Slow kinesin-dependent microtubular transport facilitates ribbon synapse assembly in developing cochlear inner hair cells.

Sensory synapses are characterized by electron-dense presynaptic specializations, so-called synaptic ribbons. In cochlear inner hair cells (IHCs), ribbons play an essential role as core active zone (AZ) organizers, where they tether synaptic vesicles, cluster calcium channels and facilitate the temporally-precise release of primed vesicles. While a multitude of studies aimed to elucidate the molecular composition and function of IHC ribbon synapses, the developmental formation of these signalling complexes remains largely elusive to date. To address this shortcoming, we performed long-term live-cell imaging of fluorescently-labelled ribbon precursors in young postnatal IHCs to track ribbon precursor motion. We show that ribbon precursors utilize the apico-basal microtubular (MT) cytoskeleton for targeted trafficking to the presynapse, in a process reminiscent of slow axonal transport in neurons. During translocation, precursor volume regulation is achieved by highly dynamic structural plasticity - characterized by regularly-occurring fusion and fission events. Pharmacological MT destabilization negatively impacted on precursor translocation and attenuated structural plasticity, whereas genetic disruption of the anterograde molecular motor Kif1a impaired ribbon volume accumulation during developmental maturation. Combined, our data thus indicate an essential role of the MT cytoskeleton and Kif1a in adequate ribbon synapse formation and structural maintenance.

Autochthonous infection with Ehrlichia Canis and Hepatozoon Canis in dogs from Serbia.

BACKGROUND: The epidemiological status concerning many canine tick-borne diseases (TBDs) in Serbia is still insufficiently known. OBJECTIVES: Our study aimed to investigate the presence of tick-borne pathogens of the family Anaplasmataceae and Hepatozoon spp., as a cause of illnesses accompanied by clinical signs that can occur in dogs with anaplasmosis, ehrlichiosis and hepatozoonosis. METHODS: Dogs are included in the study based on the presence of a minimum of three clinical and/or pathological findings that could be associated with anaplasmosis, ehrlichiosis and hepatozoonosis. During the study (April-October 2018), 11 dogs met the conditions to be included in the survey. Identification of the causative agent in the blood of diseased dogs was performed by conventional PCR followed by sequencing. RESULTS: The presence of the pathogens was confirmed in three animals (3/11, 27.3%). The presence of Ehrlichia canis was confirmed in 3-month-old female Rottweiler puppy, an 8-year old Miniature Schnauzer female was positive for Hepatozoon canis infection, while 4-year-old mixed breed male dog was co-infected with both mentioned pathogens. These are the first cases of autochthonous infection with E. canis and H. canis in dogs from Serbia confirmed by molecular methods. CONCLUSIONS: The results of our study indicate the importance of molecular methods to establish a reliable diagnosis of TBDs. Also, the confirmed presence of causative agents of canine monocytic ehrlichiosis and hepatozoonosis in Serbia appeals to veterinary practitioners that it is necessary to exclude the presence of those diseases in suspicious dogs.

Analyzing efficacy, stability, and safety of AAV-mediated optogenetic hearing restoration in mice.

AAV-mediated optogenetic neural stimulation has become a clinical approach for restoring function in sensory disorders and feasibility for hearing restoration has been indicated in rodents. Nonetheless, long-term stability and safety of AAV-mediated channelrhodopsin (ChR) expression in spiral ganglion neurons (SGNs) remained to be addressed. Here, we used longitudinal studies on mice subjected to early postnatal administration of AAV2/6 carrying fast gating ChR f-Chrimson under the control of the human synapsin promoter unilaterally to the cochlea. f-Chrimson expression in SGNs in both ears and the brain was probed in animals aged 1 mo to 2 yr. f-Chrimson was observed in SGNs at all ages indicating longevity of ChR-expression. SGN numbers in the AAV-injected cochleae declined with age faster than in controls. Investigations were extended to the brain in which viral transduction was observed across the organ at varying degrees irrespective of age without observing viral spread-related pathologies. No viral DNA or virus-related histopathological findings in visceral organs were encountered. In summary, our study demonstrates life-long (24 mo in mice) expression of f-Chrimson in SGNs upon single AAV-dosing of the cochlea.

Cabp2-Gene Therapy Restores Inner Hair Cell Calcium Currents and Improves Hearing in a DFNB93 Mouse Model.

Clinical management of auditory synaptopathies like other genetic hearing disorders is currently limited to the use of hearing aids or cochlear implants. However, future gene therapy promises restoration of hearing in selected forms of monogenic hearing impairment, in which cochlear morphology is preserved over a time window that enables intervention. This includes non-syndromic autosomal recessive hearing impairment DFNB93, caused by defects in the CABP2 gene. Calcium-binding protein 2 (CaBP2) is a potent modulator of inner hair cell (IHC) voltage-gated calcium channels CaV1.3. Based on disease modeling in Cabp2-/- mice, DFNB93 hearing impairment has been ascribed to enhanced steady-state inactivation of IHC CaV1.3 channels, effectively limiting their availability to trigger synaptic transmission. This, however, does not seem to interfere with cochlear development and does not cause early degeneration of hair cells or their synapses. Here, we studied the potential of a gene therapeutic approach for the treatment of DFNB93. We used AAV2/1 and AAV-PHP.eB viral vectors to deliver the Cabp2 coding sequence into IHCs of early postnatal Cabp2-/- mice and assessed the level of restoration of hair cell function and hearing. Combining in vitro and in vivo approaches, we observed high transduction efficiency, and restoration of IHC CaV1.3 function resulting in improved hearing of Cabp2-/- mice. These preclinical results prove the feasibility of DFNB93 gene therapy.

Utility of red-light ultrafast optogenetic stimulation of the auditory pathway.

Optogenetic stimulation of spiral ganglion neurons (SGNs) in the ear provides a future alternative to electrical stimulation used in current cochlear implants. Here, we employed fast and very fast variants of the red-light-activated channelrhodopsin (ChR) Chrimson (f-Chrimson and vf-Chrimson) to study their utility for optogenetic stimulation of SGNs in mice. The light requirements were higher for vf-Chrimson than for f-Chrimson, even when optimizing membrane expression of vf-Chrimson by adding potassium channel trafficking sequences. Optogenetic time and intensity coding by single putative SGNs were compared with coding of acoustic clicks. vf-Chrimson enabled putative SGNs to fire at near-physiological rates with good temporal precision up to 250 Hz of stimulation. The dynamic range of SGN spike rate coding upon optogenetic stimulation was narrower than for acoustic clicks but larger than reported for electrical stimulation. The dynamic range of spike timing, on the other hand, was more comparable for optogenetic and acoustic stimulation. In conclusion, f-Chrimson and vf-Chrimson are promising candidates for optogenetic stimulation of SGNs in auditory research and future cochlear implants.

Developing Fast, Red-Light Optogenetic Stimulation of Spiral Ganglion Neurons for Future Optical Cochlear Implants.

Optogenetic stimulation of type I spiral ganglion neurons (SGNs) promises an alternative to the electrical stimulation by current cochlear implants (CIs) for improved hearing restoration by future optical CIs (oCIs). Most of the efforts in using optogenetic stimulation in the cochlea so far used early postnatal injection of viral vectors carrying blue-light activated channelrhodopsins (ChRs) into the cochlea of mice. However, preparing clinical translation of the oCI requires (i) reliable and safe transduction of mature SGNs of further species and (ii) use of long-wavelength light to avoid phototoxicity. Here, we employed a fast variant of the red-light activated channelrhodopsin Chrimson (f-Chrimson) and different AAV variants to implement optogenetic SGN stimulation in Mongolian gerbils. We compared early postnatal (p8) and adult (>8 weeks) AAV administration, employing different protocols for injection of AAV-PHP.B and AAV2/6 into the adult cochlea. Success of the optogenetic manipulation was analyzed by optically evoked auditory brainstem response (oABR) and immunohistochemistry of mid-modiolar cryosections of the cochlea. In order to most efficiently evaluate the immunohistochemical results a semi-automatic procedure to identify transduced cells in confocal images was developed. Our results indicate that the rate of SGN transduction is significantly lower for AAV administration into the adult cochlea compared to early postnatal injection. SGN transduction upon AAV administration into the adult cochlea was largely independent of the chosen viral vector and injection approach. The higher the rate of SGN transduction, the lower were oABR thresholds and the larger were oABR amplitudes. Our results highlight the need to optimize viral vectors and virus administration for efficient optogenetic manipulation of SGNs in the adult cochlea for successful clinical translation of SGN-targeting gene therapy and of the oCI.

Application of Targeting-Optimized Chronos for Stimulation of the Auditory Pathway.

In the last 15 years, optogenetics has revolutionized the life sciences and enabled studies of complex biological systems such as the brain. Applying optogenetics also has great potential for restorative medicine, such as hearing restoration, by stimulating genetically modified spiral ganglion neurons of the cochlea with light. To this end, opsins with short closing kinetics are required, given the high firing rates and utmost temporal precision of spiking in these neurons. Chronos is the fastest native blue channelrhodopsin (ChR) reported so far with a closing kinetics bellow 1 ms at body temperature and an interesting candidate for the development of the future optogenetic cochlear implants. This book chapter explains in more details the development and application of Chronos with optimized membrane targeting for temporally precise optical stimulation of spiral ganglion neurons. In addition, the generation of adeno-associated virus (AAV) and AAV delivery to the cochlea of postnatal mice and the procedure to record optically evoked auditory brainstem responses are described.

Viral rhodopsins 1 are an unique family of light-gated cation channels.

Phytoplankton is the base of the marine food chain as well as oxygen and carbon cycles and thus plays a global role in climate and ecology. Nucleocytoplasmic Large DNA Viruses that infect phytoplankton organisms and regulate the phytoplankton dynamics encompass genes of rhodopsins of two distinct families. Here, we present a functional and structural characterization of two proteins of viral rhodopsin group 1, OLPVR1 and VirChR1. Functional analysis of VirChR1 shows that it is a highly selective, Na+/K+-conducting channel and, in contrast to known cation channelrhodopsins, it is impermeable to Ca2+ ions. We show that, upon illumination, VirChR1 is able to drive neural firing. The 1.4 Å resolution structure of OLPVR1 reveals remarkable differences from the known channelrhodopsins and a unique ion-conducting pathway. Thus, viral rhodopsins 1 represent a unique, large group of light-gated channels (viral channelrhodopsins, VirChR1s). In nature, VirChR1s likely mediate phototaxis of algae enhancing the host anabolic processes to support virus reproduction, and therefore, might play a major role in global phytoplankton dynamics. Moreover, VirChR1s have unique potential for optogenetics as they lack possibly noxious Ca2+ permeability.

Multichannel optogenetic stimulation of the auditory pathway using microfabricated LED cochlear implants in rodents.

When hearing fails, electrical cochlear implants (eCIs) provide the brain with auditory information. One important bottleneck of CIs is the poor spectral selectivity that results from the wide current spread from each of the electrode contacts. Optical CIs (oCIs) promise to make better use of the tonotopic order of spiral ganglion neurons (SGNs) inside the cochlea by spatially confined stimulation. Here, we established multichannel oCIs based on light-emitting diode (LED) arrays and used them for optical stimulation of channelrhodopsin (ChR)-expressing SGNs in rodents. Power-efficient blue LED chips were integrated onto microfabricated 15-µm-thin polyimide-based carriers comprising interconnecting lines to address individual LEDs by a stationary or mobile driver circuitry. We extensively characterized the optoelectronic, thermal, and mechanical properties of the oCIs and demonstrated stability over weeks in vitro. We then implanted the oCIs into ChR-expressing rats and gerbils, and characterized multichannel optogenetic SGN stimulation by electrophysiological and behavioral experiments. Improved spectral selectivity was directly demonstrated by recordings from the auditory midbrain. Long-term experiments in deafened ChR-expressing rats and in nontreated control animals demonstrated specificity of optogenetic stimulation. Behavioral studies on animals carrying a wireless oCI sound processor revealed auditory percepts. This study demonstrates hearing restoration with improved spectral selectivity by an LED-based multichannel oCI system.

µLED-based optical cochlear implants for spectrally selective activation of the auditory nerve.

Electrical cochlear implants (eCIs) partially restore hearing and enable speech comprehension to more than half a million users, thereby re-connecting deaf patients to the auditory scene surrounding them. Yet, eCIs suffer from limited spectral selectivity, resulting from current spread around each electrode contact and causing poor speech recognition in the presence of background noise. Optogenetic stimulation of the auditory nerve might overcome this limitation as light can be conveniently confined in space. Here, we combined virus-mediated optogenetic manipulation of cochlear spiral ganglion neurons (SGNs) and microsystems engineering to establish acute multi-channel optical cochlear implant (oCI) stimulation in adult Mongolian gerbils. oCIs based on 16 microscale thin-film light-emitting diodes (µLEDs) evoked tonotopic activation of the auditory pathway with high spectral selectivity and modest power requirements in hearing and deaf gerbils. These results prove the feasibility of µLED-based oCIs for spectrally selective activation of the auditory nerve.

Overloaded Adeno-Associated Virus as a Novel Gene Therapeutic Tool for Otoferlin-Related Deafness.

Hearing impairment is the most common sensory disorder in humans. So far, rehabilitation of profoundly deaf subjects relies on direct stimulation of the auditory nerve through cochlear implants. However, in some forms of genetic hearing impairment, the organ of Corti is structurally intact and therapeutic replacement of the mutated gene could potentially restore near natural hearing. In the case of defects of the otoferlin gene (OTOF), such gene therapy is hindered by the size of the coding sequence (~6 kb) exceeding the cargo capacity (<5 kb) of the preferred viral vector, adeno-associated virus (AAV). Recently, a dual-AAV approach was used to partially restore hearing in deaf otoferlin knock-out (Otof-KO) mice. Here, we employed in vitro and in vivo approaches to assess the gene-therapeutic potential of naturally-occurring and newly-developed synthetic AAVs overloaded with the full-length Otof coding sequence. Upon early postnatal injection into the cochlea of Otof-KO mice, overloaded AAVs drove specific expression of otoferlin in ~30% of all IHCs, as demonstrated by immunofluorescence labeling and polymerase chain reaction. Recordings of auditory brainstem responses and a behavioral assay demonstrated partial restoration of hearing. Together, our results suggest that viral gene therapy of DFNB9-using a single overloaded AAV vector-is indeed feasible, reducing the complexity of gene transfer compared to dual-AAV approaches.

Near physiological spectral selectivity of cochlear optogenetics.

Cochlear implants (CIs) electrically stimulate spiral ganglion neurons (SGNs) and partially restore hearing to half a million CI users. However, wide current spread from intracochlear electrodes limits spatial selectivity (i.e. spectral resolution) of electrical CIs. Optogenetic stimulation might become an alternative, since light can be confined in space, promising artificial sound encoding with increased spectral selectivity. Here we compare spectral selectivity of optogenetic, electric, and acoustic stimulation by multi-channel recordings in the inferior colliculus (IC) of gerbils. When projecting light onto tonotopically distinct SGNs, we observe corresponding tonotopically ordered IC activity. An activity-based comparison reveals that spectral selectivity of optogenetic stimulation is indistinguishable from acoustic stimulation for modest intensities. Moreover, optogenetic stimulation outperforms bipolar electric stimulation at medium and high intensities and monopolar electric stimulation at all intensities. In conclusion, we demonstrate better spectral selectivity of optogenetic over electric SGN stimulation, suggesting the potential for improved hearing restoration by optical CIs.

Toward the Optical Cochlear Implant.

When hearing fails, cochlear implants (CIs) provide open speech perception to most of the currently half a million CI users. CIs bypass the defective sensory organ and stimulate the auditory nerve electrically. The major bottleneck of current CIs is the poor coding of spectral information, which results from wide current spread from each electrode contact. As light can be more conveniently confined, optical stimulation of the auditory nerve presents a promising perspective for a fundamental advance of CIs. Moreover, given the improved frequency resolution of optical excitation and its versatility for arbitrary stimulation patterns the approach also bears potential for auditory research. Here, we review the current state of the art focusing on the emerging concept of optogenetic stimulation of the auditory pathway. Developing optogenetic stimulation for auditory research and future CIs requires efforts toward viral gene transfer to the neurons, design and characterization of appropriate optogenetic actuators, as well as engineering of multichannel optical implants.

Ultrafast optogenetic stimulation of the auditory pathway by targeting-optimized Chronos.

Optogenetic tools, providing non-invasive control over selected cells, have the potential to revolutionize sensory prostheses for humans. Optogenetic stimulation of spiral ganglion neurons (SGNs) in the ear provides a future alternative to electrical stimulation used in cochlear implants. However, most channelrhodopsins do not support the high temporal fidelity pertinent to auditory coding because they require milliseconds to close after light-off. Here, we biophysically characterized the fast channelrhodopsin Chronos and revealed a deactivation time constant of less than a millisecond at body temperature. In order to enhance neural expression, we improved its trafficking to the plasma membrane (Chronos-ES/TS). Following efficient transduction of SGNs using early postnatal injection of the adeno-associated virus AAV-PHPB into the mouse cochlea, fiber-based optical stimulation elicited optical auditory brainstem responses (oABR) with minimal latencies of 1 ms, thresholds of 5 µJ and 100 µs per pulse, and sizable amplitudes even at 1,000 Hz of stimulation. Recordings from single SGNs demonstrated good temporal precision of light-evoked spiking. In conclusion, efficient virus-mediated expression of targeting-optimized Chronos-ES/TS achieves ultrafast optogenetic control of neurons.

High frequency neural spiking and auditory signaling by ultrafast red-shifted optogenetics.

Optogenetics revolutionizes basic research in neuroscience and cell biology and bears potential for medical applications. We develop mutants leading to a unifying concept for the construction of various channelrhodopsins with fast closing kinetics. Due to different absorption maxima these channelrhodopsins allow fast neural photoactivation over the whole range of the visible spectrum. We focus our functional analysis on the fast-switching, red light-activated Chrimson variants, because red light has lower light scattering and marginal phototoxicity in tissues. We show paradigmatically for neurons of the cerebral cortex and the auditory nerve that the fast Chrimson mutants enable neural stimulation with firing frequencies of several hundred Hz. They drive spiking at high rates and temporal fidelity with low thresholds for stimulus intensity and duration. Optical cochlear implants restore auditory nerve activity in deaf mice. This demonstrates that the mutants facilitate neuroscience research and future medical applications such as hearing restoration.

A combination of NMDA and AMPA receptor antagonists retards granule cell dispersion and epileptogenesis in a model of acquired epilepsy.

Epilepsy may arise following acute brain insults, but no treatments exist that prevent epilepsy in patients at risk. Here we examined whether a combination of two glutamate receptor antagonists, NBQX and ifenprodil, acting at different receptor subtypes, exerts antiepileptogenic effects in the intrahippocampal kainate mouse model of epilepsy. These drugs were administered over 5 days following kainate. Spontaneous seizures were recorded by video/EEG at different intervals up to 3 months. Initial trials showed that drug treatment during the latent period led to higher mortality than treatment after onset of epilepsy, and further, that combined therapy with both drugs caused higher mortality at doses that appear safe when used singly. We therefore refined the combined-drug protocol, using lower doses. Two weeks after kainate, significantly less mice of the NBQX/ifenprodil group exhibited electroclinical seizures compared to vehicle controls, but this effect was lost at subsequent weeks. The disease modifying effect of the treatment was associated with a transient prevention of granule cell dispersion and less neuronal degeneration in the dentate hilus. These data substantiate the involvement of altered glutamatergic transmission in the early phase of epileptogenesis. Longer treatment with NBQX and ifenprodil may shed further light on the apparent temporal relationship between dentate gyrus reorganization and development of spontaneous seizures.

Refinement of a model of acquired epilepsy for identification and validation of biomarkers of epileptogenesis in rats.

In rodent models in which status epilepticus (SE) is used to induce epilepsy, typically most animals develop spontaneous recurrent seizures (SRS). The SE duration for induction of epileptogenesis depends on the type of SE induction. In models with electrical SE induction, the minimum duration of SE to induce epileptogenesis in >90% of animals ranges from 3-4h. A high incidence of epilepsy is an advantage in the search of antiepileptogenic treatments, whereas it is a disadvantage in the search for biomarkers of epileptogenesis, because it does not allow a comparison of potential biomarkers in animals that either develop or do not develop epilepsy. The aim of this project was the refinement of an established SE rat model so that only ~50% of the animals develop epilepsy. For this purpose, we used an electrical model of SE induction, in which a self-sustained SE develops after prolonged stimulation of the basolateral amygdala. Previous experiments had shown that the majority of rats develop SRS after 4-h SE in this model so that the SE reduced duration to 2.5h by administering diazepam. This resulted in epilepsy development in only 50% of rats, thus reaching the goal of the project. The latent period to onset of SRS wa s >2weeks in most rats. Development of epilepsy could be predicted in most rats by behavioral hyperexcitability, whereas seizure threshold did not differentiate rats that did and did not develop SRS. The refined SE model may offer a platform to identify and validate biomarkers of epileptogenesis.

Network pharmacology for antiepileptogenesis: Tolerability of multitargeted drug combinations in nonepileptic vs. post-status epilepticus mice.

Prevention of symptomatic epilepsy ("antiepileptogenesis") in patients at risk is a major unmet clinical need. Several drugs underwent clinical trials for epilepsy prevention, but none of the drugs tested was effective. Similarly, most previous preclinical attempts to develop antiepileptogenic strategies failed. In the majority of studies, drugs were given as monotherapy. However, epilepsy is a complex network phenomenon, so that it is unlikely that a single drug can halt epileptogenesis. We recently proposed multitargeted approaches ("network pharmacology") to interfere with epileptogenesis. One strategy, which, if effective, would allow a relatively rapid translation into the clinic, is developing novel combinations of clinically used drugs with diverse mechanisms that are potentially relevant for antiepileptogenesis. In order to test this strategy preclinically, we developed an algorithm for testing such drug combinations, which was inspired by the established drug development phases in humans. As a first step of this algorithm, tolerability of four rationally chosen, repeatedly administered drug combinations was evaluated by a large test battery in mice: A, levetiracetam and phenobarbital; B, valproate, losartan, and memantine; C, levetiracetam and topiramate; and D, levetiracetam, parecoxib, and anakinra. As in clinical trials, tolerability was separately evaluated before starting efficacy experiments to identify any adverse effects of the combinations that may critically limit the successful translation of preclinical findings to the clinic. Except combination B, all drug cocktails were relatively well tolerated. Based on previous studies, we expected that tolerability would be lower in the latent and chronic phases following status epilepticus in mice, but, except combinations C and D, no significant differences were determined between nonepileptic and post-status epilepticus animals. As a next step, the rationally chosen drug combinations will be evaluated for antiepileptogenic activity in mouse and rat models of symptomatic epilepsy.

Zinc deficiency dysregulates the synaptic ProSAP/Shank scaffold and might contribute to autism spectrum disorders.

Proteins of the ProSAP/Shank family act as major organizing scaffolding elements within the postsynaptic density of excitatory synapses. Deletions, mutations or the downregulation of these molecules has been linked to autism spectrum disorders, the related Phelan McDermid Syndrome or Alzheimer's disease. ProSAP/Shank proteins are targeted to synapses depending on binding to zinc, which is a prerequisite for the assembly of the ProSAP/Shank scaffold. To gain insight into whether the previously reported assembly of ProSAP/Shank through zinc ions provides a crossing point between genetic forms of autism spectrum disorder and zinc deficiency as an environmental risk factor for autism spectrum disorder, we examined the interplay between zinc and ProSAP/Shank in vitro and in vivo using neurobiological approaches. Our data show that low postsynaptic zinc availability affects the activity dependent increase in ProSAP1/Shank2 and ProSAP2/Shank3 levels at the synapse in vitro and that a loss of synaptic ProSAP1/Shank2 and ProSAP2/Shank3 occurs in a mouse model for acute and prenatal zinc deficiency. Zinc-deficient animals displayed abnormalities in behaviour such as over-responsivity and hyperactivity-like behaviour (acute zinc deficiency) and autism spectrum disorder-related behaviour such as impairments in vocalization and social behaviour (prenatal zinc deficiency). Most importantly, a low zinc status seems to be associated with an increased incidence rate of seizures, hypotonia, and attention and hyperactivity issues in patients with Phelan-McDermid syndrome, which is caused by haploinsufficiency of ProSAP2/Shank3. We suggest that the molecular underpinning of prenatal zinc deficiency as a risk factor for autism spectrum disorder may unfold through the deregulation of zinc-binding ProSAP/Shank family members.

Encoding and transducing the synaptic or extrasynaptic origin of NMDA receptor signals to the nucleus.

The activation of N-methyl-D-aspartate-receptors (NMDARs) in synapses provides plasticity and cell survival signals, whereas NMDARs residing in the neuronal membrane outside synapses trigger neurodegeneration. At present, it is unclear how these opposing signals are transduced to and discriminated by the nucleus. In this study, we demonstrate that Jacob is a protein messenger that encodes the origin of synaptic versus extrasynaptic NMDAR signals and delivers them to the nucleus. Exclusively synaptic, but not extrasynaptic, NMDAR activation induces phosphorylation of Jacob at serine-180 by ERK1/2. Long-distance trafficking of Jacob from synaptic, but not extrasynaptic, sites depends on ERK activity, and association with fragments of the intermediate filament α-internexin hinders dephosphorylation of the Jacob/ERK complex during nuclear transit. In the nucleus, the phosphorylation state of Jacob determines whether it induces cell death or promotes cell survival and enhances synaptic plasticity.