REMODELING IN THE ACTIN CORE OF THE AUDITORY HAIR CELL STEREOCILIA AS A NOVEL COMPONENT OF TEMPORARY NOISE-INDUCED HEARING LOSS

The rigid, paracrystalline actin core of auditory hair cell stereocilia is extremely stable and after initial formation must persist for the life of the cell to preserve hearing in mammals. In healthy hair cells, turnover of actin molecules occurs only in a small region near the tips of stereocilia, while the actin filaments of the shaft are stable. For decades damage to the actin core of stereocilia from acoustic trauma has only been attributed to cases of permanent noise-induced hearing loss. Here, we show that repairable actin core damage occurs in temporary noise-induced hearing loss from moderate acoustic trauma. We have found that moderate noise exposure causing a temporary hearing loss results in damage to the stereocilia actin core in the form of small, submicron breaks in the filamentous actin (F-actin) at the base of the stereocilia, and displacement of the stereocilia from its anchoring rootlet. The same damages were recapitulated in vitro after mechanical overstimulation of stereocilia bundles by fluid-jet. Despite the well-established stable nature of the F-actin within stereocilia, 24 hours after the damage we observed complete repair of this damage in vitro and only partial repair in vivo, indicating slower in vivo recovery. The mechanism of this repair appears to involve actin remodeling in the upper portion of the rootlet located within the stereocilia shaft. Our results suggest that repairable damage to the F-actin at the base of stereocilia is a novel component of temporary noise-induced hearing loss. We believe that restoration of hearing thresholds after moderate noise exposure includes the repair of this damage. Although the exact mechanism of this repair is unknown, this is the first evidence for actin cytoskeleton repair in the stereocilia of auditory hair cells which have to maintain their structure and mechanosensitivity throughout the life of an organism

Neuroblastoma Cell Lines Contain Pluripotent Tumor Initiating Cells That Are Susceptible to a Targeted Oncolytic Virus

Although disease remission can frequently be achieved for patients with neuroblastoma, relapse is common. The cancer stem cell theory suggests that rare tumorigenic cells, resistant to conventional therapy, are responsible for relapse. If true for neuroblastoma, improved cure rates may only be achieved via identification and therapeutic targeting of the neuroblastoma tumor initiating cell. Based on cues from normal stem cells, evidence for tumor populating progenitor cells has been found in a variety of cancers.Four of eight human neuroblastoma cell lines formed tumorspheres in neural stem cell media, and all contained some cells that expressed neurogenic stem cell markers including CD133, ABCG2, and nestin. Three lines tested could be induced into multi-lineage differentiation. LA-N-5 spheres were further studied and showed a verapamil-sensitive side population, relative resistance to doxorubicin, and CD133+ cells showed increased sphere formation and tumorigenicity. Oncolytic viruses, engineered to be clinically safe by genetic mutation, are emerging as next generation anticancer therapeutics. Because oncolytic viruses circumvent typical drug-resistance mechanisms, they may represent an effective therapy for chemotherapy-resistant tumor initiating cells. A Nestin-targeted oncolytic herpes simplex virus efficiently replicated within and killed neuroblastoma tumor initiating cells preventing their ability to form tumors in athymic nude mice.These results suggest that human neuroblastoma contains tumor initiating cells that may be effectively targeted by an oncolytic virus

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Neuroblastoma tumorspheres express nestin and are efficiently infected by oHSV.

<p>(A) Immunohistochemistry on tumorsphere cryosection for nestin (green) and DAPI co-stain (blue). Scale bar = 10 microns. (B, C) A neuroblastoma tumorsphere infected with rQNestin34.5 was imaged at 48 hours post-infection by (B) phase-contrast and (C) fluorescent microscopy for GFP. (D, E) rQNestin34.5-infected neuroblastoma tumorsphere at 48 hours post-infection evaluated by transmission electron microscopy showing viral nucleocapsids in the nucleus (arrows), and mature HSV particles in the cytoplasm in the process of acquiring their envelopes (arrows).</p

Stem cell-like characteristics of human neuroblastoma cell lines.

<p><i>MYCN</i>^amp, amplification of the <i>MYCN</i> gene.</p><p>DP, double positive for CD34 and CD133.</p><p>Side pop, presence of a verapamil-sensitive side population on flow cytometry with Hoescht stain.</p><p>n.d., not done.</p