Amy Nowack

Cotrafficking of SV2 and Synaptotagmin at the Synapse

Synaptic vesicles are specialized cycling endosomes that contain a unique constellation of membrane proteins. Proteins are sorted to vesicles by short amino acid sequences that serve as binding sites for clathrin adaptor proteins. Here we show that a tyrosine-based endocytosis motif in the vesicle protein SV2 is required for trafficking to synaptic vesicles of both SV2 and the calcium sensor protein synaptotagmin. Aberrant neurotransmission in cultured hippocampal neurons lacking SV2 was rescued by expression of wild-type SV2A, but not by SV2A-Y46A, a mutant containing a disrupted endocytosis motif in SV2As cytoplasmic N terminus. Neurons expressing SV2A-Y46A had significantly more SV2 on the plasma membrane, indicating reduced internalization. A screen for proteins that preferentially bound wild-type SV2A identified multiple endocytosis-related proteins, and in vitro binding studies confirmed binding to the clathrin adaptors AP2, EPS15, and amphiphysin 2/Bin1. Neurons lacking SV2 contained less synaptotagmin and had a higher proportion of synaptotagmin on the plasma membrane. Expression of either wild-type SV2A or SV2A-Y46A restored synaptotagmin expression levels; however, only wild-type SV2A restored a normal proportion of synaptotagmin on the plasma membrane. These findings indicate that SV2 influences the expression and trafficking of synaptotagmin via separate mechanisms. Synaptic vesicles immunoisolated from SV2A/B double knock-out mice had significantly less synaptotagmin than vesicles isolated from wild-type mice. Our results indicate that SV2 plays a major role in regulating the amount of synaptotagmin in synaptic vesicles and provide an explanation for the observation that synapses lacking SV2 have fewer vesicles competent for calcium-induced fusion.

SV2 regulates neurotransmitter release via multiple mechanisms

Among the proteins that mediate calcium-stimulated transmitter release, the synaptic vesicle protein 2 (SV2) stands out as a unique modulator specific to the neurons and endocrine cells of vertebrates. In synapses, SV2 regulates the expression and trafficking of the calcium sensor protein synaptotagmin, an action consistent with the reduced calcium-mediated exocytosis observed in neurons lacking SV2. Yet SV2 contains amino acid motifs consistent with it performing other actions that could regulate presynaptic functioning and that might underlie the mechanism of drug action. To test the role of these functional motifs, we performed a mutagenic analysis of SV2A and assessed the ability of mutant SV2A proteins to restore normal synaptic transmission in neurons from SV2A/B knockout mice. We report that SV2A-R231Q, harboring a mutation in a canonical transporter motif, restored normal synaptic depression (a measure of release probability and signature deficit of neurons lacking SV2). In contrast, normal synaptic depression was not restored by SV2A-W300A and SV2A-W666A, harboring mutations of conserved tryptophans in the 5th and 10th transmembrane domains. Although they did not rescue normal neurotransmission, SV2A-W300A and SV2A-W666A did restore normal levels of synaptotagmin expression and internalization. This indicates that tryptophans 300 and 666 support an essential action of SV2 that is unrelated to its role in synaptotagmin expression or trafficking. These results indicate that SV2 performs at least two actions at the synapse that contribute to neurotransmitter release.

Levetiracetam Reverses Synaptic Deficits Produced by Overexpression of SV2A

Levetiracetam is an FDA-approved drug used to treat epilepsy and other disorders of the nervous system. Although it is known that levetiracetam binds the synaptic vesicle protein SV2A, how drug binding affects synaptic functioning remains unknown. Here we report that levetiracetam reverses the effects of excess SV2A in autaptic hippocampal neurons. Expression of an SV2A-EGFP fusion protein produced a ∼1.5-fold increase in synaptic levels of SV2, and resulted in reduced synaptic release probability. The overexpression phenotype parallels that seen in neurons from SV2 knockout mice, which experience severe seizures. Overexpression of SV2A also increased synaptic levels of the calcium-sensor protein synaptotagmin, an SV2-binding protein whose stability and trafficking are regulated by SV2. Treatment with levetiracetam rescued normal neurotransmission and restored normal levels of SV2 and synaptotagmin at the synapse. These results indicate that changes in SV2 expression in either direction impact neurotransmission, and suggest that levetiracetam may modulate SV2 protein interactions.

Prosthetic implantation of the human vestibular system

Hypothesis A functional vestibular prosthesis can be implanted in human such that electrical stimulation of each semicircular canal produces canal-specific eye movements while preserving vestibular and auditory function. Background A number of vestibular disorders could be treated with prosthetic stimulation of the vestibular end organs. We have previously demonstrated in rhesus monkeys that a vestibular neurostimulator, based on the Nucleus Freedom cochlear implant, can produce canal-specific electrically evoked eye movements while preserving auditory and vestibular function. An investigational device exemption has been obtained from the FDA to study the feasibility of treating uncontrolled Ménière’s disease with the device. Methods The UW/Nucleus vestibular implant was implanted in the perilymphatic space adjacent to the three semicircular canal ampullae of a human subject with uncontrolled Ménière’s disease. Preoperative and postoperative vestibular and auditory function was assessed. Electrically evoked eye movements were measured at 2 time points postoperatively. Results Implantation of all semicircular canals was technically feasible. Horizontal canal and auditory function were largely, but not totally, lost. Electrode stimulation in 2 of 3 canals resulted in canal-appropriate eye movements. Over time, stimulation thresholds increased. Conclusion Prosthetic implantation of the semicircular canals in humans is technically feasible. Electrical stimulation resulted in canal-specific eye movements, although thresholds increased over time. Preservation of native auditory and vestibular function, previously observed in animals, was not demonstrated in a single subject with advanced Ménière’s disease.

Postural responses to electrical stimulation of the vestibular end organs in human subjects

A multichannel vestibular prosthesis that delivers electrical stimulation to the perilymph of individual semicircular canals is a potential new treatment modality for patients with vestibular deficiencies. Most research in this field has evaluated the efficacy of this approach by its ability to reproduce eye movements in response to head rotations. Our group has developed such a device and implanted it in four human subjects with intractable unilateral Meniere’s disease. This allows us to evaluate individual semicircular canal contribution to the control of balance and posture in human subjects. In this report, we demonstrate that electrical stimulation trains delivered to the perilymph of individual semicircular canals elicit postural responses specific to the particular canal stimulated, with some current spread to adjacent end organs. Modulation of stimulation current modulates the amplitude of the postural response. However, eye movements elicited by the same electrical stimuli were not consistent with postural responses in magnitude or direction in all subjects. Taken together, these findings support the feasibility of a vestibular prosthesis for the control of balance and illustrate new challenges for the development of this technology.

Auditory outcomes following implantation and electrical stimulation of the semicircular canals

We measured auditory brainstem responses (ABRs) in eight Rhesus monkeys after implantation of electrodes in the semicircular canals of one ear, using a multi-channel vestibular prosthesis based on cochlear implant technology. In five animals, click-evoked ABR thresholds in the implanted ear were within 10 dB of thresholds in the non-implanted control ear. Threshold differences in the remaining three animals varied from 18 to 69 dB, indicating mild to severe hearing losses. Click- and tone-evoked ABRs measured in a subset of animals before and after implantation revealed a comparable pattern of threshold changes. Thresholds obtained five months or more after implantation--a period in which the prosthesis regularly delivered electrical stimulation to achieve functional activation of the vestibular system--improved in three animals with no or mild initial hearing loss and increased in a fourth with a moderate hearing loss. These results suggest that, although there is a risk of hearing loss with unilateral vestibular implantation to treat balance disorders, the surgery can be performed in a manner that preserves hearing over an extended period of functional stimulation.

Longitudinal performance of an implantable vestibular prosthesis

Loss of vestibular function may be treatable with an implantable vestibular prosthesis that stimulates semicircular canal afferents with biphasic pulse trains. Several studies have demonstrated short-term activation of the vestibulo-ocular reflex (VOR) with electrical stimulation. Fewer long-term studies have been restricted to small numbers of animals and stimulation designed to produce adaptive changes in the electrically elicited response. This study is the first large consecutive series of implanted rhesus macaque to be studied longitudinally using brief stimuli designed to limit adaptive changes in response, so that the efficacy of electrical activation can be studied over time, across surgeries, canals and animals. The implantation of a vestibular prosthesis in animals with intact vestibular end organs produces variable responses to electrical stimulation across canals and animals, which change in threshold for electrical activation of eye movements and in elicited slow phase velocities over time. These thresholds are consistently lower, and the slow phase velocities higher, than those obtained in human subjects. The changes do not appear to be correlated with changes in electrode impedance. The variability in response suggests that empirically derived transfer functions may be required to optimize the response of individual canals to a vestibular prosthesis, and that this function may need to be remapped over time. This article is part of a Special Issue entitled .

Longitudinal performance of a vestibular prosthesis as assessed by electrically evoked compound action potential recording

Electrical stimulation of the vestibular end organ with a vestibular prosthesis may provide an effective treatment for vestibular loss if the stimulation remains effective over a significant period of time after implantation of the device. To assess efficacy of electrical stimulation in an animal model, we implanted 3 rhesus monkeys with a vestibular prosthesis based on a cochlear implant. We then recorded vestibular electrically evoked compound action potentials (vECAPs) longitudinally in each of the implanted canals to see how the amplitude of the response changed over time. The results suggest that vECAPs, and therefore electrical activation of vestibular afferent fibers, can remain largely stable over time following implantation.

Loss of Afferent Vestibular Input Produces Central Adaptation and Increased Gain of Vestibular Prosthetic Stimulation

ABSTRACTImplanted vestibular neurostimulators are effective in driving slow phase eye movements in monkeys and humans. Furthermore, increases in slow phase velocity and electrically evoked compound action potential (vECAP) amplitudes occur with increasing current amplitude of electrical stimulation. In intact monkeys, protracted intermittent stimulation continues to produce robust behavioral responses and preserved vECAPs. In lesioned monkeys, shorter duration studies show preserved but with somewhat lower or higher velocity behavioral responses. It has been proposed that such changes are due to central adaptive changes in the electrically elicited vestibulo-ocular reflex (VOR). It is equally possible that these differences are due to changes in the vestibular periphery in response to activation of the vestibular efferent system. In order to investigate the site of adaptive change in response to electrical stimulation, we performed transtympanic gentamicin perfusions to induce rapid changes in vestibular input in monkeys with long-standing stably functioning vestibular neurostimulators, disambiguating the effects of implantation from the effects of ototoxic lesion. Gentamicin injection was effective in producing a large reduction in natural VOR only when it was performed in the non-implanted ear, suggesting that the implanted ear contributed little to the natural rotational response before injection. Injection of the implanted ear produced a reduction in the vECAP responses in that ear, suggesting that the intact hair cells in the non-functional ipsilateral ear were successfully lesioned by gentamicin, reducing the efficacy of stimulation in that ear. Despite this, injection of both ears produced central plastic changes that resulted in a dramatically increased slow phase velocity nystagmus elicited by electrical stimulation. These results suggest that loss of vestibular afferent activity, and a concurrent loss of electrically elicited vestibular input, produces an increase in the efficacy of a vestibular neurostimulator by eliciting centrally adapted behavioral responses without concurrent adaptive increase of galvanic afferent activation in the periphery.

The Dynamics of Prosthetically Elicited Vestibulo-Ocular Reflex Function Across Frequency and Context in the Rhesus Monkey

Electrical vestibular neurostimulation may be a viable tool for modulating vestibular afferent input to restore vestibular function following injury or disease. To do this, such stimulators must provide afferent input that can be readily interpreted by the central nervous system to accurately represent head motion to drive reflexive behavior. Since vestibular afferents have different galvanic sensitivity, and different natural sensitivities to head rotational velocity and acceleration, and electrical stimulation produces aphysiological synchronous activation of multiple afferents, it is difficult to assign a priori an appropriate transformation between head velocity and acceleration and the properties of the electrical stimulus used to drive vestibular reflex function, i.e., biphasic pulse rate or pulse current amplitude. In order to empirically explore the nature of the transformation between vestibular prosthetic stimulation and vestibular reflex behavior, in Rhesus macaque monkeys we parametrically varied the pulse rate and current amplitude of constant rate and current amplitude pulse trains, and the modulation frequency of sinusoidally modulated pulse trains that were pulse frequency modulated (FM) or current amplitude modulated (AM). In addition, we examined the effects of differential eye position and head position on the observed eye movement responses. We conclude that there is a strong and idiosyncratic, from canal to canal, effect of modulation frequency on the observed eye velocities that are elicited by stimulation. In addition, there is a strong effect of initial eye position and initial head position on the observed responses. These are superimposed on the relationships between pulse frequency or current amplitude and eye velocity that have been shown previously.