Wei Li

In retinal degenerative disease, microglia and macrophages accumulate at sites of pathology and strongly influence disease progression, yet their distinct contributions remain unclear. To define the fate and function of infiltrating monocyte-derived macrophages (MDM) in retinal degeneration, we generated a CCR2-CreER mouse line on the rd10 background to enable precise monocyte-specific tracking and ablation. Infiltrating monocytes rapidly downregulated CCR2 and LY6C upon entering the retina and acquired de novo TMEM119 and P2RY12 expression, together with a ramified, microglia-like morphology. Immunohistochemistry and transcriptomic profiling showed that a subset of these cells was cleared by resident microglia. Microglia–monocyte interactions enhanced Müller cell C3 production, whereas activated microglia increased CFB and decreased CFH expression, thereby promoting complement alternative pathway activation. Selective ablation of infiltrating monocytes reduced microglial activation and phagocytosis, suppressed Müller cell C3 expression and complement deposition, lowered proinflammatory cytokine levels, and ultimately ameliorated photoreceptor degeneration. These findings identify infiltrating monocytes as key drivers of immune dysregulation and proinflammation, highlighting them as potential targets for neuroprotective therapy.

Hibernating animals can show neuroplasticity throughout the hibernation season. In ground squirrels, decreased dendritic arborization in the hippocampus, somatosensory cortex, and thalamus during deep hibernation (“torpor”) suggests that this neuroplasticity is a brain-wide phenomenon. However, the degree to which neuroplasticity occurs in the visual system is not clear. While transient retinal changes have been reported during torpor, neuroplasticity beyond the retina remains unknown. Here, we characterized hibernation-related neuroplasticity in the primary visual cortex (V1), the first cortical area to receive visual information, in the 13-lined ground squirrel ( Ictidomys tridecemlineatus ). We compared neuronal morphology in Golgi-stained samples from male and female hibernating or nonhibernating squirrels. For the hibernating squirrels, the brain tissue was sampled during two different epochs: torpor and intertorpor arousal. Dendritic arborization decreased during torpor in V1 layer 2/3 pyramidal neurons, manifesting as decreases in dendritic length, number, and complexity. These changes fully reversed during intertorpor arousal, indicating that on average dendritic arbors grew by 0.75 mm (65%) over ∼1.5 h. No morphological differences between hibernating and nonhibernating squirrels were apparent when compared 6 months after the hibernation season. We also found no neuroplastic changes in V1 layer 4 spiny stellate neurons, unlike in this cell type in the somatosensory cortex. Together, this revealed, for the first time, hibernation-related neuroplasticity in V1 in support of a brain-wide mechanism but with area-specific differences. The speed and magnitude of this naturally occurring neuroplasticity could make ground squirrel V1 a powerful translational model system for conditions requiring neuroplasticity, such as recovery from stroke.

At the blood-tissue interface, vasculature luminal surface is critical for molecular transport, signaling transduction, and cell extravasation. Here, we present a method for proteomic profiling of the vasculature luminal surface in vivo, broadly applicable to any vertebrate. Quantitative mass spectrometry revealed the luminal surface proteome of the mouse brain vasculature and its temporal evolution from development to aging. In vivo genetic perturbation found that the arginine transporter SLC7A1 and the nitric oxide synthase NOS3 are needed for blood-brain barrier integrity in neonatal but not adult mice, whereas the hyaluronan degradation enzyme HYAL2 safeguards the barrier throughout the lifespan. By characterizing the proteomic dynamics of the vasculature luminal surface, the study links the metabolism of nitric oxide and hyaluronan to blood-brain barrier integrity.

The suprachiasmatic nucleus (SCN), the central circadian pacemaker, receives photic input exclusively from intrinsically photosensitive retinal ganglion cells (ipRGCs)^1,2. However, light mainly shifts the SCN clock during night-time^3,4,5. Here we induced phase shifts in the SCN clock during the daytime in mice by activating ipRGCs using chemogenetics or violet light. Our data reveal that the inability to induce daytime shifts with light in most animals is not only attributed to the SCN, as has been proposed for decades, but also requires the limitation of ipRGC firing via depolarization block. Chemogenetic activation of ipRGCs induces large shifts during both night-time and daytime, but daytime shifts require brain circuits and neuropeptide transmitters that are dispensable for night-time shifts. Thus, propensity of ipRGCs for depolarization block not only prevents daytime shifts in mice, but also limits the magnitude of night-time shifts, suggesting that ipRGC inputs to SCN act as an integrated pacemaker across the circadian cycle.

The optomotor reflex (OMR) provides a behavioral assessment of an animal's contrast sensitivity and visual acuity. Mice or rats are typically placed directly onto a small circular platform by hand; however, handling animals like this can stimulate stress and anxiety, which introduce confounding factors when interpreting data. It has been shown that non-aversive handling methods, such as picking up mice or rats in a familiar tunnel/tube, can reduce anxiety. This is of particular interest in studies where animals display heightened stress, overactivity, or motor dysfunction, resulting in an inability to stay on the platform. A team led by Drs. Kiyoharu J. Miyagishima and Francisco M. Nadal-Nicolás have redesigned the conventional OMR platform to provide semi-closed containment. This makes it possible for the first time to record the optomotor reflex in the 13-lined ground squirrel, which is one of the few mammals that can see color. It has a visual streak with a high density of cones similar to the human macula providing an attractive model for studying effects on the cone visual system.

The optomotor reflex (OMR) provides a behavioral assessment of an animal's contrast sensitivity and visual acuity. Mice or rats are typically placed directly onto a small circular platform by hand; however, handling animals like this can stimulate stress and anxiety, which introduce confounding factors when interpreting data. It has been shown that non-aversive handling methods, such as picking up mice or rats in a familiar tunnel/tube, can reduce anxiety. This is of particular interest in studies where animals display heightened stress, overactivity, or motor dysfunction, resulting in an inability to stay on the platform. A team led by Drs. Kiyoharu J. Miyagishima and Francisco M. Nadal-Nicolás have redesigned the conventional OMR platform to provide semi-closed containment. This makes it possible for the first time to record the optomotor reflex in the 13-lined ground squirrel, which is one of the few mammals that can see color. It has a visual streak with a high density of cones similar to the human macula providing an attractive model for studying effects on the cone visual system.

We propose a standardized naming system for vertebrate visual photoreceptors (i.e., rods and cones) that reflects our current understanding of their evolutionary history. Vertebrate photoreceptors have been studied for well over a century, but a fixed nomenclature for referring to orthologous cell types across diverse species has been lacking. Instead, photoreceptors have been variably - and often confusingly - named according to morphology, presence/absence of ‘rhodopsin,’ spectral sensitivity, chromophore usage, and/or the gene family of the opsin(s) they express. Here, we propose a unified nomenclature for vertebrate rods and cones that aligns with the naming systems of other retinal cell classes and that is based on the photoreceptor’s putative ancestral derivation. This classification is informed by the functional, anatomical, developmental and molecular identities of the neuron as a whole, including the expression of deeply conserved transcription factors required for development. The proposed names will be applicable across all vertebrates and indicative of the widest-possible range of properties, including their postsynaptic wiring, and hence will allude to their common and species-specific roles in vision. Furthermore, the naming system is open-ended to accommodate the future discovery of as-yet unknown photoreceptor types.

Currently there are no effective treatments for an array of neurodegenerative disorders to a large part because cell-based models fail to recapitulate disease. Here we develop a reproducible human iPSC-based model where laser axotomy causes retrograde axon degeneration leading to neuronal cell death. Time-lapse confocal imaging revealed that damage triggers an apoptotic wave of mitochondrial fission proceeding from the site of injury to the soma. We demonstrate that this apoptotic wave is locally initiated in the axon by dual leucine zipper kinase (DLK). We find that mitochondrial fission and resultant cell death are entirely dependent on phosphorylation of dynamin related protein 1 (DRP1) downstream of DLK, revealing a mechanism by which DLK can drive apoptosis. Importantly, we show that CRISPR mediated Drp1 depletion protects mouse retinal ganglion neurons from degeneration after optic nerve crush. Our results provide a platform for studying degeneration of human neurons, pinpoint key early events in damage related neural death and provide potential focus for therapeutic intervention.

Microglia exhibit both maladaptive and adaptive roles in the pathogenesis of neurodegenerative diseases and have emerged as a cellular target for central nervous system (CNS) disorders, including those affecting the retina. Replacing maladaptive microglia, such as those impacted by aging or over-activation, with exogenous microglia that can enable adaptive functions has been proposed as a potential therapeutic strategy for neurodegenerative diseases. To investigate microglia replacement as an approach for retinal diseases, we first employed a protocol to efficiently generate human-induced pluripotent stem cell (hiPSC)-derived microglia in quantities sufficient for in vivo transplantation. These cells demonstrated expression of microglia-enriched genes and showed typical microglial functions such as LPS-induced responses and phagocytosis. We then performed xenotransplantation of these hiPSC-derived microglia into the subretinal space of adult mice whose endogenous retinal microglia have been pharmacologically depleted. Long-term analysis post-transplantation demonstrated that transplanted hiPSC-derived microglia successfully integrated into the neuroretina as ramified cells, occupying positions previously filled by the endogenous microglia and expressed microglia homeostatic markers such as P2ry12 and Tmem119. Furthermore, these cells were found juxtaposed alongside residual endogenous murine microglia for up to 8 months in the retina, indicating their ability to establish a stable homeostatic state in vivo. Following retinal pigment epithelial cell injury, transplanted microglia demonstrated responses typical of endogenous microglia, including migration, proliferation, and phagocytosis. Our findings indicate the feasibility of microglial transplantation and integration in the retina and suggest that modulating microglia through replacement may be a therapeutic strategy for treating neurodegenerative retinal diseases.

Maintaining protein homeostasis (proteostasis) requires precise control of protein folding and degradation. Failure to properly respond to stresses disrupts proteostasis, which is a hallmark of many diseases, including cataracts. Hibernators are natural cold-stress adaptors; however, little is known about how they keep a balanced proteome under conditions of drastic temperature shift. Intriguingly, we identified a reversible lens opacity phenotype in ground squirrels (GSs) associated with their hibernation-rewarming process. To understand this “cataract-reversing” phenomenon, we first established induced lens epithelial cells differentiated from GS-derived induced pluripotent stem cells, which helped us explore the molecular mechanism preventing the accumulation of protein aggregates in GS lenses. We discovered that the ubiquitin-proteasome system (UPS) played a vital role in minimizing the aggregation of the lens protein αA-crystallin (CRYAA) during rewarming. Such function was, for the first time to our knowledge, associated with an E3 ubiquitin ligase, RNF114, which appears to be one of the key mechanisms mediating the turnover and homeostasis of lens proteins. Leveraging this knowledge gained from hibernators, we engineered a deliverable RNF114 complex and successfully reduced lens opacity in rats with cold-induced cataracts and zebrafish with oxidative stress–related cataracts. These data provide new insights into the critical role of the UPS in maintaining proteostasis in cold and possibly other forms of stresses. The newly identified E3 ubiquitin ligase RNF114, related to CRYAA, offers a promising avenue for treating cataracts with protein aggregates.