Mycobacterium tuberculosis evades macrophage defenses by inhibiting plasma membrane repair.

Induction of macrophage necrosis is a strategy used by virulent Mycobacterium tuberculosis (Mtb) to avoid innate host defense. In contrast, attenuated Mtb causes apoptosis, which limits bacterial replication and promotes T cell cross-priming by antigen-presenting cells. Here we show that Mtb infection causes plasma membrane microdisruptions. Resealing of these lesions, a process crucial for preventing necrosis and promoting apoptosis, required translocation of lysosomal and Golgi apparatus-derived vesicles to the plasma membrane. Plasma membrane repair depended on prostaglandin E(2) (PGE(2)), which regulates synaptotagmin 7 (Syt-7), the calcium sensor involved in the lysosome-mediated repair mechanism. By inducing production of lipoxin A(4) (LXA(4)), which blocks PGE(2) biosynthesis, virulent Mtb prevented membrane repair and induced necrosis. Thus, virulent Mtb impairs macrophage plasma membrane repair to evade host defenses.

Adenomatous Polyposis Coli Protein Deletion in Efferent Olivocochlear Neurons Perturbs Afferent Synaptic Maturation and Reduces the Dynamic Range of Hearing.

Normal hearing requires proper differentiation of afferent ribbon synapses between inner hair cells (IHCs) and spiral ganglion neurons (SGNs) that carry acoustic information to the brain. Within individual IHCs, presynaptic ribbons show a size gradient with larger ribbons on the modiolar face and smaller ribbons on the pillar face. This structural gradient is associated with a gradient of spontaneous rates and threshold sensitivity, which is essential for a wide dynamic range of hearing. Despite their importance for hearing, mechanisms that direct ribbon differentiation are poorly defined. We recently identified adenomatous polyposis coli protein (APC) as a key regulator of interneuronal synapse maturation. Here, we show that APC is required for ribbon size heterogeneity and normal cochlear function. Compared with wild-type littermates, APC conditional knock-out (cKO) mice exhibit decreased auditory brainstem responses. The IHC ribbon size gradient is also perturbed. Whereas the normal-developing IHCs display ribbon size gradients before hearing onset, ribbon sizes are aberrant in APC cKOs from neonatal ages on. Reporter expression studies show that the CaMKII-Cre used to delete the floxed APC gene is present in efferent olivocochlear (OC) neurons, not IHCs or SGNs. APC loss led to increased volumes and numbers of OC inhibitory dopaminergic boutons on neonatal SGN fibers. Our findings identify APC in efferent OC neurons as essential for regulating ribbon heterogeneity, dopaminergic terminal differentiation, and cochlear sensitivity. This APC effect on auditory epithelial cell synapses resembles interneuronal and nerve-muscle synapses, thereby defining a global role for APC in synaptic maturation in diverse cell types. SIGNIFICANCE STATEMENT: This study identifies novel molecules and cellular interactions that are essential for the proper maturation of afferent ribbon synapses in sensory cells of the inner ear, and for normal hearing.

Selective inhibition of BET bromodomains.

Epigenetic proteins are intently pursued targets in ligand discovery. So far, successful efforts have been limited to chromatin modifying enzymes, or so-called epigenetic 'writers' and 'erasers'. Potent inhibitors of histone binding modules have not yet been described. Here we report a cell-permeable small molecule (JQ1) that binds competitively to acetyl-lysine recognition motifs, or bromodomains. High potency and specificity towards a subset of human bromodomains is explained by co-crystal structures with bromodomain and extra-terminal (BET) family member BRD4, revealing excellent shape complementarity with the acetyl-lysine binding cavity. Recurrent translocation of BRD4 is observed in a genetically-defined, incurable subtype of human squamous carcinoma. Competitive binding by JQ1 displaces the BRD4 fusion oncoprotein from chromatin, prompting squamous differentiation and specific antiproliferative effects in BRD4-dependent cell lines and patient-derived xenograft models. These data establish proof-of-concept for targeting protein-protein interactions of epigenetic 'readers', and provide a versatile chemical scaffold for the development of chemical probes more broadly throughout the bromodomain family.

Dopaminergic and cholinergic innervation in the mouse cochlea after noise-induced or age-related synaptopathy.

Cochlear synaptopathy, the loss of or damage to connections between auditory-nerve fibers (ANFs) and inner hair cells (IHCs), is a prominent pathology in noise-induced and age-related hearing loss. Here, we investigated if degeneration of the olivocochlear (OC) efferent innervation is also a major aspect of the synaptopathic ear, by quantifying the volume and spatial organization of its cholinergic and dopaminergic components, using antibodies to vesicular acetylcholine transporter (VAT) and tyrosine hydroxylase (TH), respectively. CBA/CaJ male mice were examined 1 day to 8 months after a synaptopathic noise exposure, and compared to unexposed age-matched controls and unexposed aged mice at 24-28 months. In normal ears, cholinergic lateral (L)OC terminals were denser in the apical half of the cochlea and on the modiolar side of the inner hair cells (IHCs), where ANFs of low-spontaneous rate are typically found, while dopaminergic terminals were more common in the basal third of the cochlea and, re the IHC axes, were offset towards the habenula with respect to cholinergic terminals. The noise had only small and transient effects on the density of LOC innervation, its spatial organization around the IHC axes, or the extent to which TH and VAT signal were colocalized. The synaptopathic noise also had relatively small and transient effects on cholinergic innervation density in the outer hair cell (OHC) area, which normally peaks in the 16 kHz region and falls monotonically towards higher and lower frequencies. In contrast, in the aged ears, there was massive degeneration of OHC efferents, especially in the apical half of the cochlea, where there was also significant loss of OHCs. In the IHC area, there was significant loss of cholinergic terminals in both apical and basal regions and of dopaminergic innervation in the basal half. Furthermore, the cholinergic terminals in the aged ears spread from their normal clustering near the IHC basolateral pole, where the ANF synapses are found, to positions up and down the IHC somata and regions of the neuropil closer to the habenula. This apparent migration was most striking in the apex, where the hair cell pathology was greatest, and may be a harbinger of impending hair cell death.

Cochlear Synaptic Degeneration and Regeneration After Noise: Effects of Age and Neuronal Subgroup.

In CBA/CaJ mice, confocal analysis has shown that acoustic overexposure can immediately destroy synapses between auditory-nerve fibers (ANFs) and their peripheral targets, the inner hair cells (IHCs), and that years later, a corresponding number of ANF cell bodies degenerate. In guinea pig, post-exposure disappearance of pre-synaptic ribbons can be equally dramatic, however, post-exposure recovery to near-baseline counts has been reported. Since confocal counts are confounded by thresholding issues, the fall and rise of synaptic ribbon counts could represent "regeneration," i.e., terminal retraction, re-extension and synaptogenesis, or "recovery," i.e., down- and subsequent up-regulation of synaptic markers. To clarify, we counted pre-synaptic ribbons, assessed their juxtaposition with post-synaptic receptors, measured the extension of ANF terminals, and quantified the spatial organization and size gradients of these synaptic elements around the hair cell. Present results in guinea pigs exposed as adults (14 months), along with prior results in juveniles (1 month), suggest there is post-exposure neural regeneration in the guinea pig, but not the CBA/CaJ mouse, and that this regenerative capacity extends into adulthood. The results also show, for the first time, that the acute synaptic loss is concentrated on the modiolar side of IHCs, consistent with a selective loss of the high-threshold ANFs with low spontaneous rates. The morphological similarities between the post-exposure neurite extension and synaptogenesis, seen spontaneously in the guinea pig, and in CBA/CaJ only with forced overexpression of neurotrophins, suggest that the key difference may be in the degree of sustained or injury-induced expression of these signaling molecules in the cochlea.

Synaptic migration and reorganization after noise exposure suggests regeneration in a mature mammalian cochlea.

Overexposure to intense noise can destroy the synapses between auditory nerve fibers and their hair cell targets without destroying the hair cells themselves. In adult mice, this synaptopathy is immediate and largely irreversible, whereas, in guinea pigs, counts of immunostained synaptic puncta can recover with increasing post-exposure survival. Here, we asked whether this recovery simply reflects changes in synaptic immunostaining, or whether there is actual retraction and extension of neurites and/or synaptogenesis. Analysis of the numbers, sizes and spatial distribution of pre- and post-synaptic markers on cochlear inner hair cells, in guinea pigs surviving from 1 day to 6 months after a synaptopathic exposure, shows dramatic synaptic re-organization during the recovery period in which synapse counts recover from 16 to 91% of normal in the most affected regions. Synaptic puncta move all over the hair cell membrane during recovery, translocating far from their normal positions at the basolateral pole, and auditory-nerve terminals extend towards the hair cell's apical end to re-establish contact with them. These observations provide stronger evidence for spontaneous neural regeneration in a mature mammalian cochlea than can be inferred from synaptic counts alone.

Protection from noise-induced cochlear synaptopathy by virally mediated overexpression of NT3.

Noise exposures causing only transient threshold shifts can destroy auditory-nerve synapses without damaging hair cells. Here, we asked whether virally mediated neurotrophin3 (NT3) overexpression can repair this damage. CBA/CaJ mice at 6 wks were injected unilaterally with adeno-associated virus (AAV) containing either NT3 or GFP genes, via the posterior semicircular canal, 3 wks prior to, or 5 hrs after, noise exposure. Controls included exposed animals receiving vehicle only, and unexposed animals receiving virus. Thresholds were measured 2 wks post-exposure, just before cochleas were harvested for histological analysis. In separate virus-injected animals, unexposed cochleas were extracted for qRT-PCR. The GFP reporter showed that inner hair cells (IHCs) were transfected throughout the cochlea, and outer hair cells mainly in the apex. qRT-PCR showed 4- to 10-fold overexpression of NT3 from 1-21 days post-injection, and 1.7-fold overexpression at 40 days. AAV-NT3 delivered prior to noise exposure produced a dose-dependent reduction of synaptopathy, with nearly complete rescue at some cochlear locations. In unexposed ears, NT3 overexpression did not affect thresholds, however GFP overexpression caused IHC loss. In exposed ears, NT3 overexpression increased permanent threshold shifts. Thus, although NT3 overexpression can minimize noise-induced synaptic damage, the forced overexpression may be harmful to hair cells themselves during cochlear overstimulation.