Systemic inflammation disrupts oligodendrocyte gap junctions and induces ER stress in a model of CNS manifestations of X-linked Charcot-Marie-Tooth disease
© The Author(s). 2016
Received: 16 August 2016
Accepted: 17 August 2016
Published: 1 September 2016
X-linked Charcot-Marie-Tooth disease (CMT1X) is a common form of inherited neuropathy resulting from different mutations affecting the gap junction (GJ) protein connexin32 (Cx32). A subset of CMT1X patients may additionally present with acute fulminant CNS dysfunction, typically triggered by conditions of systemic inflammation and metabolic stress. To clarify the underlying mechanisms of CNS phenotypes in CMT1X we studied a mouse model of systemic inflammation induced by lipopolysaccharide (LPS) injection to compare wild type (WT), connexin32 (Cx32) knockout (KO), and KO T55I mice expressing the T55I Cx32 mutation associated with CNS phenotypes. Following a single intraperitoneal LPS or saline (controls) injection at the age of 40–60 days systemic inflammatory response was documented by elevated TNF-α and IL-6 levels in peripheral blood and mice were evaluated 1 week after injection. Behavioral analysis showed graded impairment of motor performance in LPS treated mice, worse in KO T55I than in Cx32 KO and in Cx32 KO worse than WT. Iba1 immunostaining revealed widespread inflammation in LPS treated mice with diffusely activated microglia throughout the CNS. Immunostaining for the remaining major oligodendrocyte connexin Cx47 and for its astrocytic partner Cx43 revealed widely reduced expression of Cx43 and loss of Cx47 GJs in oligodendrocytes. Real-time PCR and immunoblot analysis indicated primarily a down regulation of Cx43 expression with secondary loss of Cx47 membrane localization. Inflammatory changes and connexin alterations were most severe in the KO T55I group. To examine why the presence of the T55I mutant exacerbates pathology even more than in Cx32 KO mice, we analyzed the expression of ER-stress markers BiP, Fas and CHOP by immunostaining, immunoblot and Real-time PCR. All markers were increased in LPS treated KO T55I mice more than in other genotypes. In conclusion, LPS induced neuroinflammation causes disruption of the main astrocyte-oligodendrocyte GJs, which may contribute to the increased sensitivity of Cx32 KO mice to LPS and of patients with CMT1X to various stressors. Moreover the presence of an intracellularly retained, misfolded CMT1X mutant such as T55I induces ER stress under inflammatory conditions, further exacerbating oligodendrocyte dysfunction and pathological changes in the CNS.
X-linked Charcot-Marie-Tooth (CMT) disease (CMT1X), associated with over 400 different mutations in the GJB1 gene, is one of the commonest forms of inherited neuropathies (http://www.molgen.ua.ac.be/CMTMutations/) [8, 28]. GJB1 encodes connexin32 (Cx32), a protein that forms gap junctions (GJs) both in Schwann cells in the peripheral nerves and in oligodendrocytes throughout the CNS, among other tissues [1, 59]. CMT1X patients present with slowly progressive weakness and atrophy usually starting in distal leg muscles, causing difficulty in running and frequently sprained ankles, typically beginning by 10 years of age. Males are earlier and more severely affected than heterozygous females who may be asymptomatic or may have a milder clinical form of the disease at an older age .
CMT1X mutations have been associated with clinical CNS phenotypes in addition to peripheral neuropathy, including chronic corticospinal tract dysfunction manifesting with spasticity, extensor plantar responses and hyperactive reflexes in patients with the A39V , T55I , M93V , R164Q , R164W , R183H , T191 frameshift  and L143P  mutations. Subclinical evidence of CNS involvement, such as abnormal brainstem auditory, visual, and motor evoked responses , is seen in a high proportion of cases [44, 45]. Acute transient encephalopathy, along with MRI changes, has been described in CMT1X patients carrying the M1L, T55I, R75W, E102del, R142W, R142Q, R164W, R164Q, C168Y and V177A mutations [49, 52, 65]. In most cases encephalopathy occurred under conditions of metabolic stress such as traveling to high attitudes , febrile illness [18, 58], hyperventilation  or concussion [2, 17]. CNS dysfunction caused by GJB1 mutations is more common in children and young adults  without correlation to the stage and severity of the peripheral neuropathy.
The cellular mechanisms leading to CNS phenotypes in a subset of CMT1X patients remain unclear. Connexins are synthesized in the endoplasmic reticulum (ER), transported to the Golgi apparatus and then inserted as hexamers into the cell membrane . Both in vivo and in vitro studies of CMT1X mutations have shown that many mutants are retained in the ER or Golgi with reduced or absent formation of GJ plaques at the cell membrane, [29, 75] and these missfolded mutants are effectively degraded by proteasomes and lysosomes . Impaired formation of GJs by mutant Cx32 molecules and the loss of Cx32 function is responsible for the development of the neuropathy in most CMT1X mutations  with the exception of rare reports of toxic mutants associated with severe neuropathy phenotypes . A gain-of-function mechanism for CNS manifestations has been considered both because patients with complete lack of the coding sequence have no subclinical CNS manifestations [16, 64] and because a disproportionate fraction of the mutations associated with the florid manifestations are represented in more than one pedigree, suggesting that specific mutations are particularly prone to cause this syndrome. Nevertheless, an ADEM-like CNS phenotype has been recently reported in a patient with start codon GJB1 mutation  leading to complete loss of Cx32 synthesis , suggesting that loss of Cx32 function alone is sufficient to account for the CNS phenotypes in CMT1X.
Although trans-dominant effects of Cx32 mutants on co-expressed oligodendrocyte connexins Cx47 and Cx29 have been hypothesized, we found no interaction in vivo of two representative Cx32 mutants showing retention in the ER (T55I) or Golgi (R75W) with other connexins expressed in oligodendrocytes . However, these mice were not subjected to stressful conditions as described in some CMT1X patients, and toxic effects only manifesting under stress conditions such as neuroinflammation may have been missed. Pro-inflammatory cytokines may disrupt glial GJs both by affecting connexins in astrocytes [10, 23], as well as in oligodendrocytes . In the case of CMT1X patients and related models, this could further exacerbate the already tenuous GJ coupling of oligodendrocytes, leading to clinical manifestations.
To further clarify the cellular mechanisms underlying the CNS phenotypes in patients with CMT1X, we used a model of systemic inflammation induced by lipopolysaccharide (LPS) injection comparing wild-type (WT) mice, Cx32 knockout (KO) mice, and Cx32 KO expressing the T55I mutant of Cx32 (KO T55I). We hypothesized that dominant effects of the T55I mutant might only become manifest under stress conditions that mimic the metabolic and inflammatory events leading to encephalopathy in CMT1X patients. We found that a combination of cellular alterations, both mutant-related but also independent from the mutant, likely contribute to disrupt oligodendrocyte integrity.
Materials and methods
Adult male (p40–p60) mice were used for this project and kept under standard housing conditions with a 12 hours (h) dark/light cycle and with food and water provided ad libitum. All experiments were performed in accordance with the animal care protocols approved by the Cyprus Government’s Chief Veterinary Officer according to EU guidelines. Three genotypic groups were used (n = 26 per group): Wild type (WT), connexin32 knockout (Cx32 KO), and Cx32 KO expressing the T55I mutant (KO T55I) associated with CMT1X. The KO T55I mutant was generated by pronuclear injection of the transgenic cassette described in  on a Bl6/N background and then crossed with the backcrossed Cx32 KO line. The WT was a 6 N and the Cx32 KO (originally generated by Prof. Klaus Willecke, University of Bonn, Bonn, Germany) was backcrossed at least 12 times to ensure a similar genetic background for the purpose of comparing the response to LPS. Only male animals were used for this study.
Determination of Genotypes
Genotypes were identified by PCR of genomic tail DNA with specific primers for the Cx32 KO allele: Exon1F (5’- GACCACTCCCCCTACACAGA-3’), Cx32R (5’- CGCTGTTGCAGCCAGGCTGG-3’) resulting in a 732 bp PCR product (94 °C × 5 min, 40 cycles of 94 °C × 30 s, 57 °C × 30 s, 72 °C × 30 s and then 72 °C × 7 min) and Exon1F (5’-GACCACTCCCCCTACACAGA-3’), NeoR2 (5’-CTCGTCCTGCAGTTCATTCA-3’) resulting in a 721 bp PCR product (94 °C × 5 min, 35 cycles of 94 °C × 30 s, 56 °C × 30 s, 72 °C × 30 s and then 72 °C × 7 min). For the KO T55I, all primers specific for the Cx32 KO were used, and in addition the CNP1F (5’- TGTGGCTTTGCCCATACATA-3’), Cx32R (5’-CGCTGTTGCAGCCAGGCTGG-3’), resulting in a 732 bp PCR product (94 °C × 5 min, 40 cycles of 94 °C × 30 s, 57 °C × 30 s, 72 °C × 30 s and then 72 °C × 7 min) and Cx32F (5’- CGCTGTTGCAGCCAGGCTGG-3’), EGFPR1 (5’- GCTGAACTTGTGGCCGTTTA-3’), resulting in a 785 bp PCR product.
Lipopolysaccharide (LPS) from Escherichia coli (E. coli) (serotype 0127:B8) was purchased from Sigma Chemical (St. Louis, MO) and 150 μl (LPS stock concentration 1 μg/mL, 7.5 mg/Kg injected LPS) were injected once intraperitonealy (i.p) to induce a systemic inflammatory response. Sterile saline i.p injection was used as control treatment for all experiments. Animals were weighted daily until the day of sacrifice (7 days after injection) and were observed for any sickness behavior.
Approximately 100 μl of blood was collected from the vein with a glass micropipette, at different time-points (just before saline/LPS injection, as well as 4, 24 and 96 h after injection). For blood collection mice were positioned under warm light for five minutes prior to blood collection in order to increase blood flow and then placed in a restraining tube so that their head and body were restrained, leaving only the tail outside the tube. Using sharp scissors, approximately 1 cm of the tail was removed and blood was collected in a capillary tube as drops appeared. Pressure was applied to stop the bleeding and for further collection the original wound was re-opened by removing the clot. Following blood collection, blood was incubated at 37 °C for 20 min in the heat block, stored overnight at 4 °C, subjected to centrifugation at 3000 rpm for 10 min and then stored in aliquots at −80 °C. Expression levels of plasma pro-inflammatory cytokines, tumor necrosis factor alpha (TNF-α) and Interleukin-6 (IL-6) were evaluated in LPS and saline treated animals in order to confirm the induced inflammatory response upon LPS injection. ELISA was performed using the Mouse TNF alpha ELISA Ready-SET-Go!® (eBioscience) for the detection of mouse TNF-α and the Mouse IL-6 ELISA Ready-SET-Go!® (eBioscience) kit for the detection of mouse IL-6.
Foot slip test
The method of Britt et al.  modified for mice was used in order to assess motor co-ordination. Approximately 4 h after injection mice were placed in a 15 × 15 × 15 cm clear plexiglass box with a floor consisting of a metal wire grid with 1.25 cm spacing with a 1.25 cm grid suspended 1.25 cm above the floor. A trial consisted of 50 steps. If a misstep resulted in the hindlimb falling through the grid but the limb was withdrawn prior to touching the floor it was scored 1; if the limb touched the floor it was scored 2. A video camera was used to film the mice. Mice were acclimated by performing a trial on each of 3 consecutive days prior to injection and for 60 min in the box prior to testing.
Four hours after saline/LPS injection mice were tested for motor balance and co-ordination. Animals were placed on a 3.5 cm diameter rod, rotating at a constant speed of 12 rpm and the time took for the animal to fall off was recorded using a timer. Mice were left to rest for 15 min and then tested at 20 rpm. Mice were trained on the rotarod for 3 trials of 3 consecutive days prior to injection. Mice were also acclimated to the testing room for 60 min before each session.
For immunoblot analysis, saline- and LPS-injected animals (n = 4 per treatment condition from each genotypic group) were sacrificed 7 days after injection and fresh tissues (brainstem and cerebellum) were harvested and lysed in ice-cold RIPA buffer (10 mM sodium phosphate pH 7.0, 150 mM NaCl, 2 mM EDTA, 50 mM sodium fluoride, 1 % NP-40, 1 % sodium deoxycholate, and 0.1 % DSD) containing a mixture of protease inhibitors (Roche). Tissues were then sonicated 5 × 5 s and protein concentrations were measured with Nanodrop at λ = 280. Proteins (100 μg) from tissue lysates were fractionated by 12 % SDS-PAGE and then transferred to a Hybond-C extra membrane (GE Healthcare Bio-Sciences), using a semi-dry transfer unit (GE Healthcare Bio-Sciences). The membrane was blocked for 1 h at room temperature (RT) with 5 % non-fat skimmed milk in Tris-buffered saline containing 0.1 % Tween-20 (TBS-T). Immunoblots were then incubated with the following antibodies: rabbit anti-Iba1 (BioCare, diluted 1:500), rabbit anti-BiP (Santa Cruz, diluted 1:1000), mouse anti-Cx43 (Millipore, diluted 1:1000), rabbit anti-Cx47 (1:20,000, ) or mouse anti-MBP (Abcam, diluted 1:5000) in 5 % non-fat skimmed milk in TBS-T, overnight at 4 °C. Following three 15 min washes in TBS-T, immunoblots were incubated for 1 h at RT with an anti-mouse or anti-rabbit HRP-conjugated secondary antiserum (Jackson ImmunoResearch Laboratories) diluted 1:3000) in 5 % non-fat skimmed milk in TBS-T. All membranes were reprobed with either GAPDH (Santa Cruz Biotechnology, diluted 1:3000) or tubulin (Developmental Studies Hybridoma Bank diluted 1:4000) to demonstrate the loading. The bound antibody was visualized by enhanced chemiluminescence system (ECL, GE Healthcare Bio-Sciences) and band intensity was quantified using Tinascan software after normalizing each band to the GAPDH or tubulin band intensity.
For immunofluorescence staining, saline- and LPS-injected animals (n = 5 per treatment condition from each genotypic group) were sacrificed 7 days after injection. Mice were anesthetized with Avertin according to institutionally approved protocols and then transcardially perfused with saline followed by addition of 4 % paraformaldehyde (PFA) in 0.1 M PB. Tissues (midbrain, brainstem, cerebellum and spinal cord) were harvested and fixed for 30 min followed by cryoprotection in 20 % sucrose in phosphate buffer (PB) overnight. Tissues were then embedded in optimum cutting temperature compound (OCT), placed in ice cold acetone and stored at −80 °C. Twelve μm thick sections were thaw-mounted onto glass slides, permeabilized in cold acetone (−20 °C for 10 min), and incubated for 1 h at RT with blocking solution (5 % bovine serum albumin - BSA) containing 0.5 % Triton-X. The primary antibodies diluted in blocking solution were incubated overnight at 4 °C: mouse Cx43 (Millipore, 1:200), CC1 (Calbiochem 1:50), GFAP (Sigma, 1:200), RT97 (Hybridoma Bank, 1:1000), MOG (Dr. Sara Piddlesden, Cardiff, 1:200) and MBP (Abcam, 1:500), and rabbit antisera against Cx47 (Invitrogen, 1:500), Iba1 (BioCare, 1:500), activated caspase-3 (BioVision, 1:100), Fas (Santa Cruz, 1:200), CHOP/GADD153 (Santa Cruz, 1:50), BiP (Santa Cruz, 1:50), Fibrinogen (Dako, 1:100) and Fibronectin (Dako, 1:500). Sections were then washed in PBS and incubated with rhodamine (TRITC) conjugated affinity purified goat anti-rabbit IgG and fluorescein (FITC) conjugated affinity purified goat anti-mouse IgG secondary antibodies (Jackson ImmunoResearch Laboratories, 1:500) for 1 h at RT. Cell nuclei were visualized with 4’, 6’-diamidino-2-phenylindole (DAPI) (Sigma-Aldrich). Slides were mounted with Dako fluorescent mounting medium and images were photographed under a Zeiss fluorescence microscope with a digital camera using the Zeiss Axiovision software (Carl Zeiss MicroImaging). For Cx47 localization some images were also taken with a Leica DMR confocal microscope (Leica Microsystems).
Assessment of inflammatory cell infiltrates
Spinal cord roots and sciatic nerves of saline and LPS injected animals were also harvested as described above in order to assess the existence of inflammatory cell infiltrates (lymphocytes and macrophages) in the peripheral nervous system with immunohistochemistry. Rabbit antiserum against T-cell marker CD3+ (Santa Cruz, diluted 1:100) and fluorescein conjugated rat antiserum against macrophage marker CD68+ (Serotec, diluted 1:50) were used as primary antibodies.
Immunostained coronal sections of brainstem, cerebellum and spinal cord were visualized and captured with an AxioHR camera. Images of Iba1, MBP, MOG, and GFAP, immunostained sections were captured at 200× final magnification whereas images of connexin and CC-1 stained sections were captured at 400× final magnification. To assess microglial activation we measured the total area of Iba1 immunofluorescence and for myelin density evaluation we measured the total area of MBP immunofluorescence with ImageJ software, and results were presented as percentage of total image area. Oligodendrocyte numbers per total area were counted in CC-1 stained sections.
Quantification of GJ plaques formed by Cx47 and Cx43
Quantification of GJ plaques (defined as a focal accumulation of connexin immunoreactivity and size set between 0.01 and 1 μm2 for Cx43, and between 0.01 and 2 μm2 for Cx47) was performed within a 9620 μm2 area using Image Pro 6.3 software in spinal cord gray and white matter and in brainstem sections stained with antibodies against Cx43 and Cx47 as described previously . The total number of GJ plaques formed by Cx43 and Cx47 was measured. In addition, we counted the total number of oligodendrocytes labeled with the CC1 antibody in the same areas, and also quantified the number of Cx47 GJ plaques per oligodendrocyte in a fixed area of 30x30μm. All results of morphometric analysis were compared between LPS and saline treated mice in each genotype as well as between genotypes.
RNA extraction and quantitative Real-Time PCR
RNA was isolated from the brainstem using the RNeasy Lipid Tissue Mini Kit (Qiagen, Germany) according to manufacturer instructions using the Qiazol Lysis Reagent followed by DNase treatment. RNA samples were quantified by spectrophotometry (Nanodrop ND_100) and subjected to reverse transcription (RT)-PCR (25 °C for 10 min, 48 °C for 30 min, and 95 °C for 5 min) using the TaqMan RT-PCR Reagents and a GeneAmp PCR System (Applied Biosystems, Singapore) (end volume of 40 μL). The expression levels of genes encoding Cx47, Cx43 and BiP were assessed by quantitative Real-Time PCR Analysis (hold at 55 °C for 2 min and at 95 °C for 10 min, followed by 40 cycles at 95 °C for 15 s and at 60 °C for 1 min) using a 7900HT Real-Time PCR System (Applied Biosystems) and Taq-Man© Gene Expression Assays: Cx43: Mm01179639_s1; Cx47 Mm00519131_s1; BIP: Mm00517691_m1, Tubulin (Mm00726185_s1) was used as endogenous “house- keeping” control gene. Each sample was loaded in triplicate and contained 250 ng of cDNA, 1 μl of TaqMan Gene Expression Assay, and 10 μl of TaqMan Gene Expression Master Mix (end volume 20 μl). Expression levels in LPS and saline control mice were calculated after normalizing cycle thresholds against tubulin and presented as the fold induction value (2–D DCt) relative to naive control mice (mean ± standard deviation).
All data were expressed as the mean, standard deviation SD; standard error of the mean (SEM), and statistical significance was assessed with the two-tailed Student’s t-test followed by Bonferroni's correction when multiple comparisons were performed, using Microsoft Excel software. A value of p < 0.05 was considered statistically significant.
Establishment of the LPS-induced systemic inflammation model
The CNS phenotypes in CMT1X patients are frequently triggered by febrile illness [2, 58, 65]. In order to study this we developed a model of LPS-induced systemic inflammation. Intraperitoneal (i.p.) injection of LPS (150 μl) induced a systemic inflammatory response characterized by sickness behavior with transient mild weight loss (1.7 ± 0.64 gr at 24 h post injection), lethargy, isolation, and reduced mobility in the first 2 days after injection, followed by recovery of mobility back to normal levels. ELISA confirmed a marked increase of two pro-inflammatory cytokines, TNF-α and IL-6, 4 h after injection in peripheral blood of Cx32 KO LPS-injected as opposed to saline-injected mice (Additional file 1: Figure S1).
LPS-induced neuroinflammation impairs motor performance and coordination
LPS induced neuroinflammation does not cause demyelination or blood-brain barrier disruption in Cx32 mutant mice
To clarify whether the observed inflammatory changes and impaired motor function could be caused by demyelination in the CNS of Cx32 mutant mice, we examined myelination by immunohistochemistry and immunoblot analysis. Immunostaining for myelin basic protein (MBP) and the axonal marker RT97, as well as for myelin oligodendrocyte glycoprotein (MOG), showed preservation of myelin immunoreactivity in different areas of the CNS, including the brain at the level of the corpus callosum (Additional file 7: Figure S5a–l), cerebellum and spinal cord (data not shown) in LPS-injected mice without significant change compared to saline-injected controls. In addition, immunoblot analysis confirmed that MBP levels assessed in brainstem lysates were not significantly altered in LPS-injected compared to control mice from all three genotypes (Additional file 7: Figure S5m–o). Thus, demyelination is unlikely to contribute to the observed phenotype of LPS-injected Cx32 mutant mice.
Given the increased CNS inflammation in Cx32 KO and KO T55I mice we also examined whether blood-brain barrier (BBB) disruption could play a role in CNS phenotypes in Cx32 mutant mice following systemic inflammation induced by LPS. We therefore examined expression of fibrinogen and fibronectin, two major BBB markers , on fixed brain tissues comparing LPS to saline treated tissues for each genotype. We found no evidence of BBB disruption in KO or KO T55I animals injected with LPS compared to WT and saline controls (Additional file 8: Figure S6).
LPS-induced systemic inflammation disrupts oligodendrocyte gap junction formation in the CNS
Although we did not observe any significant reduction in the numbers of CC1-positive oligodendrocytes in LPS tissues we further investigated by double immunostaining with CC1 and caspase-3 whether LPS induced any oligodendrocyte apoptosis in WT or Cx32 mutant mice. This analysis showed that caspase-3 immunoreactivity was not increased in the CNS of LPS-injected WT, Cx32 KO, or T55I KO mice compared to saline controls (Additional file 12: Figure S10). Thus, LPS-induced inflammation causes loss of Cx47 GJ plaques in oligodendrocytes but no oligodendrocyte loss or apoptosis, up to 1 week after injection.
LPS-induced neuroinflammation disrupts astrocyte to oligodendrocyte gap junctions
Downregulation of Cx43 precedes the loss of Cx47 in LPS-injected Cx32 mutant mice
To clarify which of the connexin partners at A/O GJs is affected first by LPS-induced neuroinflammation in Cx32 mutant mice, we examined their expression by Real-time PCR analysis. These experiments revealed that Cx43 mRNA levels were significantly reduced in CNS tissues from KO and KO T55I LPS-injected mice compared to controls, while a non-significant reduction was also observed in the WT group. Comparison of baseline Cx43 mRNA levels between the three genotypes at baseline (saline) showed no significant difference. However, among LPS-treated groups Cx43 expression was significantly more reduced in KO T55I than in simple KO and the WT groups (Fig. 5g). We also analyzed Cx47 mRNA expression levels comparing LPS-injected to control mice in all genotypic groups both at baseline and after LPS injection. Although we observed a small reduction in the expression of Cx47 in the T55I KO groups, there was no significant difference compared to WT or KO mice or in LPS compared to control groups (Fig. 5h). Thus, astrocytic Cx43 appears to be downregulated under inflammatory conditions with reduction of GJ channels. Loss of Cx43 is associated with reduction of oligodendrocyte Cx47 GJs and A/O connectivity, and diffusion of Cx47 away from the cell membrane, since Cx47 mRNA and protein levels are not reduced. These alterations appear to be more severe in the Cx32 KO T55I mutant than in the simple Cx32 KO mouse.
The expression of the ER-retained T55I mutant triggers ER-stress under inflammatory conditions
Our study provides further insights into the cellular and molecular mechanisms underlying the intriguing CNS phenotypes in patients with CMT1X. First, we show that in the absence of Cx32 in Cx32 KO mice there is a higher vulnerability to inflammation, since the associated downregulation of astrocytic Cx43 leads to reduction of Cx47-formed O/A GJ channels and impaired oligodendrocyte connectivity to the glial GJ network. This mechanism is independent from the presence Cx32 mutants. Second, we demonstrate that the presence of the ER-retained T55I mutant in a Cx32 KO background further increases oligodendrocyte vulnerability to inflammatory stress, through an increased ER-stress response. This mechanism could be additive to the disruption of A/O GJs, leading to even more severe CNS dysfunction.
The most common trigger of the CNS phenotype reported in CMT1X patients besides high altitude travel was a febrile systemic illness such as upper respiratory infection, fever of uncertain cause, pneumonia or gastroenteritis in at least half of all cases (reviewed by ). In order to reproduce the CNS phenotype induced by systemic inflammation in CMT1X patients, we generated a model of systemic inflammation induced in Cx32 mutant mice by intraperitoneal injections of LPS. We confirmed the generalized inflammatory response by detecting transient elevation in blood of two pro-inflammatory cytokines, TNF-α and IL-6, as shown in other studies . LPS activates liver cells to produce cytokines in the periphery, which in turn activate humoral and neural communication pathways [12, 55, 62] and induce glial cells in the CNS to produce the same inflammatory cytokines. Activation of microglia, the resident immune cells of the brain, results in synthesis of additional TNF-α and other cytokines and leads to persistent neuroinflammation . Likewise, in our study we observed diffusely activated microglia in different CNS areas including the cerebrum, cerebellum, brainstem and spinal cord and Iba1 immunoblots from brainstem lysates confirmed increased microglial activation induced by systemic LPS injection. Thus, LPS treatment provides a relevant model for the CNS phenotypes in CMT1X patients.
The LPS model described here was characterized by early sickness behavior and transient weight loss, similar to the phenotype reported in other models . In addition, we examined motor behavior by rotarod and foot-slip test behavioral analysis, showing a marked impairment in LPS-treated mice compared to saline controls as shown in similar studies . Moreover, the comparison of the three genotypes showed that Cx32 KO mice are more vulnerable to neuroinflammation than WT mice, and KO T55I show even more vulnerability than do simple KO mice. Thus, LPS-induced neuroinflammation affects motor performance even more in the presence of the T551 mutant, suggesting additional gain of function mechanisms that could explain the CNS phenotypes in some CMT1X patients.
Despite the diffuse response of the innate immunity in the CNS, we found no disruption of BBB, inflammatory cell infiltrates in the peripheral nervous system, oligodendrocyte loss, or demyelination in Cx32 mutant mice, indicating that these are unlikely mechanisms in CNS phenotypes occurring in CMT1X patients. Our results are therefore in agreement not only with the clinical course in CMT1X patients, who show early recovery, but also with the available MRI studies that have shown transient, nearly symmetric, diffuse T2 and fluid-attenuated inversion recovery (FLAIR) hyperintensities and restricted diffusion signal abnormalities, but no contrast enhancement and no typical demyelinating lesions [2, 49, 65]. The clinical and MRI characteristics of CNS involvement in CMT1X patients are in fact consistent with transient disruption of the gap junctional communication between oligodendrocytes and astrocytes leading to inability of these cells to regulate fluid exchange.
Astrocytes appear to play a role in the link between neuroinflammation or metabolic stress and CNS phenotypes in CMT1X as they are closely connected to oligodendrocytes through GJs. Oligodendrocytes express Cx47 throughout the CNS, and in many areas also Cx32 . Besides their participation in A/O GJs, Cx32 and Cx47 also form homotypic channels between oligodendrocytes. Cx29 is a third oligodendrocyte connexin that appears to form only hemichannels and does not participate in intercellular GJs . Astrocytes express Cx43 in both gray and white matter and Cx30 mainly in the gray matter, connecting with other astrocytes through homotypic (Cx43/Cx43 and Cx30/Cx30) GJs as well as with oligodendrocytes with heterotypic (Cx43/Cx47 and Cx30/Cx32) GJs [4, 22, 43, 48, 73]. Among these channels, Cx43/Cx47 GJs play the major role especially in the white matter.
Several experimental mouse models have been generated to clarify the role of each of these astrocyte and oligodendrocyte connexins in the CNS. Single KOs showed minimal CNS pathology leading to the overall conclusion that there is partially overlapping function of connexins in both cell types. However, when more than one connexin and type of GJ channel is disrupted, severe demyelinating pathology develops. Thus, mice lacking either Cx32 or Cx47 develop minimal CNS pathology and no obvious CNS phenotype, but loss of both oligodendrocyte connexins in Cx32/Cx47 double KO mice leads to severe and early CNS demyelination [41, 46]. Likewise, deletion of both astrocyte connexins (Cx30 and Cx43) , or at least one partner of each of the two A/O GJ types, either Cx43 and Cx32 , or Cx30 and Cx47  results in severe demyelination and vacuolation because in each case all A/O GJs are completely disrupted. In contrast to these double KO models with congenital and complete lack of oligodendrocyte GJ channels, in our LPS model induced in Cx32 deficient mice there was only partial reduction of Cx47 GJs after completed development, which did not cause demyelination. This underscores the important role of Cx47 in oligodendrocytes with expression as starting at earlier stages of their development and differentiation [41, 46], as early as P7 , including in oligodendrocyte precursor cells .
Of particular relevance to the current study and the mechanism of CNS phenotypes in CMT1X is the Cx32/Cx43 double KO model, which revealed that in the absence of Cx32 GJs (as in CMT1X patients) oligodendrocytes depend exclusively on Cx43/Cx47 GJs. When Cx43 is also disrupted, the stability of Cx47 on oligodendrocyte cell membrane is impaired leading to loss of Cx47 formed GJs, and consequently to complete disconnection of oligodendrocytes . In our LPS model we observe a clear downregulation of Cx43 in astrocytes with early reduction of mRNA levels. In contrast, there are no significant changes in Cx47 mRNA and protein levels, despite reduction of Cx47 GJ plaques and diffusion in the cytoplasm. This pattern of secondary disruption of Cx47 GJ plaques following the downregulation of astrocytic Cx43 during acute inflammation has also been shown in previous studies using the EAE model . As in the EAE model, loss of Cx47 GJs was not associated with loss of oligodendrocytes, further supporting the secondary mechanism of Cx47 diffusion into the cytoplasm . The downregulation of Cx43 in the LPS model likely reflects a non-specific astrocytic reaction to diverse CNS injury, including inflammation, and has been shown in EAE [10, 36, 61], ischemia  and abscess  models and is likely mediated by pro-inflammatory cytokines [13, 15, 21]. Thus, it is plausible to hypothesize that in CMT1X patients, in whom oligodendrocyte GJ connectivity depends only on Cx43/Cx47 GJs, downregulation of Cx43 as part of an astrocyte reaction to inflammatory or metabolic stress will disrupt Cx47 in oligodendrocytes and lead to transient encephalopathy. Our previous studies in EAE models showed that Cx43 downregulation is a transient reaction followed by re-expression at later stages , and this would be in keeping with the reversibility of CNS phenotypes in CMT1X patients. Nevertheless, inflammation could also directly affect Cx47 expression independently of astrocyte reaction and Cx43 loss, since pro-inflammatory cytokines have been shown to induce ER-stress response in oligodendrocytes .
Cx32 KO mice with or without the presence of the T55I mutant showed a worse phenotype than WT mice. One explanation is the higher CNS inflammatory load reflected in the amount of microglia activation. This would also explain why Cx43 was more severely decreased in these mutants, although not an oligodendrocyte connexin. A pro-inflammatory environment associated with elevated cytokine levels at baseline has been recently documented in Cx32/Cx47 dKO mice suggesting that connexin deficiency in oligodendrocytes drives CNS inflammation independently of external immune triggers . The exacerbated phenotype of Cx32 mutant mice following LPS treatment may also result from effects of inflammation on neurons and axons directly or indirectly, for example through astrocyte dysregulation of synaptic function , independently of the effects in oligodendrocytes. Previous studies showed exacerbated axonal loss in Cx32 KO compared to WT mice after EAE induction , which may result from disturbed signaling  and energy supply , as well as axonal neurofilament dephosphorylation  in the absence of Cx32 GJs along myelinated fibers. Thus, axons of Cx32 KO mice may have increased vulnerability to inflammatory stress.
The next question is why the presence of the T55I mutant exacerbates all CNS manifestations in our LPS model, including the inflammatory, behavioral, and connexin abnormalities. Although our previous studies in Cx32 KO T55I mice failed to show a dominant effect beyond the simple KO phenotype under normal conditions , LPS-induced neuroinflammation affected more severely the KO T55I than the “simple” KO and WT mice. Thus, the presence of the T55I mutant may render oligodendrocytes more vulnerable to inflammation. We believe that this is because the T55I mutation causes retention of misfolded Cx32 in the ER leading to ER-stress response especially under inflammatory conditions, which in turn exacerbates further oligodendrocyte homeostasis. LPS induced a significant elevation of BiP expression especially in KO T55I mice, as opposed to WT and Cx32 KO mice, although Cx32 KO mice also showed a significantly higher ER-stress response compared to WT mice. Previous studies have shown that upon TNF-α release, microglia activation and ER stress are induced in the CNS and that ER stress blockers suppress the induced inflammation . Involvement of ER stress has also been demonstrated in LPS-induced inflammation and inhibition of ER stress ameliorated LPS-induced lung injury through modulation of several cytokine pathways . In addition we found increased Fas immunoreactivity induced by LPS injection mostly in KO T55I LPS treated mice compared to simple KO or WT. ER stress induces Fas and mitochondrial apoptosis pathways through elevation of cytosolic calcium . In our study, both the ER stress marker BiP and CHOP, along with the pro-apoptotic marker Fas, were mostly elevated in the presence of the T55I mutant suggesting that under stress conditions intracellularly retained Cx32 mutants may exacerbate ER stress upon neuroinflammation, leading to the development of CNS phenotypes in CMT1X patients. This effect may be additive to the disruption of A/O GJs described above, making CNS manifestations more likely in patients with intracellularly retained mutants as initially hypothesized , although they can also occur in the absence of any Cx32 .
In conclusion, the work presented here provides evidence that LPS-induced systemic and CNS inflammation causes oligodendrocyte dysfunction associated with disruption of the main A/O GJs resulting from downregulation of astrocytic Cx43 and diffusion of Cx47. Moreover, there is an exaggerated ER stress response related to the intracellularly retained Cx32 mutant. Our study provides a relevant model and a mechanistic explanation for the development of CNS phenotypes in CMT1X. In patients with Cx32 mutations the already compromised oligodendrocyte homeostasis causing subclinical manifestations at baseline conditions, may decompensate under conditions of metabolic and inflammatory stress producing the transient encephalopathy manifestations.
This study was funded by the Muscular Dystrophy Association (MDA-218284 Grant to CKA and KAK).
The monoclonal antibody RT97 (developed by John Wood) was obtained from the Developmental Studies Hybridoma Bank developed under the auspices of the National Institute of Child Health and Human Development and maintained by Department of Biology, The University of Iowa, Iowa City.
MO performed all the experiments and wrote the paper. IS, KM, CK, AK and SK performed certain experiments. CKA designed the study and critically reviewed the manuscript. KAK designed the study, supervised all experiments, and wrote the manuscript. All authors read and approved the final manuscript.
The authors declare that they have no competing interests.
Open AccessThis article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.
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