Corrigendum: Virally mediated connexin 26 expression in postnatal scala media significantly and transiently preserves hearing in connexin 30 null mice.

[This corrects the article DOI: 10.3389/fcell.2022.900416.].

Virally Mediated Connexin 26 Expression in Postnatal Scala Media Significantly and Transiently Preserves Hearing in Connexin 30 Null Mice.

Non-sensory cells in the sensory epithelium of the cochlea are connected extensively by gap junctions. Functionally null mutations in GJB6 (encoding Cx30) cause hearing loss in humans. In this study, we injected AAV1-CB7-Gjb2 into the scala media between P0-2 in the cochlea of Gjb6 -/- mice. The injection increased Cx26 expression and significantly preserved auditory functions. However, the hearing preservation gradually declined and essentially disappeared 3 months after the injections. In contrast, the morphological preservation was still significant at 3 months post-injection. We found that the expression of Cx26, at both the mRNA and protein levels, showed substantial decreases during the 3-month period. Curiously, treatments by injecting AAV1-CB7-Gjb6 with the identical approach failed to yield any hearing preservation. Our results demonstrated the first successful cochlear gene therapy treatment in mouse models by virally expressing a companion gene of Gjb6.

Virally mediated Kcnq1 gene replacement therapy in the immature scala media restores hearing in a mouse model of human Jervell and Lange-Nielsen deafness syndrome.

Mutations in the potassium channel subunit KCNQ1 cause the human severe congenital deafness Jervell and Lange-Nielsen (JLN) syndrome. We applied a gene therapy approach in a mouse model of JLN syndrome (Kcnq1(-/-) mice) to prevent the development of deafness in the adult stage. A modified adeno-associated virus construct carrying a Kcnq1 expression cassette was injected postnatally (P0-P2) into the endolymph, which resulted in Kcnq1 expression in most cochlear marginal cells where native Kcnq1 is exclusively expressed. We also found that extensive ectopic virally mediated Kcnq1 transgene expression did not affect normal cochlear functions. Examination of cochlear morphology showed that the collapse of the Reissner's membrane and degeneration of hair cells (HCs) and cells in the spiral ganglia were corrected in Kcnq1(-/-) mice. Electrophysiological tests showed normal endocochlear potential in treated ears. In addition, auditory brainstem responses showed significant hearing preservation in the injected ears, ranging from 20 dB improvement to complete correction of the deafness phenotype. Our results demonstrate the first successful gene therapy treatment for gene defects specifically affecting the function of the stria vascularis, which is a major site affected by genetic mutations in inherited hearing loss.

Early postnatal virus inoculation into the scala media achieved extensive expression of exogenous green fluorescent protein in the inner ear and preserved auditory brainstem response thresholds.

BACKGROUND: Gene transfer into the inner ear is a promising approach for treating sensorineural hearing loss. The special electrochemical environment of the scala media raises a formidable challenge for effective gene delivery at the same time as keeping normal cochlear function intact. The present study aimed to define a suitable strategy for preserving hearing after viral inoculation directly into the scala media performed at various postnatal developmental stages. METHODS: We assessed transgene expression of green fluorescent protein (GFP) mediated by various types of adeno-associated virus (AAV) and lentivirus (LV) in the mouse cochlea. Auditory brainstem responses were measured 30 days after inoculation to assess effects on hearing. RESULTS: Patterns of GFP expression confirmed extensive exogenous gene expression in various types of cells lining the endolymphatic space. The use of different viral vectors and promoters resulted in specific cellular GFP expression patterns. AAV2/1 with cytomegalovirus promoter apparently gave the best results for GFP expression in the supporting cells. Histological examination showed normal cochlear morphology and no hair cell loss after either AAV or LV injections. We found that hearing thresholds were not significantly changed when the injections were performed in mice younger than postnatal day 5, regardless of the type of virus tested. CONCLUSIONS: Viral inoculation and expression in the inner ear for the restoration of hearing must not damage cochlear function. Using normal hearing mice as a model, we have achieved this necessary step, which is required for the treatment of many types of congenital deafness that require early intervention.

Early developmental expression of connexin26 in the cochlea contributes to its dominate functional role in the cochlear gap junctions.

Mutations in Gjb2 and Gjb6 genes, coding for connexin26 (Cx26) and Cx30 proteins, respectively, are linked to about half of all cases of human autosomal non-syndromic prelingual deafness. Molecular mechanisms of the hearing impairments, however, are unclear. Most cochlear gap junctions (GJs) are co-assembled from Cx26 and Cx30 and deletion of either one of them causes deafness. Our previous studies have shown that normal hearing is possible in the absence of the Cx30 gene when Cx26 is over-expressed. To further test unique functional requirements for various types of connexins in the hearing, we investigated whether the hearing in the conditional Cx26 (cCx26) null mice could be rescued by genetically over-expressing Cx30. Multiple lines of control and experimental mouse models were used. Auditory brainstem response (ABR) measurements showed normal hearing in targeted gene deletion mice when the deleted Cx26 or Cx30 was transgenically expressed from integrated bacterial artificial chromosome (BAC), demonstrating the effectiveness of the BAC rescue approach. In contrast, severe hearing loss was found in cCx26 null mice in which Cx30 was over-expressed. Morphology observations were consistent with the ABR data. Cochleae of cCx26 null mice with and without the transgenic over-expression of Cx30 both showed the typical immature feature of postnatal cochlear development-the closed tunnel of Corti. Immunolabeling data and Western blot quantification indicated that the Cx26 protein expression preceded that of Cx30 during the early postnatal period in the cochlea. Null expression of Cx26 may therefore uniquely result in a transient period when a total elimination of GJs in functionally-important regions of the developing cochlea is possible. We conclude that Cx26 plays an essential role in the development of the auditory sensory epithelium and its unique developmental functions required for normal hearing is not replaceable by Cx30.

Targeted connexin26 ablation arrests postnatal development of the organ of Corti.

Mutations in the gene coding for connexin26 (Cx26) is the most common cause of human nonsyndromic hereditary deafness. To investigate deafness mechanisms underlying Cx26 null mutations, we generated three independent lines of conditional Cx26 null mice. Cell differentiation and gross cochlear morphology at birth seemed normal. However, postnatal development of the organ of Corti was stalled as the tunnel of Corti and the Nuel's space were never opened. Cell degeneration was first observed in the Claudius cells around P8. Outer hair cell loss was initially observed around P13 at middle turn when inner hair cells were still intact. Massive cell death occurred in the middle turn thereafter and gradually spread to the basal turn, resulting in secondary degeneration of spiral ganglion neurons in the corresponding cochlear locations. These results demonstrated that Cx26 plays essential roles in postnatal maturation and homoeostasis of the organ of Corti before the onset of hearing.

Multidrug resistance-associated protein-overexpressing teniposide-resistant human lymphomas undergo apoptosis by a tubulin-binding agent.

Several DNA- and microtubule-binding agents are used to manage hematologic malignancies in the clinic. However, drug resistance has been a challenge, perhaps due to a few surviving cancer stem cells. Toxicity is another major impediment to successful chemotherapy, leading to an impoverished quality of life. Here, we show that a semisynthetic nontoxic tubulin-binding agent, 9-bromonoscapine (EM011), effectively inhibits growth and regresses multidrug resistance-associated protein (MRP)-overexpressing teniposide-resistant T-cell lymphoma xenografts and prolongs longevity. As expected, teniposide treatment failed to regress teniposide-resistant xenografts, rather, treated mice suffered tremendous body weight loss. Mechanistically, EM011 displays significant antiproliferative activity, perturbs cell cycle progression by arresting mitosis, and induces apoptosis in teniposide-resistant lymphoblastoid T cells both in vitro and in vivo. EM011-induced apoptosis has a mitochondrially-mediated component, which was attenuated by pretreatment with cyclosporin A. We also observed alterations of apoptosis-regulatory molecules such as inactivation of Bcl2, translocation of BAX to the mitochondrial membrane, cytochrome c release, and activation of downstream apoptotic signaling. EM011 caused DNA degradation as evident by terminal deoxynucleotidyl transferase-mediated dUTP-biotin end labeling staining of the increased concentration of 3'-DNA ends. Furthermore, the apoptotic induction was caspase dependent as shown by cleavage of the caspase substrate, poly(ADP)ribose polymerase. In addition, EM011 treatment caused a suppression of natural survival pathways such as the phosphatidylinositol-3'-kinase/Akt signaling. These preclinical findings suggest that EM011 is an excellent candidate for clinical evaluation.

Gap junction mediated intercellular metabolite transfer in the cochlea is compromised in connexin30 null mice.

Connexin26 (Cx26) and connexin30 (Cx30) are two major protein subunits that co-assemble to form gap junctions (GJs) in the cochlea. Mutations in either one of them are the major cause of non-syndromic prelingual deafness in humans. Because the mechanisms of cochlear pathogenesis caused by Cx mutations are unclear, we investigated effects of Cx30 null mutation on GJ-mediated ionic and metabolic coupling in the cochlea of mice. A novel flattened cochlear preparation was used to directly assess intercellular coupling in the sensory epithelium of the cochlea. Double-electrode patch clamp recordings revealed that the absence of Cx30 did not significantly change GJ conductance among the cochlear supporting cells. The preserved electrical coupling is consistent with immunolabeling data showing extensive Cx26 GJs in the cochlea of the mutant mice. In contrast, dye diffusion assays showed that the rate and extent of intercellular transfer of multiple fluorescent dyes (including a non-metabolizable D-glucose analogue, 2-NBDG) among cochlear supporting cells were severely reduced in Cx30 null mice. Since the sensory epithelium in the cochlea is an avascular organ, GJ-facilitated intercellular transfer of nutrient and signaling molecules may play essential roles in cellular homeostasis. To test this possibility, NBDG was used as a tracer to study the contribution of GJs in transporting glucose into the cochlear sensory epithelium when delivered systemically. NBDG uptake in cochlear supporting cells was significantly reduced in Cx30 null mice. The decrease was also observed with GJ blockers or glucose competition, supporting the specificity of our tests. These data indicate that GJs facilitate efficient uptake of glucose in the supporting cells. This study provides the first direct experimental evidence showing that the transfer of metabolically-important molecules in cochlear supporting cells is dependent on the normal function of GJs, thereby suggesting a novel pathogenesis process in the cochlea for Cx-mutation-linked deafness.

Treatment of hormone-refractory breast cancer: apoptosis and regression of human tumors implanted in mice.

Following surgery, the hormone dependence of breast tumors is exploited for therapy using antagonists such as tamoxifen, although occasional hormone-resistant clones do appear. Another chemotherapeutic strategy uses microtubule inhibitors such as taxanes. Unfortunately, these agents elicit toxicities such as leukocytopenia, diarrhea, alopecia, and peripheral neuropathies and are also associated with the emergence of drug resistance. We have previously described a tubulin-binding, natural compound, noscapine, that was nontoxic and triggered apoptosis in many cancer types albeit at 10 mumol/L or higher concentrations depending on the cell type. We now show that a synthetic analogue of noscapine, 9-bromonoscapine, is approximately 10-fold to 15-fold more potent than noscapine in inhibiting cell proliferation and induces apoptosis following G2-M arrest in hormone-insensitive human breast cancers (MDA-MB-231). Furthermore, a clear loss of mitochondrial membrane potential, release of cytochrome c, activation of the terminal caspase-3, and the cleavage of its substrates such as poly(ADP-ribose) polymerase, suggest an intrinsic apoptotic mechanism. Taken together, these data point to a mitochondrially mediated apoptosis of hormone-insensitive breast cancer cells. Human tumor xenografts in nude mice showed significant tumor volume reduction and a surprising increase in longevity without signs of obvious toxicity. Thus, our data provide compelling evidence that 9-bromonoscapine can be useful for the therapy of hormone-refractory breast cancer.

Rational design of the microtubule-targeting anti-breast cancer drug EM015.

We studied in silico docking of noscapine onto tubulin, combined with calculations of surface charge, pi-pi, van der Waals, and hydrogen bonding interactions, to rationally design a new compound, EM015. This tubulin-binding semisynthetic compound is a selective and potent anti-breast cancer agent and displays a 20-fold lower IC(50) against many tumor cells compared with our founding compound, (S)-6,7-dimethoxy-3-((R)-4-methoxy-6-methyl-5,6,7,8-tetrahydro[1,3]-dioxolo-[4,5-g]isoquinolin-5-yl)isobenzo-furan-1(3H)-one (noscapine). Furthermore, EM015 is also effective against a variety of drug-resistant cells. Surprisingly, the cell cycle profile of nontumorigenic normal cells is not affected. Many antimicrotubule cancer drugs in clinic today, particularly taxanes and Vincas, face challenges including frequent visits to the hospital for prolonged i.v. infusions, toxicities, and tumor recurrences due to drug resistance. EM015, on the other hand, is orally available, regresses breast tumor xenografts in nude mice models, and increases longevity. Furthermore, we have failed to observe any detectable toxicity in tissues, such as liver, kidney, spleen, lung, heart, and brain, as well as neurons, which are common targets of antimicrotubule drug therapy. Thus, EM015 has a great promise in the clinic.

Drug-resistant T-lymphoid tumors undergo apoptosis selectively in response to an antimicrotubule agent, EM011.

We have shown previously that EM011, a synthetic compound, binds tubulin with a higher affinity than the founding compound, noscapine, without changing total microtubule polymer mass. Now we show that EM011 is potently effective against vinblastine-resistant human lymphoblastoid line CEM/VLB100 and its parental vinblastine-sensitive line CEM. The cytotoxicity is mediated by cell cycle arrest at G2/M phase and subsequent apoptosis, as indicated by altered plasma membrane asymmetry, loss of mitochondrial transmembrane potential, activation of caspase-3, and increased DNA fragmentation. Furthermore, oral EM011 treatment of nude mice bearing human lymphoma xenografts results in pronounced tumor regression by triggering apoptosis and significantly lengthens the survival time of mice. EM011 treatment does not have obvious side effects in tissues with frequently dividing cells, such as the spleen and duodenum. In addition, EM011 does not show any toxicity in the liver, lung, heart, brain, and sciatic nerve. More importantly, EM011 does not affect hematopoiesis as determined by complete blood count profiles. These findings suggest that EM011 may be a safe and effective chemotherapeutic agent for oral treatment of drug-resistant human lymphomas.