Despite widespread localization of synucleins throughout the CNS, their exact function remains undetermined. However, with their identification in Lewy bodies, the pathological hallmark of disorder of Parkinson’s disease (Spillantini et al. 1997
) and their role in other neurodegenerative disorders, interest in the underlying physiology of synucleins remains high. The identification of synucleins within the auditory neuronal system also now raises some interesting questions regarding their possible role in neurodegenerative processes within the inner ear.
This initial work focuses on localization of the three synucleins within the mammalian organ of Corti, and our results correlate with prior localization studies of synucleins within the CNS. Prior work by other groups have shown strong labeling of the synucleins throughout the CNS, with both α- and β-synuclein migrating from the cell body to the presynaptic nerve terminals at approximately 17–20 weeks and β synuclein ultimately localizing to somatic cholinergic neurons. In contrast, γ-synuclein is localized primarily to the peripheral nervous system in both cholinergic and catecholaminergic neurons (Jakes et al. 1994
; Lavedan et al. 1998a
; Lavedan et al. 1998b
; Li et al. 2002
; Ueda et al. 1993
). Here, we identified all three synucleins, α-, β-, and γ-, within the rodent cochlea using multiple modalities (RT-PCR, Western blot, and immunofluorescence). The three synucleins all localize predominantly to the efferent synapse at the base of the outer hair cell, co-localizing both with synaptophysin (Fig. ) and ChAT (data not shown). Furthermore, there is additional weaker labeling of the base of the inner hair cell in the region of the inner spiral bundle (α- and γ-synuclein) and efferent tunnel-crossing fibers (α- and γ- synucleins). Thus, the previously described cholinergic localization of synucleins within the CNS is recapitulated in the peripheral organ of Corti. The significance of the additional labeling seen in the spiral ganglion cells (β-synuclein), and stria vascularis (α- and β-synuclein; Fig. ) is unclear.
Interestingly, most studies show that both α- and β-synuclein have similar expression patterns. In this study, β-synuclein appears to differ from both α- and γ-synuclein in at least two regards: β-synuclein was the only one to label the spiral ganglion neurons (Fig. e), and in contrast to both α- and γ-synuclein which labeled both efferent tunnel crossing fibers (medial olivocochlear fibers) and the efferent terminals, β-synuclein only labeled the efferent terminals. β-synuclein also showed the highest levels of mRNA by qPCR in the cochlea (Fig. ). Whether this might represent different functions between the synucleins remains unclear, though it would appear from these results that β-synuclein has a distinct role to play within the cochlea.
The developmental labeling pattern identified is somewhat interesting. Labeling below the developing outer hair cells was seen as early as E19 for γ-synuclein, while α- and β-synuclein did not label their respective regions until ~P10, implying two different functions for these isoforms. Studies by Fritzsch (1996
)and Karis et al. (2001
) show that the first efferent fibers can be traced towards various sensory epithelia of the inner ear by E13 and penetrate the sensory neuroepithelia by E14–E15 with individual efferent axons branching below OHCs at this time. While some studies show that the first observable efferent synapse on hair cells do not occur until approximately P0, additional studies (Bruce et al. 2000
; Emmerling et al. 1990
) suggest that the efferent axons and their growth cones may reach hair cells much earlier than this, before the first synapses can be identified (Simmons 2002
). Thus the γ-synuclein labeling we identified below the OHC at E19 and beyond might be consistent with this notion, though what the precise role of γ-synuclein would be is unclear at present. In contrast, α- and β-synuclein, not showing up until P10, may play a more traditional role in efferent synaptic function.
Within the CNS, the function of synucleins in the cochlea and their relationship to hearing remains unclear. Prior studies have shown that synucleins may play a role in synaptic function. In mature cultured primary neurons, synucleins co-localize almost exclusively with synaptophysin in the presynaptic terminals and appear earlier than synaptophysin, but later than the vesicle-associated protein synapsin I during CNS development (Hsu et al. 1998
; Jensen et al. 1999
; Murphy et al. 2000
; Withers et al. 1997
). Additional recent studies have shown that α-synuclein may be required for the genesis and/or maintenance of resting pools of presynaptic vesicles (Cabin et al. 2002
; Gureviciene et al. 2007
; Murphy et al. 2000
). Recent work has also shown that α-synuclein can potentiate Ca2+
influx through voltage-dependent Ca2+
channels (Adamczyk and Strosznajder 2006
; Ueda et al. 1997
). The identification of all three synucleins at the base of the outer hair cell at the efferent synapse, co-localizing with both synaptophysin and ChAT would therefore support a similar role for synucleins in efferent neuronal transmission in the auditory system. Perhaps through regulation of the size of the resting pool of presynaptic vesicles, as has been demonstrated in cultured neurons (Cabin et al. 2002
; Murphy et al. 2000
), synucleins may play a role in long-term, sustained release of acetylcholine in the efferent auditory system.
Prior work has shown that α-synucleins can exist in various splice forms and that variations in these splice forms may be part of the underlying pathophysiology in such neurodegenerative disorders as Parkinson’s disease.(Beyer et al. 2008
; Fuchs et al. 2008
; Muller et al. 2005
) It is thus intriguing to speculate that a similar phenomenon may be occurring in degenerative auditory processes. While synuclein isoforms were not specifically sought in this work, it would certainly be worthy of further investigation.
As this study demonstrates, γ-synuclein alone is not required for the onset or maintenance of normal hearing and outer hair cell function through P21, as demonstrated by the knockout mouse. Our studies show no phenotypic or histological differences in the inner ears of the wild-type vs. the knockout mice, while ABR thresholds and otoacoustic emissions also are unchanged in the knockout mouse through P21. This follows prior studies on this same mouse, in which no difference was observed in the number of neurons between wild-type and null mutant animals in several brain stem motor nuclei, in lumbar dorsal root ganglia, and in the trigeminal ganglion (Ninkina et al. 2003
). Lack of clear morphological and phenotypic differences were similarly observed in the CNS of α-synuclein null mutant mouse (Abeliovich et al. 2000
; Cabin et al. 2002
). Whether the α-synuclein null mutant mouse has any auditory defect is still unknown, though it is currently under study in our lab. Furthermore, in contrast to the rodent CNS (Li et al. 2002
), our data indicates that β-synuclein is the predominant mRNA species in the cochlea (Fig. ) and that perhaps a β-synuclein null-mutant mouse would show auditory defects; unfortunately a β-synuclein knockout mouse is not currently available to us for study. Based upon prior findings, it has been postulated that the close homologous protein structure as well as overlapping expression patterns of all three synucleins allow the other synucleins to compensate for loss of one isoform as seen in the null mutant mice studied to date(Ninkina et al. 2003
). This could certainly account for a lack of any auditory deficits within the cochlea in the γ-synuclein knockout mice studied here. Arguing against this proposition, however, is that prior studies have failed to demonstrate altered expression levels of the other synucleins in either the α- and γ-synuclein null mutants (Abeliovich et al. 2000
; Ninkina et al. 2003
). However, studies on double mutant mice lacking both α- and β-synuclein have demonstrated that, while synucleins are not essential components of synaptic transmission, they may contribute to the long-term regulation or maintenance of presynaptic function through their effects on transmitter release(Chandra et al. 2004
Despite not finding an obvious deficit within the inner ears of the γ-synuclein null mutant mice, the organ of Corti nonetheless could be an important model for testing synuclein function, a protein known to be involved in other neurodegenerative disorders. There are a variety of neurodegenerative processes that can affect the inner ear, most notably presbycusis or age-related hearing loss. Recent reports have linked α-synuclein to voltage-gated calcium flux, known to play a role in outer hair cell efferent signaling (Adamczyk and Strosznajder 2006
). Furthermore, Cabin et al. (2002
) have shown that, while there were no obvious phenotypic changes in an α-synuclein knockout mouse, prolonged trains of repetitive stimulation in hippocampal slice preparations depleted both docked and reserve pools of neurotransmitter vesicles, while replenishment of these vesicles were slower in the mutant synapses. Thus, the localization of synucleins predominantly to the efferent neuronal system within the inner ear raises the intriguing possibility that synuclein dysfunction could play a role in susceptibility to noise-induced hearing loss or presbycusis, two disorders that have been linked, in part, to the efferent neuronal system (Maison and Liberman 2000
; Zhu et al. 2007
). The finding of α- and β-synuclein within the stria vascularis, also a well-known site of age-related hearing loss changes (Spicer and Schulte 2005
), further supports this notion. Thus, it is possible that age-related hearing loss studies beyond P21, or studies with auditory perturbation (e.g., loud noise exposure) could reveal deficits not seen in our initial studies. Such studies are currently ongoing in our lab in an attempt to better define the role of synucleins within the inner ear.