The PNAS-Liu team revealed the important role of connexin hemichannels in alleviating neuroinflammation and overexcitability during the onset of temporal lobe epilepsy

Written by—Annie Guo, Junyu Liu

Responsible editor—Wang Sizhen, Fang Yiyi

Editor—Summer Leaf

Epilepsy is one of the most common neurological disorders and manifests as spontaneous and recurrent seizures
.
Seizures are formed by a group of highly synchronized neuronal fires, first appearing in seizure-susceptible areas and spreading to other areas of
the brain.
Although about 50 million people worldwide are suffering from epilepsy, about one-third of patients are resistant to existing antiepileptic drugs [1].

The main targets of existing antiepileptic drugs are synapses or neurons, which regulate the activity of neurons by regulating the activity of ion channels or synaptic transmission[1].

Given that such a large patient population is resistant, the discovery of new targets or mechanisms to treat epilepsy is critical
.

Astrocytes play an important role
in regulating the activity of neurons.
One of the most important ways is the release of gliotransmitters, such as glutamate, D-serine, and ATP, through connexin hemichannels [2-6]
。 During the onset of epilepsy, the increase in the degree of connexin hemichannel openness leads to the release of excess glial transmitters around neurons, further enhancing neuroinflammation and neuronal excitability [4-6].

Can connexin hemichannels be used as effective targets for the treatment of temporal lobe epilepsy? What role does inhibition of connexin hemichannels play in the pathogenesis of temporal lobe epilepsy? In response to these problems, the researchers carried out a series of studies
.

On November 2, 2022, Liu Junyu's team from City University of Hong Kong presented a presentation in the Proceedings of the National Academy of Sciences of the United States of America) published the title "Inhibition of connexin hemichannels alleviates neuroinflammation and hyperexcitability in temporal lobe epilepsy.
" Research papers
.
Dr.
Annie Guo is the first author of the paper, and Dr.
Junyu Liu and Professor Juan C.
Sáez are co-corresponding authors
.
By using a novel inhibitory drug called D4 connexin hemichannel, the paper reveals the important role
of connexin hemichannel in reducing neuroinflammation and overexcitability during the onset of temporal lobe epilepsy.

First, the researchers explored the effect of connexin hemichannel inhibitor D4 on the survival rate of epileptic animals modeled by pilocarpine.

Pretreatment with D4 increased the survival rate of epileptic mice after seven days of modeling from 46% to 74% (Figure 1A-B).

Next, to explore the effect of D4 on the neuroinflammatory response caused by epilepsy, the researchers examined GFAP and Iba1 to reflect the expression of stellate cells and microglia
.
Seven days after epileptogenesis, pilocarpine significantly increased the expression of stellate cells and microglia in the piriformis cortex and hippocampus, while pretreatment of D4 decreased the expression of stellate cells in the piriformis cortex and microglia in the piriformis cortex and hippocampus, D4 Post-treatment rescued overexpression of stellate cells and microglia in the piriformis cortex and hippocampus caused by pilocarpine (Figure 1C-F).

Figure 1: D4 improves survival in epileptic animals and reduces overexpression of stellate and microglia

(Source: Guo A, et al.
, PNAS, 2022).

Next, to explore the effects of D4 on neuronal overexcitation caused by epilepsy, the researchers conducted two experiments
.
First, the researchers recorded spontaneous inhibitory postsynaptic currents (sIPSCs) from individual pyramidal neurons in the CA1 region of the hippocampus ）
。 Three days after epileptomy, the amplitude and frequency of sIPSC in CA1 pyramidal neurons decreased, while pretreatment with D4 increased the frequency of sIPSC, illustrate D4 alleviates the decrease in inhibition of neurons caused by epilepsy (Figure 2A-C).

Second, the researchers recorded the local field potential of the primary motor cortex of anesthetized animals during major seizures
.
D4 greatly reduces electrographic seizure activity, the number of epileptic spikes, and high-amplitude spikes high-amplitude spikes) (Figure 2D-H).

Figure 2: D4 alleviates epilepsy-induced inhibitory postsynaptic current reduction and electrogram epilepsy activity
.

(Source: Guo A, et al.
, PNAS, 2022).

Next, to explore molecular events and gene expression during epilepsy onset, the researchers used real-time quantitative PCR to detect mRNA expression of synaptic and inflammation-related genes.

The researchers found that three days after modeling, synapse-related genes (including GAD1, Vglut1, HomeR1, Pvalb) were expressed in the piriformis cortex and hippocampus, while inflammation-related genes (including GFAP, CD68, TREM2, CX3CR1, TLR9, C3, NLRP3, TNF, TNF1) were expressed, while inflammation-related genes (including GFAP, CD68, TREM2, CX3CR1, TLR9, C3, NLRP3, TNF, TNF1) were expressed Expression is significantly elevated
.
Three doses of D4 post-treatment increased mRNA expression of synapse-associated genes, while both primary and triple doses of D4 post-treatment reduced mRNA expression of inflammation-related genes (Figures 3A-C).

Figure 3: Post-treatment of D4 rescued epilepsy-induced inflammation and changes in mRNA levels of synapse-related genes

(Source: Guo A, et al.
, PNAS, 2022).

Immediately afterwards, the researchers used carboxyfluorescein (CBF) for coloring experiments to explore the mechanism
of D4 action.
Carboxylfluorescein is a dye that can enter cells through the opening of the connexin halfway, and the coloring rate of carboxyfluorescein can indirectly reflect the activity
of the connexin hemichannel.
The researchers found that the total coloring rate of carboxyfluorescein, as well as in stellate cells, increased in the piriformis cortex and hippocampus of acute brain slices from epileptic animals, indicating elevated
activity of connexin hemichannels.
Furthermore, both acute brain slices and D4 treatment in animals reduced the total coloring rate of carboxyfluorescein in the piriformis cortex and hippocampus, as well as in stellate cells (Figure 4A-D, Supplementary Figure 6-7).

。 These results reflect the increased
activity of D4 inhibiting epilepsy-induced connexin hemichannels.

Figure 4: In vitro or in vivo D4 treatment inhibits elevated activity of epilepsy-induced connexin hemichannels in hippocampal brain slices

(Source: Guo A, et al.
, PNAS, 2022).

In order to explore the mechanism of action between D4 and different types of connexin halfchannels and gap junction channels, the researchers conducted a series of molecular docking experiments
。 The researchers compared the binding site between D4 and Cx43 ligent protein hemichannels or gap junction channels or Cx39 ligent protein hemichannels and binding affinity ( binding affinity), found with respect to the Cx43 slot junction channel or Cx39 ligent protein hemichannel, D4 with There is a stronger binding affinity between Cx43 connexin hemichannels (Figure 5A-E).

Figure 5: Molecular model of Cx43 and Cx39 junction channels and binding to D4

(Source: Guo A, et al.
, PNAS, 2022).