Publications by authors named "King-Ho Cheung"

42 Publications

TFEB, a master regulator of autophagy and biogenesis, unexpectedly promotes apoptosis in response to the cyclopentenone prostaglandin 15d-PGJ2.

Acta Pharmacol Sin 2021 Aug 20. Epub 2021 Aug 20.

Mr. and Mrs. Ko Chi Ming Centre for Parkinson's Disease Research, School of Chinese Medicine, Hong Kong Baptist University, Hong Kong SAR, China.

Transcriptional factor EB (TFEB), a master regulator of autophagy and lysosomal biogenesis, is generally regarded as a pro-survival factor. Here, we identify that besides its effect on autophagy induction, TFEB exerts a pro-apoptotic effect in response to the cyclopentenone prostaglandin 15-deoxy-∆--prostaglandin J2 (15d-PGJ2). Specifically, 15d-PGJ2 promotes TFEB translocation from the cytoplasm into the nucleus to induce autophagy and lysosome biogenesis via reactive oxygen species (ROS) production rather than mTORC1 inactivation. Surprisingly, TFEB promotes rather than inhibits apoptosis in response to 15d-PGJ2. Mechanistically, ROS-mediated TFEB translocation into the nucleus transcriptionally upregulates the expression of ATF4, which is required for apoptosis elicited by 15d-PGJ2. Additionally, inhibition of TFEB activation by ROS scavenger N-acetyl cysteine or inhibition of protein synthesis by cycloheximide effectively compromises ATF4 upregulation and apoptosis in response to 15d-PGJ2. Collectively, these results indicate that ROS-induced TFEB activation exerts a novel role in promoting apoptosis besides its role in regulating autophagy in response to 15d-PGJ2. This work not only evidences how TFEB is activated by 15d-PGJ2, but also unveils a previously unexplored role of ROS-dependent activation of TFEB in modulating cell apoptosis in response to 15d-PGJ2.
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http://dx.doi.org/10.1038/s41401-021-00711-7DOI Listing
August 2021

Qingyangshen mitigates amyloid-β and Tau aggregate defects involving PPARα-TFEB activation in transgenic mice of Alzheimer's disease.

Phytomedicine 2021 Oct 12;91:153648. Epub 2021 Jul 12.

Mr. & Mrs. Ko Chi-Ming Centre for Parkinson's Disease Research, School of Chinese Medicine, Hong Kong Baptist University, Hong Kong SAR, China. Electronic address:

Background: Alzheimer's disease (AD) is the most common neurodegenerative disease. Deposition of amyloid β plaques (Aβ) and neurofibrillary tangles (NFTs) is the key pathological hallmark of AD. Accumulating evidence suggest that impairment of autophagy-lysosomal pathway (ALP) plays key roles in AD pathology.

Purpose: The present study aims to assess the neuroprotective effects of Qingyangshen (QYS), a Chinese herbal medicine, in AD cellular and animal models and to determine its underlying mechanisms involving ALP regulation.

Methods: QYS extract was prepared and its chemical components were characterized by LC/MS. Then the pharmacokinetics and acute toxicity of QYS extract were evaluated. The neuroprotective effects of QYS extract were determined in 3XTg AD mice, by using a series of behavioral tests and biochemical assays, and the mechanisms were examined in vitro.

Results: Oral administration of QYS extract improved learning and spatial memory, reduced carboxy-terminal fragments (CTFs), amyloid precursor protein (APP), Aβ and Tau aggregates, and inhibited microgliosis and astrocytosis in the brains of 3XTg mice. Mechanistically, QYS extract increased the expression of PPARα and TFEB, and promoted ALP both in vivo and in vitro.

Conclusion: QYS attenuates AD pathology, and improves cognitive function in 3XTg mice, which may be mediated by activation of PPARα-TFEB pathway and the subsequent ALP enhancement. Therefore, QYS may be a promising herbal material for further anti-AD drug discovery.
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http://dx.doi.org/10.1016/j.phymed.2021.153648DOI Listing
October 2021

Lysosomal TPCN (two pore segment channel) inhibition ameliorates beta-amyloid pathology and mitigates memory impairment in Alzheimer disease.

Autophagy 2021 Jul 27:1-19. Epub 2021 Jul 27.

School of Chinese Medicine and Mr. And Mrs. Ko Chi Ming Centre for Parkinson's Disease Research, Hong Kong Baptist University, Hong Kong, China.

Abbreviations: Aβ: β-amyloid; AD: Alzheimer disease; AIF1/IBA1: allograft inflammatory factor 1; ALP: autophagy-lysosomal pathway; APP: amyloid beta precursor protein; ATP6V1B1/V-ATPase V1b1: ATPase H+ transporting V1 subunit B1; AVs: autophagy vacuoles; BAF: bafilomycin A; CFC: contextual/cued fear conditioning assay; CHX: Ca/H exchanger; CTF-β: carboxy-terminal fragment derived from β-secretase; CTSD: cathepsin D; fAD: familial Alzheimer disease; GFAP: glial fibrillary acidic protein; LAMP1: lysosomal associated membrane protein 1; LTP: long-term potentiation; MCOLN1/TRPML1: mucolipin 1; MAP1LC3B/LC3B: microtubule associated protein 1 light chain 3 beta; MAPT: microtubule associated protein tau; MWM: Morris water maze; NFT: neurofibrillary tangles; PFC: prefrontal cortex; PSEN1: presenilin 1; SQSTM1/p62: sequestosome 1; TBS: theta burst stimulation; TEM: transmission electronic microscopy; TPCN2/TPC2: two pore segment channel 2; WT: wild-type; V-ATPase: vacuolar type H-ATPase.
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http://dx.doi.org/10.1080/15548627.2021.1945220DOI Listing
July 2021

Guidelines for the use and interpretation of assays for monitoring autophagy (4th edition).

Autophagy 2021 Jan 8;17(1):1-382. Epub 2021 Feb 8.

University of Crete, School of Medicine, Laboratory of Clinical Microbiology and Microbial Pathogenesis, Voutes, Heraklion, Crete, Greece; Foundation for Research and Technology, Institute of Molecular Biology and Biotechnology (IMBB), Heraklion, Crete, Greece.

In 2008, we published the first set of guidelines for standardizing research in autophagy. Since then, this topic has received increasing attention, and many scientists have entered the field. Our knowledge base and relevant new technologies have also been expanding. Thus, it is important to formulate on a regular basis updated guidelines for monitoring autophagy in different organisms. Despite numerous reviews, there continues to be confusion regarding acceptable methods to evaluate autophagy, especially in multicellular eukaryotes. Here, we present a set of guidelines for investigators to select and interpret methods to examine autophagy and related processes, and for reviewers to provide realistic and reasonable critiques of reports that are focused on these processes. These guidelines are not meant to be a dogmatic set of rules, because the appropriateness of any assay largely depends on the question being asked and the system being used. Moreover, no individual assay is perfect for every situation, calling for the use of multiple techniques to properly monitor autophagy in each experimental setting. Finally, several core components of the autophagy machinery have been implicated in distinct autophagic processes (canonical and noncanonical autophagy), implying that genetic approaches to block autophagy should rely on targeting two or more autophagy-related genes that ideally participate in distinct steps of the pathway. Along similar lines, because multiple proteins involved in autophagy also regulate other cellular pathways including apoptosis, not all of them can be used as a specific marker for autophagic responses. Here, we critically discuss current methods of assessing autophagy and the information they can, or cannot, provide. Our ultimate goal is to encourage intellectual and technical innovation in the field.
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http://dx.doi.org/10.1080/15548627.2020.1797280DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7996087PMC
January 2021

Rectifying Attenuated Store-Operated Calcium Entry as a Therapeutic Approach for Alzheimer's Disease.

Curr Alzheimer Res 2020 ;17(12):1072-1087

School of Chinese Medicine and Mr. and Mrs. Ko Chi Ming Centre for Parkinson's Disease Research, Hong Kong Baptist University, 7 Baptist University Road, Kowloon Tong, Kowloon, Hong Kong, China.

Alzheimer's disease (AD) is the most common neurodegenerative disorder. Although the pathological hallmarks of AD have been identified, the derived therapies cannot effectively slow down or stop disease progression; hence, it is likely that other pathogenic mechanisms are involved in AD pathogenesis. Intracellular calcium (Ca) dyshomeostasis has been consistently observed in AD patients and numerous AD models and may emerge prior to the development of amyloid plaques and neurofibrillary tangles. Thus, intracellular Ca disruptions are believed to play an important role in AD development and could serve as promising therapeutic intervention targets. One of the disrupted intracellular Ca signaling pathways manifested in AD is attenuated storeoperated Ca entry (SOCE). SOCE is an extracellular Ca entry mechanism mainly triggered by intracellular Ca store depletion. Maintaining normal SOCE function not only provides a means for the cell to replenish ER Ca stores but also serves as a cellular signal that maintains normal neuronal functions, including excitability, neurogenesis, neurotransmission, synaptic plasticity, and gene expression. However, normal SOCE function is diminished in AD, resulting in disrupted neuronal spine stability and synaptic plasticity and the promotion of amyloidogenesis. Mounting evidence suggests that rectifying diminished SOCE in neurons may intervene with the progression of AD. In this review, the mechanisms of SOCE disruption and the associated pathogenic impacts on AD will be discussed. We will also highlight the potential therapeutic targets or approaches that may help ameliorate SOCE deficits for AD treatment.
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http://dx.doi.org/10.2174/1567205018666210119150613DOI Listing
January 2020

Correction to: InsPR-SEC5 interaction on phagosomes modulates innate immunity to Candida albicans by promoting cytosolic Ca elevation and TBK1 activity.

BMC Biol 2020 Nov 2;18(1):158. Epub 2020 Nov 2.

NHFPC Key Laboratory of Reproduction Regulation, Shanghai Institute of Planned Parenthood Research, 2140 Xie Tu Road, Shanghai, 200032, China.

An amendment to this paper has been published and can be accessed via the original article.
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http://dx.doi.org/10.1186/s12915-020-00900-6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7604923PMC
November 2020

NRBF2 is a RAB7 effector required for autophagosome maturation and mediates the association of APP-CTFs with active form of RAB7 for degradation.

Autophagy 2021 05 16;17(5):1112-1130. Epub 2020 Jun 16.

State Key Laboratory of Quality Research in Chinese Medicine, Institute of Chinese Medical Sciences, University of Macau, Macau SAR, China.

NRBF2 is a component of the class III phosphatidylinositol 3-kinase (PtdIns3K) complex. Our previous study has revealed its role in regulating ATG14-associated PtdIns3K activity for autophagosome initiation. In this study, we revealed an unknown mechanism by which NRBF2 modulates autophagosome maturation and APP-C-terminal fragment (CTF) degradation. Our data showed that NRBF2 localized at autolysosomes, and loss of NRBF2 impaired autophagosome maturation. Mechanistically, NRBF2 colocalizes with RAB7 and is required for generation of GTP-bound RAB7 by interacting with RAB7 GEF CCZ1-MON1A and maintaining the GEF activity. Specifically, NRBF2 regulates CCZ1-MON1A interaction with PI3KC3/VPS34 and CCZ1-associated PI3KC3 kinase activity, which are required for CCZ1-MON1A GEF activity. Finally, we showed that NRBF2 is involved in APP-CTF degradation and amyloid beta peptide production by maintaining the interaction between APP and the CCZ1-MON1A-RAB7 module to facilitate the maturation of APP-containing vesicles. Overall, our study revealed a pivotal role of NRBF2 as a new RAB7 effector in modulating autophagosome maturation, providing insight into the molecular mechanism of NRBF2-PtdIns3K in regulating RAB7 activity for macroautophagy/autophagy maturation and Alzheimer disease-associated protein degradation.. 3xTg AD, triple transgenic mouse for Alzheimer disease; Aβ, amyloid beta peptide; Aβ amyloid beta peptide 1-40; Aβ amyloid beta peptide 1-42; AD, Alzheimer disease; APP, amyloid beta precursor protein; APP-CTFs, APP C-terminal fragments; ATG, autophagy related; ATG5, autophagy related 5; ATG7, autophagy related 7; ATG14, autophagy related 14; CCD, coiled-coil domain; CCZ1, CCZ1 homolog, vacuolar protein trafficking and biogenesis associated; CHX, cycloheximide; CQ, chloroquine; DAPI, 4',6-diamidino-2-phenylindole; dCCD, delete CCD; dMIT, delete MIT; FYCO1, FYVE and coiled-coil domain autophagy adaptor 1; FYVE, Fab1, YGL023, Vps27, and EEA1; GAP, GTPase-activating protein; GDP, guanine diphosphate; GEF, guanine nucleotide exchange factor; GTP, guanine triphosphate; GTPase, guanosine triphosphatase; HOPS, homotypic fusion and vacuole protein sorting; ILVs, endosomal intralumenal vesicles; KD, knockdown; KO, knockout; LAMP1, lysosomal associated membrane protein 1; MAP1LC3/LC3, microtubule associated protein 1 light chain 3; MLVs, multilamellar vesicles; MON1A, MON1 homolog A, secretory trafficking associated; NRBF2, nuclear receptor binding factor 2; PtdIns3K, class III phosphatidylinositol 3-kinase; PtdIns3P, phosphatidylinositol-3-phosphate; RILP, Rab interacting lysosomal protein; SNARE, soluble -ethylmaleimide-sensitive factor attachment protein receptor; SQSTM1/p62, sequestosome 1; UVRAG, UV radiation resistance associated; VPS, vacuolar protein sorting; WT, wild type.
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http://dx.doi.org/10.1080/15548627.2020.1760623DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8143228PMC
May 2021

A Curcumin Derivative Activates TFEB and Protects Against Parkinsonian Neurotoxicity .

Int J Mol Sci 2020 Feb 22;21(4). Epub 2020 Feb 22.

Mr. & Mrs. Ko Chi-Ming Centre for Parkinson's Disease Research, School of Chinese Medicine, Hong Kong Baptist University, Hong Kong SAR, China.

bstract TFEB (transcription factor EB), which is a master regulator of autophagy and lysosome biogenesis, is considered to be a new therapeutic target for Parkinson's disease (PD). However, only several small-molecule TFEB activators have been discovered and their neuroprotective effects in PD are unclear. In this study, a curcumin derivative, named E4, was identified as a potent TFEB activator. Compound E4 promoted the translocation of TFEB from cytoplasm into nucleus, accompanied by enhanced autophagy and lysosomal biogenesis. Moreover, TFEB knockdown effectively attenuated E4-induced autophagy and lysosomal biogenesis. Mechanistically, E4-induced TFEB activation is mainly through AKT-MTORC1 inhibition. In the PD cell models, E4 promoted the degradation of α-synuclein and protected against the cytotoxicity of MPP (1-methyl-4-phenylpyridinium ion) in neuronal cells. Overall, the TFEB activator E4 deserves further study in animal models of neurodegenerative diseases, including PD.
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http://dx.doi.org/10.3390/ijms21041515DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7073207PMC
February 2020

A stress response p38 MAP kinase inhibitor SB202190 promoted TFEB/TFE3-dependent autophagy and lysosomal biogenesis independent of p38.

Redox Biol 2020 05 28;32:101445. Epub 2020 Jan 28.

Mr. and Mrs. Ko Chi Ming Centre for Parkinson's Disease Research, School of Chinese Medicine, Hong Kong Baptist University, Hong Kong SAR, China; School of Chinese Medicine, Hong Kong Baptist University, Hong Kong SAR, China; Institute for Research and Continuing Education, Hong Kong Baptist University, Shenzhen, China. Electronic address:

TFEB (transcription factor EB) and TFE3 (transcription factor E3) are "master regulators" of autophagy and lysosomal biogenesis. The stress response p38 mitogen-activated protein (MAP) kinases affect multiple intracellular responses including inflammation, cell growth, differentiation, cell death, senescence, tumorigenesis, and autophagy. Small molecule p38 MAP kinase inhibitors such as SB202190 are widely used in dissection of related signal transduction mechanisms including redox biology and autophagy. Here, we initially aimed to investigate the links between p38 MAP kinase and TFEB/TFE3-mediated autophagy and lysosomal biogenesis. Unexpectedly, we found that only SB202190, rather than several other p38 inhibitors, promotes TFEB and TFE3 to translocate from the cytosol into the nucleus and subsequently enhances autophagy and lysosomal biogenesis. In addition, siRNA-mediated Tfeb and Tfe3 knockdown effectively attenuated SB202190-induced gene expression and lysosomal biogenesis. Mechanistical studies showed that TFEB and TFE3 activation in response to SB202190 is dependent on PPP3/calcineurin rather than on the inhibition of p38 or MTOR signaling, the main pathway for regulating TFEB and TFE3 activation. Importantly, SB202190 increased intracellular calcium levels, and calcium chelator BAPTAP-AM blocked SB202190-induced TFEB and TFE3 activation as well as autophagy and lysosomal biogenesis. Moreover, endoplasmic reticulum (ER) calcium is required for TFEB and TFE3 activation in response to SB202190. In summary, we identified a previously uncharacterized role of SB202190 in activating TFEB- and TFE3-dependent autophagy and lysosomal biogenesis via ER calcium release and subsequent calcium-dependent PPP3/calcineurin activation, leading to dephosphorylation of TFEB and TFE3. Given the importance of p38 MAP kinase invarious conditions including oxidative stress, the findings collectively indicate that SB202190 should not be used as a specific inhibitor for elucidating the p38 MAP kinase biological functions due to its potential effect on activating autophagy-lysosomal axis.
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http://dx.doi.org/10.1016/j.redox.2020.101445DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7264467PMC
May 2020

Targeting Aggrephagy for the Treatment of Alzheimer's Disease.

Cells 2020 01 28;9(2). Epub 2020 Jan 28.

Mr. and Mrs. Ko Chi Ming Centre for Parkinson's Disease Research, School of Chinese Medicine, Hong Kong Baptist University, Hong Kong, China.

Alzheimer's disease (AD) is one of the most common neurodegenerative diseases in older individuals with specific neuropsychiatric symptoms. It is a proteinopathy, pathologically characterized by the presence of misfolded protein (Aβ and Tau) aggregates in the brain, causing progressive dementia. Increasing studies have provided evidence that the defect in protein-degrading systems, especially the autophagy-lysosome pathway (ALP), plays an important role in the pathogenesis of AD. Recent studies have demonstrated that AD-associated protein aggregates can be selectively recognized by some receptors and then be degraded by ALP, a process termed aggrephagy. In this study, we reviewed the role of aggrephagy in AD development and discussed the strategy of promoting aggrephagy using small molecules for the treatment of AD.
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http://dx.doi.org/10.3390/cells9020311DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7072705PMC
January 2020

NeuroDefend, a novel Chinese medicine, attenuates amyloid-β and tau pathology in experimental Alzheimer's disease models.

J Food Drug Anal 2020 01 8;28(1):132-146. Epub 2019 Oct 8.

Mr. & Mrs. Ko Chi-Ming Centre for Parkinson's Disease Research, School of Chinese Medicine, Hong Kong Baptist University, Hong Kong Special Administrative Region of China. Electronic address:

Alzheimer's disease (AD) is the most common age-related neurodegenerative disorder. Amyloid-β (Aβ) and hyper-phosphorylated tau accumulation are accountable for the progressive neuronal loss and cognitive impairments usually observed in AD. Currently, medications for AD offer moderate symptomatic relief but fail to cure the disease; hence development of effective and safe drugs is urgently needed for AD treatment. In this study, we investigated a Chinese medicine (CM) formulation named NeuroDefend (ND), for reducing amyloid β (Aβ) and tau pathology in transgenic AD mice models. Regular oral administration of ND improved cognitive function and memory in 3XTg-AD and 5XFAD mice. In addition, ND reduced beta-amyloid precursor protein (APP), APP C-terminal fragments (CTF-β/α), Aβ and 4G8 positive Aβ burden in 3XTg-AD and 5XFAD mice. Furthermore, ND efficiently reduced the levels of insoluble phospho-tau protein aggregates and AT8 positive phospho tau neuron load in 3XTg-AD mice. Hence, ND could be a promising candidate for the treatment of AD in humans.
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http://dx.doi.org/10.1016/j.jfda.2019.09.004DOI Listing
January 2020

A small molecule transcription factor EB activator ameliorates beta-amyloid precursor protein and Tau pathology in Alzheimer's disease models.

Aging Cell 2020 02 19;19(2):e13069. Epub 2019 Dec 19.

Mr. and Mrs. Ko Chi Ming Centre for Parkinson's Disease Research, School of Chinese Medicine, Hong Kong Baptist University, Hong Kong SAR, China.

Accumulating studies have suggested that targeting transcription factor EB (TFEB), an essential regulator of autophagy-lysosomal pathway (ALP), is promising for the treatment of neurodegenerative disorders, including Alzheimer's disease (AD). However, potent and specific small molecule TFEB activators are not available at present. Previously, we identified a novel TFEB activator named curcumin analog C1 which directly binds to and activates TFEB. In this study, we systematically investigated the efficacy of curcumin analog C1 in three AD animal models that represent beta-amyloid precursor protein (APP) pathology (5xFAD mice), tauopathy (P301S mice) and the APP/Tau combined pathology (3xTg-AD mice). We found that C1 efficiently activated TFEB, enhanced autophagy and lysosomal activity, and reduced APP, APP C-terminal fragments (CTF-β/α), β-amyloid peptides and Tau aggregates in these models accompanied by improved synaptic and cognitive function. Knockdown of TFEB and inhibition of lysosomal activity significantly inhibited the effects of C1 on APP and Tau degradation in vitro. In summary, curcumin analog C1 is a potent TFEB activator with promise for the prevention or treatment of AD.
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http://dx.doi.org/10.1111/acel.13069DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6996953PMC
February 2020

Mitochondrial Calcium Signaling as a Therapeutic Target for Alzheimer's Disease.

Curr Alzheimer Res 2020 ;17(4):329-343

School of Chinese Medicine, Hong Kong Baptist University, 7 Baptist University Road, Kowloon Tong, Kowloon, Hong Kong, China.

Mitochondria absorb calcium (Ca2+) at the expense of the electrochemical gradient generated during respiration. The influx of Ca2+ into the mitochondrial matrix helps maintain metabolic function and results in increased cytosolic Ca2+ during intracellular Ca2+ signaling. Mitochondrial Ca2+ homeostasis is tightly regulated by proteins located in the inner and outer mitochondrial membranes and by the cross-talk with endoplasmic reticulum Ca2+ signals. Increasing evidence indicates that mitochondrial Ca2+ overload is a pathological phenotype associated with Alzheimer's Disease (AD). As intracellular Ca2+ dysregulation can be observed before the appearance of typical pathological hallmarks of AD, it is believed that mitochondrial Ca2+ overload may also play an important role in AD etiology. The high mitochondrial Ca2+ uptake can easily compromise neuronal functions and exacerbate AD progression by impairing mitochondrial respiration, increasing reactive oxygen species formation and inducing apoptosis. Additionally, mitochondrial Ca2+ overload can damage mitochondrial recycling via mitophagy. This review will discuss the molecular players involved in mitochondrial Ca2+ dysregulation and the pharmacotherapies that target this dysregulation. As most of the current AD therapeutics are based on amyloidopathy, tauopathy, and the cholinergic hypothesis, they achieve only symptomatic relief. Thus, determining how to reestablish mitochondrial Ca2+ homeostasis may aid in the development of novel AD therapeutic interventions.
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http://dx.doi.org/10.2174/1567205016666191210091302DOI Listing
August 2021

Balancing mTOR Signaling and Autophagy in the Treatment of Parkinson's Disease.

Int J Mol Sci 2019 Feb 8;20(3). Epub 2019 Feb 8.

Mr. and Mrs. Ko Chi Ming Centre for Parkinson's Disease Research, School of Chinese Medicine, Hong Kong Baptist University, Hong Kong SAR 999077, China.

The mammalian target of rapamycin (mTOR) signaling pathway plays a critical role in regulating cell growth, proliferation, and life span. mTOR signaling is a central regulator of autophagy by modulating multiple aspects of the autophagy process, such as initiation, process, and termination through controlling the activity of the unc51-like kinase 1 (ULK1) complex and vacuolar protein sorting 34 (VPS34) complex, and the intracellular distribution of TFEB/TFE3 and proto-lysosome tubule reformation. Parkinson's disease (PD) is a serious, common neurodegenerative disease characterized by dopaminergic neuron loss in the substantia nigra pars compacta (SNpc) and the accumulation of Lewy bodies. An increasing amount of evidence indicates that mTOR and autophagy are critical for the pathogenesis of PD. In this review, we will summarize recent advances regarding the roles of mTOR and autophagy in PD pathogenesis and treatment. Further characterizing the dysregulation of mTOR pathway and the clinical translation of mTOR modulators in PD may offer exciting new avenues for future drug development.
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http://dx.doi.org/10.3390/ijms20030728DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6387269PMC
February 2019

Calcium signaling in Alzheimer's disease & therapies.

Biochim Biophys Acta Mol Cell Res 2018 11 29;1865(11 Pt B):1745-1760. Epub 2018 Jul 29.

School of Chinese Medicine, Hong Kong Baptist University, 7 Baptist University Road, Kowloon Tong, Kowloon, Hong Kong, China. Electronic address:

Alzheimer's disease (AD) is the most common type of dementia and is characterized by the accumulation of amyloid (Aβ) plaques and neurofibrillary tangles in the brain. Much attention has been given to develop AD treatments based on the amyloid cascade hypothesis; however, none of these drugs had good efficacy at improving cognitive functions in AD patients suggesting that Aβ might not be the disease origin. Thus, there are urgent needs for the development of new therapies that target on the proximal cause of AD. Cellular calcium (Ca) signals regulate important facets of neuronal physiology. An increasing body of evidence suggests that age-related dysregulation of neuronal Ca homeostasis may play a proximal role in the pathogenesis of AD as disrupted Ca could induce synaptic deficits and promote the accumulation of Aβ plaques and neurofibrillary tangles. Given that Ca disruption is ubiquitously involved in all AD pathologies, it is likely that using chemical agents or small molecules specific to Ca channels or handling proteins on the plasma membrane and membranes of intracellular organelles to correct neuronal Ca dysregulation could open up a new approach to AD prevention and treatment. This review summarizes current knowledge on the molecular mechanisms linking Ca dysregulation with AD pathologies and discusses the possibility of correcting neuronal Ca disruption as a therapeutic approach for AD.
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http://dx.doi.org/10.1016/j.bbamcr.2018.07.018DOI Listing
November 2018

InsPR-SEC5 interaction on phagosomes modulates innate immunity to Candida albicans by promoting cytosolic Ca elevation and TBK1 activity.

BMC Biol 2018 04 27;16(1):46. Epub 2018 Apr 27.

NHFPC Key Laboratory of Reproduction Regulation, Shanghai Institute of Planned Parenthood Research, 2140 Xie Tu Road, Shanghai, 200032, China.

Background: Candida albicans (C. albicans) invasion triggers antifungal innate immunity, and the elevation of cytoplasmic Ca levels via the inositol 1,4,5-trisphosphate receptor (InsPR) plays a critical role in this process. However, the molecular pathways linking the InsPR-mediated increase in Ca and immune responses remain elusive.

Results: In the present study, we find that during C. albicans phagocytosis in macrophages, exocyst complex component 2 (SEC5) promotes InsPR channel activity by binding to its C-terminal α-helix (H1), increasing cytosolic Ca concentrations ([Ca]). Immunofluorescence reveals enriched InsPR-SEC5 complex formation on phagosomes, while disruption of the InsPR-SEC5 interaction by recombinant H1 peptides attenuates the InsPR-mediated Ca elevation, leading to impaired phagocytosis. Furthermore, we show that C. albicans infection promotes the recruitment of Tank-binding kinase 1 (TBK1) by the InsPR-SEC5 interacting complex, leading to the activation of TBK1. Subsequently, activated TBK1 phosphorylates interferon regulatory factor 3 (IRF-3) and mediates type I interferon responses, suggesting that the InsPR-SEC5 interaction may regulate antifungal innate immune responses not only by elevating cytoplasmic Ca but also by activating the TBK1-IRF-3 pathway.

Conclusions: Our data have revealed an important role of the InsPR-SEC5 interaction in innate immune responses against C. albicans.
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http://dx.doi.org/10.1186/s12915-018-0507-6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5921305PMC
April 2018

Familial Alzheimer's disease-associated presenilin 1 mutants promote γ-secretase cleavage of STIM1 to impair store-operated Ca2+ entry.

Sci Signal 2016 09 6;9(444):ra89. Epub 2016 Sep 6.

School of Biomedical Sciences, Li Ka Shing Faculty of Medicine, University of Hong Kong, Hong Kong, China. State Key Laboratory of Brain and Cognitive Sciences, Li Ka Shing Faculty of Medicine, University of Hong Kong, Hong Kong, China. HKU-Shenzhen Institute of Research and Innovation, Shenzhen, Guangdong, China.

Some forms of familial Alzheimer's disease (FAD) are caused by mutations in presenilins (PSs), catalytic components of a γ-secretase complex that cleaves target proteins, including amyloid precursor protein (APP). Calcium (Ca(2+)) dysregulation in cells with these FAD-causing PS mutants has been attributed to attenuated store-operated Ca(2+) entry [SOCE; also called capacitative Ca(2+) entry (CCE)]. CCE occurs when STIM1 detects decreases in Ca(2+) in the endoplasmic reticulum (ER) and activates ORAI channels to replenish Ca(2+) stores in the ER. We showed that CCE was attenuated by PS1-associated γ-secretase activity. Endogenous PS1 and STIM1 interacted in human neuroblastoma SH-SY5Y cells, patient fibroblasts, and mouse primary cortical neurons. Forms of PS1 with FAD-associated mutations enhanced γ-secretase cleavage of the STIM1 transmembrane domain at a sequence that was similar to the γ-secretase cleavage sequence of APP. Cultured hippocampal neurons expressing mutant PS1 had attenuated CCE that was associated with destabilized dendritic spines, which were rescued by either γ-secretase inhibition or overexpression of STIM1. Our results indicate that γ-secretase activity may physiologically regulate CCE by targeting STIM1 and that restoring STIM1 may be a therapeutic approach in AD.
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http://dx.doi.org/10.1126/scisignal.aaf1371DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5384262PMC
September 2016

Mechanistic study of TRPM2-Ca(2+)-CAMK2-BECN1 signaling in oxidative stress-induced autophagy inhibition.

Autophagy 2016 08 31;12(8):1340-54. Epub 2016 May 31.

a Department of Biomedical Sciences , City University of Hong Kong , Hong Kong , China.

Reactive oxygen species (ROS) have been commonly accepted as inducers of autophagy, and autophagy in turn is activated to relieve oxidative stress. Yet, whether and how oxidative stress, generated in various human pathologies, regulates autophagy remains unknown. Here, we mechanistically studied the role of TRPM2 (transient receptor potential cation channel subfamily M member 2)-mediated Ca(2+) influx in oxidative stress-mediated autophagy regulation. On the one hand, we demonstrated that oxidative stress triggered TRPM2-dependent Ca(2+) influx to inhibit the induction of early autophagy, which renders cells more susceptible to death. On the other hand, oxidative stress induced autophagy (and not cell death) in the absence of the TRPM2-mediated Ca(2+) influx. Moreover, in response to oxidative stress, TRPM2-mediated Ca(2+) influx activated CAMK2 (calcium/calmodulin dependent protein kinase II) at levels of both phosphorylation and oxidation, and the activated CAMK2 subsequently phosphorylated BECN1/Beclin 1 on Ser295. Ser295 phosphorylation of BECN1 in turn decreased the association between BECN1 and PIK3C3/VPS34, but induced binding between BECN1 and BCL2. Clinically, acetaminophen (APAP) overdose is the most common cause of acute liver failure worldwide. We demonstrated that APAP overdose also activated ROS-TRPM2-CAMK2-BECN1 signaling to suppress autophagy, thereby causing primary hepatocytes to be more vulnerable to death. Inhibiting the TRPM2-Ca(2+)-CAMK2 cascade significantly mitigated APAP-induced liver injury. In summary, our data clearly demonstrate that oxidative stress activates the TRPM2-Ca(2+)-CAMK2 cascade to phosphorylate BECN1 resulting in autophagy inhibition.
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http://dx.doi.org/10.1080/15548627.2016.1187365DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4968236PMC
August 2016

Impaired mitochondrial function due to familial Alzheimer's disease-causing presenilins mutants via Ca(2+) disruptions.

Cell Calcium 2016 05 5;59(5):240-50. Epub 2016 Mar 5.

Department of Physics, University of South Florida, Tampa, FL 33620, United States. Electronic address:

Mutants in presenilins (PS1 or PS2) is the major cause of familial Alzheimer's disease (FAD). FAD causing PS mutants affect intracellular Ca(2+) homeostasis by enhancing the gating of inositol trisphosphate (IP3) receptor (IP3R) Ca(2+) release channel on the endoplasmic reticulum, leading to exaggerated Ca(2+) release into the cytoplasm. Using experimental IP3R-mediated Ca(2+) release data, in conjunction with a computational model of cell bioenergetics, we explore how the differences in mitochondrial Ca(2+) uptake in control cells and cells expressing FAD-causing PS mutants affect key variables such as ATP, reactive oxygen species (ROS), NADH, and mitochondrial Ca(2+). We find that as a result of exaggerated cytosolic Ca(2+) in FAD-causing mutant PS-expressing cells, the rate of oxygen consumption increases dramatically and overcomes the Ca(2+) dependent enzymes that stimulate NADH production. This leads to decreased rates in proton pumping due to diminished membrane potential along with less ATP and enhanced ROS production. These results show that through Ca(2+) signaling disruption, mutant PS leads to mitochondrial dysfunction and potentially to cell death.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5088788PMC
http://dx.doi.org/10.1016/j.ceca.2016.02.013DOI Listing
May 2016

Characterization of Two-Pore Channel 2 by Nuclear Membrane Electrophysiology.

Sci Rep 2016 Feb 3;6:20282. Epub 2016 Feb 3.

School of Biomedical Sciences, LKS Faculty of Medicine, University of Hong Kong, Hong Kong, China.

Lysosomal calcium (Ca(2+)) release mediated by NAADP triggers signalling cascades that regulate many cellular processes. The identification of two-pore channel 2 (TPC2) as the NAADP receptor advances our understanding of lysosomal Ca(2+) signalling, yet the lysosome is not amenable to traditional patch-clamp electrophysiology. Previous attempts to record TPC2 single-channel activity put TPC2 outside its native environment, which not reflect TPC2's true physiological properties. To test the feasibility of using nuclear membrane electrophysiology for TPC2 channel characterization, we constructed a stable human TPC2-expressing DT40TKO cell line that lacks endogenous InsP3R and RyR (DT40TKO-hTPC2). Immunostaining revealed hTPC2 expression on the ER and nuclear envelope. Intracellular dialysis of NAADP into Fura-2-loaded DT40TKO-hTPC2 cells elicited cytosolic Ca(2+) transients, suggesting that hTPC2 was functionally active. Using nuclear membrane electrophysiology, we detected a ~220 pS single-channel current activated by NAADP with K(+) as the permeant ion. The detected single-channel recordings displayed a linear current-voltage relationship, were sensitive to Ned-19 inhibition, were biphasically regulated by NAADP concentration, and regulated by PKA phosphorylation. In summary, we developed a cell model for the characterization of the TPC2 channel and the nuclear membrane patch-clamp technique provided an alternative approach to rigorously investigate the electrophysiological properties of TPC2 with minimal manipulation.
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http://dx.doi.org/10.1038/srep20282DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4738322PMC
February 2016

Analyzing and Quantifying the Gain-of-Function Enhancement of IP3 Receptor Gating by Familial Alzheimer's Disease-Causing Mutants in Presenilins.

PLoS Comput Biol 2015 Oct 6;11(10):e1004529. Epub 2015 Oct 6.

Department of Physics, University of South Florida, Tampa, Florida, United States of America.

Familial Alzheimer's disease (FAD)-causing mutant presenilins (PS) interact with inositol 1,4,5-trisphosphate (IP3) receptor (IP3R) Ca(2+) release channels resulting in enhanced IP3R channel gating in an amyloid beta (Aβ) production-independent manner. This gain-of-function enhancement of IP3R activity is considered to be the main reason behind the upregulation of intracellular Ca(2+) signaling in the presence of optimal and suboptimal stimuli and spontaneous Ca(2+) signals observed in cells expressing mutant PS. In this paper, we employed computational modeling of single IP3R channel activity records obtained under optimal Ca(2+) and multiple IP3 concentrations to gain deeper insights into the enhancement of IP3R function. We found that in addition to the high occupancy of the high-activity (H) mode and the low occupancy of the low-activity (L) mode, IP3R in FAD-causing mutant PS-expressing cells exhibits significantly longer mean life-time for the H mode and shorter life-time for the L mode, leading to shorter mean close-time and hence high open probability of the channel in comparison to IP3R in cells expressing wild-type PS. The model is then used to extrapolate the behavior of the channel to a wide range of IP3 and Ca(2+) concentrations and quantify the sensitivity of IP3R to its two ligands. We show that the gain-of-function enhancement is sensitive to both IP3 and Ca(2+) and that very small amount of IP3 is required to stimulate IP3R channels in the presence of FAD-causing mutant PS to the same level of activity as channels in control cells stimulated by significantly higher IP3 concentrations. We further demonstrate with simulations that the relatively longer time spent by IP3R in the H mode leads to the observed higher frequency of local Ca(2+) signals, which can account for the more frequent global Ca(2+) signals observed, while the enhanced activity of the channel at extremely low ligand concentrations will lead to spontaneous Ca(2+) signals in cells expressing FAD-causing mutant PS.
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http://dx.doi.org/10.1371/journal.pcbi.1004529DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4595473PMC
October 2015

Vacuolin-1 potently and reversibly inhibits autophagosome-lysosome fusion by activating RAB5A.

Autophagy 2014 30;10(11):1895-905. Epub 2014 Oct 30.

a Department of Biomedical Sciences ; City University of Hong Kong ; Hong Kong , China.

Autophagy is a catabolic lysosomal degradation process essential for cellular homeostasis and cell survival. Dysfunctional autophagy has been associated with a wide range of human diseases, e.g., cancer and neurodegenerative diseases. A large number of small molecules that modulate autophagy have been widely used to dissect this process and some of them, e.g., chloroquine (CQ), might be ultimately applied to treat a variety of autophagy-associated human diseases. Here we found that vacuolin-1 potently and reversibly inhibited the fusion between autophagosomes and lysosomes in mammalian cells, thereby inducing the accumulation of autophagosomes. Interestingly, vacuolin-1 was less toxic but at least 10-fold more potent in inhibiting autophagy compared with CQ. Vacuolin-1 treatment also blocked the fusion between endosomes and lysosomes, resulting in a defect in general endosomal-lysosomal degradation. Treatment of cells with vacuolin-1 alkalinized lysosomal pH and decreased lysosomal Ca(2+) content. Besides marginally inhibiting vacuolar ATPase activity, vacuolin-1 treatment markedly activated RAB5A GTPase activity. Expression of a dominant negative mutant of RAB5A or RAB5A knockdown significantly inhibited vacuolin-1-induced autophagosome-lysosome fusion blockage, whereas expression of a constitutive active form of RAB5A suppressed autophagosome-lysosome fusion. These data suggest that vacuolin-1 activates RAB5A to block autophagosome-lysosome fusion. Vacuolin-1 and its analogs present a novel class of drug that can potently and reversibly modulate autophagy.
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http://dx.doi.org/10.4161/auto.32200DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4502727PMC
September 2015

General anesthetic isoflurane modulates inositol 1,4,5-trisphosphate receptor calcium channel opening.

Anesthesiology 2014 Sep;121(3):528-37

From the Department of Anesthesiology and Critical Care (J.D.J., Y.P., H.W.) and Department of Physiology (D.-O.D.M., K.-H.C., H.V., J.K.F.), Perelman School of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania. Current address: Department of Physiology, University of Hong Kong, Hong Kong, China (K.-H.C.).

Background: Pharmacological evidence suggests that inhalational general anesthetics induce neurodegeneration in vitro and in vivo through overactivation of inositol trisphosphate receptor (InsP3R) Ca-release channels, but it is not clear whether these effects are due to direct modulation of channel activity by the anesthetics.

Methods: Using single-channel patch clamp electrophysiology, the authors examined the gating of rat recombinant type 3 InsP3R (InsP3R-3) Ca-release channels in isolated nuclei (N = 3 to 15) from chicken lymphocytes modulated by isoflurane at clinically relevant concentrations in the absence and presence of physiological levels of the agonist inositol 1,4,5-trisphosphate (InsP3). The authors also examined the effects of isoflurane on InsP3R-mediated Ca release from the endoplasmic reticulum and changes in intracellular Ca concentration ([Ca]i).

Results: Clinically relevant concentrations (approximately 1 minimal alveolar concentration) of the commonly used general anesthetic, isoflurane, activated InsP3R-3 channels with open probability similar to channels activated by 1 µM InsP3 (Po ≈ 0.2). This isoflurane modulation of InsP3R-3 Po depended biphasically on [Ca]i. Combination of isoflurane with subsaturating levels of InsP3 in patch pipettes resulted in at least two-fold augmentations of InsP3R-3 channel Po compared with InsP3 alone. These effects were not noted in the presence of saturating [InsP3]. Application of isoflurane to DT40 cells resulted in a 30% amplification of InsP3R-mediated [Ca]i oscillations, whereas InsP3-induced increase in [Ca]i and cleaved caspase-3 activity were enhanced by approximately 2.5-fold.

Conclusion: These results suggest that the InsP3R may be a direct molecular target of isoflurane and plays a role in the mechanisms of anesthetic-mediated pharmacological or neurotoxic effects.
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http://dx.doi.org/10.1097/ALN.0000000000000316DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4281525PMC
September 2014

Role of STIM1 in survival and neural differentiation of mouse embryonic stem cells independent of Orai1-mediated Ca2+ entry.

Stem Cell Res 2014 Mar 27;12(2):452-66. Epub 2013 Dec 27.

Department of Physiology, University of Hong Kong, Hong Kong, China. Electronic address:

Store-operated Ca(2+) entry (SOCE) is an important Ca(2+) influx pathway in non-excitable cells. STIM1, an ER Ca(2+) sensor, and Orai1, a plasma membrane Ca(2+) selective channel, are the two essential components of the Ca(2+) release activated channel (CRAC) responsible for SOCE activity. Here we explored the role of STIM1 and Orai1 in neural differentiation of mouse embryonic stem (ES) cells. We found that STIM1 and Orai1 were expressed and functionally active in ES cells, and expressions of STIM1 and Orai1 were dynamically regulated during neural differentiation of mouse ES cells. STIM1 knockdown inhibited the differentiation of mouse ES cells into neural progenitors, neurons, and astrocytes. In addition, STIM1 knockdown caused severe cell death and markedly suppressed the proliferation of neural progenitors. Surprisingly, Orai1 knockdown had little effect on neural differentiation of mouse ES cells, but the neurons derived from Orai1 knockdown ES cells, like those from STIM1 knockdown cells, had defective SOCE. Taken together, our data indicate that STIM1 is involved in both early neural differentiation of ES cells and survival of early differentiated ES cells independent of Orai1-mediated SOCE.
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http://dx.doi.org/10.1016/j.scr.2013.12.005DOI Listing
March 2014

Nuclear patch-clamp electrophysiology of Ca2+ channels.

Cold Spring Harb Protoc 2013 Sep 1;2013(9):885-91. Epub 2013 Sep 1.

Department of Physiology, Perelman School of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania 19104, USA.

Patch-clamping the outer or inner nuclear membrane of isolated nuclei is very similar to patch-clamping the plasma membrane of isolated cells. This protocol describes in detail all the steps required to successfully obtain nuclear membrane patches, in various configurations, from both the outer and inner nuclear membranes of isolated nuclei.
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http://dx.doi.org/10.1101/pdb.prot073064DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3984033PMC
September 2013

Isolating nuclei from cultured cells for patch-clamp electrophysiology of intracellular Ca(2+) channels.

Cold Spring Harb Protoc 2013 Sep 1;2013(9):880-4. Epub 2013 Sep 1.

Department of Physiology, Perelman School of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania 19104, USA.

Nuclear patch-clamp experiments can be performed with intact nuclei or with nuclei from which the outer nuclear membrane has been removed. This protocol presents procedures for harvesting different types of cultured cells, isolating nuclei, and exposing the inner nuclear membrane by agitating in the presence of sodium citrate. Particulars about obtaining and maintaining the cells of interest in culture are not described here. However, care should be taken not to allow the cells to grow beyond a density of 2-3 × 10(6) cells/mL because this may decrease both the cell viability and the success rate of detecting active inositol 1,4,5-trisphosphate receptor (InsP3R) channels in nuclear patches.
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http://dx.doi.org/10.1101/pdb.prot073056DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3979423PMC
September 2013

Patch-clamp electrophysiology of intracellular Ca2+ channels.

Cold Spring Harb Protoc 2013 Sep 1;2013(9):787-97. Epub 2013 Sep 1.

Department of Physiology, Perelman School of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania 19104, USA.

The modulation of cytoplasmic free Ca(2+) concentration ([Ca(2+)]i) is a universal intracellular signaling pathway that regulates numerous cellular physiological processes. Ubiquitous intracellular Ca(2+)-release channels localized to the endoplasmic/sarcoplasmic reticulum-inositol 1,4,5-trisphosphate receptor (InsP3R) and ryanodine receptor (RyR) channels-play a central role in [Ca(2+)]i signaling in all animal cells. Despite their intracellular localization, electrophysiological studies of the single-channel permeation and gating properties of these Ca(2+)-release channels using the powerful patch-clamp approach have been possible by application of this technique to isolated nuclei because the channels are present in membranes of the nuclear envelope. Here we provide a concise description of how nuclear patch-clamp experiments have been used to study single-channel properties of different InsP3R channels in the outer nuclear membrane. We compare this with other methods for studying intracellular Ca(2+) release. We also briefly describe application of the technique to InsP3R channels in the inner nuclear membrane and to channels in the outer nuclear membrane of HEK293 cells expressing recombinant RyR.
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http://dx.doi.org/10.1101/pdb.top066217DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3977788PMC
September 2013

DNA-loaded chitosan oligosaccharide nanoparticles with enhanced permeability across Calu-3 cells.

J Drug Target 2013 May 12;21(5):474-86. Epub 2013 Mar 12.

Women's Hospital, School of Medicine of Zhejiang University, Hangzhou, China.

Chitosan oligosaccharide (oligoCS) is a low molecular weight chitosan and its potential for DNA delivery is described here. DNA-loaded oligoCS nanoparticles were prepared by ionic gelation using thiamine pyrophosphate (TPP) as cross-linker. The nanoparticles with oligoCS:DNA: TPP weight ratio of 50:1:25 were approximately 170 nm in diameter with a zeta potential of +40 mV, and were used in the permeability study. The cytotoxicity of oligoCS solutions and nanoparticles was evaluated by MTT assay. The concentrations that exhibited minimal cytotoxicity were employed to investigate their effect on trans-epithelial electrical resistance (TEER) and cellular uptake across the Calu-3 cell layer which was used as a nasal epithelial model. OligoCS nanoparticles were able to cause a significant and reversible decrease in TEER and promote efficient cellular uptake. In addition, the oligoCS nanoparticles were able to enhance paracellular permeability to a greater extent than oligoCS solutions at an equivalent concentration. However, the oligoCS nanoparticles were too large to cross the cell layers through the paracellular route. The transcellular pathway appeared to be the major mechanism of the transportation of oligoCS nanoparticles across the cell layers. OligoCS nanoparticles also allowed efficient DNA incorporation, thereby providing the possibility of controlled nucleic acids release and absorption across epithelial surface.
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http://dx.doi.org/10.3109/1061186X.2013.766885DOI Listing
May 2013

Calcium homeostasis modulator 1 (CALHM1) is the pore-forming subunit of an ion channel that mediates extracellular Ca2+ regulation of neuronal excitability.

Proc Natl Acad Sci U S A 2012 Jul 18;109(28):E1963-71. Epub 2012 Jun 18.

Department of Physiology, University of Pennsylvania, Philadelphia, PA 19104, USA.

Extracellular Ca(2+) (Ca(2+)(o)) plays important roles in physiology. Changes of Ca(2+)(o) concentration ([Ca(2+)](o)) have been observed to modulate neuronal excitability in various physiological and pathophysiological settings, but the mechanisms by which neurons detect [Ca(2+)](o) are not fully understood. Calcium homeostasis modulator 1 (CALHM1) expression was shown to induce cation currents in cells and elevate cytoplasmic Ca(2+) concentration ([Ca(2+)](i)) in response to removal of Ca(2+)(o) and its subsequent addback. However, it is unknown whether CALHM1 is a pore-forming ion channel or modulates endogenous ion channels. Here we identify CALHM1 as the pore-forming subunit of a plasma membrane Ca(2+)-permeable ion channel with distinct ion permeability properties and unique coupled allosteric gating regulation by voltage and [Ca(2+)](o). Furthermore, we show that CALHM1 is expressed in mouse cortical neurons that respond to reducing [Ca(2+)](o) with enhanced conductance and action potential firing and strongly elevated [Ca(2+)](i) upon Ca(2+)(o) removal and its addback. In contrast, these responses are strongly muted in neurons from mice with CALHM1 genetically deleted. These results demonstrate that CALHM1 is an evolutionarily conserved ion channel family that detects membrane voltage and extracellular Ca(2+) levels and plays a role in cortical neuronal excitability and Ca(2+) homeostasis, particularly in response to lowering [Ca(2+)](o) and its restoration to normal levels.
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http://dx.doi.org/10.1073/pnas.1204023109DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3396471PMC
July 2012

Calpain-cleaved type 1 inositol 1,4,5-trisphosphate receptor (InsP(3)R1) has InsP(3)-independent gating and disrupts intracellular Ca(2+) homeostasis.

J Biol Chem 2011 Oct 22;286(41):35998-36010. Epub 2011 Aug 22.

Department of Emergency Medicine, University of Pennsylvania, Philadelphia, Pennsylvania 19104. Electronic address:

The type 1 inositol 1,4,5-trisphosphate receptor (InsP(3)R1) is a ubiquitous intracellular Ca(2+) release channel that is vital to intracellular Ca(2+) signaling. InsP(3)R1 is a proteolytic target of calpain, which cleaves the channel to form a 95-kDa carboxyl-terminal fragment that includes the transmembrane domains, which contain the ion pore. However, the functional consequences of calpain proteolysis on channel behavior and Ca(2+) homeostasis are unknown. In the present study we have identified a unique calpain cleavage site in InsP(3)R1 and utilized a recombinant truncated form of the channel (capn-InsP(3)R1) corresponding to the stable, carboxyl-terminal fragment to examine the functional consequences of channel proteolysis. Single-channel recordings of capn-InsP(3)R1 revealed InsP(3)-independent gating and high open probability (P(o)) under optimal cytoplasmic Ca(2+) concentration ([Ca(2+)](i)) conditions. However, some [Ca(2+)](i) regulation of the cleaved channel remained, with a lower P(o) in suboptimal and inhibitory [Ca(2+)](i). Expression of capn-InsP(3)R1 in N2a cells reduced the Ca(2+) content of ionomycin-releasable intracellular stores and decreased endoplasmic reticulum Ca(2+) loading compared with control cells expressing full-length InsP(3)R1. Using a cleavage-specific antibody, we identified calpain-cleaved InsP(3)R1 in selectively vulnerable cerebellar Purkinje neurons after in vivo cardiac arrest. These findings indicate that calpain proteolysis of InsP(3)R1 generates a dysregulated channel that disrupts cellular Ca(2+) homeostasis. Furthermore, our results demonstrate that calpain cleaves InsP(3)R1 in a clinically relevant injury model, suggesting that Ca(2+) leak through the proteolyzed channel may act as a feed-forward mechanism to enhance cell death.
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http://dx.doi.org/10.1074/jbc.M111.254177DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3195633PMC
October 2011
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