Tumor Necrosis Factor-α Potentiates Intraneuronal Ca2+ Signaling via Regulation of the Inositol 1,4,5-Trisphosphate Receptor

Inflammatory events have long been implicated in initiating and/or propagating the pathophysiology associated with a number of neurological diseases. In addition, defects in Ca2+-handling processes, which shape membrane potential, influence gene transcription, and affect neuronal spiking patterns, have also been implicated in disease progression and cognitive decline. The mechanisms underlying the purported interplay that exists between neuroinflammation and Ca2+ homeostasis have yet to be defined. Herein, we describe a novel neuron-intrinsic pathway in which the expression of the type-1 inositol 1,4,5-trisphosphate receptor is regulated by the potent pro-inflammatory cytokine tumor necrosis factor-α. Exposure of primary murine neurons to tumor necrosis factor-α resulted in significant enhancement of Ca2+ signals downstream of muscarinic and purinergic stimulation. An increase in type-1 inositol 1,4,5-trisphosphate receptor mRNA and protein steady-state levels following cytokine exposure positively correlated with this alteration in Ca2+ homeostasis. Modulation of Ca2+ responses arising from this receptor subtype and its downstream effectors may exact significant consequences on neuronal function and could underlie the compromise in neuronal activity observed in the setting of chronic neuroinflammation, such as that associated with Parkinson disease and Alzheimer disease. Inflammatory events have long been implicated in initiating and/or propagating the pathophysiology associated with a number of neurological diseases. In addition, defects in Ca2+-handling processes, which shape membrane potential, influence gene transcription, and affect neuronal spiking patterns, have also been implicated in disease progression and cognitive decline. The mechanisms underlying the purported interplay that exists between neuroinflammation and Ca2+ homeostasis have yet to be defined. Herein, we describe a novel neuron-intrinsic pathway in which the expression of the type-1 inositol 1,4,5-trisphosphate receptor is regulated by the potent pro-inflammatory cytokine tumor necrosis factor-α. Exposure of primary murine neurons to tumor necrosis factor-α resulted in significant enhancement of Ca2+ signals downstream of muscarinic and purinergic stimulation. An increase in type-1 inositol 1,4,5-trisphosphate receptor mRNA and protein steady-state levels following cytokine exposure positively correlated with this alteration in Ca2+ homeostasis. Modulation of Ca2+ responses arising from this receptor subtype and its downstream effectors may exact significant consequences on neuronal function and could underlie the compromise in neuronal activity observed in the setting of chronic neuroinflammation, such as that associated with Parkinson disease and Alzheimer disease. Although the brain was once believed to be an immunoprivileged site, it is presently evident that immune-related activities, including inflammation, underlie normative and pathological brain function. The physiological events that govern the transition of inflammatory reactions associated with self-limited events to chronic activation of inflammatory cascades found in the setting of neurodegeneration are of considerable interest. Various neurological disorders, including Parkinson disease, Alzheimer disease, and traumatic brain injury, display inflammation-related hallmarks, including cytokine and chemokine expression, the activation of central nervous system-residing macrophage-related cells (microglia), and in some cases, immune cell infiltration from peripheral sites via the brain microvasculature (reviewed in Ref. 1McGeer E.G. Klegeris A. McGeer P.L. Neurobiol. Aging. 2005; 26: 94-97Crossref PubMed Scopus (148) Google Scholar). The roles these processes play in disease pathogenesis are, however, less clearly defined. Tumor necrosis factor-α (TNF-α) 2The abbreviations used are: TNF-α, tumor necrosis factor-α; [Ca2+]c, cytosolic Ca2+; CCh, carbamylcholine; IP3, inositol 1,4,5-trisphosphate; JNK, c-Jun N-terminal kinase; MCP-1, monocyte chemoattractant protein-1; NFκB, nuclear factor-κB; RyR, ryanodine receptor; SERCA, sarcoendoplasmic reticulum ATPase; CPA, cyclopiazonic acid; BHK, baby hamster kidney; RT, reverse transcription; qRT, quantitative PCR; PMCA, Ca2+ clearance at the plasma membrane; PBS, phosphate-buffered saline; ER, endoplasmic reticulum. 2The abbreviations used are: TNF-α, tumor necrosis factor-α; [Ca2+]c, cytosolic Ca2+; CCh, carbamylcholine; IP3, inositol 1,4,5-trisphosphate; JNK, c-Jun N-terminal kinase; MCP-1, monocyte chemoattractant protein-1; NFκB, nuclear factor-κB; RyR, ryanodine receptor; SERCA, sarcoendoplasmic reticulum ATPase; CPA, cyclopiazonic acid; BHK, baby hamster kidney; RT, reverse transcription; qRT, quantitative PCR; PMCA, Ca2+ clearance at the plasma membrane; PBS, phosphate-buffered saline; ER, endoplasmic reticulum. a prototypical pro-inflammatory cytokine, has been studied primarily in the context of immune cell/tumor cell biology and induction of innate immunity and apoptosis (2Carswell E.A. Old L.J. Kassel R.L. Green S. Fiore N. Williamson B. Proc. Natl. Acad. Sci. U. S. A. 1975; 72: 3666-3670Crossref PubMed Scopus (3738) Google Scholar, 3Ortaldo J.R. Mason L.H. Mathieson B.J. Liang S.M. Flick D.A. Herberman R.B. Nature. 1986; 321: 700-702Crossref PubMed Scopus (98) Google Scholar, 4Laster S.M. Wood J.G. Gooding L.R. J. Immunol. 1988; 141: 2629-2634PubMed Google Scholar). In the central nervous system, TNF-α is produced by brain-resident astrocytes, microglia, and neurons in response to numerous intrinsic and extrinsic stimuli (5Hanisch U.K. Glia. 2002; 40: 140-155Crossref PubMed Scopus (1285) Google Scholar). Radioactive binding assays have also indicated the presence of TNF receptors in several regions in the brain, including the cortex (6Kinouchi K. Brown G. Pasternak G. Donner D.B. Biochem. Biophys. Res. Commun. 1991; 181: 1532-1538Crossref PubMed Scopus (168) Google Scholar). However, its role within the central nervous system has been controversial. TNF-α has been shown in some studies to lead to positive outcomes, as in the case of remyelination (7Arnett H.A. Mason J. Marino M. Suzuki K. Matsushima G.K. Ting J.P. Nat. Neurosci. 2001; 4: 1116-1122Crossref PubMed Scopus (802) Google Scholar), and its ability to prevent neurons from apoptosis when exposed to amyloid-β, a possible causative agent of Alzheimer disease (8Barger S.W. Horster D. Furukawa K. Goodman Y. Krieglstein J. Mattson M.P. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 9328-9332Crossref PubMed Scopus (561) Google Scholar). Alternatively, TNF-α induces neuronal apoptosis though an excitotoxic mechanism mediated through alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors (9Gelbard H.A. Dzenko K.A. DiLoreto D. del Cerro C. del Cerro M. Epstein L.G. Dev. Neurosci. 1993; 15: 417-422Crossref PubMed Scopus (153) Google Scholar) and activates microglia, which may lead to the propagation of various neurological diseases (5Hanisch U.K. Glia. 2002; 40: 140-155Crossref PubMed Scopus (1285) Google Scholar). Few studies have detailed the effects of TNF-α on ionic signaling cascades within neuronal cells, and most of these have focused on the modulation of plasma membrane ion channels. For example, it was shown that TNF-α can cause the insertion of Ca2+-permeable alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors in the plasma membrane of hippocampal pyramidal neurons (10Ogoshi F. Yin H.Z. Kuppumbatti Y. Song B. Amindari S. Weiss J.H. Exp. Neurol. 2005; 193: 384-393Crossref PubMed Scopus (123) Google Scholar) and that TNF-α can increase N-methyl-d-aspartic acid receptor activity in mouse cortical neurons (11Jara J.H. Singh B.B. Floden A.M. Combs C.K. J. Neurochem. 2007; 100: 1407-1420Crossref PubMed Scopus (62) Google Scholar). These events both result in an increase in neuronal stress and possibly death. Our present study examined the effects of TNF-α on intracellular ion channels, specifically the inositol 1,4,5-trisphosphate (IP3R) and ryanodine (RyR) receptors. We posited that changes in channel expression and/or activity contribute to alterations in neuronal functioning observed during neuroinflammation. Modulation of responses arising from these receptors may have significant consequences for neuronal function and survival, ranging from membrane hyperpolarization (12Stutzmann G.E. Smith I. Caccamo A. Oddo S. Laferla F.M. Parker I. J. Neurosci. 2006; 26: 5180-5189Crossref PubMed Scopus (260) Google Scholar) to the alteration of global protein expression (13Dolmetsch R.E. Xu K. Lewis R.S. Nature. 1998; 392: 933-936Crossref PubMed Scopus (1671) Google Scholar). To this end, we report a novel neuro-intrinsic pathway through which TNF-α regulates type-1 IP3R expression at least partially through c-Jun N-terminal protein kinase (JNK) activation. Perturbation of Ca2+ signaling through activation of this IP3R subtype and its downstream effectors may significantly impact neuronal function and could ultimately compromise neuronal activity in the setting of chronic neuroinflammation. Primary Cell Cultures—Baby hamster kidney (BHK) cells were maintained as previously described (14Lu B. Federoff H.J. Hum. Gene Ther. 1995; 6: 421-430Crossref Scopus (56) Google Scholar). Neuronal cultures were obtained from E14.5 cortical homogenates by microdissection and trypsin digestion. Neurons were then plated on polyethyleneimine-coated coverslips at a density of 3 × 105 cells/ml in B-27-supplemented Neurobasal Media (with 0.5 mm glutamine plus glutamic acid). One-half of the supplemented neurobasal media was changed every 4 days. Transwell co-cultures were established by culturing glia obtained from P0 C57BL/6 mice on plastic inserts (Corning). P0 cortices were microdissected, mechanically homogenized, and grown in minimal essential medium with 10% fetal bovine serum for 12 days post-dissection (Invitrogen). Microglial cultures were obtained by gentle agitation of the T-75 flask, removal of the media, centrifugation, and plating at a density of 5 × 104 cells/insert in 5% fetal bovine serum. After 1 week of growth, supplemented minimal essential medium was removed from the inserts, and cocultures were established for 4 days before murine recombinant TNF-α addition (11 neuronal days in vitro). Ca2+ Imaging—Days 12, 13, and 14 in vitro (24, 48, and 72 h, respectively) neuronal cultures were removed from their culturing media and loaded with Fura2-AM (Teflabs, 1 μm) in HEPES-buffered physiological saline solution (in mm): 5.5 glucose, 0.56 MgCl2, 4.7 KCl, 1 Na2HPO4, 10 HEPES, 1.2 CaCl2, pH 7.4. Ryanodine (100 μm) or Ru360 (10 μm) was loaded in conjunction with Fura2-AM for the RyR and mitochondrial blocking experiments, respectively. Coverslips were mounted in an imaging chamber and transferred to an inverted Nikon microscope with a 40× oil immersion objective lens (numerical aperture, 1.3). During experiments, HEPES-buffered solution, or an La3+-Ca2+ reduced solution (300 μm La3+ with 0.5 mm Ca2+), and agonists were perfused using a gravity-fed system (Warner Instruments), and excitation at 340 nm/380 nm was provided by a monochromator illumination system under the of was used to nm from at a of 1 in from 340 nm excitation and in nm excitation to an increase in intracellular During addition, the from was for and these were then for of from was a and of the Ca2+ signals were using was from neuronal cultures by the of was to using the was using an to the gene and by to a the gene of interest. were on a and and/or mRNA was as a for IP3R primary neurons × were mechanically from and through a in mm mm 1 mm mm pH × 4 was then to and the were transferred to The homogenates were then at × for 1 at 4 in a were in of mm KCl, 10 mm 5 mm 1 mm mm pH For cell receptor primary neurons × were mechanically with a in and through a were with were then by an A. H.J. Cell Res. PubMed Scopus Google Scholar), and of was then to on a 5% for the or a for the receptors. were transferred to and with a type-1 IP3R IP3R and a or a were then used in with for protein TNF-α and media was removed from neuronal cultures at 48, and 72 and for the levels of murine TNF-α by in with Media were for TNF-α activity to a previously described Williamson S. S. PubMed Scopus (123) Google Scholar). that binding and minimal of the recombinant gene was cells with in the was and neuronal media described was the were using and examined for activity using the system and a was from the cultures were exposed to murine recombinant TNF-α for 48, and 72 and then with in a saline solution was to the Coverslips were then on an microscope with nm excitation from a and on a and were using of was using and or of was on as with of were Ca2+ in Primary Neuronal with the impact of the pro-inflammatory cytokine, TNF-α, on endoplasmic reticulum Ca2+ we established neuronal cultures from C57BL/6 mouse on The cell of these cultures was through a neuronal nuclear the presence of primary the of protein minimal it was evident that the primary neurons processes during the of the culturing a to that previously R.E. Federoff H.J. Ther. 2002; PubMed Scopus Google Scholar). Ca2+ levels in neurons were then examined by imaging following and with The of murine recombinant TNF-α was on previously (8Barger S.W. Horster D. Furukawa K. Goodman Y. Krieglstein J. Mattson M.P. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 9328-9332Crossref PubMed Scopus (561) Google Scholar), as as of neuronal in the presence of the cytokine of the neurons with the muscarinic was to exposure to the pro-inflammatory cytokine to modulation of Ca2+ with was used to RyR was with cytokine TNF-α to a significant in by of Ca2+ signals at and exposure with a to levels at 72 cytokine and result was by an of for which the of TNF-α was significant in the The of neurons that to μm were also and significant was observed in the of cells In addition, levels of and cultures were by the of the before addition and were observed to with TNF-α addition of RyR was also obtained by the addition of mm In to the observed alterations in the Ca2+ in Ca2+ signals was observed exposure as a function of or with TNF-α exposure was significant in the of cells that to mm a on previously (12Stutzmann G.E. Smith I. Caccamo A. Oddo S. Laferla F.M. Parker I. J. Neurosci. 2006; 26: 5180-5189Crossref PubMed Scopus (260) Google Scholar, B. 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Google Scholar), as a positive and by following TNF-α and at levels through 12 increase in was significant from to of the of IP3R were used to levels of the receptors in neurons or neurons exposed to TNF-α for 12, and The of for the neuronal of the the type-1 was significantly at 12 The IP3R a the for the IP3R with TNF-α exposure was used as an for and for with experiments, its expression in response to TNF-α was to levels of type-1 IP3R lead to steady-state levels of with the the levels of the type-1 IP3R at and exposure were and the of receptor to protein was significantly and following TNF-α protein expression was used as a its expression was in these and its it to be observed on the IP3R and was also for the IP3R and to the significant in protein cytokine the in type-1 IP3R mRNA at 12 and protein at and correlated with the observed increase in Ca2+ signals at the and of IP3R and Ca2+ of TNF-α influence on IP3R protein expression and Ca2+ alterations was observed with at and and a to at the In an to the for TNF-α and activity was by an and an in vitro activity in which a was cells The of TNF-α in the was the However, the activity of the TNF-α in the media from primary neuronal cultures for a significant increase in activity from cultures for and 72 to the These TNF-α was during the of and an as to Ca2+ enhancement and type-1 IP3R protein levels were by 72 in primary neuronal cultures with the TNF-α on IP3R and Ca2+ primary neurons grown in were found to both the type-1 and TNF receptor and are both to to JNK, we examined the of a of JNK, on the observed in type-1 IP3R and Ca2+ was to the cultures 1 to cytokine addition Xu A. S. Y. A.M. Proc. Natl. Acad. Sci. U. S. A. 2001; PubMed Scopus Google Scholar). was from TNF-α plus and primary neuronal cultures and by as described with to the levels of the gene Exp. Cell Res. PubMed Scopus Google Scholar). To the of this at a the levels of mRNA were TNF-α exposure in the presence and of the K. U. K. M. S. M. U.K. Glia. 2005; PubMed Scopus Google Scholar). significant induction of was observed that was by with 10 μm of the μm) was then to cytokine exposure and for its ability to the enhancement type-1 IP3R steady-state levels was to the increase in type-1 IP3R expression at a of 10 μm this to the IP3R and to a of Ca2+ channels, was of the on type-1 RyR levels The protein of the type-1 IP3R was also and the 10 μm in the presence of TNF-α was found to receptor expression to levels were to with muscarinic signals to Primary C57BL/6 neurons were with TNF-α and 10 μm with a of the or with as a and to Ca2+ In with the and protein with the the observed with TNF-α exposure The of the was by its effects on Ca2+ was observed modulation of signals with TNF-α and the was found to Ca2+ signals In the of the that TNF-α type-1 IP3R steady-state levels and muscarinic Ca2+ homeostasis through a The present study a novel neuronal pathway in which the expression of the IP3R can be regulated by TNF-α through the activation of a signaling of neuronal exposure to murine recombinant TNF-α resulted in an increase in type-1 IP3R mRNA and protein and was shown to Ca2+ signals downstream of receptor stimulation. this increase in was to be of possible alterations in Ca2+ Ca2+ and Ca2+ Our that enhancement of type-1 IP3R protein levels the alterations observed in muscarinic TNF-α to the or TNF activates a which in steady-state levels of type-1 IP3R mRNA at 12 and protein at and We that the of IP3R protein levels at 72 to of recombinant TNF-α in the However, it is also that alterations in TNF receptor and expression could be a role in the of this We also the of IP3R such as J. 1993; PubMed Google Scholar, J. PubMed Scopus Google Scholar, I. J. Neurosci. PubMed Google Scholar), binding Google Scholar), modulation of and/or modulation by binding (reviewed in Ref. I. Cell 2005; PubMed Scopus Google Scholar), these have been shown to affect Ca2+ is also possible that TNF-α may the of through the modulation of These mechanisms as the of the Ca2+ enhancement correlated with IP3R protein and changes in levels of IP3R protein have been shown previously to Ca2+ responses to the receptor K. PubMed Scopus Google Scholar, S. Exp. Res. PubMed Scopus Google Scholar). is a which also central nervous inflammatory cells We to the presence of could affect the signaling of TNF-α within the brain in such a as to the or its pro-inflammatory Our in this inflammatory were a However, in may as of or signals that may neuronal Ca2+ and S. Mattson M.P. J. 2005; PubMed Scopus Google Scholar) previously that of the gene in mouse IP3R expression and Ca2+ which was the result of a of an by the We that this increase in IP3R expression may be a result of a in by the a that these also observed an increase in activity following The ability of TNF-α to a response on receptor may lead to a of Ca2+ signals during Our in vitro study has that and of TNF-α may cause the enhancement of Ca2+ signals to be However, of TNF-α within the brain may cause a chronic enhancement of muscarinic alterations in of the receptor to downstream may play a role in the inflammatory to an or chronic to or downstream of that are for the enhancement of IP3R by L.J. Gene 1998; PubMed Scopus Google Scholar) has sites within the type-1 IP3R that the may The of these sites be the of this a of that can this TNF-α, can be which may the physiological role of this signaling during inflammatory events within the The long physiological consequences of the of TNF-α IP3R expression have yet to be is that the enhancement of muscarinic signals a in a of the for cell death. is also possible that inflammatory possibly cytokine, or an could cause the to a an pathway to death. this is the the enhancement of IP3R steady-state levels could the changes in Ca2+ signals for of with Floden and A.M. S. Combs C.K. J. Neurosci. 2005; PubMed Scopus Google Scholar) have shown that TNF-α neuronal cell death. However, in the presence of the cytokine can apoptosis via the increase in IP3R expression could of a physiological mechanism of a An increase in the of muscarinic agonists could lead to a and it to excitotoxic by a is that both of these mechanisms could on the with the to or its to the in TNF receptor expression and the activity of may the for neuronal are at for and in neurological diseases underlying chronic neuroinflammation. We and of for of for for the and of for on IP3R protein and for on the with