RESUMEN
Currently, there are no approved medicines available for the treatment of hearing loss. However, research over the past two decades has contributed to a growing understanding of the pathological mechanisms in the cochlea that result in hearing difficulties. The concept that a loss of the synapses connecting inner hair cells with the auditory nerve (cochlear synaptopathy) contributes to hearing loss has gained considerable attention. Both animal and human post-mortem studies support the idea that these synapses (ribbon synapses) are highly vulnerable to noise, ototoxicity, and the aging process. Their degeneration has been suggested as an important factor in the speech-in-noise difficulties commonly experienced by those suffering with hearing loss. Neurotrophins such as brain derived neurotrophic factor (BDNF) have the potential to restore these synapses and provide improved hearing function. OTO-413 is a sustained exposure formulation of BDNF suitable for intratympanic administration that in preclinical models has shown the ability to restore ribbon synapses and provide functional hearing benefit. A phase 1/2 clinical trial with OTO-413 has provided initial proof-of-concept for improved speech-in-noise hearing performance in subjects with hearing loss. Key considerations for the design of this clinical study, including aspects of the speech-in-noise assessments, are discussed.
Asunto(s)
Sordera , Pérdida Auditiva , Animales , Factor Neurotrófico Derivado del Encéfalo , Cóclea , Audición , Humanos , Modelos AnimalesRESUMEN
The neurotrophin growth factors bind and activate two types of cell surface receptors: the tropomyosin receptor kinase (Trk) family and p75. TrkA, TrkB, and TrkC are bound preferentially by nerve growth factor, brain-derived neurotrophic factor, and neurotrophin 3 (NT3), respectively, to activate neuroprotective signals. The p75 receptors are activated by all neurotrophins, and paradoxically in neurodegenerative disease p75 is upregulated and mediates neurotoxic signals. To test neuroprotection strategies, we engineered NT3 to broadly activate Trk receptors (mutant D) or to reduce p75 binding (mutant RK). We also combined these features in a molecule that activates TrkA, TrkB, and TrkC but has reduced p75 binding (mutant DRK). In neurodegenerative disease mouse models in vivo, the DRK protein is a superior therapeutic agent compared with mutant D, mutant RK, and wild-type neurotrophins and protects a broader range of stressed neurons. This work rationalizes a therapeutic strategy based on the biology of each type of receptor, avoiding activation of p75 toxicity while broadly activating neuroprotection in stressed neuronal populations expressing different Trk receptors. SIGNIFICANCE STATEMENT: The neurotrophins nerve growth factor, brain-derived neurotrophic factor, and neurotrophin 3 each can activate a tropomyosin receptor kinase (Trk) A, TrkB, or TrkC receptor, respectively, and all can activate a p75 receptor. Trks and p75 mediate opposite signals. We report the engineering of a protein that activates all Trks, combined with low p75 binding, as an effective therapeutic agent in vivo.
Asunto(s)
Factores de Crecimiento Nervioso/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Neuroprotección/fisiología , Ingeniería de Proteínas/métodos , Receptor trkA/metabolismo , Receptores de Factores de Crecimiento/metabolismo , Animales , Axotomía/efectos adversos , Neuropatías Diabéticas/tratamiento farmacológico , Neuropatías Diabéticas/genética , Neuropatías Diabéticas/metabolismo , Relación Dosis-Respuesta a Droga , Células HEK293 , Humanos , Masculino , Ratones , Ratones Endogámicos C57BL , Células 3T3 NIH , Factores de Crecimiento Nervioso/administración & dosificación , Factores de Crecimiento Nervioso/genética , Proteínas del Tejido Nervioso/genética , Neuroprotección/efectos de los fármacos , Nervio Óptico/efectos de los fármacos , Nervio Óptico/metabolismo , Receptor trkA/genética , Receptores de Factores de Crecimiento/genéticaRESUMEN
Neurotrophins and their mimetics are potential treatments for hearing disorders because of their trophic effects on spiral ganglion neurons (SGNs) whose connections to hair cells may be compromised in many forms of hearing loss. Studies in noise or ototoxin-exposed animals have shown that local delivery of NT-3 or BDNF has beneficial effects on SGNs and hearing. We evaluated several TrkB or TrkC monoclonal antibody agonists and small molecules, along with BDNF and NT-3, in rat cochlea ex vivo models. The TrkB agonists BDNF and a monoclonal antibody, M3, had the greatest effects on SGN survival, neurite outgrowth and branching. In organotypic cochlear explants, BDNF and M3 enhanced synapse formation between SGNs and inner hair cells and restored these connections after excitotoxin-induced synaptopathy. Loss of these synapses has recently been implicated in hidden hearing loss, a condition characterized by difficulty hearing speech in the presence of background noise. The unique profile of M3 revealed here warrants further investigation, and the broad activity profile of BDNF observed underpins its continued development as a hearing loss therapeutic.
Asunto(s)
Anticuerpos Monoclonales/inmunología , Factor Neurotrófico Derivado del Encéfalo/farmacología , Cóclea/citología , Pérdida Auditiva/patología , Neuritas/metabolismo , Receptor trkA/agonistas , Sinapsis/metabolismo , Animales , Línea Celular , Supervivencia Celular , Modelos Animales de Enfermedad , Pérdida Auditiva/inmunología , Humanos , Neuritas/efectos de los fármacos , Neuritas/inmunología , Ratas , Receptor trkA/inmunología , Sinapsis/efectos de los fármacos , Sinapsis/inmunologíaRESUMEN
NMDA receptors, which regulate synaptic strength and are implicated in learning and memory, consist of several subtypes with distinct subunit compositions and functional properties. To enable spatiotemporally defined, rapid and reproducible manipulation of function of specific subtypes, we engineered a set of photoswitchable GluN subunits ('LiGluNs'). Photo-agonism of GluN2A or GluN2B elicits an excitatory drive to hippocampal neurons that can be shaped in time to mimic synaptic activation. Photo-agonism of GluN2A at single dendritic spines evokes spine-specific calcium elevation and expansion, the morphological correlate of LTP. Photo-antagonism of GluN2A alone, or in combination with photo-antagonism of GluN1a, reversibly blocks excitatory synaptic currents, prevents the induction of long-term potentiation and prevents spine expansion. In addition, photo-antagonism in vivo disrupts synaptic pruning of developing retino-tectal projections in larval zebrafish. By providing precise and rapidly reversible optical control of NMDA receptor subtypes, LiGluNs should help unravel the contribution of specific NMDA receptors to synaptic transmission, integration and plasticity.
Asunto(s)
Luz , Receptores de N-Metil-D-Aspartato/metabolismo , Receptores de N-Metil-D-Aspartato/efectos de la radiación , Potenciales de Acción , Animales , Hipocampo/fisiología , Neuronas/fisiología , Receptores de N-Metil-D-Aspartato/genética , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Pez Cebra/embriologíaRESUMEN
In the vibrant field of optogenetics, optics and genetic targeting are combined to commandeer cellular functions, such as the neuronal action potential, by optically stimulating light-sensitive ion channels expressed in the cell membrane. One broadly applicable manifestation of this approach are covalently attached photochromic tethered ligands (PTLs) that allow activating ligand-gated ion channels with outstanding spatial and temporal resolution. Here, we describe all steps towards the successful development and application of PTL-gated ion channels in cell lines and primary cells. The basis for these experiments forms a combination of molecular modeling, genetic engineering, cell culture, and electrophysiology. The light-gated glutamate receptor (LiGluR), which consists of the PTL-functionalized GluK2 receptor, serves as a model.
Asunto(s)
Optogenética/métodos , Receptores de Ácido Kaínico/genética , Animales , Técnicas de Cultivo de Célula , Disección , Fenómenos Electrofisiológicos , Glutamatos/metabolismo , Células HEK293 , Hipocampo/citología , Hipocampo/fisiología , Humanos , Mutagénesis , Mutación , Neuronas/citología , Neuronas/metabolismo , Técnicas de Placa-Clamp , Reacción en Cadena de la Polimerasa , Ratas , Receptores de Ácido Kaínico/metabolismo , Transfección , Receptor de Ácido Kaínico GluK2RESUMEN
Genetically targeted light-activated ion channels and pumps make it possible to determine the role of specific neurons in neuronal circuits, information processing and behavior. We developed a K+-selective ionotropic glutamate receptor that reversibly inhibits neuronal activity in response to light in dissociated neurons and brain slice and also reversibly suppresses behavior in zebrafish. The receptor is a chimera of the pore region of a K+-selective bacterial glutamate receptor and the ligand-binding domain of a light-gated mammalian kainate receptor. This hyperpolarizing light-gated channel, HyLighter, is turned on by a brief light pulse at one wavelength and turned off by a pulse at a second wavelength. The control is obtained at moderate intensity. After optical activation, the photocurrent and optical silencing of activity persists in the dark for extended periods. The low light requirement and bi-stability of HyLighter represent advantages for the dissection of neural circuitry.
Asunto(s)
Neuronas/fisiología , Procesos Fotoquímicos , Canales de Potasio/metabolismo , Receptores de Glutamato/metabolismo , Animales , Animales Modificados Genéticamente , Línea Celular , Quimera , Potenciales Evocados , Hipocampo/fisiología , Humanos , Luz , Maleimidas/metabolismo , Mutagénesis Sitio-Dirigida , Técnicas de Placa-Clamp , Ratas , Transfección , Pez CebraRESUMEN
Advances in optics, genetics, and chemistry have enabled the investigation of brain function at all levels, from intracellular signals to single synapses, whole cells, circuits, and behavior. Until recent years, these research tools have been utilized in an observational capacity: imaging neural activity with fluorescent reporters, for example, or correlating aberrant neural activity with loss-of-function and gain-of-function pharmacological or genetic manipulations. However, optics, genetics, and chemistry have now combined to yield a new strategy: using light to drive and halt neuronal activity with molecular specificity and millisecond precision. Photostimulation of neurons is noninvasive, has unmatched spatial and temporal resolution, and can be targeted to specific classes of neurons. The optical methods developed to date encompass a broad array of strategies, including photorelease of caged neurotransmitters, engineered light-gated receptors and channels, and naturally light-sensitive ion channels and pumps. In this review, we describe photostimulation methods, their applications, and opportunities for further advancement.
Asunto(s)
Neuronas/fisiología , Óptica y Fotónica/métodos , Animales , Transducción de SeñalRESUMEN
Photoswitched tethered ligands (PTLs) can be used to remotely control protein function with light. We have studied the geometric and conformational factors that determine the efficacy of PTL gating in the ionotropic glutamate receptor iGluR6 using a family of photoiosomerizable MAG (maleimide-azobenzene-glutamate) PTLs that covalently attach to the clamshell ligand-binding domain. Experiments and molecular dynamics simulations of the modified proteins show that optical switching depends on 2 factors: (i) the relative occupancy of the binding pocket in the 2 photoisomers of MAG and (ii) the degree of clamshell closure that is possible given the disposition of the MAG linker. A synthesized short version of MAG turns the channel on in either the cis or trans state, depending on the point of attachment. This yin/yang optical control makes it possible for 1 wavelength of light to elicit action potentials in one set of neurons, while deexciting a second set of neurons in the same preparation, whereas a second wavelength has the opposite effect. The ability to generate opposite responses with a single PTL and 2 versions of a target channel, which can be expressed in different cell types, paves the way for engineering opponency in neurons that mediate opposing functions.
Asunto(s)
Luz , Nanotecnología/métodos , Receptores de Glutamato/metabolismo , Animales , Compuestos Azo/metabolismo , Línea Celular , Simulación por Computador , Ácido Glutámico/metabolismo , Humanos , Activación del Canal Iónico , Ligandos , Maleimidas/metabolismo , Modelos Moleculares , Neuronas/metabolismo , Técnicas de Placa-Clamp , Conformación Proteica , Quinoxalinas , Ratas , Receptores de Glutamato/química , Estereoisomerismo , VolumetríaRESUMEN
A key technical barrier to furthering our understanding of complex neural networks has been the lack of tools for the simultaneous spatiotemporal control and detection of activity in a large number of neurons. Here, we report an all-optical system for achieving this kind of parallel and selective control and detection. We do this by delivering spatiotemporally complex optical stimuli through a digital micromirror spatiotemporal light modulator to cells expressing the light-activated ionotropic glutamate receptor (LiGluR), which have been labeled with a calcium dye to provide a fluorescent report of activity. Reliable and accurate spatiotemporal stimulation was obtained on HEK293 cells and cultured rat hippocampal neurons. This technique should be adaptable to in vivo applications and could serve as an optical interface for communicating with complex neural circuits.
Asunto(s)
Neuronas/fisiología , Animales , Calcio/fisiología , Línea Celular , Células Cultivadas , Hipocampo/fisiología , Humanos , Riñón/citología , Riñón/fisiología , Luz , Ratas , Receptores AMPA/fisiología , Receptores de Glutamato Metabotrópico/fisiología , Receptores de Glutamato Metabotrópico/efectos de la radiaciónRESUMEN
The analysis of cell signaling requires the rapid and selective manipulation of protein function. We have synthesized photoswitches that covalently modify target proteins and reversibly present and withdraw a ligand from its binding site due to photoisomerization of an azobenzene linker. We describe here the properties of a glutamate photoswitch that controls an ion channel in cells. Affinity labeling and geometric constraints ensure that the photoswitch controls only the targeted channel, and enables spatial patterns of light to favor labeling in one location over another. Photoswitching to the activating state places a tethered glutamate at a high (millimolar) effective local concentration near the binding site. The fraction of active channels can be set in an analog manner by altering the photostationary state with different wavelengths. The bistable photoswitch can be turned on with millisecond-long pulses at one wavelength, remain on in the dark for minutes, and turned off with millisecond long pulses at the other wavelength, yielding sustained activation with minimal irradiation. The system provides rapid, reversible remote control of protein function that is selective without orthogonal chemistry.
Asunto(s)
Sistema Libre de Células , Canales Iónicos/metabolismo , Luz , Receptores de Ácido Kaínico/metabolismo , Sitios de Unión , Investigación Biomédica/métodos , Modelos Biológicos , Proteínas/fisiología , Receptores de Ácido Kaínico/efectos de la radiaciónRESUMEN
The ability to stimulate select neurons in isolated tissue and in living animals is important for investigating their role in circuits and behavior. We show that the engineered light-gated ionotropic glutamate receptor (LiGluR), when introduced into neurons, enables remote control of their activity. Trains of action potentials are optimally evoked and extinguished by 380 nm and 500 nm light, respectively, while intermediate wavelengths provide graded control over the amplitude of depolarization. Light pulses of 1-5 ms in duration at approximately 380 nm trigger precisely timed action potentials and EPSP-like responses or can evoke sustained depolarizations that persist for minutes in the dark until extinguished by a short pulse of approximately 500 nm light. When introduced into sensory neurons in zebrafish larvae, activation of LiGluR reversibly blocks the escape response to touch. Our studies show that LiGluR provides robust control over neuronal activity, enabling the dissection and manipulation of neural circuitry in vivo.
Asunto(s)
Conducta Animal/fisiología , Iluminación/métodos , Neuronas/fisiología , Receptores de Ácido Kaínico/fisiología , Potenciales de Acción/fisiología , Potenciales de Acción/efectos de la radiación , Animales , Animales Modificados Genéticamente , Animales Recién Nacidos , Conducta Animal/efectos de la radiación , Células Cultivadas , Cisteína/genética , Relación Dosis-Respuesta en la Radiación , Estimulación Eléctrica/métodos , Potenciales Postsinápticos Excitadores , Hipocampo/citología , Larva , Leucina/genética , Mutación , Neuronas/efectos de los fármacos , Neuronas/efectos de la radiación , Técnicas de Placa-Clamp/métodos , Estimulación Física/métodos , Ratas , Receptores de Ácido Kaínico/genética , Transfección/métodos , Pez Cebra , Receptor de Ácido Kaínico GluK2Asunto(s)
Ácido Glutámico/farmacología , Ácido Glutámico/efectos de la radiación , Receptores de Glutamato/efectos de los fármacos , Rayos Ultravioleta , Potenciales de Acción/efectos de los fármacos , Potenciales de Acción/efectos de la radiación , Animales , Células Cultivadas , Relación Dosis-Respuesta a Droga , Ácido Glutámico/análogos & derivados , Humanos , Conformación Molecular , Neuronas/efectos de los fármacos , Neuronas/fisiología , Fotoquímica , Ratas , Receptores de Glutamato/metabolismo , Relación Estructura-ActividadRESUMEN
Because ice-I is less dense than water, the formation of an ice nucleus in an isochoric (constant volume) chamber will cause an increase in pressure. This analysis shows that the energy required to overcome such a pressure increase makes homogeneous ice nucleation thermodynamically improbable in an isochoric system at temperatures above -109 degrees C. By suppressing ice nucleation, isochoric cooling is expected to significantly promote vitrification. Because water has a higher freezing temperature and a lower glass-transition temperature than physiological solutions, this analysis represents a scenario for avoiding ice crystallization during the preservation of biological substances. While isochoric cryopreservation has not yet been put into practice, this theoretical, first-order analysis suggests that if attainable it could make organ preservation significantly more effective and practical.
Asunto(s)
Criopreservación/métodos , Congelación , Conservación de Tejido/métodos , Frío , Hielo , Transición de Fase , PresiónRESUMEN
Many wheelchair users must travel in motor vehicles while seated in their wheelchairs. The safety features of seat assemblies are key to motor vehicle occupant crash protection. Seating system properties such as strength, stiffness, and energy absorbance have been shown to have significant influence on risk of submarining. This study investigated the effects of wheelchair seat stiffness and energy absorption properties on occupant risk of submarining during a frontal motor vehicle 20 g/30 mph impact using a validated computer crash simulation model. The results indicate that wheelchair-seating stiffness and energy absorption characteristics influence occupant kinematics associated with the risk of submarining. Softer seat surfaces and relatively high energy absorption/permanent deformation were found to produce pelvis excursion trajectories associated with increased submarining risk. Findings also suggest that the current American National Standards Institute/Rehabilitation Engineering and Assistive Technology Society of North America (ANSI/RESNA) WC-19 seating integrity may not adequately assess submarining risk.