Mechanisms of rod photoreceptor motility in development and following transplantation

To establish the mature neuroepithelial tissue architecture, newborn neurons often migrate from their place of birth, usually at the apical neuroepithelial limit, towards their target destination. Even before neurons are born, the nucleus of the mitotic neuronal progenitor cell migrates within the apico-basal cellular extent in synchrony with the cell cycle (interkinetic nuclear migration or IKNM) – a migratory pattern so far only observed in mitotic epithelial progenitor cells. Rod photoreceptors, too, are born at the apical limit of the retinal neuroepithelium. They then populate a layer directly adjacent (outer nuclear layer or ONL), but not the more basal retinal layers. How rod photoreceptors become enriched specifically within the ONL is presently ill-defined. Here, it was identified that rod photoreceptor somata of the developing mouse retina are constantly pushed basally (presumably caused by proximal progenitor IKNM events). To become enriched apically within the (presumptive) ONL despite this, the post-mitotic rod photoreceptors utilised an IKNM-like migratory behaviour more typically associated with dividing cells: rod somata actively migrated apically, driven by microtubule-associated dynein I motors. Another microtubule-associated motor protein, KIF1A, acts as a molecular brake during basal displacement, preventing ectopic basal positions. Rod somata oscillate between apical and basal motions at least from P1 up until ~P10. Whether this involves a component of glial-guided migration could not be established beyond reasonable doubt. Nonetheless, this is the first report of an oscillatory, IKNM-like migration behaviour occurring within a post-mitotic neuronal cell population. Rod photoreceptors have also been assumed to migrate into the adult neural retina following sub-retinal transplantation for cell-replacement therapeutic purposes, although this has not been directly observed. Here, time-lapse footage for the first time showed rod photoreceptors migrating from the sub-retinal space into the host retina. This supports the notion that photoreceptor cell replacement therapy could become a clinically viable treatment option

Tracking neuronal motility in live murine retinal explants

The developing retina undergoes dynamic organizational changes involving significant intra-retinal motility of the encompassing cells. Here, we present a protocol for tracking retinal cell motility in live explanted mouse retinae. Although originally applied to rod and cone photoreceptors, this strategy is applicable to any fluorescently labeled cell in mouse retinae and other similar experimental retinal models. Careful tissue handling is critical for the successful acquisition of high-quality live imaging data. Further instructions for semi-automated in silico data handling are provided. For complete details on the use and execution of this protocol, please refer to Aghaizu et al. (2021)

Repeated nuclear translocations underlie photoreceptor positioning and lamination of the outer nuclear layer in the mammalian retina

In development, almost all stratified neurons must migrate from their birthplace to the appropriate neural layer. Photoreceptors reside in the most apical layer of the retina, near their place of birth. Whether photoreceptors require migratory events for fine-positioning and/or retention within this layer is not well understood. Here, we show that photoreceptor nuclei of the developing mouse retina cyclically exhibit rapid, dynein-1-dependent translocation toward the apical surface, before moving more slowly in the basal direction, likely due to passive displacement by neighboring retinal nuclei. Attenuating dynein 1 function in rod photoreceptors results in their ectopic basal displacement into the outer plexiform layer and inner nuclear layer. Synapse formation is also compromised in these displaced cells. We propose that repeated, apically directed nuclear translocation events are necessary to ensure retention of post-mitotic photoreceptors within the emerging outer nuclear layer during retinogenesis, which is critical for correct neuronal lamination

A primary rodent triculture model to investigate the role of glia-neuron crosstalk in regulation of neuronal activity

Neuroinflammation and hyperexcitability have been implicated in the pathogenesis of neurodegenerative disease, and new models are required to investigate the cellular crosstalk involved in these processes. We developed an approach to generate a quantitative and reproducible triculture system that is suitable for pharmacological studies. While primary rat cells were previously grown in a coculture medium formulated to support only neurons and astrocytes, we now optimised a protocol to generate tricultures containing neurons, astrocytes and microglia by culturing in a medium designed to support all three cell types and adding exogenous microglia to cocultures. Immunocytochemistry was used to confirm the intended cell types were present. The percentage of ramified microglia in the tricultures decreases as the number of microglia present increases. Multi-electrode array recordings indicate that microglia in the triculture model suppress neuronal activity in a dose-dependent manner. Neurons in both cocultures and tricultures are responsive to the potassium channel blocker 4-aminopyridine, suggesting that neurons remained viable and functional in the triculture model. Furthermore, suppressed neuronal activity in tricultures correlates with decreased densities of dendritic spines and of the postsynaptic protein Homer1 along dendrites, indicative of a direct or indirect effect of microglia on synapse function. We thus present a functional triculture model, which, due to its more complete cellular composition, is a more relevant model than standard cocultures. The model can be used to probe glia-neuron interactions and subsequently aid the development of assays for drug discovery, using neuronal excitability as a functional endpoint

Dysregulated Wnt Signalling in the Alzheimer's Brain

The Wnt signalling system is essential for both the developing and adult central nervous system. It regulates numerous cellular functions ranging from neurogenesis to blood brain barrier biology. Dysregulated Wnt signalling can thus have significant consequences for normal brain function, which is becoming increasingly clear in Alzheimer’s disease (AD), an age-related neurodegenerative disorder that is the most prevalent form of dementia. AD exhibits a range of pathophysiological manifestations including aberrant amyloid precursor protein processing, tau pathology, synapse loss, neuroinflammation and blood brain barrier breakdown, which have been associated to a greater or lesser degree with abnormal Wnt signalling. Here we provide a comprehensive overview of the role of Wnt signalling in the CNS, and the research that implicates dysregulated Wnt signalling in the ageing brain and in AD pathogenesis. We also discuss the opportunities for therapeutic intervention in AD via modulation of the Wnt signalling pathway, and highlight some of the challenges and the gaps in our current understanding that need to be met to enable that goal