations has been challenging with prior methodologies. We recently described the development of a Gaussia luciferase (GLuc)-based reporter protein responsive to ER calcium depletion (GLuc-SERCaMP) and investigated the effect of a high fat diet on ER calcium homeostasis. METHODS: A GLuc-based reporter cell line was treated with palmitate, a free fatty acid. Rats intrahepatically injected with GLuc-SERCaMP reporter were fed a cafeteria diet or high fat diet. The liver and plasma were examined for established markers of steatosis and compared to plasma levels of SERCaMP activity. RESULTS: Palmitate induced GLuc-SERCaMP release in vitro, indicating ER calcium depletion. Consumption of a cafeteria diet or high fat pellets correlated with alterations to hepatic ER calcium homeostasis in rats, shown by increased GLuc-SERCaMP release. Access to ad lib high fat pellets also led to a corresponding decrease in microsomal calcium ATPase activity and an increase in markers of hepatic steatosis. In addition to GLuc-SERCaMP, we have also identified endogenous proteins (endogenous SERCaMPs) with a similar response to ER calcium depletion. We demonstrated the release of an endogenous SERCaMP, thought to be a liver esterase, during access to a high fat diet. Attenuation of both GLuc-SERCaMP and endogenous SERCaMP was observed during dantrolene administration. CONCLUSIONS: Here we describe the use of a reporter for in vitro and in vivo models of high fat diet. Our results support the theory that dietary fat intake correlates with a decrease in ER calcium levels in the liver and suggest a high fat diet alters the ER proteome. Lay summary: ER calcium dysregulation was observed in rats fed a cafeteria diet or high fat pellets, with fluctuations in sensor release correlating with fat intake. Attenuation of sensor release, as well as food intake was observed during administration of dantrolene, a drug that stabilizes ER calcium. The study describes a novel technique for liver research and provides insight into cellular processes that may contribute to the pathogenesis of obesity and fatty liver disease.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}
BACKGROUND & AIMS: Disruption to endoplasmic reticulum (ER) calcium homeostasis has been implicated in obesity, however, the ability to longitudinally monitor ER calcium fluctuations has been challenging with prior methodologies. We recently described the development of a Gaussia luciferase (GLuc)-based reporter protein responsive to ER calcium depletion (GLuc-SERCaMP) and investigated the effect of a high fat diet on ER calcium homeostasis. METHODS: A GLuc-based reporter cell line was treated with palmitate, a free fatty acid. Rats intrahepatically injected with GLuc-SERCaMP reporter were fed a cafeteria diet or high fat diet. The liver and plasma were examined for established markers of steatosis and compared to plasma levels of SERCaMP activity. RESULTS: Palmitate induced GLuc-SERCaMP release in vitro, indicating ER calcium depletion. Consumption of a cafeteria diet or high fat pellets correlated with alterations to hepatic ER calcium homeostasis in rats, shown by increased GLuc-SERCaMP release. Access to ad lib high fat pellets also led to a corresponding decrease in microsomal calcium ATPase activity and an increase in markers of hepatic steatosis. In addition to GLuc-SERCaMP, we have also identified endogenous proteins (endogenous SERCaMPs) with a similar response to ER calcium depletion. We demonstrated the release of an endogenous SERCaMP, thought to be a liver esterase, during access to a high fat diet. Attenuation of both GLuc-SERCaMP and endogenous SERCaMP was observed during dantrolene administration. CONCLUSIONS: Here we describe the use of a reporter for in vitro and in vivo models of high fat diet. Our results support the theory that dietary fat intake correlates with a decrease in ER calcium levels in the liver and suggest a high fat diet alters the ER proteome. Lay summary: ER calcium dysregulation was observed in rats fed a cafeteria diet or high fat pellets, with fluctuations in sensor release correlating with fat intake. Attenuation of sensor release, as well as food intake was observed during administration of dantrolene, a drug that stabilizes ER calcium. The study describes a novel technique for liver research and provides insight into cellular processes that may contribute to the pathogenesis of obesity and fatty liver disease.
Wires, Emily S; Henderson, Mark J; Yan, Xiaokang; Bäck, Susanne; Trychta, Kathleen A; Lutrey, Molly H; Harvey, Brandon K
Longitudinal monitoring of Gaussia and Nano luciferase activities to concurrently assess ER calcium homeostasis and ER stress in vivo Journal Article
In: PLOS ONE, vol. 12, no. 4, pp. 1-17, 2017.
@article{10.1371/journal.pone.0175481,
title = {Longitudinal monitoring of Gaussia and Nano luciferase activities to concurrently assess ER calcium homeostasis and ER stress in vivo},
author = {Emily S Wires and Mark J Henderson and Xiaokang Yan and Susanne Bäck and Kathleen A Trychta and Molly H Lutrey and Brandon K Harvey},
url = {https://pubmed.ncbi.nlm.nih.gov/28403212/},
doi = {10.1371/journal.pone.0175481},
year = {2017},
date = {2017-01-01},
urldate = {2017-01-01},
journal = {PLOS ONE},
volume = {12},
number = {4},
pages = {1-17},
publisher = {Public Library of Science},
abstract = {The endoplasmic reticulum (ER) is essential to many cellular processes including protein processing, lipid metabolism and calcium storage. The ability to longitudinally monitor ER homeostasis in the same organism would offer insight into progressive molecular and cellular adaptations to physiologic or pathologic states, but has been challenging. We recently described the creation of a Gaussia luciferase (GLuc)-based secreted ER calcium-modulated protein (SERCaMP or GLuc-SERCaMP) to longitudinally monitor ER calcium homeostasis. Here we describe a complementary tool to measure the unfolded protein response (UPR), utilizing a UPRE-driven secreted Nano luciferase (UPRE-secNLuc) to examine the activating transcription factor-6 (ATF6) and inositol-requiring enzyme 1 (IRE1) pathways of the UPR. We observed an upregulation of endogenous ATF6- and XBP1-regulated genes following pharmacologically-induced ER stress that was consistent with responsiveness of the UPRE sensor. Both GLuc and NLuc-based reporters have favorable properties for in vivo studies, however, they are not easily used in combination due to overlapping substrate activities. We describe a method to measure the enzymatic activities of both reporters from a single sample and validated the approach using culture medium and rat blood samples to measure GLuc-SERCaMP and UPRE-secNLuc. Measuring GLuc and NLuc activities from the same sample allows for the robust and quantitative measurement of two cellular events or cell populations from a single biological sample. This study is the first to describe the in vivo measurement of UPRE activation by sampling blood, using an approach that allows concurrent interrogation of two components of ER homeostasis.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}
The endoplasmic reticulum (ER) is essential to many cellular processes including protein processing, lipid metabolism and calcium storage. The ability to longitudinally monitor ER homeostasis in the same organism would offer insight into progressive molecular and cellular adaptations to physiologic or pathologic states, but has been challenging. We recently described the creation of a Gaussia luciferase (GLuc)-based secreted ER calcium-modulated protein (SERCaMP or GLuc-SERCaMP) to longitudinally monitor ER calcium homeostasis. Here we describe a complementary tool to measure the unfolded protein response (UPR), utilizing a UPRE-driven secreted Nano luciferase (UPRE-secNLuc) to examine the activating transcription factor-6 (ATF6) and inositol-requiring enzyme 1 (IRE1) pathways of the UPR. We observed an upregulation of endogenous ATF6- and XBP1-regulated genes following pharmacologically-induced ER stress that was consistent with responsiveness of the UPRE sensor. Both GLuc and NLuc-based reporters have favorable properties for in vivo studies, however, they are not easily used in combination due to overlapping substrate activities. We describe a method to measure the enzymatic activities of both reporters from a single sample and validated the approach using culture medium and rat blood samples to measure GLuc-SERCaMP and UPRE-secNLuc. Measuring GLuc and NLuc activities from the same sample allows for the robust and quantitative measurement of two cellular events or cell populations from a single biological sample. This study is the first to describe the in vivo measurement of UPRE activation by sampling blood, using an approach that allows concurrent interrogation of two components of ER homeostasis.
