Ganglion Cells In Mice Research Articles

Environmental Light Controls the Daily Organization of Breathing by Activating Brn3b-expressing Intrinsically Photosensitive Retinal Ganglion Cells in Mice.

Rhythmic, daily fluctuations in minute ventilation are controlled by the endogenous circadian clock located in the suprachiasmatic nucleus (SCN). While light serves as a potent synchronizer for the SCN, it also influences physiology and behavior by activating Brn3b-expressing, intrinsically photosensitive retinal ganglion cells (ipRGCs). It is currently unclear the extent to which the external light environment shapes daily ventilatory patterns independent of the SCN. To determine the relative influence of environmental light versus circadian timing on the organization of daily rhythms in minute ventilation, we used whole-body plethysmography to measure the breathing of mice housed on a non-entraining T28 cycle (14 h light:14 h dark). Using this protocol, we found that minute ventilation exhibits a ~28-h rhythm with a peak at dark onset that coincides with the light:dark cycle and the animals' locomotor activity. To determine if this 28-h rhythm in minute ventilation was mediated by Brn3b-expressing ipRGCs, we measured the breathing of Brn3bDTA mice housed under the T28 cycle. Brn3bDTA mice lack the Brn3b-expressing ipRGCs that project to many non-SCN brain regions. We found that despite rhythmic light cues occurring on a 28-h basis, Brn3bDTA mice exhibited 24-h rhythms in minute ventilation, locomotor activity, and core body temperature consistent with organization by the SCN. The 24-h minute ventilation rhythm of Brn3bDTA mice was found to be driven predominantly by tidal volume rather than respiratory rate. These data indicate that the external light:dark cycle can directly drive daily patterns in minute ventilation by way of Brn3b-expressing ipRGCs. In addition, these data strongly suggest that the activation of Brn3b-expressing ipRGCs principally organizes daily patterns in breathing and locomotor activity when light:dark cues are presented in opposition to endogenous clock timing.

Piezo1 and Piezo2 channels in retinal ganglion cells and the impact of Piezo1 stimulation on light-dependent neural activity.

Activation of Piezo1 excites retinal ganglion cells, paralleling the early neurodegenerative progression in glaucoma mouse models and retinal degeneration.Piezo1 and Piezo2 were expressed in axons and soma of retinal ganglion cells in mice, rats, and human iPSC-RGCs.Functional assays confirmed Piezo1 in soma and neurites of neurons. Sustained elevation of Piezo1 and Piezo2 occurred after a single transient stretch may enhance damage from repeated traumatic nerve injury.

Inflammatory response of both retinas and superior colliculi to unilateral optic nerve axotomy

Objective: Unilateral optic nerve crush (ONC) induces a bilateral inflammatory reaction affecting both injured and contralateral un‐injured retinas. However, few works have investigated the spread of inflammation along visual pathway. Our aim was to study the glial response and the underlying molecular changes in both retinas and the superior colliculi (SCi), the main projection areas of retinal ganglion cells in mice. In this species, >90% of RGCs project to the contralateral SC (cSCi).Methods: The left optic nerve of adult pigmented C57Bl/6 male mice were crushed (ONC) at 0.5 mm from the optic disk or underwent surgery without crushing (Sham). One group of intact mice was used as controls. Two studies were carried out:1) At 1, 3, 9 and 30 days post lesion (dpl) both retinas and SCi were freshly dissected and their RNA extracted. Using qPCR we measured the expression levels of TGF‐β1, IL‐1β, TNF‐α, Iba1, GFAP, MHC II and TSPO in retinas and SCi. In SCi extracts we also studied Casp3, CXCR1, Lcn2, IL‐6, CD206, IL‐4, and AQ4. Hptr and Gapdh were used as houskeepings. Intact retinas and SCi were used as controls.2) Three, 9 and 30 days after ONC or sham‐surgery immunodetection of microglia and macroglial cells was carried out in sagittal brain sections. The area occupied by GFAP and MHC II signal as well as the density of Iba1+ and CD68+ cells was quantified in both SCi. Intact brains were used as controls.Results: Sham surgery alone modified gene expression in both retinas and SCi compared to intact animals. Therefore, to isolate the effect of ONC, we compared ONC vs. Sham. After ONC, TNF‐α upregulated early in both retinas and IL‐β remained overexpressed in injured retina at day 30. GFAP overexpression was exclusively observed in injured retina while Iba1 overexpression was presented in both. TSPO and MHCII and Casp3 overexpression was observed only in the cSC.Anatomical microglial activation (rounded somas and CD68 expression) was observed in the cSC from day 3 onwards after ONC. In the ipsilateral SC (iSC) microglial cells did not show signs of activation. Astrocytes hypertrophied in both SCi from day 9 after ONC, but this response was also observed in Sham‐animals.Conclusions: Sham surgery promotes an inflammatory and glial response that must be considered in experimental designs. Unilateral ONC triggers a bilateral response which extends anterogradely and retrogradely along the visual pathway.

Development of an AAV-based model of tauopathy targeting retinal ganglion cells and the mouse visual pathway to study the role of microglia in Tau pathology

Tauopathy is a typical feature of Alzheimer's disease of major importance because it strongly correlates with the severity of cognitive deficits experienced by patients. During the pathology, it follows a characteristic spatiotemporal course which takes its origin in the transentorhinal cortex, and then gradually invades the entire forebrain. To study the mechanisms of tauopathy, and test new therapeutic strategies, it is necessary to set-up relevant and versatile in vivo models allowing to recapitulate tauopathy. With this in mind, we have developed a model of tauopathy by overexpression of the human wild-type Tau protein in retinal ganglion cells in mice (RGCs). This overexpression led to the presence of hyperphosphorylated forms of the protein in the transduced cells as well as to their progressive degeneration. The application of this model to mice deficient in TREM2 (Triggering Receptor Expressed on Myeloid cells-2, an important genetic risk factor for AD) as well as to 15-month-old mice showed that microglia actively participate in the degeneration of RGCs. Surprisingly, although we were able to detect the transgenic Tau protein up to the terminal arborization of RGCs at the level of the superior colliculi, spreading of the transgenic Tau protein to post-synaptic neurons was detected only in aged animals. This suggests that there may be neuron-intrinsic- or microenvironment mediators facilitating this spreading that appear with aging.

Long Non-Coding RNA-X-Inactive Specific Transcript Promotes the Retinal Ganglion Cell Survival After Optic Nerve Crush Injury by Upregulating miR-36

Retinal ganglion cells (RGC) axons participate in the construction of optic nerve, and prevent the damage of RGC during acute optic nerve injury. IncRNA-XIST is crucial for RGC apoptosis. Our study intends to assess IncRNA-XIST’s role in the regulation of RGC apoptosis in an attempt to provide a theoretical basis for treating optic nerve crush injury. Two genotypes of mice (wild-type and miR-36 KO) were used to establish an optic nerve crush injury model to investigate the regulatory role of IncRNA-XIST gene in RGCs apoptosis. These mice were then randomly assigned into control group (WT), injury group, and XIST/injury group. The changes of apoptotic genes and proteins in retinal ganglion cells were analyzed by qPCR, WB and TUNEL staining. In wild-type mice, RGC apoptosis was significantly increased after optic nerve compression injury, and the expression of Bax and Bad was significantly increased. When the LncRNA-XIST gene was overexpressed before retinal crush injury, the apoptosis of retinal ganglion cells was significantly reduced, and Bax and Bad levels were decreased as compared with model group of optic nerve injury. The results showed that in wild-type mice, overexpression of IncRNA-XIST gene promoted the survival of RGC after optic nerve crush injury. In addition, upregulation of IncRNA-xist expression in miR-36 KO mice did not reduce retinal ganglion cell apoptosis and alter the apoptotic protein expression after optic nerve crush injury. Defects of miR-36 alone or overexpression of XIST gene do not cause morphological damage of retina in mice. In mouse ganglion cells, miR-36 expression was up-regulated in both injured cells and overexpressed XIST gene. However, up-regulation of miR-36 caused by overexpression of XIST gene was more obvious. In addition, in vivo studies of wild-type mice, it was found that overexpression of XIST reduced retinal ganglion cell apoptosis, and this effect was abolished in miR-36 KO mice. In conclusion, lncRNA-XIST reduces ganglion cell apoptosis by upregulating miR-36 and promotes the survival of RGC after nerve crush injury.

Light responses and amacrine cell modulation of morphologically-identified retinal ganglion cells in the mouse retina

By analyzing light-evoked spike responses, cation currents (ΔIC) and chloride currents (ΔICl) of over 100 morphologically-identified retinal ganglion cells (GCs) in dark-adapted mouse retina, we found there are at least 14 functionally- and morphologically-distinct types of RGCs. These cells can be divided into 5 groups based on their patterns of spike response to whole field light steps (SRWFLS), a GC identification scheme commonly used in studies with extracellular recording techniques. We also found that all GCs in the mouse retina express strychnine-sensitive glycine receptors, and receive light-elicited chloride current (ΔICl) accompanied by a conductance increase from narrow-field, glycinergic amacrine cells. As the dark membrane potential of RGC are near the chloride-equilibrium potential, mouse GCs’ spike responses are mediated primarily by bipolar cells inputs, and modulated by “shunting inhibition” from narrow-field amacrine cells. Analysis of strychnine actions on light-evoked cation current ΔIC (bipolar cell inputs) in GCs suggests that narrow-field amacrine cells modulate GCs by sending ON-OFF crossover feedback signals to presynaptic bipolar cell axon terminals via sign-inverting glycinergic synapses, and the feedback signals are synergistic to the bipolar cell light responses. Therefore narrow-field amacrine cells enhance light-evoked bipolar cell inputs to GCs by presynaptic “synergistic addition”, besides the abovementioned postsynaptic “shunting inhibition” in GCs.

Use of a tissue clearing technique combined with retrograde trans-synaptic viral tracing to evaluate changes in mouse retinorecipient brain regions following optic nerve crush.

Successful establishment of reconnection between retinal ganglion cells and retinorecipient regions in the brain is critical to optic nerve regeneration. However, morphological assessments of retinorecipient regions are limited by the opacity of brain tissue. In this study, we used an innovative tissue cleaning technique combined with retrograde trans-synaptic viral tracing to observe changes in retinorecipient regions connected to retinal ganglion cells in mice after optic nerve injury. Specifically, we performed light-sheet imaging of whole brain tissue after a clearing process. We found that pseudorabies virus 724 (PRV724) mostly infected retinal ganglion cells, and that we could use it to retrogradely trace the retinorecipient regions in whole tissue-cleared brains. Unexpectedly, PRV724-traced neurons were more widely distributed compared with data from previous studies. We found that optic nerve injury could selectively modify projections from retinal ganglion cells in the hypothalamic paraventricular nucleus, intergeniculate leaflet, ventral lateral geniculate nucleus, central amygdala, basolateral amygdala, Edinger-Westphal nucleus, and oculomotor nucleus, but not the superior vestibular nucleus, red nucleus, locus coeruleus, gigantocellular reticular nucleus, or facial nerve nucleus. Our findings demonstrate that the tissue clearing technique, combined with retrograde trans-synaptic viral tracing, can be used to objectively and comprehensively evaluate changes in mouse retinorecipient regions that receive projections from retinal ganglion cells after optic nerve injury. Thus, our approach may be useful for future estimations of optic nerve injury and regeneration.

Retracted: Effects of Ultrasound Contrast Agent-Mediated Nerve Growth Factor on Apoptosis of Retinal Ganglion Cells in Mice with Glaucoma.

[This retracts the article DOI: 10.1155/2021/6084496.].

Scalable Three-Dimensional Recording Electrodes for Probing Biological Tissues.

Electrophysiological recording technologies can provide critical insight into the function of the nervous system and other biological tissues. Standard silicon-based probes have limitations, including single-sided recording sites and intrinsic instabilities due to the probe stiffness. Here, we demonstrate high-performance neural recording using double-sided three-dimensional (3D) electrodes integrated in an ultraflexible bioinspired open mesh structure, allowing electrodes to sample fully the 3D interconnected tissue of the brain. In vivo electrophysiological recording using 3D electrodes shows statistically significant increases in the number of neurons per electrode, average spike amplitudes, and signal to noise ratios in comparison to standard two-dimensional electrodes, while achieving stable detection of single-neuron activity over months. The capability of these 3D electrodes is further shown for chronic recording from retinal ganglion cells in mice. This approach opens new opportunities for a comprehensive 3D interrogation, stimulation, and understanding of the complex circuitry of the brain and other electrogenic tissues in live animals over extended time periods.

Differential effects of experimental glaucoma on intrinsically photosensitive retinal ganglion cells in mice.

Glaucoma is a group of eye diseases characterized by retinal ganglion cell (RGC) loss and optic nerve damage. Studies, including this study, support that RGCs degenerate and die in a type-specific manner following the disease insult. Here we specifically examined one RGC type, the intrinsically photosensitive retinal ganglion cell (ipRGC), and its associated functional deficits in a mouse model of experimental glaucoma. We induced chronic ocular hypertension (OHT) by laser photocoagulation and then characterized the survival of ipRGC subtypes. We found that ipRGCs suffer significant loss, similar to the general RGC population, but ipRGC subtypes are differentially affected following chronic OHT. M4 ipRGCs, which are involved in pattern vision, are susceptible to chronic OHT. Correspondingly, mice with chronic OHT experience reduced contrast sensitivity and visual acuity. By comparison, M1 ipRGCs, which project to the suprachiasmatic nuclei to regulate circadian rhythmicity, exhibit almost no cell loss following chronic OHT. Accordingly, we observed that circadian re-entrainment and circadian rhythmicity are largely not disrupted in OHT mice. Our study demonstrates the link between subtype-specific ipRGC survival and behavioral deficits in glaucomatous mice. These findings provide insight into glaucoma-induced visual behavioral deficits and their underlying mechanisms.

Effects of Ultrasound Contrast Agent-Mediated Nerve Growth Factor on Apoptosis of Retinal Ganglion Cells in Mice with Glaucoma.

With an increasing incidence in recent years, glaucoma (GL) has gradually become a global public health problem for humans of all ages. Nerve growth factor (NGF) eye drops, with well-documented stable effect in the treatment of GL, can be potentiated by the administration of NGF drugs via ultrasound contrast agent (UCA). This study analyzed the efficacy of NGF+UCA on GL mice and the influencing mechanism on retinal ganglion cells and further explored the pathological changes of GL mice under different UCA irradiation duration. In this study, we established GL mouse models and treated the mouse with NGF+UCA. The effect of NGF+UCA on intraocular pressure in mice was observed; the flash visual evoked potential of mice was compared; the changes of retinal structure, inflammation index, and oxidative stress index were observed, and autophagic protein levels were tested. Finally, the influence of UCA irradiation duration on GL symptoms was observed. The results showed that the intraocular pressure of mice decreased greatly, while their flash visual evoked potential and nervous layer of retina increased, and their ganglion cells showed stronger proliferation activity and weaker apoptosis and autophagy, indicating that UCA-mediated NGF can strongly improve the pathological condition of GL mice. In addition, PI3K/AKT pathway-associated proteins were inhibited in retina under the intervention of NGF+UCA, which further suggests that the influence of UCA-mediated NGF on GL is achieved by inhibiting autophagy of retinal ganglion cells and enhancing their apoptosis via the PI3K/AKT signaling pathway. Moreover, we found that in the treatment of GL, three weeks of UCA irradiation and six weeks caused no significant difference in the pathological manifestations and ganglion cells of mice, while after six weeks of irradiation, the level of NLRP3 in mice increased. In conclusion, UCA-mediated NGF can significantly improve the pathological condition of GL mice and improve the apoptosis of retinal ganglion cells by inhibiting autophagy, which is associated with the inhibition of the PI3K/AKT signal pathway. In terms of selection of UCA irradiation duration, three weeks of irradiation is enough to yield good clinical results.

Nonlinear Spatial Integration Underlies the Diversity of Retinal Ganglion Cell Responses to Natural Images.

How neurons encode natural stimuli is a fundamental question for sensory neuroscience. In the early visual system, standard encoding models assume that neurons linearly filter incoming stimuli through their receptive fields, but artificial stimuli, such as contrast-reversing gratings, often reveal nonlinear spatial processing. We investigated to what extent such nonlinear processing is relevant for the encoding of natural images in retinal ganglion cells in mice of either sex. We found that standard linear receptive field models yielded good predictions of responses to flashed natural images for a subset of cells but failed to capture the spiking activity for many others. Cells with poor model performance displayed pronounced sensitivity to fine spatial contrast and local signal rectification as the dominant nonlinearity. By contrast, sensitivity to high-frequency contrast-reversing gratings, a classical test for nonlinear spatial integration, was not a good predictor of model performance and thus did not capture the variability of nonlinear spatial integration under natural images. In addition, we also observed a class of nonlinear ganglion cells with inverse tuning for spatial contrast, responding more strongly to spatially homogeneous than to spatially structured stimuli. These findings highlight the diversity of receptive field nonlinearities as a crucial component for understanding early sensory encoding in the context of natural stimuli.SIGNIFICANCE STATEMENT Experiments with artificial visual stimuli have revealed that many types of retinal ganglion cells pool spatial input signals nonlinearly. However, it is still unclear how relevant this nonlinear spatial integration is when the input signals are natural images. Here we analyze retinal responses to natural scenes in large populations of mouse ganglion cells. We show that nonlinear spatial integration strongly influences responses to natural images for some ganglion cells, but not for others. Cells with nonlinear spatial integration were sensitive to spatial structure inside their receptive fields, and a small group of cells displayed a surprising sensitivity to spatially homogeneous stimuli. Traditional analyses with contrast-reversing gratings did not predict this variability of nonlinear spatial integration under natural images.

Protective effects of human umbilical cord mesenchymal stem cells on retinal ganglion cells in mice with acute ocular hypertension.

To observe the protective effect of human umbilical cord mesenchymal stem cells (hucMSCs) on retinal ganglion cells (RGCs) injury in mice with acute ocular hypertension (AOH). Fifty-six adult male C57BL/6 mice were randomly divided into four groups: normal group, AOH group, hucMSCs group, normal saline (NS) group. Left eye of mice was induced by 90 mm Hg intraocular pressure for 1h to establish AOH model. hucMSCs 1×105/µL, 1 µL or NS 1 µL was injected into the vitreous body the next day. CM-Dil fluorescent dye was used to label the 3rd generation of hucMSCs, for tracing the cells in the vitreous cavity of mice. Seven days after the model established, hematoxylin-eosin (HE) staining was used to observe the thickness of the inner retina layer in four groups. Numbers and loss rate of RGCs were evaluated by counting Brn-3a positive cells stained by immunofluorescencein. On the 7th day after AOH established, labeled hucMSCs were found in the vitreous cavity. HE staining showed that the thickness of retinal inner layer in AOH group was significantly lower than that in normal group and hucMSCs group (P<0.05), same as that in NS group (P>0.05). Compared with AOH group, the RGCs in normal group was significantly higher; RGCs number increased in hucMSCs group and the loss rate was lower (P<0.05). Injection of NS had no protective effect on RGCs. In AOH mouse model, vitreous injection of hucMSCs have shown a protection for RGCs.

Intravitreal Co-Administration of GDNF and CNTF Confers Synergistic and Long-Lasting Protection against Injury-Induced Cell Death of Retinal Ganglion Cells in Mice

We have recently demonstrated that neural stem cell-based intravitreal co-administration of glial cell line-derived neurotrophic factor (GDNF) and ciliary neurotrophic factor (CNTF) confers profound protection to injured retinal ganglion cells (RGCs) in a mouse optic nerve crush model, resulting in the survival of ~38% RGCs two months after the nerve lesion. Here, we analyzed whether this neuroprotective effect is long-lasting and studied the impact of the pronounced RGC rescue on axonal regeneration. To this aim, we co-injected a GDNF- and a CNTF-overexpressing neural stem cell line into the vitreous cavity of adult mice one day after an optic nerve crush and determined the number of surviving RGCs 4, 6 and 8 months after the lesion. Remarkably, we found no significant decrease in the number of surviving RGCs between the successive analysis time points, indicating that the combined administration of GDNF and CNTF conferred lifelong protection to injured RGCs. While the simultaneous administration of GDNF and CNTF stimulated pronounced intraretinal axon growth when compared to retinas treated with either factor alone, numbers of regenerating axons in the distal optic nerve stumps were similar in animals co-treated with both factors and animals treated with CNTF only.

Endogenous Apelin Is Protective Against Age-Associated Loss of Retinal Ganglion Cells in Mice.

Age-associated loss of retinal ganglion cells (RGCs) causes visual deficits, but there is not yet any therapeutic agent to prevent the loss of these cells. Herein, we report that apelin, an endogenous peptide ligand of APJ receptor, is protective against the age-related loss of RGCs in mice. The mRNA expression of apelin was reduced in the retina of old mice compared with that in young mice, whereas retinal APJ expression increased with age. Immunofluorescence staining showed that APJ was present in RGCs and their surrounding cells expressed apelin. In addition, both functional and histological analyses demonstrated that apelin deficiency accelerated the loss of RGCs associated with age in mice. These results suggest that endogenous apelin plays a protective role against the degeneration of RGCs and that the apelinergic axis may be a new target for preventing age-related visual impairment.

In vitro study on signal transduction in mice spiral ganglion cell stimulated by multi-wavelength laser based on calcium imaging

Objective: To research the auditory nerve transduction effects under multi-wavelength pulsed laser stimulations within a safe and acceptable signal range. Methods: The real-time detection of intracellular calcium concentration was adopted by specific fluorescent indicator staining based on calcium imager. The spiral ganglion cells of mice were cultured in vitro. After fluorescent indicating, morphologic observation under optical microscope, Fura-2 calcium ion fluorescence excitation, intact morphology cells selection, fixing the optical fiber, the spiral ganglion cells were irradiated by different wavelength laser, including visible light (450 nm) and near infrared light (808 nm,1 065 nm). The intracellular calcium concentration was monitored by calcium ion imaging. Results: When 450 nm laser stimulated spiral ganglion cells, the intracellular calcium concentration was strongly increased, however, for other wavelength laser stimulation, there was no obvious relative response. And the sensitivity expression of the nerve cells under laser was related with the location of laser fiber. Cells closer to the fiber produced more obvious changes in calcium ion concentration, while for cells farther away from the fiber, the change amplitudes were weaker although the number of changes in calcium ion concentration was consistent. Conclusion: The spiral ganglion cells of mice can induce a signal transduction response under the action of laser, and the response has laser wavelength selectivity.

加齢に伴う網膜神経節細胞の脱落に対する内因性アペリンの保護効果

加齢に伴う網膜神経節細胞の脱落に対する内因性アペリンの保護効果

Numbers of Retinal Ganglion Cells in Mice with Genetic Defects in Different A1 Adrenoreceptor Subtypes

Objective. To perform a comparative study of the numbers of retinal ganglion cells (RGC) in mice with genetic defects affecting different subtypes of α1 adrenoreceptors. Materials and methods. Studies were run on 36 male laboratory mice aged over 18 months with genetic defects affecting one α1 adrenoreceptor subtype (α1a, α1b, α1d). The control group consisted of intact laboratory mice (C57Bl/6NTac) of the same age and sex distribution. An immunofluorescent method was used for differential visualization of cells in retinal wholemount preparations using the Brn3a marker allowing these cells to be counted. Results. Mice with genetic defects of α1b and α1d adrenoreceptors had more and mice with defective α1a adrenoreceptors had fewer RGC than intact mice of the same age and sex. The total numbers of retinal cells in mice of all the study strains were essentially identical. Conclusions. The results obtained here provide evidence that each α1 adrenoreceptor subtype (α1a, α1b, α1d) makes a specific contribution during the process of forming retinal topography.

New MiniPromoter Ple345 (NEFL) Drives Strong and Specific Expression in Retinal Ganglion Cells of Mouse and Primate Retina

Retinal gene therapy is leading the neurological gene therapy field, with 32 ongoing clinical trials of recombinant adeno-associated virus (rAAV)–based therapies. Importantly, over 50% of those trials are using restricted promoters from human genes. Promoters that restrict expression have demonstrated increased efficacy and can limit the therapeutic to the target cells thereby reducing unwanted off-target effects. Retinal ganglion cells are a critical target in ocular gene therapy; they are involved in common diseases such as glaucoma, rare diseases such as Leber's hereditary optic neuropathy, and in revolutionary optogenetic treatments. Here, we used computational biology and mined the human genome for the best genes from which to develop a novel minimal promoter element(s) designed for expression in restricted cell types (MiniPromoter) to improve the safety and efficacy of retinal ganglion cell gene therapy. Gene selection included the use of the first available droplet-based single-cell RNA sequencing (Drop-seq) dataset, and promoter design was bioinformatically driven and informed by a wide range of genomics datasets. We tested seven promoter designs from four genes in rAAV for specificity and quantified expression strength in retinal ganglion cells in mouse, and then the single best in nonhuman primate retina. Thus, we developed a new human-DNA MiniPromoter, Ple345 (NEFL), which in combination with intravitreal delivery in rAAV9 showed specific and robust expression in the retinal ganglion cells of the nonhuman-primate rhesus macaque retina. In mouse, we also developed MiniPromoters expressing in retinal ganglion cells, the hippocampus of the brain, a pan neuronal pattern in the brain, and peripheral nerves. As single-cell transcriptomics such as Drop-seq become available for other cell types, many new opportunities for additional novel restricted MiniPromoters will present.

A Ser75-to-Asp phospho-mimicking mutation in Src accelerates ageing-related loss of retinal ganglion cells in mice

Src knockout mice show no detectable abnormalities in central nervous system (CNS) post-mitotic neurons, likely reflecting functional compensation by other Src family kinases. Cdk1- or Cdk5-dependent Ser75 phosphorylation in the amino-terminal Unique domain of Src, which shares no homology with other Src family kinases, regulates the stability of active Src. To clarify the roles of Src Ser75 phosphorylation in CNS neurons, we established two types of mutant mice with mutations in Src: phospho-mimicking Ser75Asp (SD) and non-phosphorylatable Ser75Ala (SA). In ageing SD/SD mice, retinal ganglion cell (RGC) number in whole retinas was significantly lower than that in young SD/SD mice in the absence of inflammation and elevated intraocular pressure, resembling the pathogenesis of progressive optic neuropathy. By contrast, SA/SA mice and wild-type (WT) mice exhibited no age-related RGC loss. The age-related retinal RGC number reduction was greater in the peripheral rather than the mid-peripheral region of the retina in SD/SD mice. Furthermore, Rho-associated kinase activity in whole retinas of ageing SD/SD mice was significantly higher than that in young SD/SD mice. These results suggest that Src regulates RGC survival during ageing in a manner that depends on Ser75 phosphorylation.