Assessment of blood-brain barrier leakage and brain oxygenation in Connexin-32 knockout mice with systemic neuroinflammation using pulse electron paramagnetic resonance imaging techniques.

PURPOSE: The determination of blood-brain barrier (BBB) integrity and partial pressure of oxygen (pO2) in the brain is of substantial interest in several neurological applications. This study aimed to assess the feasibility of using trityl OX071-based pulse electron paramagnetic resonance imaging (pEPRI) to provide a quantitative estimate of BBB integrity and pO2 maps in mouse brains as a function of neuroinflammatory disease progression. METHODS: Five Connexin-32 (Cx32)-knockout (KO) mice were injected with lipopolysaccharide to induce neuroinflammation for imaging. Three wild-type mice were also used to optimize the imaging procedure and as control animals. An additional seven Cx32-KO mice were used to establish the BBB leakage of trityl using the colorimetric assay. All pEPRI experiments were performed using a preclinical instrument, JIVA-25 (25 mT/720 MHz), at times t = 0, 4, and 6 h following lipopolysaccharide injection. Two pEPRI imaging techniques were used: (a) single-point imaging for obtaining spatial maps to outline the brain and calculate BBB leakage using the signal amplitude, and (b) inversion-recovery electron spin echo for obtaining pO2 maps. RESULTS: A statistically significant change in BBB leakage was found using pEPRI with the progression of inflammation in Cx32 KO animals. However, the change in pO2 values with the progression of inflammation for these animals was not statistically significant. CONCLUSIONS: For the first time, we show the ability of pEPRI to provide pO2 maps in mouse brains noninvasively, along with a quantitative assessment of BBB leakage. We expect this study to open new queries from the field to explore the pathology of many neurological diseases and provide a path to new treatments.

Knock-in mouse models for CMTX1 show a loss of function phenotype in the peripheral nervous system.

The X-linked form of Charcot-Marie-Tooth disease (CMTX1) is the second most common form of CMT. In this study we used CRISPR/Cas9 to develop new "knock-in" models of CMTX1 that are more representative of the spectrum of mutations seen with CMTX1 than the Cx32 knockout (KO) mouse model used previously. We compared mice of four genotypes - wild-type, Cx32KO, p.T55I, and p.R75W. Sciatic motor conduction velocity slowing was the most robust electrophysiologic indicator of neuropathy, showing reductions in the Cx32KO by 3 months and in the p.T55I and p.R75W mice by 6 months. At both 6 and 12 months, all three mutant genotypes showed reduced four limb and hind limb grip strength compared to WT mice. Performance on 6 and 12 mm width balance beams revealed deficits that were most pronounced at on the 6 mm balance beam at 6 months of age. There were pathological changes of myelinated axons in the femoral motor nerve in all three mutant lines by 3 months of age, and these became more pronounced at 6 and 12 months of age; sensory nerves (femoral sensory and the caudal nerve of the tail) appeared normal at all ages examined. Our results demonstrate that mice can be used to show the pathogenicity of human GJB1 mutations, and these new models for CMTX1 should facilitate the preclinical work for developing treatments for CMTX1.

Activation of the unfolded protein response by Connexin47 mutations associated with Pelizaeus-Merzbacher-like disease.

Pelizaeus-Merzbacher-like disease type 1 (PMLD1) is a hypomyelinating disorder arising in patients with mutations in GJC2, encoding Connexin47 (Cx47). PMLD1 causes nystagmus, cerebellar ataxia, spasticity and changes in CNS white matter detected by MRI. At least one mutation (p.I33M) yields a much milder phenotype, spastic paraplegia type 44 (SPG44). Cx47 contributes to gap junction communication channels between oligodendrocytes (OLs), the myelinating cells in the central nervous system (CNS), and between OLs and astrocytes. Prior studies in cell lines have shown that PMLD1 mutants such as p.P87S display defective protein trafficking, intracellular retention in the ER and loss-of-function. Here we show that when expressed in primary OLs, three PMLD1 associated mutants (p.P87S, p.Y269D and p.M283T) show ER retention of Cx47 and evidence of activation of the cellular stress (unfolded protein response, UPR) and apoptotic pathways. On the other hand, the milder SPG44 associated mutation p.I33M shows a wild-type-like subcellular distribution and no activation of the UPR or apoptotic pathways. These studies provide new insight into a potential element of toxic gain of function underlying the mechanism of PMLD1 that should help guide future therapeutic approaches.

AAV1.NT-3 gene therapy for X-linked Charcot-Marie-Tooth neuropathy type 1.

X-linked Charcot-Marie-Tooth neuropathy (CMTX) is caused by mutations in the gene encoding Gap Junction Protein Beta-1 (GJB1)/Connexin32 (Cx32) in Schwann cells. Neurotrophin-3 (NT-3) is an important autocrine factor supporting Schwann cell survival and differentiation and stimulating axon regeneration and myelination. Improvements in these parameters have been shown previously in a CMT1 model, TremblerJ mouse, with NT-3 gene transfer therapy. For this study, scAAV1.tMCK.NT-3 was delivered to the gastrocnemius muscle of 3-month-old Cx32 knockout (KO) mice. Measurable levels of NT-3 were found in the serum at 6-month post gene delivery. The outcome measures included functional, electrophysiological and histological assessments. At 9-months of age, NT-3 treated mice showed no functional decline with normalized compound muscle action potential amplitudes. Myelin thickness and nerve conduction velocity significantly improved compared with untreated cohort. A normalization toward age-matched wildtype histopathological parameters included increased number of Schmidt-Lanterman incisures, and muscle fiber diameter. Collectively, these findings suggest a translational application to CMTX1.

Investigating oligodendrocyte connexins: Heteromeric interactions between Cx32 and mutant or wild-type forms of Cx47 do not contribute to or modulate gap junction function.

Oligodendrocytes express two gap junction forming connexins, connexin 32 (Cx32) and Cx47; therefore, formation of heteromeric channels containing both Cx47 and Cx32 monomers might occur. Mutations in Cx47 cause both Pelizaeus-Merzbacher-like disease Type 1 (PMLD1) and hereditary spastic paraparesis Type 44 (SPG44) and heteromer formation between these mutants and Cx32 may contribute to the pathogenesis of these disorders. Here, we utilized electrophysiological and antibody-based techniques to examine this possibility. When cells expressing both Cx32 and Cx47 were paired with cells expressing either Cx32 or Cx47, properties were indistinguishable from those produced by cells expressing homotypic Cx32 or Cx47 channels. Similarly, pairing cells expressing both Cx32 and Cx47 with cells expressing Cx30 or Cx43 produced channels indistinguishable from heterotypic Cx32/Cx30 or Cx47/Cx43 channels, respectively. The same assessments were performed on cells expressing Cx32 and four mutant forms of Cx47 (p.I33M associated with SPG44 or p.P87S, p.Y269D or p.M283T associated with PMLD1). None of these mutants showed a functional effect on Cx32. Immunostained cells co-expressing Cx32WT (wild type) and Cx47WT showed a Pearson correlation coefficient close to zero, suggesting that any overlap was due to chance. p.Y269D showed a statistically significant negative correlation with Cx32, suggesting that Cx32 and this mutant overlap less than expected by chance. Co-immunoprecipitation of Cx32 with Cx47WT and mutants show only very low levels of co-immunoprecipitated protein. Overall, our data suggest that interactions between PMLD1 or SPG44 mutants and Cx32 gap junctions do not contribute to the pathogenesis of these disorders.

Effects of early crush on aging wild type and Connexin 32 knockout mice: Evidence for a neuroprotective state in CMT1X mouse nerve.

The long-term sequelae of nerve injury as well as age-related neurodegeneration have been documented in numerous studies, however the role of Cx32 in these processes is not well understood. There is a need for better understanding of the molecular mechanisms that underlie long-term suboptimal nerve function and for approaches to prevent or improve it. In this communication we describe our studies using whole animal electrophysiology to examine the long-term sequelae of sciatic nerve crush in both WT and Cx32KO mice, a model of X-linked Charcot Marie Tooth disease, a subtype of inherited peripheral neuropathies. We present results from electrical nerve recordings done 14 to 27 days and 18 to 20 months after a unilateral sciatic nerve crush performed on 35 to 37-day old mice. Contrary to expectations, we find that whereas crush injury leads to a degradation of WT nerve function relative to uninjured nerves at 18 to 20 months, previously crushed Cx32KO nerves perform at the same level as their uninjured counterparts. Thus, 18 to 20 months after injury, WT nerves perform below the level of normal (uninjured) WT nerves in both motor and sensory nerve function. In contrast, measures of nerve function in Cx32KO mice are degraded for sensory axons but exhibit no additional dysfunction in motor axons. Early nerve injury has no negative electrophysiologic effect on the Cx32 KO motor nerves. Based on our prior demonstration that the transcriptomic profile of uninjured Cx32KO and injured WT sciatic nerves are very similar, the lack of an additional effect of crush on Cx32KO motor nerve parameters suggests that Cx32 knockout may implement a form of neuroprotection that limits the effects of subsequent injury.

Acetylation of C-terminal lysines modulates protein turnover and stability of Connexin-32.

BACKGROUND: The gap junction protein, Connexin32 (Cx32), is expressed in various tissues including liver, exocrine pancreas, gastrointestinal epithelium, and the glia of the central and peripheral nervous system. Gap junction-mediated cell-cell communication and channel-independent processes of Cx32 contribute to the regulation of physiological and cellular activities such as glial differentiation, survival, and proliferation; maintenance of the hepatic epithelium; and axonal myelination. Mutations in Cx32 cause X-linked Charcot-Marie-Tooth disease (CMT1X), an inherited peripheral neuropathy. Several CMT1X causing mutations are found in the cytoplasmic domains of Cx32, a region implicated in the regulation of gap junction assembly, turnover and function. Here we investigate the roles of acetylation and ubiquitination in the C-terminus on Cx32 protein function. Cx32 protein turnover, ubiquitination, and response to deacetylase inhibitors were determined for wild-type and C-terminus lysine mutants using transiently transfected Neuro2A (N2a) cells. RESULTS: We report here that Cx32 is acetylated in transfected N2a cells and that inhibition of the histone deacetylase, HDAC6, results in an accumulation of Cx32. We identified five lysine acetylation targets in the C-terminus. Mutational analysis demonstrates that these lysines are involved in the regulation of Cx32 ubiquitination and turnover. While these lysines are not required for functional Cx32 mediated cell-cell communication, BrdU incorporation studies demonstrate that their relative acetylation state plays a channel-independent role in Cx32-mediated control of cell proliferation. CONCLUSION: Taken together these results highlight the role of post translational modifications and lysines in the C-terminal tail of Cx32 in the fine-tuning of Cx32 protein stability and channel-independent functions.

Regulatory role of oligodendrocyte gap junctions in inflammatory demyelination.

Gap junctions (GJs) coupling oligodendrocytes to astrocytes and to other oligodendrocytes are formed mainly by connexin47 (Cx47) and a smaller portion by connexin32 (Cx32). Mutations in both connexins cause inherited demyelinating disorders, but their expression is also disrupted in multiple sclerosis (MS). To clarify whether the loss of either Cx47 or Cx32 could modify the outcome of inflammation and myelin loss, we induced experimental autoimmune encephalomyelitis (EAE) in fully backcrossed Cx32 knockout (KO) and Cx47KO mice and compared their outcome with wild type (WT, C57BI/6 N) mice. Cx47KO EAE mice developed the most severe phenotype assessed by clinical scores and behavioral testing, followed by Cx32KO and WT mice. Cx47KO more than Cx32KO EAE mice developed more microglial activation, myelin, and axonal loss than did WT mice. Oligodendrocyte apoptosis and precursor proliferation was also higher in Cx47KO than in Cx32KO or WT EAE mice. Similarly, blood-spinal cord barrier (BSCB) disruption and inflammatory infiltrates of macrophages, T- and B-cells were more severe in Cx47KO than either Cx32KO or WT EAE groups. Finally, expression profiling revealed that several proinflammatory cytokines were higher at the peak of inflammation in the Cx47KO mice and persisted at later stages of EAE in contrast to reduction of their levels in WT EAE mice. Thus, loss of oligodendrocyte GJs aggravates BSCB disruption and inflammatory myelin loss, likely due to dysregulation of proinflammatory cytokines. This mechanism may play an important role in MS brain with reduced connexin expression, as well as in patients with inherited mutations in oligodendrocyte connexins and secondary inflammation.

Alterations at Arg76 of human connexin 46, a residue associated with cataract formation, cause loss of gap junction formation but preserve hemichannel function.

The connexins are members of a family of integral membrane proteins that form gap junction channels between apposed cells and/or hemichannels across the plasma membranes. The importance of the arginine at position 76 (Arg76) in the structure and/or function of connexin 46 (Cx46) is highlighted by its conservation across the entire connexin family and the occurrence of pathogenic mutations at this (or the corresponding homologous) residue in a number of human diseases. Two mutations at Arg76 in Cx46 are associated with cataracts in humans, highlighting the importance of this residue. We examined the expression levels and macroscopic and single-channel properties of human Cx46 and compared them with those for two pathogenic mutants, namely R76H and R76G. To gain further insight into the role of charge at this position, we generated two additional nonnaturally occurring mutants, R76K (charge conserving) and R76E (charge inverting). We found that, when expressed exogenously in Neuro2a cells, all four mutants formed membrane hemichannels, inducing membrane permeability at levels comparable to those recorded in cells expressing the wild-type Cx46. In contrast, the number of gap-junction plaques and the magnitude of junctional coupling were reduced by all four mutations. To gain further insight into the role of Arg76 in the function of Cx46, we performed homology modeling of Cx46 and in silico mutagenesis of Arg76 to Gly, His, or Glu. Our studies suggest that the loss of interprotomeric interactions has a significant effect on the extracellular domain conformation and dynamics, thus affecting the hemichannel docking required for formation of cell-cell channels.

Rapid and Specific Immunomagnetic Isolation of Mouse Primary Oligodendrocytes.

The efficient and robust isolation and culture of primary oligodendrocytes (OLs) is a valuable tool for the in vitro study of the development of oligodendroglia as well as the biology of demyelinating diseases such as multiple sclerosis and Pelizaeus-Merzbacher-like disease (PMLD). Here, we present a simple and efficient selection method for the immunomagnetic isolation of stage three O4+ preoligodendrocytes cells from neonatal mice pups. Since immature OL constitute more than 80% of the rodent-brain white matter at postnatal day 7 (P7) this isolation method not only ensures high cellular yield, but also the specific isolation of OLs already committed to the oligodendroglial lineage, decreasing the possibility of isolating contaminating cells such as astrocytes and other cells from the mouse brain. This method is a modification of the techniques reported previously, and provides oligodendrocyte preparation purity above 80% in about 4 h.

Loss of Coupling Distinguishes GJB1 Mutations Associated with CNS Manifestations of CMT1X from Those Without CNS Manifestations.

CMT1X, an X-linked inherited neuropathy, is caused by mutations in GJB1, which codes for Cx32, a gap junction protein expressed by Schwann cells and oligodendrocytes. Many GJB1 mutations cause central nervous system (CNS) abnormality in males, including stable subclinical signs and, less often, short-duration episodes characterized by motor difficulties and altered consciousness. However, some mutations have no apparent CNS effects. What distinguishes mutations with and without CNS manifestations has been unclear. Here we studied a total of 14 Cx32 mutations, 10 of which are associated with florid episodic CNS clinical syndromes in addition to peripheral neuropathy. The other 4 mutations exhibit neuropathy without clinical or subclinical CNS abnormalities. These "PNS-only" mutations (Y151C, V181M, R183C and L239I) form gap junction plaques and produce levels of junctional coupling similar to those for wild-type Cx32. In contrast, mutants with CNS manifestations (F51L, E102del, V139M, R142Q, R142W, R164W T55I, R164Q and C168Y) either form no morphological gap junction plaques or, if they do, produce little or no detectable junctional coupling. Thus, PNS and CNS abnormalities may involve different aspects of connexin function.

A new mutation in GJC2 associated with subclinical leukodystrophy.

Recessive mutations in GJC2, the gene-encoding connexin 47 (Cx47), cause Pelizaeus-Merzbacher-like disease type 1, a severe dysmyelinating disorder. One recessive mutation (p.Ile33Met) has been associated with a much milder phenotype--hereditary spastic paraplegia type 44. Here, we present evidence that a novel Arg98Leu mutation causes an even milder phenotype--a subclinical leukodystrophy. The Arg98Leu mutant forms gap junction plaques in HeLa cells comparable to wild-type Cx47, but electrical coupling was 20-fold lower in cell pairs expressing Arg98Leu than for cell pairs expressing wild-type Cx47. On the other hand, coupling between Cx47Arg98Leu and Cx43WT expressing cells did not show such reductions. Single channel conductance and normalized steady-state junctional conductance-junctional voltage (G(j)-V(j)) relations differed only slightly from those for wild-type Cx47. Our data suggest that the minimal phenotype in this patient results from a reduced efficiency of opening of Cx47 channels between oligodendrocyte and oligodendrocyte with preserved coupling between oligodendrocyte and astrocyte, and support a partial loss of function model for the mild Cx47 associated disease phenotypes.

Exome sequencing identification of a GJB1 missense mutation in a kindred with X-linked spinocerebellar ataxia (SCA-X1).

We undertook a gene identification and molecular characterization project in a large kindred originally clinically diagnosed with SCA-X1. While presenting with ataxia, this kindred also had some unique peripheral nervous system features. The implicated region on the X chromosome was delineated using haplotyping. Large deletions and duplications were excluded by array comparative genomic hybridization. Exome sequencing was undertaken in two affected subjects. The single identified X chromosome candidate variant was then confirmed to co-segregate appropriately in all affected, carrier and unaffected family members by Sanger sequencing. The variant was confirmed to be novel by comparison with dbSNP, and filtering for a minor allele frequency of <1% in 1000 Genomes project, and was not present in the NHLBI Exome Sequencing Project or a local database at the BCM HGSC. Functional experiments on transfected cells were subsequently undertaken to assess the biological effect of the variant in vitro. The variant identified consisted of a previously unidentified non-synonymous variant, GJB1 p.P58S, in the Connexin 32/Gap Junction Beta 1 gene. Segregation studies with Sanger sequencing confirmed the presence of the variant in all affected individuals and one known carrier, and the absence of the variant in unaffected members. Functional studies confirmed that the p.P58S variant reduced the number and size of gap junction plaques, but the conductance of the gap junctions was unaffected. Two X-linked ataxias have been associated with genetic loci, with the first of these recently characterized at the molecular level. This represents the second kindred with molecular characterization of X-linked ataxia, and is the first instance of a previously unreported GJB1 mutation with a dominant and permanent ataxia phenotype, although different CNS deficits have previously been reported. This pedigree has also been relatively unique in its phenotype due to the presence of central and peripheral neural abnormalities. Other X-linked SCAs with unique features might therefore also potentially represent variable phenotypic expression of other known neurological entities.

Functional requirement for a highly conserved charged residue at position 75 in the gap junction protein connexin 32.

Charcot Marie Tooth disease (CMT) is a group of inherited disorders characterized clinically by exclusively or predominantly peripheral nerve dysfunction. CMT1X, the most common form of X-linked CMT is caused by mutations in connexin 32 (Cx32). In this work, we used dual whole cell patch clamp recording to examine the functional effects of mutations at the Arg(75) position. This residue is highly conserved among members of the connexin family, and disease-causing mutations have been identified at this (or the corresponding) position in Cx26, Cx43, and Cx46. Thus, a better understanding of the effects of mutations of this position in Cx32 may have relevance to pathogenesis of a number of different human diseases. All three mutants associated with CMT1X (R75P, R75Q, and R75W) showed very low levels of coupling similar to those of the cells transfected with vector alone. Heterotypic pairing with Cx32 WT showed that the absence of coupling for these mutants in the homotypic configuration could be explained by shifts in their hemichannel G(j)-V(j) relations. Examination of the expression levels and gating characteristics of seven additional mutants (R75A, R75D, R75E, R75H, R75K, R75L, and R75V) at this position suggest that the positive charge at position 75 in Cx32 is required for normal channel function but not for gap junction assembly. Our studies also suggest that disease treatment strategies for CMT1X, which correct trafficking abnormalities in Cx32, may be ineffective for the group of mutations also conferring changes in gating properties of Cx32 channels.

Hereditary spastic paraplegia is a novel phenotype for GJA12/GJC2 mutations.

Recessive mutations in GJA12/GJC2, the gene that encodes the gap junction protein connexin47 (Cx47), cause Pelizaeus-Merzbacher-like disease (PMLD), an early onset dysmyelinating disorder of the CNS, characterized by nystagmus, psychomotor delay, progressive spasticity and cerebellar signs. Here we describe three patients from one family with a novel recessively inherited mutation, 99C>G (predicted to cause an Ile>Met amino acid substitution; I33M) that causes a milder phenotype. All three had a late-onset, slowly progressive, complicated spastic paraplegia, with normal or near-normal psychomotor development, preserved walking capability through adulthood, and no nystagmus. MRI and MR spectroscopy imaging were consistent with a hypomyelinating leukoencephalopathy. The mutant protein forms gap junction plaques at cell borders similar to wild-type (WT) Cx47 in transfected cells, but fails to form functional homotypic channels in scrape-loading and dual whole-cell patch clamp assays. I33M forms overlapping gap junction plaques and functional channels with Cx43, however, I33M/Cx43 channels open only when a large voltage difference is applied to paired cells. These channels probably do not function under physiological conditions, suggesting that Cx47/Cx43 channels between astrocytes and oligodendrocytes are disrupted, similar to the loss-of-function endoplasmic reticulum-retained Cx47 mutants that cause PMLD. Thus, GJA12/GJC2 mutations can result in a milder phenotype than previously appreciated, but whether I33M retains a function of Cx47 not directly related to forming functional gap junction channels is not known.

Properties of human connexin 31, which is implicated in hereditary dermatological disease and deafness.

The connexins are a family of at least 20 homologous proteins in humans that form aqueous channels connecting the interiors of coupled cells and mediating electrical and chemical communication. Mutations in the gene for human connexin 31 (hCx31) are associated with disorders of the skin and auditory system. Alterations in functional properties of Cx31 junctions are likely to play a role in these diseases; nonetheless, little is known about the properties of the wild-type channels. Here we show that hCx31 channels, like other connexin channels, are gated by voltage and close at low pH and when exposed to long-chain alkanols. Single-channel conductance of the fully open channel is approximately 85 pS, and it is permeable to Lucifer yellow, Alexa Fluor(350), ethidium bromide, and DAPI, which have valences of -2, -1, +1, and +2, respectively. In contrast to what has been reported for mouse Cx31, hCx31 appears to form functional heterotypic channels with all four connexins tested, Cx26, Cx30, Cx32, and Cx45. These findings provide an important first step in evaluating the pathogenesis of inherited human diseases associated with mutations in the gene for Cx31.