Cerebellar neurodegeneration in the absence of microRNAs.

Genome-encoded microRNAs (miRNAs) are potent regulators of gene expression. The significance of miRNAs in various biological processes has been suggested by studies showing an important role of these small RNAs in regulation of cell differentiation. However, the role of miRNAs in regulation of differentiated cell physiology is not well established. Mature neurons express a large number of distinct miRNAs, but the role of miRNAs in postmitotic neurons has not been examined. Here, we provide evidence for an essential role of miRNAs in survival of differentiated neurons. We show that conditional Purkinje cell-specific ablation of the key miRNA-generating enzyme Dicer leads to Purkinje cell death. Deficiency in Dicer is associated with progressive loss of miRNAs, followed by cerebellar degeneration and development of ataxia. The progressive neurodegeneration in the absence of Dicer raises the possibility of an involvement of miRNAs in neurodegenerative disorders.

Is otospiralin inner ear specific? Evidence for its expression in mouse brain.

The small protein otospiralin has initially been identified as an inner ear specific molecule. However, compelling evidence from high throughput sequencing projects suggested that otospiralin is likely expressed in the central nervous system. Here, we tested this hypothesis using a combination of molecular biology, immunological, and histological techniques, and found that otospiralin is expressed in numerous regions of the central nervous system in mouse. In situ hybridization and immunohistochemistry revealed that otospiralin is widely expressed in neuronal cell bodies and glia. Ultrastructural observations in the cerebral cortex located the small protein in close proximity to membranous organelles in perikarya, the inner face of post-synaptic neuronal membranes, and in astrocytic processes. These results are in agreement with the predicted structure of the protein which revealed a single N-terminal transmembrane helix domain followed by a C-terminus cytosolic tail. Interestingly, 2 weeks after a mechanical trauma in the cerebral cortex, otospiralin expression increased in reactive astrocytes located within the vicinity of the site of injury, but not in neurons. Collectively, our observations suggest that otospiralin is possibly involved in signaling pathways, and could play a role in repair mechanisms subsequent to an injury in the central nervous system.

Localization in stereocilia, plasma membrane, and mitochondria suggests diverse roles for NMHC-IIa within cochlear hair cells.

NMHC-IIa, a nonmuscle myosin heavy chain isoform encoded by MYH9, is expressed in sensory hair cells and its dysfunction is associated with syndromic and nonsyndromic hearing loss. In this study, we investigate the ultrastructural distribution of NMHC-IIa within murine hair cells to elucidate its potential role in hair cell function. Using previously characterized anti-mouse NMHC-IIa antibody detected with immunogold labelling, NMHC-IIa was observed in the stereocilia, in the cytosol along the plasma membrane, and within mitochondria. Within stereocilia, presence of NMHC-IIa is observed throughout its length along the actin core, from the center to the periphery and at its base in the cuticular plate, suggesting a potential role in structural support. Within the sensory hair cells, NMHC-IIa was distributed throughout the cytoplasm and along the plasma membrane. A novel finding of this study is the localization of NMHC-IIa within the mitochondria, with the majority of the label along its inner membrane folds. The presence of NMHC-IIa within heterogeneous areas of the hair cell suggests that it may play different functional roles in these distinct regions. Thus, mutant NMHC-IIa may cause hearing loss by affecting hair cell dysfunction through structural and or functional disruption of its stereocilia, plasma membrane, and/or mitochondria.

Role of local immunoglobulin E specific for Alternaria alternata in the pathogenesis of nasal polyposis.

OBJECTIVE/HYPOTHESIS: The role of fungal pathogens in the etiology of nasal polyposis remains unclear. The aim of this study was to determine whether there was a correlation between the presence of Alternaria-specific immunoglobulin (Ig)E antibodies, eosinophilic inflammation, and the development of nasal polyps. STUDY DESIGN: Prospective study. METHODS: Serum and nasal tissue homogenates from 21 patients with manifestations of chronic sinusitis with nasal polyps were compared with specimens from 13 chronic sinusitis patients without polyps and 8 healthy controls. The Phadia ImmunoCAP and enzyme-linked immunosorbent assay were used to quantify levels of total IgE and Alternaria-specific (IgE, IgG, and IgA) antibodies. Eosinophil cationic protein (ECP) and tryptase levels were measured in tissue homogenates, whereas the inflammatory response was evaluated using tissue eosinophil counts in tissue samples. RESULTS: Serum analysis revealed no difference in the levels of total IgE and Alternaria-specific IgE, IgG, and IgA antibodies between the study groups. In contrast, the levels of Alternaria-specific IgE in tissue with polyps were significantly higher than in nonpolyp tissue. Increases in total tissue IgE paralleled increased levels of Alternaria-specific IgG and IgA antibodies in chronic sinusitis with nasal polyps as compared with control groups. A positive correlation was found between Alternaria-specific IgE and ECP in tissue. Increased mean levels of ECP corresponded to increased eosinophil counts in the group of patients with polyps. CONCLUSIONS: Alternaria-specific IgE and eosinophilic inflammation in nasal tissue correlates with the incidence of nasal polyps irrespective of specific IgE antibodies in serum. Together, the correlation between the local immune responses and the eosinophilic inflammation in nasal polyps suggests a possible role of Alternaria in the pathogenesis of nasal polyposis.

Robust axonal sprouting and synaptogenesis in organotypic slice cultures of rat cerebellum exposed to increased potassium chloride.

Organotypic slices of the rat cerebellum, cultured in physiological levels [K+]o (5 mM) for 14 days, loose the majority of granule cells in the anterior lobe resulting in few axons and atypical Purkinje cell dendrites with vacant spines. When the culture medium was switched from 5 mM to 20, 30 or 40 mM [K+]o during the last 7 days of cultures, slices developed axons with numerous vesicle-filled boutons that made synaptic contact with Purkinje cell spines. Most boutons had one or two spine profile contacts, while some were unusually large. Enlarged boutons abutted Purkinje cell somata or their dendrites, causing intervening spines to invaginate terminals to form rosette synaptic complexes. Calbindin immuno-labeling excluded Purkinje cell axonal collaterals as the source of rosette boutons and suggested a granule cell origin. Quantification of vacant spines as compared to those on boutons revealed a threshold for potassium, between 10 and 20 mM, where the number of synaptic spines increased and vacant spines decreased drastically. These findings suggest that elevated [K+]o triggers an activity-dependent plasticity in rat cerebellar slice cultures by promoting axonal sprouting with formation of vesicle-filled boutons and synaptogenesis on open receptor sites of Purkinje cell spines.

Cochlear implantation in human immunodeficiency virus-infected patients.

OBJECTIVE: To evaluate the efficacy of cochlear implants in human immunodeficiency virus-infected individuals and correlate these results with a proposed pathophysiological mechanism of human immunodeficiency virus-associated hearing loss. STUDY DESIGN: Retrospective case series and temporal bone analysis of deceased human immunodeficiency virus-positive patients. SETTING: Tertiary care hospital. PATIENTS: Seven human immunodeficiency virus-positive individuals with profound sensorineural hearing loss. INTERVENTION: Cochlear implantation at New York University Medical Center. METHODS: The surgical outcomes and complications were analyzed. Additionally, electron microscopic and immunohistochemical findings of cadaver temporal bone specimens of other known human immunodeficiency virus-positive individuals were reviewed. The performance results of the human immunodeficiency virus-positive cochlear implant patients were then correlated with the previously hypothesized pathophysiological mechanism of human immunodeficiency virus-associated hearing loss. RESULTS: The patients had a varied performance with cochlear implantation, and as a group performance was good. There were no surgical complications or postoperative complications. The good performance of these patients supports the hypothesis that the mechanism of human immunodeficiency virus-associated deafness involves infiltration, malfunction, and premature degeneration of the hair cells and supportive cells of the cochlea. CONCLUSIONS: Human immunodeficiency virus-positive individuals benefit from cochlear implantation without increased surgical risk.

Direct identification of proteins from T47D cells and murine brain tissue by matrix-assisted laser desorption/ionization post-source decay/collision-induced dissociation.

The purpose of this study is to determine the feasibility of the direct matrix-assisted laser desorption/ionization (MALDI) identification of proteins in fixed T47D breast cancer cells and murine brain tissues. The ability to identify proteins from cells and tissue may lead to biomarkers that effectively predict the onset of defined disease states, and their dynamic behavior could be an important hint for drug target discoveries. Direct tissue application of trypsin allows protein identification in cells and tissues, while maintaining spatial integrity and intracellular organization. Using a chemical printer, matrix was co-registered on trypsinized human T47D breast cancer cells and cryo-preserved sections of murine brain tissue, followed by MALDI post-source decay (PSD) or MALDI collision-induced dissociation (CID), respectively. Mass-to-charge (m/z) data from the cells and brain tissues were processed using Mascot software interrogation of the National Center for Biotechnology Information (NCBI) database. Histone H2B was identified from cultured T47D human breast cancer cells. Tubulin beta2 was identified from mouse brain cortex following an induced stroke. These results suggest that MALDI PSD/CID, combined with bioinformatics, can be used for the direct identification of proteins from cells and tissues. Refinements in preparation techniques may improve this approach to provide a tool for quantitative proteomics and clinical analysis.

Complementary expression and heterophilic interactions between IgLON family members neurotrimin and LAMP.

Neurotrimin (Ntm) and the limbic system-associated membrane protein (LAMP) are members of the IgLON (LAMP, OBCAM, Ntm) family of glycorylphosphatidylinositol anchored neural cell adhesion molecules. We previously reported that LAMP and Ntm promote adhesion and neurite outgrowth via a homophilic mechanism, suggesting that these proteins promote the formation of specific neuronal circuits by homophilic interactions. In this report, we have further characterized the expression and binding specificity of Ntm. Using a newly generated monoclonal antibody to Ntm, we demonstrated that this protein is largely expressed in a complementary pattern to that of LAMP in the nervous system, with co-expression at a few sites. Ntm is expressed at high levels in sensory-motor cortex and, of particular note, is transiently expressed in neurons of cortical barrel fields and corresponding thalamic "barreloids." Binding of a recombinant, soluble form of Ntm to CHO cells expressing either Ntm or LAMP demonstrates that Ntm and LAMP interact both homophilically and heterophilically. In contrast to conventional growth-promoting activity of Ig superfamily members, LAMP strongly inhibits the outgrowth of Ntm-expressing dorsal root ganglion (DRG) neurons in a heterophilic manner. These anatomical and functional data support the concept that homophilic and heterophilic interactions between IgLON family members are likely to play a role in the specification of neuronal projections via growth promoting and inhibiting effects, respectively.