Graham Atkin

Spontaneous hair cell regeneration in the mouse utricle following gentamicin ototoxicity

Whereas most epithelial tissues turn-over and regenerate after a traumatic lesion, this restorative ability is diminished in the sensory epithelia of the inner ear; it is absent in the cochlea and exists only in a limited capacity in the vestibular epithelium. The extent of regeneration in vestibular hair cells has been characterized for several mammalian species including guinea pig, rat, and chinchilla, but not yet in mouse. As the fundamental model species for investigating hereditary disease, the mouse can be studied using a wide variety of genetic and molecular tools. To design a mouse model for vestibular hair cell regeneration research, an aminoglycoside-induced method of complete hair cell elimination was developed in our lab and applied to the murine utricle. Loss of utricular hair cells was observed using scanning electron microscopy, and corroborated by a loss of fluorescent signal in utricles from transgenic mice with GFP-positive hair cells. Regenerative capability was characterized at several time points up to six months following insult. Using scanning electron microscopy, we observed that as early as two weeks after insult, a few immature hair cells, demonstrating the characteristic immature morphology indicative of regeneration, could be seen in the utricle. As time progressed, larger numbers of immature hair cells could be seen along with some mature cells resembling surface morphology of type II hair cells. By six months post-lesion, numerous regenerated hair cells were present in the utricle, however, neither their number nor their appearance was normal. A BrdU assay suggested that at least some of the regeneration of mouse vestibular hair cells involved mitosis. Our results demonstrate that the vestibular sensory epithelium in mice can spontaneously regenerate, elucidate the time course of this process, and identify involvement of mitosis in some cases. These data establish a road map of the murine vestibular regenerative process, which can be used for elucidating the molecular events that govern this process.

Ubiquitin pathways in neurodegenerative disease

Control of proper protein synthesis, function, and turnover is essential for the health of all cells. In neurons these demands take on the additional importance of supporting and regulating the highly dynamic connections between neurons that are necessary for cognitive function, learning, and memory. Regulating multiple unique synaptic protein environments within a single neuron while maintaining cell health requires the highly regulated processes of ubiquitination and degradation of ubiquitinated proteins through the proteasome. In this review, we examine the effects of dysregulated ubiquitination and protein clearance on the handling of disease-associated proteins and neuronal health in the most common neurodegenerative diseases.

Chronic excitotoxicity in the guinea pig cochlea induces temporary functional deficits without disrupting otoacoustic emissions

Brief cochlear excitotoxicity produces temporary neural swelling and transient deficits in auditory sensitivity; however, the consequences of long-lasting excitotoxic insult have not been tested. Chronic intra-cochlear infusion of the glutamate agonist AMPA (a-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid) resulted in functional deficits in the sound-evoked auditory brainstem response, as well as in behavioral measures of hearing. The electrophysiological deficits were similar to those observed following acute infusion of AMPA into the cochlea; however, the concentration-response curve was significantly shifted as a consequence of the slower infusion rate used with chronic cochlear administration. As observed following acute excitotoxic insult, complete functional recovery was evident within 7 days of discontinuing the AMPA infusion. Distortion product otoacoustic emissions were not affected by chronic AMPA infusion, suggesting that trauma to outer hair cells did not contribute to AMPA-induced deficits in acoustic sensitivity. Results from the current experiment address the permanence of deficits induced by chronic (14 day) excitotoxic insult as well as deficits in psychophysical detection of longer duration acoustic signals.

Expression of Myh9 in the mammalian cochlea: Localization within the stereocilia

Mutations of non‐muscle myosin Type IIA or MYH9 are linked to syndromic or nonsyndromic hearing loss. The biologic function of MYH9 in the auditory organ and the pathophysiology of its dysfunction remain to be determined. The mouse represents an excellent model for investigating the biologic role of MYH9 in the cells and tissues affected by its dysfunction. A primary step toward the understanding of the role of MYH9 in hearing and its dysfunction is the documentation of its cellular and sub‐cellular localization within the cochlea, the auditory organ. We describe the localization of Myh9 within the mouse cochlea using a polyclonal anti‐Myh9‐antibody, generated against an 18 amino acid long peptide corresponding to the sequence at the C‐terminus of mouse Myh9. The anti‐Myh9 antibody identified a single, specific, immunoreactive band of 220 kDa in immunoblot analysis of homogenate from a variety of different mouse tissues. The Myh9 antibody cross‐reacts with the rat but not the human orthologue. Myh9 is expressed predominantly within the spiral ligament as well as in the sensory hair cells of the organ of Corti. Confocal microscopy of cochlear surface preparations, identified Myh9 within the inner and outer hair cells and their stereocilia. Localization of Myh9 within the stereocilia raises the possibility that mutations of MYH9 may effect hearing loss though disruption of the stereocilia structure.

F-box only protein 2 (Fbxo2) regulates amyloid precursor protein levels and processing.

Background: The amyloid precursor protein (APP) is strongly implicated in the pathogenesis of Alzheimer disease. Results: The loss of Fbxo2, a brain-enriched substrate adaptor for ubiquitin ligases, leads to increased neuronal APP levels and processing. Conclusion: Fbxo2 regulates APP levels in a brain region-specific manner. Significance: These findings suggest a novel mechanism for ubiquitin-mediated regulation of amyloid-β production. The amyloid precursor protein (APP) is an integral membrane glycoprotein whose cleavage products, particularly amyloid-β, accumulate in Alzheimer disease (AD). APP is present at synapses and is thought to play a role in both the formation and plasticity of these critical neuronal structures. Despite the central role suggested for APP in AD pathogenesis, the mechanisms regulating APP in neurons and its processing into cleavage products remain incompletely understood. F-box only protein 2 (Fbxo2), a neuron-enriched ubiquitin ligase substrate adaptor that preferentially binds high-mannose glycans on glycoproteins, was previously implicated in APP processing by facilitating the degradation of the APP-cleaving β-secretase, β-site APP-cleaving enzyme. Here, we sought to determine whether Fbxo2 plays a similar role for other glycoproteins in the amyloid processing pathway. We present in vitro and in vivo evidence that APP is itself a substrate for Fbxo2. APP levels were decreased in the presence of Fbxo2 in non-neuronal cells, and increased in both cultured hippocampal neurons and brain tissue from Fbxo2 knock-out mice. The processing of APP into its cleavage products was also increased in hippocampi and cultured hippocampal neurons lacking Fbxo2. In hippocampal slices, this increase in cleavage products was accompanied by a significant reduction in APP at the cell surface. Taken together, these results suggest that Fbxo2 regulates APP levels and processing in the brain and may play a role in modulating AD pathogenesis.

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.

Basic science breaks through: New therapeutic advances in Parkinson's disease

Parkinsons disease (PD) is the second most common neurodegenerative disease and is typically associated with progressive motor dysfunction, although PD patients also exhibit a variety of non‐motor symptoms. The neuropathological hallmark of PD is intraneuronal inclusions containing primarily α‐Synuclein (α‐Syn), and several lines of evidence point to α‐Syn as a key contributor to disease progression. Thus, basic research in the field of PD is largely focused on understanding the pathogenic properties of α‐Syn. Over the past 2 y, these studies helped to identify several novel therapeutic strategies that have the potential to slow PD progression; such strategies include sequestration of extracellular α‐Syn through immunotherapy, reduction of α‐Syn multimerization or intracellular toxicity, and attenuation of the neuroinflammatory response. This review describes these and other putative therapeutic strategies, together with the basic science research that led to their identification. The current breadth of novel targets for the treatment of PD warrants cautious optimism in the fight against this devastating disease.

Loss of F-box Only Protein 2 (Fbxo2) Disrupts Levels and Localization of Select NMDA Receptor Subunits, and Promotes Aberrant Synaptic Connectivity

NMDA receptors (NMDARs) play an essential role in some forms of synaptic plasticity, learning, and memory. Therefore, these receptors are highly regulated with respect to their localization, activation, and abundance both within and on the surface of mammalian neurons. Fundamental questions remain, however, regarding how this complex regulation is achieved. Using cell-based models and F-box Only Protein 2 (Fbxo2) knock-out mice, we found that the ubiquitin ligase substrate adaptor protein Fbxo2, previously reported to facilitate the degradation of the NMDAR subunit GluN1 in vitro, also functions to regulate GluN1 and GluN2A subunit levels in the adult mouse brain. In contrast, GluN2B subunit levels are not affected by the loss of Fbxo2. The loss of Fbxo2 results in greater surface localization of GluN1 and GluN2A, together with increases in the synaptic markers PSD-95 and Vglut1. These synaptic changes do not manifest as neurophysiological differences or alterations in dendritic spine density in Fbxo2 knock-out mice, but result instead in increased axo-dendritic shaft synapses. Together, these findings suggest that Fbxo2 controls the abundance and localization of specific NMDAR subunits in the brain and may influence synapse formation and maintenance.

Developmental expression of BK channels in chick cochlear hair cells

BackgroundCochlear hair cells are high-frequency sensory receptors. At the onset of hearing, hair cells acquire fast, calcium-activated potassium (BK) currents, turning immature spiking cells into functional receptors. In non-mammalian vertebrates, the number and kinetics of BK channels are varied systematically along the frequency-axis of the cochlea giving rise to an intrinsic electrical tuning mechanism. The processes that control the appearance and heterogeneity of hair cell BK currents remain unclear.ResultsQuantitative PCR results showed a non-monotonic increase in BK α subunit expression throughout embryonic development of the chick auditory organ (i.e. basilar papilla). Expression peaked near embryonic day (E) 19 with six times the transcript level of E11 sensory epithelia. The steady increase in gene expression from E11 to E19 could not explain the sudden acquisition of currents at E18-19, implicating post-transcriptional mechanisms. Protein expression also preceded function but progressed in a sequence from diffuse cytoplasmic staining at early ages to punctate membrane-bound clusters at E18. Electrophysiology data confirmed a continued refinement of BK trafficking from E18 to E20, indicating a translocation of BK clusters from supranuclear to subnuclear domains over this critical developmental age.ConclusionsGene products encoding BK α subunits are detected up to 8 days before the acquisition of anti-BK clusters and functional BK currents. Therefore, post-transcriptional mechanisms seem to play a key role in the delayed emergence of calcium-sensitive currents. We suggest that regulation of translation and trafficking of functional α subunits, near voltage-gated calcium channels, leads to functional BK currents at the onset of hearing.