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. 2008 Dec;9(4):452-63.
doi: 10.1007/s10162-008-0134-y. Epub 2008 Jul 30.

Localization of synucleins in the mammalian cochlea

O Akil  1 , C M Weber, S N Park, N Ninkina, V Buchman, L R Lustig
Affiliations

Localization of synucleins in the mammalian cochlea

O Akil et al. J Assoc Res Otolaryngol. 2008 Dec.

Abstract

Synucleins are widely expressed synaptic proteins within the central nervous system that have been implicated in such neurodegenerative disorders as Parkinson's disease. In this study, an initial characterization of all three synucleins, alpha-, beta-, and gamma-synuclein, within the cochlea was undertaken. Reverse transcriptase-polymerase chain reaction (PCR) demonstrated all three synuclein mRNA species within microdissected cochlear tissue. Quantitative PCR suggests that beta-synuclein is the most abundantly expressed form, followed by gamma- and then alpha-synuclein. Western blot analysis similarly demonstrates all three synuclein proteins within microdissected cochlear tissue. Immunofluorescence localizes the three synucleins predominantly to the efferent neuronal system at the efferent outer hair cell synapse, with some additional localization within the efferent tunnel-crossing fibers (alpha- and gamma-synuclein), spiral ganglion (beta-synuclein), inner spiral bundle (gamma-synuclein), and stria vascularis (alpha- > beta-synuclein). Developmentally, gamma-synuclein can be seen in the region of the outer hair cells by E19, while alpha- and beta-synuclein do not clearly appear there until approximately P10. Additional studies in a null-mutant gamma-synuclein mouse show no histological changes in the organ of Corti with normal hair cell and spiral ganglion cell counts, and normal ABR and DPOAE thresholds in wild-type vs mutant littermates. Together, these results localize synucleins to the efferent cholinergic neuronal auditory system, pointing to a role in normal auditory function, and raising the potential implications for their role in auditory neurodegenerative disorders. However, gamma-synuclein alone is not required for the development and maintenance of normal hearing through P21. Whether overlapping roles of the other synucleins help compensate for the loss of gamma-synuclein remains to be determined.

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Figures

FIG. 1.
FIG. 1.
RT-PCR of synucleins within the Cochlea. RT-PCR was used to detect the presence of α-, β-, and γ-synuclein mRNA in microdissected rodent (rat—left panel, mouse—three central panels) cochlear tissue. Controls lacking reverse-transcriptase (denoted with a ‘-’) are included. Both an α- and γ-synuclein knockout (KO) mouse were also studied, demonstrating the correct phenotype of the null-mutant mouse. Quantitative PCR (qPCR—right panel) was used to assess relative mRNA differences of the three synucleins (α, β, γ) within microdissected cochlear tissue. WT Wild-type, KO knockout mice.
FIG. 2.
FIG. 2.
Western blot of synucleins in the cochlea. Western blot analysis of several tissue types was used to assess for the presence of the three synuclein proteins (α, β, γ), as demonstrated by the band at ~20 kD (arrow). In lane 1, mice spinal cord, served as a positive control for α- and β-synuclein. α and β-Synuclein were both identified in rat cochlear tissue (lane 2). All three synucleins are identified in mouse cochlear tissue (lane 3). In the α-synuclein knockout mouse (lane 4), the cochlear tissue appropriate detects β- but not α-synuclein. Similarly, in the γ-synuclein knockout mouse (lane 5), as expected, the cochlear tissues detect α- and β- but not γ-synuclein. These later studies demonstrate the specificity of the antibodies used for subsequent studies as well as the correct phenotype of the null-mutant mice.
FIG. 3.
FIG. 3.
Immunofluorescence of synucleins within the mouse cochlea. Single or double-label immunofluorescence localizes the three synucleins within the cochlea, predominantly to the efferent neuronal system and outer hair cell synaptic region. Outlines of relevant cochlear structures are provided in A (I Inner hair cell, O outer hair cell, D Deiters cell, TC tunnel of Corti), while the orientation of all organ of Corti (A–D, F–I) are similar. α-Synuclein localizes predominantly in the base of the outer hair cells and the efferent tunnel-crossing fibers (A, D, G, H) and more weakly in the base of the inner hair cell in the region of the inner spiral bundle, as well as within the stria vascularis (K). In contrast, β-synuclein was most prominently localized to the synapse at the base of the outer hair cells (B, I), spiral ganglion (E), and more weakly within the stria vascularis (l). Antibodies against γ-synuclein most strongly label the efferent synapse at the base of the outer hair cells with some labeling of the efferent tunnel-crossing fibers (C), and co-localizes with α-synuclein at the efferent synapse near the base of the outer hair cell (G), but shows no staining within the stria vascularis nor spiral ganglion (not shown). γ-Synuclein is also closely approximated to the efferent synaptic marker synaptophysin with a nearly overlapping staining pattern (F). In the γ-synuclein null-mutant mouse (H–J), there is no apparent change in the labeling pattern of α-synuclein (H) or β-synuclein (I), while γ-synuclein appropriately shows no labeling in this mouse tissue (J). Together, these studies localize the three synucleins predominantly to the efferent neuronal system within the rodent organ of Corti with localization at the efferent outer hair cell synapse, with some additional localization within the efferent tunnel-crossing fibers (α- and γ-synuclein), spiral ganglion (β-synuclein), and stria vascularis (α- > β-synuclein).
FIG. 4.
FIG. 4.
Developmental expression of synucleins in the cochlea. Synuclein expression was assessed at E16, E19, P1, P5, P10, and P15 to determine onset of α-synuclein (left column), β-synuclein (middle column), and γ-synuclein (right column). These studies demonstrate that γ-synuclein has the earliest onset, first detectable in the region below the outer hair cells at approximately E19 (arrow, E19, γ-synuclein). In contrast, immunofluorescent labeling of α- or β-synuclein cannot be clearly seen until P10 (arrows, P10, α- and β-synuclein). Diffuse γ-synuclein label can also be seen below the base of the developing inner hair cell at the earliest time point studied (E16), becoming more localized through P15, but disappearing by P21 (compare to Fig. 3c and f), an area corresponding to the developing inner spiral bundle. A linear labeling of unclear significance along the basilar membrane was consistently seen between E19 and P10 in the α- and β-synuclein sections, but disappearing by P15 for both (asterisks, P5). To assist with orientation, relevant structures are outlined that are identifiable for all three synucleins; these include developing inner hair cell nuclei (“I”, P0 onward), tunnel of Corti (“TC”, P5-P15), outer pillar cell (“OPC”, P15 only), outer hair cell nuclei (“O”, E16 onward) and Deiters’ cell nucleus (“D”, P15 only). The orientation for all figures is the same.
FIG. 5.
FIG. 5.
Null-mutant γ-synuclein histology and surface preparation. Light microscopy (top eight panels) and surface preparation (bottom four panels) from the γ-synuclein null mutant mouse and wild-type littermate. Organ of Corti histology (upper four panels) and spiral ganglion region (middle four panels) from both the apex and base of the cochlea are included for comparison. These studies demonstrate no significant histological differences between the wild-type (WT) and knockout (KO) mice; each show normal light microscopy morphology and spiral ganglion cells. Similarly, surface preparations from these same mice (WT and KO) from both the apex and base demonstrate normal inner and outer hair cells.
FIG. 6.
FIG. 6.
Auditory physiology in the γ-synuclein null mutant mouse. Auditory brainstem response (ABR, A) threshold testing as well as distortion product otoacoustic emissions (DPOAE, B) testing was used to test hearing in the γ-synuclein knockout mouse compared to the wild-type littermate. ABR testing of both broadband clicks and tone-burst stimuli (A) demonstrates no significant differences in hearing between the wild-type (WT n = 6) and knockout (KO) mice (n = 10). Similarly, DPOAE testing (B) demonstrated no significant difference between the wild-type (WT) and knockout (KO) mice. The reference noise floor (nf KO, nf WT) of each DPOAE tracing is included.
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