Benjamin K. August

The kinocilium of auditory hair cells and evidence for its morphogenetic role during the regeneration of stereocilia and cuticular plates

SummaryAuditory hair cells that survive mechanical injury in culture begin their recovery by reforming the kinocilium. This study is based on cultures of the organ of Corti of newborn mice and two control animals. The axonemal patterns were examined in 165 kinocilia in cross-section. In the immature and regenerating kinocilium, one of the normally peripheral doublets is frequently located inward, forming the modified 8 + 1 (double) form; the distribution of the remainingmicrotubules is irregular. As the cell matures, the 9 + 0 form predominates. Overall, 34–61% of auditory kinocilia consist of 9 + 0 microtubules. The 9+2 (single) form, previously thought to characterize the organelle, occurs only in about 3–14%, whereas the remaining population comprises the modified 8 + 1 (double) form. Normally, the kinocilium lasts only about 10 postnatal days; however, post-traumatic hair cells reform their kinocilia regardless of age. Concomitant with the regrowth of the kinocilium, the basal body and its cilium take a central location in the cuticular plate, stereocilia regrow, and the cytoplasmic area adjacent to the basal body displays pericentriolar fibrous densities, growth vesicles, and microtubules, all surrounded by actin filaments. Pericentriolar bodies nucleate microtubules. Involvement of microtubules is seen in the alignment of actin filaments and in the formation of the filamentous matrix of the cuticular plate. We propose that reformation of the kinocilium in recovering post-traumatic hair cells indicates the possible role of its basal body in the morphogenesis and differentiation of cuticular plates and stereocilia.

Influence of neurotrophins on the synaptogenesis of inner hair cells in the deaf Bronx waltzer (bv) mouse organ of Corti in culture.

The Bronx waltzer (bv) deaf mouse is characterized by massive degeneration of the primary auditory receptors, the inner hair cells, which occurs during the time of expected afferent synaptogenesis. The process is associated with degeneration and protracted division of the normally postmitotic afferent spiral ganglion neurons. To investigate the potential role of neurotrophins in the afferent synaptogenesis of inner hair cells, we exposed bv newborn cochleas in organotypic culture to brain‐derived neurotrophic factor (BDNF), neurotrophin‐3 (NT‐3) and nerve growth factor (NGF), and also to gamma aminobutyric acid (GABA), for up to 8 days. The study was done using light and electron microscopy. Only about 20% of the inner hair cells survived in culture, regardless of the treatment, similar to the number in the intact mutant in our colony. Depending on the exogenous treatment, this population consisted of either innervated ultrastructurally normal cells or denervated dedifferentiated cells wrapped—in lieu of nerve endings—by the supporting inner phalangeal and border cells. In the control and GABA cultures, inner hair cells were mostly denervated. BDNF and NT‐3 alone or combined increased synaptogenesis and hair cell survival only during the first 3 days (by about 10%); however, the cells became denervated by 8 postnatal (PN). Only NGF induced stable innervation and differentiation of neurosensory relationships, including supernumerary innervation characteristic of the intact bv. Denervation among the remaining 20% of inner hair cells induced a reactive wrapping by inner phalangeal and border cells which evidently extended inner hair cell survival. Immunocytochemical studies of these reactive supporting cells were done in the intact (8 PN) mutant cochlea. The supporting cells that provide sustenance to the denervated inner hair cells displayed strong BDNF (and possibly NT‐3) immunoreactivity. Subsequently, we revealed the presence of all three neurotrophins in the inner hair cell region of the developing (1–8 PN) cochlea of the normal ICR mouse. The inner hair cells expressed all three neurotrophins; BDNF prevailed in the inner phalangeal cells, NT‐3 in the pillar cells and inner phalangeal cells, and NGF in the pillar cells. In conclusion: initially, the 80% loss of inner hair cells is apparently caused by their failed afferent synaptogenesis. Exogenous neurotrophins influence synaptogenesis in the bv in culture, but NGF alone is successful in promoting stable neurosensory relationships. The presence of neurotrophins in supporting cells in the normal and degenerating cochlea indicates their role in the sustenance of inner hair cells.

COMPOUND SYNAPSES WITHIN THE GABAERGIC INNERVATION OF THE AUDITORY INNER HAIR CELLS IN THE ADOLESCENT MOUSE

Ultrastructural investigation of the γ‐aminobutyric acid (GABA) component of the inner spiral bundle in adolescent mice revealed a pathway of glutamic acid decarboxylase (GAD)‐positive and ‐negative fibers and vesiculated endings that contact inner hair cells and their afferents through a complex of axosomatic and axodendritic synapses. Ultrastructural details were investigated by using conventional electron microscopy. Several synaptic arrangements were observed: Main axosomatic synapses form between vesiculated endings and individual or adjoining inner hair cells (interreceptor synapses). Spinous synapses form on long, spinelike processes that protrude from inner hair cells to reach distant efferent endings. The efferent endings associate with inner hair cells and their synaptic afferents through compound synapses—serial, “converging,” and triadic—otherwise characteristic of sensory relay nuclei. Serial synapses form by the sequential presynaptic alignment of the efferent→receptor→afferent components. Converging synapses result from the simultaneous apposition of a receptor ribbon synapse and a presynaptic efferent terminal on a recipient afferent dendrite. Triadic synapses comprise a vesiculated efferent ending in contact with an inner hair cell and with its synaptic afferent. Additionally, efferent endings may form simple axodendritic and axoaxonal synapses with GAD‐negative vesiculated endings. The combination of different synaptic arrangements leads to short chains of compound synapses. It is assumed that these synaptic patterns seen in the adolescent mouse represent adult synaptology. The patterns of synaptic connectivity suggest an integrative role for the GABA/GAD lateral efferent system, and imply its involvement in the pre‐ and postsynaptic modulation of auditory signals. J. Comp. Neurol. 377:423–442, 1997.

Presynaptic fibres of spiral neurons and reciprocal synapses in the organ of Corti in culture

SummaryIsolated segments of the newborn mouse organ of Corti were explanted together with the spiral ganglion components. Within the innervation provided by the spiral neurons, we observed presynaptic vesiculated nerve endings that form reciprocal ribbon-afferent/efferent synapses with inner hair cells. These intracochlear presynaptic fibres are characteristically located between adjoining inner hair cells, on the modiolar side, low and close to the supporting cells. The presynaptic fibres display different modes of synaptic connectivity, forming repetitive reciprocal synapses on single inner hair cells or on adjoining hair cells, or connecting adjoining inner hair cells through simultaneous efferent synapses. Many presynaptic fibres exhibit a distinctive ultrastructure: defined clusters of synaptic vesicles, dense core vesicles, coated vesicles, and mitochondria. These organelles occur focally at the synaptic sites; beyond the efferent synaptic specializations, the endings appear quite nondescript and afferent-like.We believe that the reciprocal synapses, although observed in cultures of the organ of Corti, represent real intracochlear synaptic arrangements providing a feedback mechanism between the primary sensory receptors and a special class of spiral ganglion cells that have yet to be recognized in the organin situ.

Caveolin-3 Overexpression Attenuates Cardiac Hypertrophy via Inhibition of T-type Ca2+ Current Modulated by Protein Kinase Cα in Cardiomyocytes.

Background: Ventricular remodeling altered caveolin-3 expression, and Ca2+ signaling is associated with cardiac hypertrophy. Results: Cardiomyocyte-specific caveolin-3 overexpression prevented cardiac hypertrophy by inhibiting the T-type Ca2+ current and hyperactivation of calcineurin-dependent nuclear factor of activated T-cell signaling. Conclusion: Caveolin-3 expression is essential for protective Ca2+ signaling in pathological cardiac hypertrophy. Significance: Caveolin-3 overexpression in heart may be used as a therapeutic strategy for treatment of many cardiovascular diseases. Pathological cardiac hypertrophy is characterized by subcellular remodeling of the ventricular myocyte with a reduction in the scaffolding protein caveolin-3 (Cav-3), altered Ca2+ cycling, increased protein kinase C expression, and hyperactivation of calcineurin/nuclear factor of activated T cell (NFAT) signaling. However, the precise role of Cav-3 in the regulation of local Ca2+ signaling in pathological cardiac hypertrophy is unclear. We used cardiac-specific Cav-3-overexpressing mice and in vivo and in vitro cardiac hypertrophy models to determine the essential requirement for Cav-3 expression in protection against pharmacologically and pressure overload-induced cardiac hypertrophy. Transverse aortic constriction and angiotensin-II (Ang-II) infusion in wild type (WT) mice resulted in cardiac hypertrophy characterized by significant reduction in fractional shortening, ejection fraction, and a reduced expression of Cav-3. In addition, association of PKCα and angiotensin-II receptor, type 1, with Cav-3 was disrupted in the hypertrophic ventricular myocytes. Whole cell patch clamp analysis demonstrated increased expression of T-type Ca2+ current (ICa, T) in hypertrophic ventricular myocytes. In contrast, the Cav-3-overexpressing mice demonstrated protection from transverse aortic constriction or Ang-II-induced pathological hypertrophy with inhibition of ICa, T and intact Cav-3-associated macromolecular signaling complexes. siRNA-mediated knockdown of Cav-3 in the neonatal cardiomyocytes resulted in enhanced Ang-II stimulation of ICa, T mediated by PKCα, which caused nuclear translocation of NFAT. Overexpression of Cav-3 in neonatal myocytes prevented a PKCα-mediated increase in ICa, T and nuclear translocation of NFAT. In conclusion, we show that stable Cav-3 expression is essential for protecting the signaling mechanisms in pharmacologically and pressure overload-induced cardiac hypertrophy.

Abortive synaptogenesis as a factor in the inner hair cell degeneration in the Bronx Waltzer (bv) mutant mouse

The bronx waltzer (vb) mutation in the mouse results in the degeneration of most but not all of the primary auditory receptors, the inner hair cells, and their afferent neurons. We analyzed the ultrastructure of 94 inner hair cells in the intact postnatal mutant mouse and in neonatal cochleas in culture to understand the pathogenesis of hair cell death and to detect factors that may prevent it. The vb spiral neurons of the bronx waltzer display two distinctive features: some of them continue to divide mitotically for at least seven postnatal days, and the type I radial fibers that innervate inner hair cells display a deficiency in immunoexpression of GAD. The growing endings of spiral neurons converge around the inner hair cells or, in their absence, invade the outer hair cell region. Their profuse sprouting among inner spiral sulcus cells contributes to the characteristic ultrastructural picture of the bv cochlea. During the first three days after birth, 40% of the inner hair cells appear normal and innervated, 40% are mostly denervated and degenerating, and 20% are immature, with minimal or no neuronal appositions. However, in mutants 6 days and older only a few inner hair cells survive, and these show either normal or superfluous afferent innervation and axosomatic GABAergic efferent innervation. Degeneration of inner hair cells begins with a distention of the nuclear envelope and the ribosomal endoplasmic reticulum. The outer nuclear membrane eventually breaks, and exudate fills the cell interior. The cellular edema leads to cell death. We propose that success or failure in synaptic acquisition is a decisive factor in the survival or decline of the mutant inner hair cells. We also suggest that the developmental delay in maturation of the spiral ganglion neurons (type I) and the failure in their synaptogenesis may be caused by an impairment in neurotrophin (NT3/BDNF) synthesis by their mutant receptor cells.

A role for tumor protein TPD52 phosphorylation in endo-membrane trafficking during cytokinesis.

Tumor protein D52 is expressed at high levels in exocrine cells containing large secretory granules where it regulates Ca(2+)-dependent protein secretion; however, D52 expression is also highly induced in multiple cancers. The present study investigated a role for the Ca(2+)-dependent phosphorylation of D52 at the single major phospho-acceptor site serine 136 on cell division. Ectopic expression of wild type D52 (D52wt) and the phosphomutants serine 136/alanine (S136A) or serine 136/glutamate (S136/E) resulted in significant multinucleation of cells. D52wt and S136/E each resulted in a greater than 2-fold increase in multinucleated cells compared to plasmid-transfected controls whereas the S136/A phospho-null mutant caused a 9-fold increase in multinucleation at 48h post-transfection. Electron microscopy revealed D52 expression induced a marked accumulation of vesicles along the mid-line between nuclei where the final stages of cell abscission normally occurs. Supporting this, D52wt strongly colocalized on vesicular structures containing the endosomal regulatory protein vesicle associated membrane protein 8 (VAMP 8) and this colocalization significantly increased with elevations in cellular Ca(2+). As VAMP 8 is known to be necessary for the endo-membrane fusion reactions that mediate the final stages of cytokinesis, these data indicate that D52 expression and phosphorylation at serine 136 play an important role in supporting the Ca(2+)-dependent membrane trafficking events necessary for cytokinesis in rapidly proliferating cancer cells.

Early to Late Endosome Trafficking Controls Secretion and Zymogen Activation in Rodent and Human Pancreatic Acinar Cells.

Background & Aims Pancreatic acinar cells have an expanded apical endosomal system, the physiologic and pathophysiologic significance of which is still emerging. Phosphatidylinositol-3,5-bisphosphate [PI(3,5)P2] is an essential phospholipid generated by phosphatidylinositol 3-phosphate 5-kinase (PIKfyve), which phosphorylates phosphatidylinositol-3-phosphate (PI3P). PI(3,5)P2 is necessary for maturation of early endosomes (EE) to late endosomes (LE). Inhibition of EE to LE trafficking enhances anterograde endosomal trafficking and secretion at the plasma membrane by default through a recycling endosome (RE) intermediate. We assessed the effects of modulating PIKfyve activity on apical trafficking and pancreatitis responses in pancreatic acinar cells. Methods Inhibition of EE to LE trafficking was achieved using pharmacologic inhibitors of PIKfyve, expression of dominant negative PIKfyve K1877E, or constitutively active Rab5-GTP Q79L. Anterograde endosomal trafficking was manipulated by expression of constitutively active and dominant negative Rab11a mutants. The effects of these agents on secretion, endolysosomal exocytosis of lysosome associated membrane protein (LAMP1), and trypsinogen activation in response to supramaximal cholecystokinin (CCK-8), bile acids, and cigarette toxin was determined. Results PIKfyve inhibition increased basal and stimulated secretion. Adenoviral overexpression of PIKfyve decreased secretion leading to cellular death. Expression of Rab5-GTP Q79L or Rab11a-GTP Q70L enhanced secretion. Conversely, dominant-negative Rab11a-GDP S25N reduced secretion. High-dose CCK inhibited endolysosomal exocytosis that was reversed by PIKfyve inhibition. PIKfyve inhibition blocked intracellular trypsin accumulation and cellular damage responses to supramaximal CCK-8, tobacco toxin, and bile salts in both rodent and human acini. Conclusions These data demonstrate that EE-LE trafficking acutely controls acinar secretion and the intracellular activation of zymogens, leading to the pathogenicity of acute pancreatitis.

Apoptosis of inner hair cells caused by laser ablation of their spiral ganglion neurons in cultures of the mouse organ of Corti.

Laser beam ablation of spiral ganglion neurons was performed in seven organotypic cultures of the newborn mouse cochlea between 5 and 8 days in vitro, with a recovery period of from 18 hours to 3 days. Direct somatic injury (laser or mechanical) inflicted on hair cells does not necessarily cause their death; many of them survive, repair damage and re-establish their neurosensory connections. By contrast, laser irradiation and ablation of their afferent spiral ganglion neurons causes a most spectacular degeneration of sensory cells within 18–48 hours after the insult. Ultrastructurally, the degenerated hair cells—characteristically the inner hair cells—display “dark-cell vacuolar degeneration” that combines the signs of apoptotic death (the peripheral condensation of nuclear chromatin and nuclear pyknosis) with signs of cell edema, vacuolization and necrosis. The ultimate condensation of the cytoplasm gives the dead cells a jet black appearance. The irradiated spiral ganglion neurons die displaying similar pathological characteristics. The extent and locus of inner hair cell degeneration correspond to that of ablated spiral ganglion neurons: ultimately the ablation of one neuron causes degeneration of a single inner hair cell within the closest radial segment of the afferent innervation. The elimination of spiral ganglion neurons by mechanical means does not affect hair cell survival.It is inferred that the laser pulse acts as a stimulus depolarizing the neuronal membrane of the spiral ganglion neurons and their radial fibers and causing the excitotoxic death of their synaptic sensory cells through excessive stimulation of the glutamatergic receptors. Reciprocal pre-and postsynaptic synapses between the afferent dendrites and inner hair cells in culture could possibly serve as entryways of the stimulus. The pathogenesis of this apparent transsynaptically-induced apoptotic death of inner hair cells will be further examined in culture.

Terminal dendritic sprouting and reactive synaptogenesis in the postnatal organ of Corti in culture

Synaptogenesis in the organ of Corti between the primary receptors, the inner hair cells, and the peripheral processes of their afferent spiral ganglion neurons in the mouse lasts for 5 days postnatally (Sobkowicz et al. [1986] J. Neurocytol. 15:693–714). The transplantation of the organ into culture at the fifth postnatal day induces a reactive sprouting of dendritic terminals and an extensive formation of new ribbon synapses within 24 hours. This reactive synaptogenesis differs strikingly from the primary synaptogenesis and has been seen thus far only in the inner hair cells. The synaptically engaged neuronal endings sprout a multitude of filopodia that intussuscept the inner hair cells. The filopodial tips contain a heavy electron‐dense matter that appears to attract the synaptic ribbons, which form new synaptic contacts with the growing processes. The intensity of the filopodial growth and synaptogenesis subsides in about 3 days; the filopodia undergo resorption, leaving behind fibrous cytoplasmic plaques mostly stored in the supranuclear part of the hair cells. However, occasional filopodial growth and formation of new synaptic connections continued. The data demonstrate that any disruption or disturbance of the initial synaptic contacts between the inner hair cells and their afferent neurons caused by transplantation results in prompt synaptic reacquisition. Furthermore, we suggest that the transitory phase of terminal sprouting and multiribbon synapse formation manifests a trophic dependence that develops postnatally between the synaptic cells. J. Comp. Neurol. 397:213–230, 1998.