Site-specific gains and losses of heterochromatin accelerate the age-related neurodegeneration through the cascading destruction of KDM3B-centered epigenomic network

Epigenetic alterations explained by the “loss of heterochromatin” model have been proposed as a universal mechanism of aging, but the region-specific changes of heterochromatin during aging are unclear. Here, we examine age-dependent transcriptomic profiling of mouse retinal neurons to identify epigenetic regulators involved in heterochromatin loss. RNA sequencing analysis revealed gradual down-regulation of Kdm3b during retinal aging. Disruption of Kdm3b (Kdm3b+/-) in 12-month-old mouse retina decreased the number of cone photoreceptors and changed the morphology of cone ribbon synapses. Integration of transcriptome profiling with epigenomic analysis demonstrated gain of heterochromatin feature in synapse assembly and vesicle transport genes via the accumulation of H3K9 mono- and di-methylation. However, the loss of heterochromatin in apoptotic genes exacerbated retinal neurodegeneration. We propose that this KDM3B-centered epigenomic network is crucial for maintaining cone photoreceptor homeostasis via the modulation of gene-set specific heterochromatin features during aging.

Temporal and Isoform-Specific Expression of CTBP2 Is Evolutionarily Conserved Between the Developing Chick and Human Retina

Complex transcriptional gene regulation allows for multifaceted isoform production during retinogenesis, and novel isoforms transcribed from a single locus can have unlimited potential to code for diverse proteins with different functions. In this study, we explored the CTBP2/RIBEYE gene locus and its unique repertoire of transcripts that are conserved among vertebrates. We studied the transcriptional coregulator (CTBP2) and ribbon synapse-specific structural protein (RIBEYE) in the chicken retina by performing comprehensive histochemical and sequencing analyses to pinpoint cell and developmental stage-specific expression of CTBP2/RIBEYE in the developing chicken retina. We demonstrated that CTBP2 is widely expressed in retinal progenitors beginning in early retinogenesis but becomes limited to GABAergic amacrine cells in the mature retina. Inversely, RIBEYE is initially epigenetically silenced in progenitors and later expressed in photoreceptor and bipolar cells where they localize to ribbon synapses. Finally, we compared CTBP2/RIBEYE regulation in the developing human retina using a pluripotent stem cell derived retinal organoid culture system. These analyses demonstrate that similar regulation of the CTBP2/RIBEYE locus during chick and human retinal development is regulated by different members of the K50 homeodomain transcription factor family.

Cell Type-Selective Loss of Peroxisomal β-Oxidation Impairs Bipolar Cell but Not Photoreceptor Survival in the Retina

Retinal degeneration is a common feature in peroxisomal disorders leading to blindness. Peroxisomes are present in the different cell types of the retina; however, their precise contribution to retinal integrity is still unclear. We previously showed that mice lacking the central peroxisomal β-oxidation enzyme, multifunctional protein 2 (MFP2), develop an early onset retinal decay including photoreceptor cell death. To decipher the function of peroxisomal β-oxidation in photoreceptors, we generated cell type selective Mfp2 knockout mice, using the Crx promotor targeting photoreceptors and bipolar cells. Surprisingly, Crx-Mfp2−/− mice maintained photoreceptor length and number until the age of 1 year. A negative electroretinogram was indicative of preserved photoreceptor phototransduction, but impaired downstream bipolar cell signaling from the age of 6 months. The photoreceptor ribbon synapse was affected, containing free-floating ribbons and vesicles with altered size and density. The bipolar cell interneurons sprouted into the ONL and died. Whereas docosahexaenoic acid levels were normal in the neural retina, levels of lipids containing very long chain polyunsaturated fatty acids were highly increased. Crx-Pex5−/− mice, in which all peroxisomal functions are inactivated in photoreceptors and bipolar cells, developed the same phenotype as Crx-Mfp2−/− mice. In conclusion, the early photoreceptor death in global Mfp2−/− mice is not driven cell autonomously. However, peroxisomal β-oxidation is essential for the integrity of photoreceptor ribbon synapses and of bipolar cells.

Electrocochleography in Auditory Neuropathy Related to Mutations in the OTOF or OPA1 Gene

Auditory Neuropathy (AN) is a hearing disorder characterized by disruption of temporal coding of acoustic signals in auditory nerve fibers resulting in the impairment of auditory perceptions that rely on temporal cues. Mutations in several nuclear and mitochondrial genes have been associated to the most well-known forms of AN. Underlying mechanisms include both pre-synaptic and post-synaptic disorders affecting inner hair cell (IHC) depolarization, neurotransmitter release from ribbon synapses, spike initiation in auditory nerve terminals, loss of nerve fibers and impaired conduction, all occurring in the presence of normal physiological measures of outer hair cell (OHC) activities (otoacoustic emissions [OAEs] and cochlear microphonic [CM]). Disordered synchrony of auditory nerve activity has been suggested as the basis of both the profound alterations of auditory brainstem responses (ABRs) and impairment of speech perception. We will review how electrocochleography (ECochG) recordings provide detailed information to help objectively define the sites of auditory neural dysfunction and their effect on inner hair cell receptor summating potential (SP) and compound action potential (CAP), the latter reflecting disorders of ribbon synapses and auditory nerve fibers.

Auditory-Nerve Responses in Mice with Noise-Induced Cochlear Synaptopathy

Cochlear synaptopathy is the noise-induced or age-related loss of ribbon synapses between inner hair cells (IHCs) and auditory nerve fibers (ANFs), first reported in CBA/CaJ mice. Recordings from single ANFs in anaesthesized, noise-exposed guinea pigs suggested that neurons with low spontaneous rates (SRs) and high thresholds are more vulnerable than low-threshold, high-SR fibers. However, there is extensive post-exposure regeneration of ANFs in guinea pigs, but not in mice. Here, we exposed CBA/CaJ mice to octave-band noise and recorded sound-evoked and spontaneous activity from single ANFs at least 2 weeks later. Confocal analysis of cochleae immunostained for pre- and post-synaptic markers confirmed the expected loss of 40 - 50% of ANF synapses in the basal half of the cochlea, however, our data were not consistent with a selective loss of low-SR fibers. Rather they suggested a loss of both SR groups in synaptopathic regions. Single-fiber thresholds and frequency tuning recovered to pre-exposure levels however, response to tone bursts showed increased peak and steady-state firing rates as well as decreased jitter in first-spike latencies. This apparent gain-of-function increased the robustness of tone-burst responses in the presence of continuous masking noise. This study suggests that the nature of noise-induced synaptic damage varies between different species and that, in mouse, the noise-induced hyperexcitability seen in central auditory circuits is also observed at the level of the auditory nerve.

Multivesicular Release Increases the Efficiency of Information Transmission at Sensory Synapses

The statistics of vesicle release determine how information is transferred in neural circuits. The classical model is of Poisson synapses releasing vesicles independently but ribbon synapses transmit early sensory signals by multivesicular release (MVR) when two or more vesicles are coordinated as a single synaptic event. To investigate the impact of MVR on the spike code we used leaky integrate-and-fire models with inputs simulating the statistics of vesicle release measured experimentally from retinal bipolar cells. Comparing these with models of independent release we find that MVR increases spike generation and the efficiency of information transfer (bits per spike) over a range of conditions that mimic retinal ganglion cells of different time-constant receiving different number of synaptic inputs of different strengths. When a single input drives a neuron with short time-constant, as occurs when hair cells transmit auditory signals, MVR increases information transfer whenever spike generation requires depolarization greater than that caused by a single vesicle. This study demonstrates how presynaptic integration of vesicles by MVR can compensate for less effective summation post-synaptically to increase the efficiency with which sensory information is transmitted at the synapse.

Macrophages Are Dispensable for Postnatal Pruning of the Cochlear Ribbon Synapses

Ribbon synapses of cochlear hair cells undergo pruning and maturation before the hearing onset. In the central nervous system (CNS), synaptic pruning was mediated by microglia, the brain-resident macrophages, via activation of the complement system. Whether a similar mechanism regulates ribbon synapse pruning is currently unknown. In this study, we report that the densities of cochlear macrophages surrounding hair cells were highest at around P8, corresponding well to the completion of ribbon synaptic pruning by P8–P9. Surprisingly, using multiple genetic mouse models, we found that postnatal pruning of the ribbon synapses and auditory functions were unaffected by the knockout of the complement receptor 3 (CR3) or by ablations of macrophages expressing either LysM or Cx3cr1. Our results suggest that unlike microglia in the CNS, macrophages in the cochlea do not mediate pruning of the cochlear ribbon synapses.

Synaptic Release Potentiation at Aging Auditory Ribbon Synapses

Age-related hidden hearing loss is often described as a cochlear synaptopathy that results from a progressive degeneration of the inner hair cell (IHC) ribbon synapses. The functional changes occurring at these synapses during aging are not fully understood. Here, we characterized this aging process in IHCs of C57BL/6J mice, a strain which is known to carry a cadherin-23 mutation and experiences early hearing loss with age. These mice, while displaying a large increase in auditory brainstem thresholds due to 50% loss of IHC synaptic ribbons at middle age (postnatal day 365), paradoxically showed enhanced acoustic startle reflex suggesting a hyperacusis-like response. The auditory defect was associated with a large shrinkage of the IHCs' cell body and a drastic enlargement of their remaining presynaptic ribbons which were facing enlarged postsynaptic AMPAR clusters. Presynaptic Ca2+ microdomains and the capacity of IHCs to sustain high rates of exocytosis were largely increased, while on the contrary the expression of the fast-repolarizing BK channels, known to negatively control transmitter release, was decreased. This age-related synaptic plasticity in IHCs suggested a functional potentiation of synaptic transmission at the surviving synapses, a process that could partially compensate the decrease in synapse number and underlie hyperacusis.