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Ion Channels in Health and Disease 1st Edition Geoffrey S.

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Damage Associated Molecular Patterns in Human Diseases

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CONTRIBUTORS

Elena Adinolfi
Department of Morphology, Surgery and Experimental Medicine, Section of Pathology,
Oncology and Experimental Biology, University of Ferrara, Ferrara, Italy
Yun-Long Bai
Harbin Medical University, Harbin, PR China
Shaherin Basith
National Leading Research Laboratory of Molecular Modeling & Drug Design (NLRL),
College of Pharmacy and Graduate School of Pharmaceutical Sciences, Ewha Womans
University, Seoul, Korea
Ben-Zhi Cai
Harbin Medical University, Harbin, PR China
Sun Choi
National Leading Research Laboratory of Molecular Modeling & Drug Design (NLRL),
College of Pharmacy and Graduate School of Pharmaceutical Sciences, Ewha Womans
University, Seoul, Korea
Nuria Comes
Universitat de Barcelona; Institut d’Investigacions Biomediques August Pi i Sunyer
(IDIBAPS), Barcelona, Spain
Minghua Cui
National Leading Research Laboratory of Molecular Modeling & Drug Design (NLRL),
College of Pharmacy and Graduate School of Pharmaceutical Sciences, Ewha Womans
University, Seoul, Korea
Diego Currò
Institute of Pharmacology, School of Medicine, Catholic University of the Sacred Heart,
Rome, Italy
Diego Dal Ben
School of Pharmacy, Medicinal Chemistry Unit, University of Camerino, Camerino, Italy
Joanne O. Davidson
Department of Physiology, Faculty of Medical and Health Sciences, Auckland, New Zealand
Elena De Marchi
Department of Morphology, Surgery and Experimental Medicine, Section of Pathology,
Oncology and Experimental Biology, University of Ferrara, Ferrara, Italy
De-Li Dong
Harbin Medical University, Harbin, PR China
Xavier Gasull
Universitat de Barcelona; Institut d’Investigacions Biomediques August Pi i Sunyer
(IDIBAPS), Barcelona, Spain

ix
x Contributors

Jonathan P. Giblin
Universitat de Barcelona; Institut d’Investigacions Biomediques August Pi i Sunyer
(IDIBAPS), Barcelona, Spain
Vijayakumar Gosu
National Leading Research Laboratory of Molecular Modeling & Drug Design (NLRL),
College of Pharmacy and Graduate School of Pharmaceutical Sciences, Ewha Womans
University, Seoul, Korea
Colin R. Green
Department of Ophthalmology and New Zealand National Eye Centre, Auckland,
New Zealand
Alistair J. Gunn
Department of Physiology, Faculty of Medical and Health Sciences, Auckland, New Zealand
Katherine C. Gunn
Department of Physiology, Faculty of Medical and Health Sciences, Auckland, New Zealand
Sunhye Hong
National Leading Research Laboratory of Molecular Modeling & Drug Design (NLRL),
College of Pharmacy and Graduate School of Pharmaceutical Sciences, Ewha Womans
University, Seoul, Korea
Yuri Kim
Department of Ophthalmology and New Zealand National Eye Centre, Auckland,
New Zealand
Cesar O. Lara-Figueroa
Laboratorio Nacional de Canalopatı́as, Instituto de Fisiologı́a Celular, Universidad Nacional
Autónoma de Mexico, Mexico City, Mexico
Arlet Loza-Huerta
Laboratorio Nacional de Canalopatı́as, Instituto de Fisiologı́a Celular, Universidad Nacional
Autónoma de Mexico, Mexico City, Mexico
Elisa Orioli
Department of Morphology, Surgery and Experimental Medicine, Section of Pathology,
Oncology and Experimental Biology, University of Ferrara, Ferrara, Italy
Anthony R. Phillips
School of Biological Sciences, University of Auckland, Auckland, New Zealand
Arturo Picones
Laboratorio Nacional de Canalopatı́as, Instituto de Fisiologı́a Celular, Universidad Nacional
Autónoma de Mexico, Mexico City, Mexico
Pedro Segura-Chama
Laboratorio Nacional de Canalopatı́as, Instituto de Fisiologı́a Celular, Universidad Nacional
Autónoma de Mexico, Mexico City, Mexico
Olaf Strauss
Charite University Medicine, Berlin, Germany
Contributors xi

Wahyu Surya
School of Biological Sciences, Nanyang Technological University, Singapore
Janet To
School of Biological Sciences, Nanyang Technological University, Singapore
Jaume Torres
School of Biological Sciences, Nanyang Technological University, Singapore
Nelson S. Yee
Program of Experimental Therapeutics, Penn State Hershey Cancer Institute, Penn State
Milton S. Hershey Medical Center; Pennsylvania State University College of Medicine,
Hershey, PA, United States
PREFACE

Ion channels are integrated membrane proteins forming pores through

which ions can pass and travel between cells and extracellular space. The
transport through ion channels is passive which means that it does not
require participation of metabolic energy and occurs down their electro-
chemical gradient only. Most channels are selective for ne ion type only.
Typically, the pore is so small that ions pass through in a single-file order.
Although the human genome sequencing identified more than 400 putative
ion channels, only a very small number of these hypothetically identified
channels have been cloned and functionally characterized. The widespread
tissue distribution of ion channels, along with the multiple physiological
consequences of their opening and closing, makes targeting of ion channels
very promising targets for development of therapeutics. The validation of
ion channels as drug targets provides an enormous market opportunity
for their reemergence as key targets in drug discovery. However, in spite
of some important drugs, currently in clinical use, which target ion channels,
this call of proteins remain significantly underexploited in drug discovery.
Furthermore, many existing drugs are poorly selective with significant
toxicities or suboptimal efficacy.
This is second of the two parts of thematic volumes of the Advances in
Protein Chemistry and Structural Biology focusing on recent developments in
ion channels studies and their potential as promising therapeutic targets.
While the first part of these thematic volumes (Volume 103) focuses more
specifically on ion channels as promising treatment targets in neurological
and psychiatric disorders, the second part is dedicated on particular ion
channel types (eg, connexin hemichannels, P2X7, TRPV1, TRPM8, virus
channels) and their role as therapeutic targets in inflammation, pancreatic
cancer, eye disorders, and cardiovascular and gastrointestinal diseases. The
contribution of automated technologies to ion channel drug discovery is also
discussed in detail in this second part of the two volumes.
The first article in this volume considers the central nervous immune sys-
tem from the perspective that modulating connexin hemichannel opening
can prevent tissue damage arising from excessive and uncontrolled inflam-
mation. Authors discuss connexin channel roles in microglia, astrocytes, and
endothelial cells in both acute and chronic inflammatory conditions, and in
particular describe the role of connexin hemichannels in the inflammasome

xiii
xiv Preface

pathway where they contribute to both its activation and its spread to neigh-
boring cells. The second chapter describes structural features of P2X7 recep-
tor, chemical properties of its agonist, antagonist, and allosteric modulators,
and summarizes recent advances on P2X7 as therapeutic target in infectious,
inflammatory, autoimmune, neurological, musculoskeletal, and cancer
disorders. The use of P2X7 single nucleotide polymorphisms as diagnostic
biomarkers for the development of tailored therapies is also discussed in this
second article. The third chapter in this volume is dedicated to the transient
receptor potential vanilloid 1 (TRPV1) ion channel. Here authors give an
in-depth mechanistic outlook on the TRPV1 channel, and more specifi-
cally, they explore the TRPV1 structure, activation, modulation, ligands,
and its therapeutic targeting. Another major objective of this third review
is to highlight the fact that TRPV1 channel can be treated as an effective
therapeutic target for treating several pain- and nonpain-related physiolog-
ical and pathological states. The fourth article in this volume provides an
extensive review and discussion of the transient receptor potential
melastatin-subfamily member 8 (TRPM8) ion channel as a potential bio-
marker and target in cancer.
The next group of articles are focused on the role of different ion channel
types in disorders. In the fifth article, authors review the roles of different ion
channels expressed in ocular tissues that are involved in ocular disease path-
ophysiologies and those whose deletion or pharmacological modulation
leads to a specific diseases of the eye. These include pathologies such as
retinitis pigmentosa, macular degeneration, achromatopsia, glaucoma, cata-
racts, dry eye, or keratoconjunctivitis among others. Several of disease-
associated ion channels are discussed as potential targets for pharmacological
intervention or other therapeutic approaches, thus highlighting the impor-
tance of these channels in ocular physiology and pathophysiology. The sixth
chapter summarizes the physiological and pathological roles of the three
types of Ca2+-activated K+ channels (KCa) in cardiovascular system and
put forward the possibility of KCa channels as potential therapeutic target
for cardiovascular diseases. The next seventh article discusses extensively
the modulation of potassium channels in the smooth muscles as a therapeutic
strategy for disorders of the gastrointestinal tract. The eighth chapter in the
volume is centered on virus channels and their role as therapeutic targets.
Since the discovery that certain small viral membrane proteins, collectively
termed as viroporins, can permeabilize host cellular membranes and also
behave as ion channels, attempts have been made to link this feature to
specific biological roles. Most viroporins identified so far are virulence
Preface xv

factors, and interest has focused toward the discovery of channel inhibitors
that would have a therapeutic effect, or be used as research tools to under-
stand the biological roles of viroporin ion channel activity.
The final ninth article explores the use of automated instrumentation for
the study of ion channel functionality (and dysfunctionality), particularly in
the search for novel pharmacological agents with therapeutic purposes.
Their application has now reached out beyond the industrial setting, its
original natural enclave, and is making its way into a growing number of
academic labs and core facilities. This review presents and discusses the
increasing contributions accomplished by a variety of different key auto-
mated technologies which have revolutionized the strategies to approach
the discovery and development of new drugs targeting ion channels.
Considering the very high number of genetically predicted ion channels,
this big group of proteins remain relatively underexploited in terms of their
functions, involvement in disorders, and possibilities for therapeutic inter-
ventions. Taking into account the recent advances in biomedical knowledge
and automated technologies available for searching novel pharmacological
agents with therapeutic effects, the aim of this second volume dedicated
to ion channels as therapeutic targets is to promote further research in the
structure, function, and regulation of different families of ion channels
which would result in designing new efficient targeted drugs with signifi-
cantly fewer adverse effects.

DR. ROSSEN DONEV

Biomed Consult
United Kingdom
 CHAPTER ONE

Role of Hemichannels in CNS

Inflammation and the
Inflammasome Pathway
Yuri Kim*, Joanne O. Davidson†, Katherine C. Gunn†,
Anthony R. Phillips{, Colin R. Green*, Alistair J. Gunn†,1
*Department of Ophthalmology and New Zealand National Eye Centre, Auckland, New Zealand
†
Department of Physiology, Faculty of Medical and Health Sciences, Auckland, New Zealand
{
School of Biological Sciences, University of Auckland, Auckland, New Zealand
1
Corresponding author: e-mail address: [email protected]

Contents
1. Introduction 2
2. Role of Connexin Hemichannels in the Inflammatory Cascade of the CNS 4
2.1 Acute Inflammatory Response 4
2.2 Microglia in Acute Inflammation 5
2.3 Astrocytes in Acute Inflammation 5
2.4 Endothelial Cells in Acute Inflammation 9
2.5 Chronic Inflammation 10
3. Connexin Hemichannels and Their Roles in Response to Injury and Inflammation 12
3.1 Linking Connexin Hemichannels to the Inflammasome Pathway 17
3.2 Animal Models Show Therapeutic Benefits of Connexin Hemichannel Block 19
4. Conclusion 25
References 26

Abstract
Neurodegenerative, cardiovascular, and metabolic disorders, once triggered, share a
number of common features, including sustained inflammatory cell activation and vas-
cular disruption. These shared pathways are induced independently of any genetic pre-
disposition to the disease or the precise external stimulus. Glial cells respond to injury
with an innate immune response that includes release of proinflammatory cytokines
and chemokines. Vascular endothelial cells may also be affected, leading to opening
of the blood–brain barrier that facilitates invasion by circulating inflammatory cells.
Inflammation can trigger acute neural injury followed by chronic inflammation that
plays a key role in neurodegenerative conditions. Gap junction channels normally allow
direct cell-to-cell communication. They are formed by the docking of two
hemichannels, one contributed by each of the neighboring cells. While the opening
probability of these channels is tightly controlled under resting conditions,
hemichannels can open in response to injury or inflammatory factors, forming a large,

Advances in Protein Chemistry and Structural Biology, Volume 104 # 2016 Elsevier Inc. 1
ISSN 1876-1623 All rights reserved.
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2 Yuri Kim et al.

relatively nonselective membrane pore. In this review, we consider the CNS immune
system from the perspective that modulating connexin hemichannel opening can pre-
vent tissue damage arising from excessive and uncontrolled inflammation. We discuss
connexin channel roles in microglia, astrocytes, and endothelial cells in both acute and
chronic inflammatory conditions, and in particular describe the role of connexin
hemichannels in the inflammasome pathway where they contribute to both its activa-
tion and its spread to neighboring cells. Finally, we describe the benefits of hemichannel
block in animal models of brain injury.

1. INTRODUCTION
Neurodegenerative, cardiovascular, and metabolic disorders can trig-
ger a number of shared processes, including sustained inflammatory cell acti-
vation and vascular disruption (with accompanying tissue ischemia), that in
effect resemble persistent, nonhealing “wounds.” These shared processes
occur regardless of genetic predisposition to the disease or the initial external
stimulus (such as circulating inflammatory cytokines or an ischemic event)
(Green & Nicholson, 2008).
The central nervous system (CNS), which consists of the brain and the
spinal cord, has a continuous surveillance mechanism to detect and eliminate
mediators of infection and/or injury (Boulay et al., 2015). Even in their qui-
escent state, microglial cells continuously monitor their environment, as the
primary resident immune cells of the CNS (Nimmerjahn, Kirchhoff, &
Helmchen, 2005). Infection or sterile injury (eg, stroke) stimulates microglia
to transform to an activated phenotype and to recruit neighboring immune
cells in an attempt to clear the immune privileged site of injury and debris
(Davalos et al., 2005). The response of the microglia to injury and/or infec-
tion is usually very rapid and precedes that of other cells of the CNS
(Kreutzberg, 1996). Activated microglial cells are primed to release
proinflammatory cytokines and chemokines to trigger the innate immune
response—the host’s first line of defense against injury and/or infection
( Janzer & Raff, 1987). Activated microglia communicate with surrounding
CNS cells, including astrocytes and pericytes that project their end-feet
around vessels to form a highly specialized neurovascular unit (Bertossi
et al., 1993; Mulligan & MacVicar, 2004). The neurovascular unit, together
with endothelial cells, maintains the integrity of the blood–brain barrier
(BBB) that protects the brain from infiltration of foreign molecules and
immune cells ( Janzer & Raff, 1987).
CNS Inflammation and the Inflammasome Pathway 3

If the innate immune response is unsuccessful in clearing the stimulus,

short-term elevation in proinflammatory mediators can progress to chronic
inflammation that plays a key role in neurodegenerative conditions (eg,
Alzheimer’s disease (AD)) (reviewed in Hagberg et al., 2015). Inflammation
is a double-edged sword; innate immune response is an essential defense
mechanism (Arnett et al., 2001; Mason, Suzuki, Chaplin, & Matsushima,
2001), but hyperactive immune cells exacerbate the damage to both the
inflamed tissue and its surrounding regions (Lehnardt et al., 2003). This sug-
gests that it may be possible to improve outcomes of many conditions by
modulating excessive or uncontrolled inflammatory responses.
Gap junction channels allow direct cell-to-cell communication. CNS
gap junctions play multiple roles; critically they link glial cells to form a large
intercellular network than can dissipate molecules such as K+ or glutamate
and so to maintain homeostatic balance (Wallraff et al., 2006). Gap junction
channels are formed by two connexons (or hemichannels), one contributed
by each of the neighboring cells, that dock to form a direct pathway linking
the cytoplasm of the adjacent cells and allowing exchange of ions and
molecules of up to about 1.5 kDa (Harris, 2007; Simpson, Rose, &
Loewenstein, 1977). Each connexon is an assembly of six connexin subunits.
Of the 21 connexin isoforms in the human genome, named according to
their theoretical molecular mass in kDa (Willecke et al., 2002), 11 have been
reported to be expressed in the CNS. Isoform expression varies with brain
region, cell type, and stage of development. Connexin (Cx)43 is the pre-
dominant connexin found in astrocytes, along with Cx30 (Rash et al.,
2001). Astrocytes are linked by gap junctions to other astrocytes and to
oligodendrocytes, but not to mature neurons (Rash et al., 2001). Cx43 is
also abundant at the astrocytic end-foot processes that surround blood
vessels, and form the functional BBB, and on the astrocytic processes in close
proximity to chemical synapses (Chew, Johnson, Green, & Danesh-Meyer,
2010). Cx43 is also expressed in normal capillary endothelium and at low
levels in microglia under resting conditions (Eugenin et al., 2001; Pepper
et al., 1992).
To be able to detect infection and injury, cells in the CNS are highly
sensitive to changes in its microenvironment. One of the key channels on
the surface of CNS cells is the undocked connexin hemichannel. While
the opening probability of these channels is tightly controlled under resting
conditions (Bukauskas et al., 2000; Contreras, Saez, Bukauskas, & Bennett,
2003), hemichannels are triggered to open in response to a number of fac-
tors. These include proinflammatory cytokines (Retamal et al., 2007),
4 Yuri Kim et al.

metabolic inhibition (Contreras et al., 2002; John, Kondo, Wang,

Goldhaber, & Weiss, 1999), changes to Ca2+ concentration (increase in
cytoplasmic Ca2+ concentration or decrease in extracellular concentration)
(De Vuyst et al., 2009; Li et al., 1996; Thimm, Mechler, Lin, Rhee, & Lal,
2005), or directly by infectious material such as gram-positive bacterial cell
wall proteins (peptidoglycan (PGN)) (Robertson, Lang, Lambert, & Martin,
2010). While it is generally believed that gap junction coupling is crucial
for cell survival (Krysko, Leybaert, Vandenabeele, & D’Herde, 2005),
uncontrolled opening of a large, nonselective hemichannel has a potent
capacity to drive inflammatory cytokine release by degrading ionic and
metabolic gradients that lead to cell death (Takeuchi et al., 2006).
In this review, we consider the CNS immune system from a perspective
that modulating connexin hemichannels can prevent tissue damage arising
from excessive and uncontrolled inflammation, and discuss connexin
hemichannel roles in the inflammasome pathway.

2. ROLE OF CONNEXIN HEMICHANNELS IN THE

INFLAMMATORY CASCADE OF THE CNS
2.1 Acute Inflammatory Response
Acute inflammation is a rapid, innate response (over a period of hours) that is
triggered by microbial infection or tissue damage. The innate immune
response is initiated when microglia sense pathogen-associated molecular
patterns (PAMPs) or tissue-derived endogenous “damage”-associated
molecular patterns (DAMPs) (eg, proinflammatory cytokines, ATP,
chemokines, and reactive oxygen species) (Nimmerjahn et al., 2005;
Zhang et al., 2010). Once detected, activated microglia recruit other
CNS-derived macrophages to clear the inflammatory stimuli (reviewed in
Prinz, Priller, Sisodia, & Ransohoff, 2011).
Cx43 hemichannels are highly expressed in CNS cells, including
microglia, astrocytes, and the endothelium, and are highly sensitive to
DAMPs and PAMPs (Contreras et al., 2002; De Vuyst et al., 2009; John
et al., 1999; Li et al., 1996; Retamal et al., 2007; Robertson et al., 2010;
Thimm et al., 2005; Ye, Wyeth, Baltan-Tekkok, & Ransom, 2003).
Whereas Cx43 hemichannels are tightly controlled under resting condi-
tions, the presence of DAMPs and PAMPs can trigger the opening of the
relatively large and nonspecific pore (10 Å) to release small cytosolic com-
pounds (Cotrina et al., 1998). Under these injury conditions, pattern recog-
nition receptors are also triggered, including the NOD-like receptors of the
CNS Inflammation and the Inflammasome Pathway 5

inflammasome complex (reviewed in Takeuchi & Akira, 2010). If the innate

inflammatory response is unsuccessful in clearing the stimulus, the acquired
immune response promotes infiltration of T and B cells from the peripheral
nervous system (Metcalf, Baxter, Nilaratanakul, & Griffin, 2013), leading to
vascular leak and after a sufficiently severe stimulus, neuronal cell death.
Adaptive immunity can restore and repair the injured tissue or lead to
chronic inflammation (reviewed in Hagberg et al., 2015).

2.2 Microglia in Acute Inflammation

Microglial glial cells can dynamically switch between surveying (“resting”)
and activated states (reviewed in Kreutzberg, 1996). In its “resting” pheno-
type, the microglial cell has a ramified morphology with a low level of Cx43
(Garg, Md Syed, & Kielian, 2005). However, cues such as brain injury or
infection readily activate microglia and are associated with upregulation
of Cx43 expression (Eugenin et al., 2001). Activated microglia also prolif-
erate and migrate to the site of injury within the first 24 h from the onset
(Eugenin et al., 2001). For example, Staphylococcus aureus-derived PGN
induces Cx43 upregulation and gap junction coupling in addition to
upregulation of tumor necrosis factor (TNF)-α and interleukin (IL)-1β
(Garg et al., 2005). Microglial accumulation at the site of injury peaks after
approximately 2–4 d but in some settings can persist for many weeks (Mcrae,
Gilland, Bona, & Hagberg, 1995).

2.3 Astrocytes in Acute Inflammation

Astrocytes abundantly express Cx43 under basal conditions (Giaume &
McCarthy, 1996). However, astrocytes show altered Cx43 expression in
response to proinflammatory cytokines. In mouse cortical astrocyte cultures,
treatment with a combination of IL-1β and TNF-α for 24 h in vitro reduced
total Cx43 expression on the cell membranes compared to untreated con-
trols (Retamal et al., 2007). This combination of proinflammatory cytokines
is required to reduce Cx43 expression and cell–cell coupling, as TNF-α or
IL-1β alone at 24 h had no significant effect compared to control (Meme
et al., 2006).
Lipopolysaccharide (LPS), a major component of the cell walls of gram-
negative bacteria (Bolon, Peng, Kidder, & Tyml, 2008; Frost, Nystrom, &
Lang, 2002; Smolinski & Pestka, 2003), can also reduce Cx43 expression,
through induction of proinflammatory cytokines (TNF-α and IL-6).
Short-term exposure of rat cortical astrocytes to LPS (1–6 h) did not affect
6 Yuri Kim et al.

Cx43 expression, but a longer exposure for 10–72 h was associated with a
significant reduction in Cx43 via the ubiquitin–proteasome pathway
(Liao, Jeng, Wang, Wang, & Wu, 2013; Liao, Wang, Chen, Wang, &
Wu, 2010). Spinal cord astrocytes also displayed a time-dependent reduction
in Cx43 mRNA and protein in response to 24 and 48 h exposure to TNF-α
and IFN-γ (Zhang, Morioka, Nakashima-Hisaoka, & Nakata, 2013). Strik-
ingly, the combination of TNF-α, and IFN-γ was associated with reduced
Cx43 mRNA expression as early as 3-h postincubation (Zhang, Morioka,
et al., 2013). Similarly to brain astrocytes, a change in Cx43 expression in
the spinal cord was attributed to enhanced degradation via the ubiquitin–
proteasome pathway (Zhang, Morioka, Kitamura, Hisaoka-Nakashima, &
Nakata, 2015). It is important to appreciate, however, that there is a
distinction between reduced cell–cell coupling and increased hemichannel
opening under such conditions, as will become apparent.
Studies examining the overall effect of injury in vivo report significant
upregulation of Cx43. For example, upregulation of Cx43 immunoreactiv-
ity was observed at 1–2, 4, 8, and 24 h after ischemia–reperfusion retinal
injury, and colocalized with astrocytes, Muller cells and vascular endothelial
cells (Danesh-Meyer et al., 2012). Similarly, spinal cord injury was associated
with upregulation of astrocytic Cx43 expression in the peritraumatic regions
(Cronin, Anderson, Cook, Green, & Becker, 2008; Theriault, Frankenstein,
Hertzberg, & Nagy, 1997). In light-damaged retina in the rat, upregulation
of Cx43 in the choroid has been reported to colocalize with nitrotyrosine, a
marker of oxidative stress, and with inflammatory cells (Guo, Tran, Green,
Danesh-Meyer, & Acosta, 2014).
Under normal conditions, in vitro a rapid yet balanced translation and
internalization of cell surface Cx43 is maintained, with a half-life of approx-
imately 1–2 h (Laird, Puranam, & Revel, 1991). Turnover is dynamic and
amenable to external cues. The apparent discrepancies in the expression of
Cx43 could be attributed to the reactive status of the inflammatory cascade.
The major proinflammatory cytokines, TNF-α and IL-1, are detected as
early as 1 h after injury, before neuronal death (Liu et al., 1994; Wang
et al., 1994). Clinical studies have correlated the severity of injury with
increased cytokine levels in postmortem brain tissue (Griffin et al., 1994;
Krupinski, Kumar, Kumar, & Kaluza, 1996). Astrocytes are particularly sen-
sitive to TNF-α, which directly elevates intracellular Ca2+ concentration
and induces cell depolarization (Koller, Thiem, & Siebler, 1996; Koller,
Trimborn, Von Giesen, Schroeter, & Arendt, 2001). Additionally, IL-1β
promotes the release of vascular endothelial growth factor from activated
CNS Inflammation and the Inflammasome Pathway 7

astrocytes that can enhance the permeability of the BBB and leakage of
plasma protein-rich fluid (Argaw et al., 2012). Proinflammatory cytokines
uncouple the astrocytic gap junction network but enhance hemichannel
activity (Meme et al., 2006; Retamal et al., 2007). In the brain TNF-α
and IL-1β have been identified as the most potent inhibitors of astrocyte–
astrocyte communication (Meme et al., 2006).
While it is clear that gap junctions help to maintain neuronal homeostasis
under normal conditions (Wallraff et al., 2006), a role for gap junctions
in inflammation has been debated, particularly as to whether uncoupled
gap junctions affect the overall viability of the CNS. “Bystander death”
effect was initially termed by Freeman et al. (1993) to describe how
Ganciclovir-sensitive tumor cells could also induce apoptosis in a coculture
of Ganciclovir-insensitive tumor cells (Freeman et al., 1993). However,
Cx43 gene knockout model studies in the CNS provided an argument
against the “bystander” death in CNS injury by demonstrating that loss of
Cx43 did not confer protection and indeed exacerbated the size of the injury
(Nakase, Sohl, Theis, Willecke, & Naus, 2004; Siushansian, Bechberger,
Cechetto, Hachinski, & Naus, 2001). In support, inhibiting gap junction
channels following injury compromises astrocytic function leading to gluta-
mate cytotoxicity and increased neuronal injury (Ozog, Siushansian, &
Naus, 2002). Hence, the alternate hypothesis suggests that gap junctions pre-
vent the accumulation of toxic metabolites and synchronize tissues follow-
ing injury through “spatial-buffering.” It is possible that both prodeath and
prosurvival roles of gap junctions in astrocytes are significant, but are highly
dependent on the time from the onset of injury or infection. While gap
junctions may initially propagate the injury signals to neighboring cells,
recent evidence in the literature associates long-term inhibition of gap junc-
tion communication with enhanced secondary lesion volume.
Conversely, under normal conditions Cx43 hemichannels are predom-
inantly in the closed state (Contreras et al., 2003), but are triggered to open
after injury. Uncontrolled exchange between the cytoplasm and the extra-
cellular space via open Cx43 hemichannels contributes to astrocytic cell
death (Contreras et al., 2002). Cx43 gap junction channels and hemi-
channels are oppositely regulated during acute inflammation. The adverse
outcome with inflammation is likely to reflect both uncoupled gap junc-
tions and open hemichannels. Proinflammatory cytokines increase astrocyte
permeability to the extracellular space through Cx43 hemichannels while
intercellular communication is reduced (Retamal et al., 2007). This has
direct implications for the metabolic status of astrocytes during acute
8 Yuri Kim et al.

inflammation as cytokines increase glucose uptake but reduce intercellular

exchange (Retamal et al., 2007). Furthermore, elevation in intracellular
Ca2+ and depolarization are observed in astrocytes after exposure to
TNF-α (Koller et al., 1996, 2001).
Enhanced Cx43 hemichannel activity also elevates purinergic signaling
mediators, including adenosine 5´-triphosphate (ATP), in the extracellular
space (Stout, Costantin, Naus, & Charles, 2002). ATP release can occur in
an uncontrolled manner, as occurs during necrosis (Iyer et al., 2009), but
there is evidence for controlled release of ATP into the extracellular
space by connexin hemichannels (Gomes, Srinivas, Van Driessche,
Vereecke, & Himpens, 2005; Stout et al., 2002) and pannexin channels
(Chekeni et al., 2010). Intracellular ATP is crucial for energy transfer
(Lipmann, 1941) but Cx43 hemichannel-mediated ATP release has been
shown to activate the purinergic receptor, P2RX7, leading to reactive
astrocytes and microglia in the peritraumatic regions of a spinal cord injury
(Cotrina & Nedergaard, 2009; Huang et al., 2012). Interestingly, post-
traumatic ATP release was not present in Cx43 knockout mice, providing
further support for Cx43-dependent ATP release (Huang et al., 2012).
A rise in extracellular ATP is also reported during ischemia (Melani
et al., 2006) and epilepsy initiated seizures (During & Spencer, 1992).
ATP acts as a potent activator of the ATP-gated purinoceptors expressed
on the surface of astrocytes and microglia (Salter & Hicks, 1994), and
also leukocytes (Eltzschig et al., 2006) and endothelial cells (Yamamoto
et al., 2000). Furthermore, ATP induces its own release through connexin
hemichannels and subsequent activation of purinergic receptors (Orellana,
Martinez, & Retamal, 2013).
A key mechanism that links the ATP-purinergic pathway and inflamma-
tion is P2RX7-mediated maturation and secretion of IL-1β from microglia
(Di Virgilio, Sanz, Chiozzi, & Falzoni, 1999). Recently an intracellular mul-
tiprotein unit termed the “inflammasome” has been discovered that medi-
ates this pivotal interaction between signaling molecules and release of IL-1β
(Martinon, Agostini, Meylan, & Tschopp, 2004). In particular, ATP is
thought to trigger inflammasome activation in CNS damage (Hanamsagar,
Torres, & Kielian, 2011; Hoegen et al., 2011). Inflammasomes typically have
the NLR protein as an intracellular signaling molecule that can sense
pathogen- or host-derived signals (Wen, Miao, & Ting, 2013). Activated
NLR protein binds to apoptosis-associated speck-like protein containing a
CARD (ASC), which in turn activates cysteine protease caspase-1 to activate
cytokines IL-1β and IL-18 into their active forms to mediate cell death
CNS Inflammation and the Inflammasome Pathway 9

(pyroptosis) (Schroder & Tschopp, 2010). Inflammation-induced cell death

could be attributed to a feed-forward mechanism caused by Cx43
hemichannel-mediated release of ATP that subsequently induces chronic acti-
vation of neuronal P2RX7 and further opening of Cx43 hemichannels. This
raises a possible role for connexin hemichannels as a “switch” for the
inflammasome signaling cascade by contributing significantly to extracellular
ATP in both injury and postinjury.

2.4 Endothelial Cells in Acute Inflammation

The BBB is a highly specialized endothelial structure that separates the
neurons from blood-derived components (Stewart & Wiley, 1981).
A key feature of CNS is that BBB forms a selective permeable barrier
to allow nutrient exchange while restricting the infiltration of pathogens
and immune cells into the brain (Eugenin, Clements, Zink, & Berman,
2011). The barrier properties of BBB are dependent on the integrity of
specialized pericytes and astrocytes that project their end-feet toward the
blood vessels to form a neurovascular unit (Warth, Kroger, & Wolburg,
2004). Astrocytes are also important for the regulation of blood flow
(Takano et al., 2006) and metabolite transfer to neurons (Magistretti
et al., 1994). In acute inflammation, resting endothelial cells acquire an
altered phenotype termed rapid type I activation that is associated with
increased endothelial permeability and vasodilation in the vascular bed
of an inflamed region (reviewed in Pober & Sessa, 2007). Leakage of
plasma protein-rich fluid can cause swelling within the confined bound-
aries of the cranium, and if inflammation is not resolved, edema can cause
an increase in intracranial pressure that compromises cerebral perfusion,
with increased risk of cell death.
The BBB vascular endothelium abundantly expresses four connexin
isoforms, Cx37, Cx40, Cx43, and Cx45 (De Bock et al., 2011, 2013;
Haefliger, Nicod, & Meda, 2004; Hakim, Jackson, & Segal, 2008;
Nagasawa et al., 2006), whereby Cx37 and Cx40 are widely distributed
and Cx43 is localized in areas of disturbed blood flow (Gabriels & Paul,
1998). Upregulation of Cx43 mRNA and protein was observed following
6 h exposure to PGN-induced IL-6 in an endothelial cell line, in contrast to
the astrocytes that were reported to have reduced Cx43 expression in the
acute phase of inflammation. Similarly to astrocytes, however, Cx43
hemichannel-mediated ATP release from the cells was induced following
15 min of exposure to PGN (Robertson et al., 2010).
10 Yuri Kim et al.

As in any vessel wound, injury to the BBB compromises vessel integrity

whereby the endothelium becomes permeable to plasma proteins within the
first 24 h from the onset of injury (Barzo, Marmarou, Fatouros, Corwin, &
Dunbar, 1996; Noble & Wrathall, 1989). The vascular leakiness is caused by
impaired intracellular Ca2+ transmission in the endothelium (De Bock et al.,
2012) as the rise in intracellular Ca2+ concentration ([Ca2+]i) causes a drop in
the transendothelial electrical resistance that facilitates circulating blood
derived microglia to enter across the endothelial barrier (Lu et al., 2008).
Connexin hemichannels are Ca2+-permeable channels that have bimodal
dependence on [Ca2+]i (<500 nM) for opening, which underpins Ca2+
oscillations in primary brain endothelial cells (De Bock et al., 2012).
Calcium oscillation is triggered by bradykinin, an inflammatory peptide,
and is rapidly inhibited by Gap26 and Gap27 peptides (within minutes),
reflecting the fast nature of endothelial cell hemichannel activity
(Romanov et al., 2007). Gap27 also inhibited bradykinin-mediated perme-
ability in in vitro and in vivo experiments (De Bock et al., 2012). Bradykinin-
induced [Ca2+]i oscillations were interrupted with Cx37/43 knockdown,
further confirming a role for connexin channels in endothelial cells
(De Bock et al., 2012). The Ca2+ oscillation were associated with purinergic
signaling in response to bradykinin. Hemichannel-independent Ca2+ oscil-
lations were also suggested with the use of ATP (De Bock et al., 2012).
Gap junctions in the blood vessel wall regulate intercellular communi-
cation across different cell types at the barrier junction. The cross-talk
between astroglia and the endothelial cells is crucial for maintaining BBB
integrity (Ezan et al., 2012). Under normal conditions, the end-feet of
the astrocytes are opposed to the outer surface of the endothelium
(Mathiisen, Lehre, Danbolt, & Ottersen, 2010). However, an absence of
astroglial connexins compromises the integrity of BBB by increasing the
hydrostatic vascular pressure and shear stress (Ezan et al., 2012).

2.5 Chronic Inflammation

Chronic conditions are characterized by persistent inflammation that can
result from inefficient clearing of debris and recurrent inflammatory signals
(reviewed in Perry, Cunningham, & Holmes, 2007). In the CNS, it is also
characterized by “glial scarring” in the injured area (McKeon, Schreiber,
Rudge, & Silver, 1991). Glial scarring is characterized by dense accumula-
tion of astrocytes with extensive production of abnormal extracellular matrix
(Gallo & Bertolotto, 1990). This is often seen as a barrier to restoration of
CNS Inflammation and the Inflammasome Pathway 11

axons and neurites (McKeon et al., 1991). An example of a chronic CNS

inflammatory condition is AD, which is a form of late-onset dementia char-
acterized by progressive decline in cognitive function. A key pathological
feature of the disease is the accumulation of β-amyloid (Aβ) plaques and neu-
rofibrillary tangles that lead to synaptic depression and memory decline
(Hsieh et al., 2006; Shankar et al., 2007). While Aβ and neurofibrillary tan-
gles are the classic biomarkers for AD, current trends in the literature focus
on circulating cytokines, chemokines, and growth factors as early bio-
markers (reviewed in Humpel, 2011). It is probable that recurrent inflam-
mation could evoke or predispose an individual for chronic inflammation as
aging is the biggest risk factor for AD (Lindsay et al., 2002). Interestingly,
ischemic stroke and traumatic brain injuries are also risk factors for AD.
A correlation between acute ischemic stroke and the disease was suggested
by observations that the extent of tissue damage is more severe in postmor-
tem brain tissue of AD donors with cerebral infarct compared to AD tissue
alone (Snowdon et al., 1997). Cerebral ischemia was reported to promote
Aβ deposition, neuronal loss, and expression of phosphorylated tau protein
that was associated with an enhanced inflammatory response (Li, Wang,
Zhang, Fang, & Zhou, 2011). This is consistent with epidemiological studies
that report a higher incidence of AD with cerebral ischemia (Esiri, Nagy,
Smith, Barnetson, & Smith, 1999).
Aβ also enhances Cx43 hemichannel activity in microglia, astrocytes, and
neurons (Nagy, Li, Hertzberg, & Marotta, 1996; Orellana et al., 2011). Sup-
pression of inflammation using steroidal antiinflammatory drugs (NSAIDs) is
modestly protective against neurodegeneration including AD (Etminan,
Gill, & Samii, 2003) and also Parkinson’s disease (Chen, Green, Danesh-
Meyer, & Rupenthal, 2015; Chen et al., 2005) although the exact mechanism
of the NSAIDS on chronic degeneration is unknown. While connexin chan-
nel blockers have not previously been considered a major therapeutic target
for chronic CNS disease indications, they could be used to modulate the
inflammation that is present in neurodegenerative tissues.
In excised human epileptic mesial temporal lobe, and samples from
donors who had been diagnosed with Alzheimer’s or Parkinson’s disease,
increased astrocytosis and Cx43 expression correlates with disease progres-
sion. Reactive astrocytic proliferation is characteristic of the hippocampus of
mesial temporal lobe epilepsy tissue, and is associated with a highly signifi-
cant increase in astrocytic Cx43 protein levels (Fonseca, Green, &
Nicholson, 2002). Cx43 levels are increased in regions where there are
misfolded (Aβ) proteins and reactive astrocytes. These are regions where
12 Yuri Kim et al.

there are high levels of circulating cytokines and free radicals involved in
the complement system (McGeer & McGeer, 2001). Increased Cx43
expression also occurs in parallel with astrocytosis in the caudate nucleus
of Huntington diseased human brain (Vis et al., 1998), and in the striatum
of patients with Parkinson’s disease (Rufer et al., 1996). The loss of
pigmented dopaminergic cells in the substantia nigra pars compacta is asso-
ciated with gliosis indicative of neuroinflammation (Langston et al., 1999),
and in Parkinson’s disease donor brains, major histocompatibility complex
proteins expressed by microglia are upregulated (McGeer, Itagaki, &
McGeer, 1988). Further signs of inflammation associated with Parkinson’s
disease are high levels of inflammatory cytokines, including TNF-α,
IL-1β, and IL-6, in the cerebral spinal fluid (Hunot & Hirsch, 2003). The
presence of these cytokines, in addition to reported T-cell infiltration, indi-
cates a compromised BBB in neurodegenerative diseases accompanied
by chronic inflammation.

3. CONNEXIN HEMICHANNELS AND THEIR ROLES IN

RESPONSE TO INJURY AND INFLAMMATION
Early functional studies relied upon the use of nonspecific compounds
to regulate connexin-based membrane channels. These included fatty
acids (for example, arachidonic, doxyl stearic, oleic, palmitoleic, oleamide),
volatile anesthetics (halothane and ethane), alcohols (octanol and heptanol),
steroids such as 18β-glycyrrhetinic acid and its water soluble derivative car-
benoxolone, or quinine and derivatives (such as mefloquinine) (Herve &
Sarrouilhe, 2005). However, these are nonselective reagents that not only
block multiple membrane channels, but cannot discriminate between gap
junction channel and connexin hemichannel roles. Indeed drugs such as
carbenoxolone are notorious for their limited potency and selectivity
( Juszczak & Swiergiel, 2009; Rozental, Srinivas, & Spray, 2001). Quinine
and its derivatives such mefloquine may likewise show partial selectivity in
blocking some connexin isoforms but have also been associated with neu-
ropsychiatric symptoms. Gene knockout animal studies can be difficult to
interpret; again both gap junction communication and hemichannel num-
bers are affected. As noted earlier, long-term blockade of gap junctions
appears to be deleterious after ischemic brain injury, likely owing to disrup-
tion of the astrocytic syncytium (Nakase, Fushiki, & Naus, 2003; Nakase
et al., 2004). A functioning gap junction coupled astrocytic syncytium is
essential for neuronal support, providing for metabolite spreading and the
CNS Inflammation and the Inflammasome Pathway 13

elimination of by-products (Dienel & Cruz, 2003). Loss of this syncytium

can, for example, contribute to the onset of demyelinating diseases such
as multiple sclerosis or neuromylitis optica (Masaki, 2015).
There is increasing evidence that connexin hemichannels are the patho-
logical pores associated with the spread of CNS injury after ischemic stroke
or hypoxic injury (Davidson, Drury, et al., 2014; Davidson, Green,
Nicholson, Bennet, & Gunn, 2013; Davidson et al., 2012; Orellana et al.,
2010), spinal cord trauma, and retinal stroke (Danesh-Meyer et al., 2012;
O’Carroll, Alkadhi, Nicholson, & Green, 2008; O’Carroll, Gorrie,
Velamoor, Green, & Nicholson, 2013), the onset of demyelinating diseases
referred to in the previous paragraph (Masaki, 2015) or denervation leading
to muscle degeneration (Cea et al., 2013). Pannexin channels, similar in struc-
ture to connexin hemichannels, may also contribute to damage spread after
CNS injury (Thompson et al., 2008) although the relationship between con-
nexin hemichannels and pannexin channels, or the relative contribution of each
channel type, remains to be determined. The fact that the sum of the parts
appears to exceed the whole, with blockade of either showing a marked reduc-
tion in lesion spread, may suggest a functional relationship between these two
channels types.
Gap junction channels are formed by the docking of two hemichannels
(or connexons) at areas of close plasma membrane apposition between
cells. Because hemichannels are continuously recruited from surrounding
unopposed plasma membrane areas, migrating within the plasma membrane
to sites of close membrane apposition adjacent to existing gap junction
plaques (Goodenough & Paul, 2009; Laird, 2006; Thevenin et al., 2013),
they potentially offer a signaling pathway between the intra- and extra-
cellular environment (Evans, De Vuyst, & Leybaert, 2006; Wang et al.,
2013). However, while connexin hemichannels are said to be active under
“normal” physiological conditions, there are few studies in which cells
under completely “normal” conditions exhibit spontaneous hemichannel
opening. Purinergic signaling by regulated release of ATP from hemi-
channels, for example, requires a triggering event such as mechanical
stress, quinine-induced hemichannel opening, lowered extracellular Ca2+,
or the application of transmembrane voltages (Hofer & Dermietzel, 1998;
Kang et al., 2008; Li et al., 1996; Stout et al., 2002). Although increased
intracellular Ca2+ up to about 500 nM opens hemichannels, their opening
is inhibited at higher intracellular calcium levels (Wang et al., 2012).
Nonetheless, lowered extracellular calcium also induces nicotinamide
adenine dinucleotide (NAD+) perfusion through Cx43 hemichannels
14 Yuri Kim et al.

(Bruzzone, Guida, Zocchi, Franco, & De Flora, 2001). The large drop
in extracellular Ca2+ concentrations associated with pathological condi-
tions such as ischemia is sufficient to open hemichannels, and oxidative stress,
which causes depolarization of the cell membrane, also triggers hemichannel
opening (Ramachandran, Xie, John, Subramaniam, & Lal, 2007).
An elegant study from Orellana et al. (2010) demonstrated opening of
Cx43 hemichannels after hypoxia in cultured astrocytes. Increased dye
uptake was reported in Cx43-containing astrocytes, but not in Cx43-
deficient astrocytes. Known blockers of Cx43 hemichannels prevented
dye uptake and astrocytic demise (Orellana et al., 2010). Other examples
are hemichannel opening in rat cortical astrocytes, shown by intracellular
uptake of dyes in response to metabolic inhibition of glycolytic and
oxidative metabolism using the chemical inhibitors antimycin A and
iodoacetic acid (Contreras et al., 2002). In isolated ventricular myocytes,
metabolic inhibition induced by the glycolytic inhibitor sodium iodoacetate
and the mitochondrial uncoupler, carbonyl cyanide m-chlorophenyl-
hydrazone induced hemichannel opening (Kondo, Wang, John, Weiss, &
Goldhaber, 2000) and in astrocytes, hemichannel opening was triggered
by treatment with proinflammatory cytokines such as IL-1β or TNF-α,
or by LPS stimulation of microglia cocultured with astrocytes (Froger
et al., 2010; Retamal et al., 2007). Cx43 hemichannels play a role in intercel-
lular Ca2+ wave propagation in astrocytes to which both gap junction
channels and hemichannels contribute (Leybaert & Sanderson, 2012;
Scemes & Giaume, 2006), but even here an external stimulus is required
to trigger the wave. Triggers include local release of messengers such
as ATP, endothelin-1, glutamate or nitric oxide, mechanical force or
pressure ejection, microinjection of Bax (a proapoptotic member of the
Bcl-2 family), or a local decrease in extracellular calcium (for review, see
Giaume, Leybaert, Naus, & Saez, 2013; Leybaert & Sanderson, 2012).
In the cochlea of the ear, Cx26 hemichannels may contribute to signa-
ling, probably owing to physiologically low Ca2+ levels in the cochlea
endolymph (Bosher & Warren, 1978). However, recent evidence suggests
that it may be pannexin channels playing this role, since deletion of both
Cx26 and Cx30, the main connexin isoforms in the cochlea, had no effect
on ATP release and endocochlear potential generation (Chen, Zhu, Liang,
Chen, & Zhao, 2015). Conversely, deletion of Pannexin1 in the cochlear
lateral wall almost abolished ATP release under physiological conditions,
implying that it is the pannexin channel which is responsible for signaling
in the cochlea.
CNS Inflammation and the Inflammasome Pathway 15

One exception where there is evidence of a “normal” physiological role

for hemichannels may be in the retina where horizontal cells interact with
bipolar cells, and ephaptic signaling is an essential step in contrast enhance-
ment. Retina Cx55 knockout zebrafish have reduced hemichannel signaling
and the animals show reduced contrast sensitivity in behavioral assays
(Klaassen et al., 2011). The connexins involved are members of a special group
of connexins, encoded by the GJA9 and GJA10 genes (reviewed in Klaassen,
Fahrenfort, & Kamermans, 2012). Hemichannel-mediated feedback has also
been demonstrated from horizontal cells to cones in the goldfish retina
(Fahrenfort et al., 2009). Cx26 is said to form physiologically functional
hemichannels on horizontal cell dendrites in carp (Kamermans et al.,
2001), and to allow feedback between horizontal cells and cones in the turtle
retina (Pottek et al., 2003), such that blocking of those hemichannels abolished
feedback-mediated responses. However, other studies have indicated that
Cx26 is not present (nor Cx36) in the mouse horizontal cell and those authors
concluded that the mechanism of inhibition is restricted to the teleost retina,
or a different connexin serves this function in mammals (Deans & Paul, 2001).
So even here, a “normal” physiological role for connexin hemichannels in
mammalian tissues remains controversial. In contrast, when there is
hemichannel-mediated release of ATP from rat retinal cells (Muller glial cells),
it is in response to osmotic stress and glutamate (Voigt et al., 2015).
What is meant by “normal” physiological roles for hemichannels may
come down to semantics. As mentioned earlier, unregulated opening of such
a large and relatively nonspecific channel, linking the cell cytoplasm directly
with the extracellular space will compromise ionic gradients required for
resting membrane potentials and membrane transport as well as permitting
unregulated movement of metabolites and second messenger molecules.
Connexin hemichannels seem to be activated primarily by pathological
stimuli, and thereby drive processes such as cell edema, cell death, and
inflammation (Chandrasekhar & Bera, 2012; Danesh-Meyer et al., 2012;
Decrock et al., 2009; Kar, Batra, Riquelme, & Jiang, 2012; Vinken,
2015). The hemichannel under those terms becomes predominantly a
pathological pore (Decrock et al., 2009).
Connexin protein expression commonly increases as a result of injury, in
many cases two- to threefold within a few hours. In a rat retina ischemia–
reperfusion model, Cx43 labeling was significantly increased within 1–2 h,
and more than double within 4 h postischemia (Danesh-Meyer et al., 2012).
In that model ischemia is induced by raising intraocular pressure. In other
recent examples, instillation of bacterial endotoxin into the lung increases
16 Yuri Kim et al.

Cx43 levels, as assessed by both immunohistochemistry and Western blotting

(Kandasamy, Escue, Manna, Adebiyi, & Parthasarathi, 2015). In a bright light-
damaged albino rat model of retinal degeneration Cx43 expression in the cho-
roid, assessed by Western blot, increased by greater than triple the control at
6 h post light exposure (Guo et al., 2014). In other CNS models, Cx43 pro-
tein expression is more than doubled within 2 h after rat spinal cord injury
(O’Carroll, Gorrie, et al., 2013), Cx43 mRNA levels quadruple within 4 h
of brain ischemia in the term-equivalent fetal sheep (Davidson et al.,
2012), and Cx43 increases after traumatic brain injury, again assessed by both
immunohistochemistry and Western blotting (Li, Zhang, Zhang, Zhou, &
Meng, 2015). Increased connexin expression means there will be greater
numbers of unopposed hemichannels in the plasma membrane, at least until
the newly expressed connexons dock with those of a neighboring cell. Fur-
thermore, at the same time that these hemichannels may be showing increased
opening probability under pathological conditions, whereas cell-to-cell cou-
pling (gap junction communication) can be reduced. As noted earlier, factors
that oppositely influence channel opening in this manner include kinase-
activating stimuli (LPS and basic FGF or FGF-1) (De Vuyst et al., 2007;
Schalper et al., 2012), proinflammatory cytokines (TNF-α and IL-1β)
(Retamal et al., 2007), intercellular Ca2+ concentration changes (De Vuyst
et al., 2006), or metabolic inhibition (Contreras et al., 2002). Interestingly,
quinine has been evoked as a connexin hemichannel activator, triggering
ATP release and calcium signaling in astrocytes and in C6 cells expressing
Cx43 (Stout et al., 2002), but is also used to block gap junction channels
(Medina-Ceja, Pardo-pena, & Ventura-Mejia, 2014; Rubinos, Sanchez,
Verselis, & Srinivas, 2012; Ventura-Mejia & Medina-Ceja, 2014).
Hemichannel opening under pathological conditions contributes to tis-
sue edema and cell swelling as cells can no longer osmoregulate. This can
lead directly to cell rupture, or by mediating release of regulatory substances
or inflammatory cytokines, act as trigger for cell death processes in the
affected cells but also in neighboring tissue. Quist, Rhee, Lin, and Lal
(2000) showed that extracellular Ca2+ concentration changes have a signif-
icant effect on cell volume. In connexin null cells volume change did not
occur, but after Cx43 transfection cell swelling occurred in the presence
of a lowered extracellular Ca2+ ion concentration. Swelling was prevented
by gap junctional channel blockers such as oleamide and beta-glycyrrhetinic
acid and was reversible; the cell volume returned to normal when extra-
cellular calcium levels were raised again. Cell swelling and rupture has
also been associated with hemichannel opening after cardiac ischemia
CNS Inflammation and the Inflammasome Pathway 17

(Rodriguez-Sinovas, Sanchez, Fernandez-Sanz, Ruiz-Meana, & Garcia-

Dorado, 2012), and blood vessel endothelial cell rupture has been directly
attributed to hemichannel opening after ischemia, both in vitro and in vivo
(Danesh-Meyer et al., 2012; Zhang et al., 2014). Hemichannel-blocking
Cx43 mimetic peptides added to endothelial cells in vitro prevented
hypoxia-activated endothelial cell death, as did systemic delivery after retinal
ischemia–reperfusion (Danesh-Meyer et al., 2012). After damage to the
optic nerve (a white matter tract) or spinal cord (a white and gray matter
tract), hemichannel opening is a major cause of tissue swelling. This has been
demonstrated in ex vivo models (optic nerve or spinal cord segments in
organotypic culture) and in vivo (spinal cord crush injury), where gap junc-
tion channel modulators prevented tissue swelling (Cronin et al., 2008;
Danesh-Meyer & Green, 2008; O’Carroll et al., 2008).
The possibility should not be excluded that hemichannels may also have
positive roles. Recent studies show that Cx43 hemichannels may have a role
in basal glutamatergic synaptic transmission and plasticity in hippocampal
astrocytes (Chever, Lee, & Rouach, 2014). Paracrine ATP signaling mediated
by hemichannels may compensate for lost gap junction communication and
stimulate the recovery of injured tissues by its mitogenic actions (Pearson,
Dale, Llaudet, & Mobbs, 2005; Thevananther et al., 2004). ATP from
polymorphonuclear leukocytes amplifies chemotactic signals for directed cell
orientation and feedback (Eltzschig, Macmanus, & Colgan, 2008). In endo-
thelial cells, bacterial PGNs elicit a potent proinflammatory response. As with
leukocytes, ATP is released from the endothelial cells but inhibition of
hemichannel activity using carbenoxolone prevents ATP induction of IL-6
and Toll-like receptor 2 (TLR2) mRNA expression (Robertson et al.,
2010). Hemichannel opening under pathological conditions, including acute
injury and chronic disease has been extensively reviewed elsewhere (Bosch &
Kielian, 2014; Castellano & Eugenin, 2014; Chen, Green, et al., 2015;
Davidson, Green, Bennet, & Gunn, 2014; Davidson, Green, Bennet, et al.,
2013; De Vuyst et al., 2011; Eugenin, 2014; Evans, Bultynck, & Leybaert,
2012; Kielian, 2008; Mallard et al., 2014; O’Carroll et al., 2013; Zhang,
O’Carroll, et al., 2013), but in the following sections we again question
whether this response is really necessary or beneficial.

3.1 Linking Connexin Hemichannels to the Inflammasome

Pathway
As described earlier, Cx43 gap junction coupling is suppressed, whereas
Cx43 protein expression and hemichannel opening are both increased in
18 Yuri Kim et al.

response to injury and inflammation. This is associated with ATP release

(Castellano & Eugenin, 2014; Eltzschig et al., 2008; Robertson et al.,
2010) and also Ca2+ efflux (De Bock et al., 2011; Evans et al., 2006) that
directly contributes to the release of inflammatory cytokines such as
TNF-α and IL-1β (Froger et al., 2010; Retamal et al., 2007) and the
induction of IL-6 and TLR2 mRNA expression, which play a role in the
initiation of early innate immune responses in at least the endothe-
lium (Robertson et al., 2010). Inflammasomes are specialized signaling
platforms that are critical for the innate immune system and inflammatory
responses (Choi & Ryter, 2014). There are a number of pathways to
inflammasome activation. Those of direct relevance to hemichannel
activity include:
1. Bacterial- or viral-derived proinflammatory factors such as LPS, PGN, or
streptococcus hemolysin/cytolysin. Bacterial-derived proinflammatory
factors such as LPS or PGN stimulate TLR and NOD-like receptors to
induce translocation of NF-κβ, activation of MAPK signaling, and
transcription of genes encoding inflammatory cytokines (Choi &
Ryter, 2014). Or as described earlier, bacterial PGNs elicit a potent
proinflammatory response with induction of IL-6 and TLR2 mRNA
expression (Robertson et al., 2010).
2. Extracellular ATP, or ATP release from stimulated cells (spreading to
neighboring cells), binding to purinergic receptors (such as P2X7YR),
is a potent activator of NLRP3 inflammasome activation, and subse-
quently IL-1β and Il-18 release. Extracellular ATP also strongly acceler-
ates IL-1β processing and secretion. Mice that are P2Y7 deficient, for
example, are not sensitized to allergens and so do not release IL-1β in
response to LPS or ATP (Desai & Leitinger, 2014). ATP release triggers
the induction of IL-6 and TLR2 in endothelial cells (Robertson
et al., 2010).
3. Ca2+ efflux is a signal for NLRP3 inflammasome formation. In some
cases a dual signal is proposed, with a primary signal inducing transcrip-
tion of proinflammatory IL-β1 and, for example, NLRP3. The NLRP
family members (NLRP1, NLRP3, and IPAF) as well as AIM2 can form
inflammasome complexes, activating inflammatory caspase-1, which
catalyzes proinflammatory cytokine maturation, and so release of mature
cytokines such as IL-β1 and IL-18, as well as further ATP release. Mono-
cytes may only need a one-step activation since LPS is sufficient on its
own to induce secretion of both mature IL-1β and ATP (Shenderov
et al., 2014).
CNS Inflammation and the Inflammasome Pathway 19

As a specific example, Cea and colleagues showed that a lack of Cx43/45

hemichannels attenuates inflammasome activation in skeletal muscle (reduc-
ing atrophy) (Cea et al., 2013). Membrane channel-mediated permeability
was slightly reduced by pannexin channel blockade, but completely
inhibited by connexin hemichannel block. Pannexin channels have been
proposed to play a role in the inflammasome pathway although more
recently it has been suggested that Pannexin1 is required for ATP release
during apoptosis, but not for inflammasome activation (Qu et al., 2011),
which would leave connexin hemichannels as the prime therapeutic target.
In perinatal infection/inflammation as seen in preterm fetal sheep exposed to
LPS, which is associated with release of proinflammatory cytokines, partic-
ularly TNF-α, led to reduced maturation of brain activity and white matter
injury TNF-α is known to increase hemichannel activity (Mallard et al.,
2014) with ATP and cytokine release, and calcium waves, triggering further
injury (inflammasome activation) in neighboring cells. These roles for con-
nexin hemichannels in both the initiation and propagation of the
inflammasome pathway are illustrated in Fig. 1. Connexin hemichannel
blockers may have a role in the limiting the initiation and propagation of
the inflammasome process.

3.2 Animal Models Show Therapeutic Benefits of Connexin

Hemichannel Block
Intervention is possible at several levels. In the first instance, both acute and
chronic wounds can lead to endothelial die back and vascular leak associated
with hemichannel opening. Compromised blood–brain or blood–retina
structural barrier causes vessel leak, which induces infiltration of both
tissue-derived (activated microglia) and blood-derived inflammatory cells
(neutrophils/leukocytes). Intervention in these cases aims first to reduce ves-
sel leak, and secondarily to reduce inflammation and the inflammasome
pathway. In some scenarios, however, there is no vessel leak component
and intervention targets the inflammatory response in affected tissues.
Finally, it is possible to directly target neuronal connexin hemichannels
and still show benefit.
Following spinal cord injury in rat models, lesion spread over the first
24 h postinjury is attributed to the gap junction mediated bystander effect
and the opening of connexin hemichannels (Cronin et al., 2008;
O’Carroll et al., 2008; O’Carroll, Gorrie, et al., 2013). Hemichannel open-
ing causes tissue swelling, inflammation (with blood–borne inflammatory
cells entering the lesion, astrocytosis, and microglia activation). Vessel leak,
Figure 1 See legend on opposite page.
CNS Inflammation and the Inflammasome Pathway 21

determined using fluorescent tagged bovine serum albumin as a tracer,

occurs 4–5 mm to either side of a crush injury site. Reduced expression
of Cx43 using topically applied antisense oligodeoxynucleotides, or hemi-
channel block using topically or systemically delivered Cx43 mimetic pep-
tides, significantly reduces these adverse events. Both were given as a single
application and showed sustained downstream benefits. The end result was
significantly improved behavioral outcomes over the following weeks
(Cronin et al., 2008; O’Carroll, Gorrie, et al., 2013). In the O’Carroll,
Becker, et al. (2013) and O’carroll, Gorrie, et al. (2013) study where
hemichannels were modulated with mimetic peptides delivered using an
intrathecal catheter, levels of the inflammatory cytokines TNF-α and
IL1-β were reduced by about 50% relative to untreated, injured spinal cords

Figure 1 Connexin43 hemichannel-mediated inflammation in the central nervous sys-

tem. Nucleotides (ATP and ADP) are released into the extracellular space during cellular
injury, necrosis, apoptosis, and pyroptosis. Multiple cell types release nucleotides,
including reactive inflammatory cells, astrocytes, microglia, and neurons in the CNS.
Several molecular pathways have been implicated in the release of nucleotides and ions
into the extracellular space of which Cx43 hemichannels play a significant role. Extra-
cellular ATP and AMP activate cell surface P2 purinergic receptors that are grouped into
metabotropic P2Y receptors (P2YRs; GPCRs with seven transmembrane-spanning
motifs) or nucleotide-gated ionotropic P2X receptors (P2XRs), which are ion channels.
ATP and AMP nucleotides are further catalyzed to adenosine, which activates cell sur-
face P1 purinergic receptors. Binding of ATP to the P2X channels trigger a flux of Na+,
Ca2+, and K+ ions across the pore. Activated P1 and P2 receptors trigger the intracellular
signaling molecule, NLRP3, which binds to ASC. ASC then interacts with protease
caspase-1 (procaspase1) to form the inflammasome complex. The inflammasome com-
plex activates caspase-1 to cleave proinflammatory cytokines to their active forms, IL-1β
and IL-18, which can mediate a type of programmed cell death known as pyroptosis
(Schroder & Tschopp, 2010). In addition to the purinergic receptor-mediated pathway
via nucleotides, pathogen-associated molecular patterns (PAMPs) and cell-derived
damage-associated molecular patterns (DAMPs) act as signaling molecules to bind to
specific receptors, for example, the Toll-like receptors (TLRs), NOD-like receptors
(NOD-R), and TNF receptor 1 (TNFR-1). Activation of TLRs, NOD-Rs, and TNFR-1 initiates
the proinflammatory nuclear factor (NF-κβ) pathway to initiate cytokine release. Mito-
chondria are also at the center of the cell death pathway that can be triggered by stimuli
such as DAMPs. This subsequently leads to elevation of reactive oxygen species (ROS)
and the activation of the NF-κβ pathway. Inflammation alters gene expression, including
upregulation of Cx43 mRNA that subsequently results in enhanced numbers of
hemichannels at the cell surface where increased hemichannel opening leads to further
ATP release and regulates the release of inflammatory cytokines. These cytokines may
not pass directly through hemichannels, but hemichannel block significantly reduces
their levels relative to untreated tissues.
22 Yuri Kim et al.

at 8 h postinjury. The treatment not only reduced inflammation but nearly

doubled counts of surviving motor neurons adjacent to the lesion site, and at
3.5 and 7 mm distant to it.
In the eye, we have used a high intraocular pressure model of rat retinal
ischemia–reperfusion (Sun et al., 2007) to investigate Cx43 hemichannel
roles after acute CNS ischemia–reperfusion. In this model a cannula is
inserted into the anterior chamber of the rat eye and the intraocular pressure
is raised to 120 mmHg to occlude retinal blood flow for 1 h. The cannula is
then removed and reperfusion confirmed by ophthalmoscopy. Cx43 is
upregulated as early as 60 min following reperfusion, reaching statistical sig-
nificance within 1–2 h after injury and peaking in the retinal nerve fiber
layer at 8 h postinjury (Danesh-Meyer et al., 2012). Interestingly, ische-
mia–reperfusion did not cause uniform pan-retinal inflammation. Instead
astrocytosis was patchy and adjacent to blood vessels. Intraperitoneal injec-
tion of Evans blue dye, an azo dye that binds to serum albumin and is used to
quantify vascular endothelial integrity, revealed extravascular dye leakage
within an hour of reperfusion, peaking at 4 h postischemia and continuing
at 24 h postinjury, the longest reperfusion period analyzed. The vessel leak
therefore correlated with both Cx43 expression, and the patchy inflamma-
tory response. An intraperitoneal injection of a Cx43 hemichannel blocking
peptidomimetic, Peptide5 (O’Carroll et al., 2008), reduced vascular leak by
up to 86% while also reducing the inflammation. Assessed 1 and 3 wk later,
this one-off treatment at the point of reperfusion, resulted in significant spar-
ing of retinal ganglion cells, with a two-thirds reduction in neuronal loss
compared to untreated or scrambled peptide treated animals. In two repeat
studies, the same Cx43 peptidomimetic was delivered by intravitreal injec-
tion instead of systemically. The peptides were delivered in their native
form, or C12–C12 lipo aminoacid modified to increase stability, or encap-
sulated into slow-release poly(lactic-co-glycolic) acid nanoparticles (Chen,
Green, Wang, Danesh-Meyer, & Rupenthal, 2014; Chen, Green, et al.,
2015). Intravitreal peptide delivery again resulted in significantly reduced
Cx43 levels compared to untreated controls, to levels close to that of the
normal retina, reduced vessel leak, and over 50% reduction in neuronal loss
relative to controls.
Another recognized model of retinal degeneration is the bright light
damage model in which the albino rat is exposed to intense fluorescent light
over a 24-h period (Noell, Walker, Kang, & Berman, 1966). The light dam-
age leads to retinal degeneration that resembles that of patients with age-
related macular degeneration, albeit within an acute time frame.
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