Expression patterns of CYP26A1, FGF8, CDKN1A, and NPVF in the developing rhesus monkey retina.

The fovea centralis (fovea) is a specialized region of the primate retina that plays crucial roles in high-resolution visual acuity and color perception. The fovea is characterized by a high density of cone photoreceptors and no rods, and unique anatomical properties that contribute to its remarkable visual capabilities. Early histological analyses identified some of the key events that contribute to foveal development, but the mechanisms that direct the specification of this area are not understood. Recently, the expression of the retinoic acid-metabolizing enzyme CYP26A1 has become a hallmark of some of the retinal specializations found in vertebrates, including the primate fovea and the high-acuity area in avian species. In chickens, the retinoic acid pathway regulates the expression of FGF8 to then direct the development of a rod-free area. Similarly, high levels of CYP26A1, CDKN1A, and NPVF expression have been observed in the primate macula using transcriptomic approaches. However, which retinal cells express these genes and their expression dynamics in the developing primate eye remain unknown. Here, we systematically characterize the expression patterns of CYP26A1, FGF8, CDKN1A, and NPVF during the development of the rhesus monkey retina, from early stages of development in the first trimester until the third trimester (near term). Our data suggest that some of the markers previously proposed to be fovea-specific are not enriched in the progenitors of the rhesus monkey fovea. In contrast, CYP26A1 is expressed at high levels in the progenitors of the fovea, while it localizes in a subpopulation of macular Müller glia cells later in development. Together these data provide invaluable insights into the expression dynamics of several molecules in the nonhuman primate retina and highlight the developmental advancement of the foveal region.

α-Synuclein-dependent increases in PIP5K1γ drive inositol signaling to promote neurotoxicity.

Anomalous aggregation of α-synuclein (α-Syn) is a pathological hallmark of many degenerative synucleinopathies including Lewy body dementia (LBD) and Parkinson's disease (PD). Despite its strong link to disease, the precise molecular mechanisms that link α-Syn aggregation to neurodegeneration have yet to be elucidated. Here, we find that elevated α-Syn leads to an increase in the plasma membrane (PM) phosphoinositide PI(4,5)P2, which precipitates α-Syn aggregation and drives toxic increases in mitochondrial Ca2+ and reactive oxygen species leading to neuronal death. Upstream of this toxic signaling pathway is PIP5K1γ, whose abundance and localization is enhanced at the PM by α-Syn-dependent increases in ARF6. Selective inhibition of PIP5K1γ or knockout of ARF6 in neurons rescues α-Syn aggregation and cellular phenotypes of toxicity. Collectively, our data suggest that modulation of phosphoinositide metabolism may be a therapeutic target to slow neurodegeneration for PD and other related neurodegenerative disorders.

Regulation of young-adult neurogenesis and neuronal differentiation by neural cell adhesion molecule 2 (NCAM2).

Adult neurogenesis persists in mammals in the neurogenic zones, where newborn neurons are incorporated into preexisting circuits to preserve and improve learning and memory tasks. Relevant structural elements of the neurogenic niches include the family of cell adhesion molecules (CAMs), which participate in signal transduction and regulate the survival, division, and differentiation of radial glial progenitors (RGPs). Here we analyzed the functions of neural cell adhesion molecule 2 (NCAM2) in the regulation of RGPs in adult neurogenesis and during corticogenesis. We characterized the presence of NCAM2 across the main cell types of the neurogenic process in the dentate gyrus, revealing different levels of NCAM2 amid the progression of RGPs and the formation of neurons. We showed that Ncam2 overexpression in adult mice arrested progenitors in an RGP-like state, affecting the normal course of young-adult neurogenesis. Furthermore, changes in Ncam2 levels during corticogenesis led to transient migratory deficits but did not affect the survival and proliferation of RGPs, suggesting a differential role of NCAM2 in adult and embryonic stages. Our data reinforce the relevance of CAMs in the neurogenic process by revealing a significant role of Ncam2 levels in the regulation of RGPs during young-adult neurogenesis in the hippocampus.

NPC1-dependent alterations in KV2.1-CaV1.2 nanodomains drive neuronal death in models of Niemann-Pick Type C disease.

Lysosomes communicate through cholesterol transfer at endoplasmic reticulum (ER) contact sites. At these sites, the Niemann Pick C1 cholesterol transporter (NPC1) facilitates the removal of cholesterol from lysosomes, which is then transferred to the ER for distribution to other cell membranes. Mutations in NPC1 result in cholesterol buildup within lysosomes, leading to Niemann-Pick Type C (NPC) disease, a progressive and fatal neurodegenerative disorder. The molecular mechanisms connecting NPC1 loss to NPC-associated neuropathology remain unknown. Here we show both in vitro and in an animal model of NPC disease that the loss of NPC1 function alters the distribution and activity of voltage-gated calcium channels (CaV). Underlying alterations in calcium channel localization and function are KV2.1 channels whose interactions drive calcium channel clustering to enhance calcium entry and fuel neurotoxic elevations in mitochondrial calcium. Targeted disruption of KV2-CaV interactions rescues aberrant CaV1.2 clustering, elevated mitochondrial calcium, and neurotoxicity in vitro. Our findings provide evidence that NPC is a nanostructural ion channel clustering disease, characterized by altered distribution and activity of ion channels at membrane contacts, which contribute to neurodegeneration.

Notch directs telencephalic development and controls neocortical neuron fate determination by regulating microRNA levels.

The central nervous system contains a myriad of different cell types produced from multipotent neural progenitors. Neural progenitors acquire distinct cell identities depending on their spatial position, but they are also influenced by temporal cues to give rise to different cell populations over time. For instance, the progenitors of the cerebral neocortex generate different populations of excitatory projection neurons following a well-known sequence. The Notch signaling pathway plays crucial roles during this process, but the molecular mechanisms by which Notch impacts progenitor fate decisions have not been fully resolved. Here, we show that Notch signaling is essential for neocortical and hippocampal morphogenesis, and for the development of the corpus callosum and choroid plexus. Our data also indicate that, in the neocortex, Notch controls projection neuron fate determination through the regulation of two microRNA clusters that include let-7, miR-99a/100 and miR-125b. Our findings collectively suggest that balanced Notch signaling is crucial for telencephalic development and that the interplay between Notch and miRNAs is essential for the control of neocortical progenitor behaviors and neuron cell fate decisions.

The E3 Ubiquitin Ligase CRL5 Regulates Dentate Gyrus Morphogenesis, Adult Neurogenesis, and Animal Behavior.

The dentate gyrus (DG) is an essential part of the hippocampal formation and participates in the majority of hippocampal functions. The DG is also one of the few structures in the mammalian central nervous system that produces adult-born neurons and, in humans, alterations in adult neurogenesis are associated with stress and depression. Given the importance of DG in hippocampal function, it is imperative to understand the molecular mechanisms driving DG development and homeostasis. The E3 ubiquitin ligase Cullin-5/RBX2 (CRL5) is a multiprotein complex involved in neuron migration and localization in the nervous system, but its role during development and in the adult DG remain elusive. Here, we show that CRL5 participates in mossy fiber pruning, DG layering, adult neurogenesis, and overall physical activity in mice. During DG development, RBX2 depletion causes an overextension of the DG mossy fiber infrapyramidal bundle (IPB). We further demonstrate that the increased activity in Reelin/DAB1 or ARF6 signaling, observed in RBX2 knockout mice, is not responsible for the lack of IPB pruning. Knocking out RBX2 also affects granule cell and neural progenitor localization and these defects were rescued by downregulating the Reelin/DAB1 signaling. Finally, we show that absence of RBX2 increases the number neural progenitors and adult neurogenesis. Importantly, RBX2 knockout mice exhibit higher levels of physical activity, uncovering a potential mechanism responsible for the increased adult neurogenesis in the RBX2 mutant DG. Overall, we present evidence of CRL5 regulating mossy fiber pruning and layering during development and opposing adult neurogenesis in the adult DG.

RapID Cell Counter: Semi-Automated and Mid-Throughput Estimation of Cell Density within Diverse Cortical Layers.

Tracking and quantifying the abundance and location of cells in the developing brain is essential in neuroscience research, enabling a greater understanding of mechanisms underlying nervous system morphogenesis. Widely used experimental methods to quantify cells labeled with fluorescent markers, such as immunohistochemistry (IHC), in situ hybridization, and expression of transgenes via stable lines or transient in utero electroporations (IUEs), depend on accurate and consistent quantification of images. Current methods to quantify fluorescently-labeled cells rely on labor-intensive manual counting approaches, such as the Fiji plugin Cell Counter, which requires custom macros to enable higher-throughput analyses. Here, we present RapID Cell Counter, a semi-automated cell-counting tool with an easy-to-implement graphical user interface (GUI), which facilitates quick and consistent quantifications of cell density within user-defined boundaries that can be divided into equally-partitioned segments. Compared with the standard manual counting approach, we show that RapID matched accuracy and consistency and only required ∼10% of user time relative to manual counting methods, when quantifying the distribution of fluorescently-labeled neurons in mouse IUE experiments. Using RapID, we recapitulated previously published work focusing on two genes, SRGAP2 and CUL5, important for projection neuron (PN) migration in the neocortex and used it to quantify PN displacement in a mouse knock-out model of RBX2 Moreover, RapID is capable of quantifying other cell types in the brain with complex cell morphologies, including astrocytes and dopaminergic neurons. We propose RapID as an efficient method for neuroscience researchers to process fluorescently-labeled brain images in a consistent, accurate, and mid-throughput manner.

IP3R-driven increases in mitochondrial Ca2+ promote neuronal death in NPC disease.

Ca2+ is the most ubiquitous second messenger in neurons whose spatial and temporal elevations are tightly controlled to initiate and orchestrate diverse intracellular signaling cascades. Numerous neuropathologies result from mutations or alterations in Ca2+ handling proteins; thus, elucidating molecular pathways that shape Ca2+ signaling is imperative. Here, we report that loss-of-function, knockout, or neurodegenerative disease-causing mutations in the lysosomal cholesterol transporter, Niemann-Pick Type C1 (NPC1), initiate a damaging signaling cascade that alters the expression and nanoscale distribution of IP3R type 1 (IP3R1) in endoplasmic reticulum membranes. These alterations detrimentally increase Gq-protein coupled receptor-stimulated Ca2+ release and spontaneous IP3R1 Ca2+ activity, leading to mitochondrial Ca2+ cytotoxicity. Mechanistically, we find that SREBP-dependent increases in Presenilin 1 (PS1) underlie functional and expressional changes in IP3R1. Accordingly, expression of PS1 mutants recapitulate, while PS1 knockout abrogates Ca2+ phenotypes. These data present a signaling axis that links the NPC1 lysosomal cholesterol transporter to the damaging redistribution and activity of IP3R1 that precipitates cell death in NPC1 disease and suggests that NPC1 is a nanostructural disease.

NPC1 regulates the distribution of phosphatidylinositol 4-kinases at Golgi and lysosomal membranes.

Cholesterol and phosphoinositides (PI) are two critically important lipids that are found in cellular membranes and dysregulated in many disorders. Therefore, uncovering molecular pathways connecting these essential lipids may offer new therapeutic insights. We report that loss of function of lysosomal Niemann-Pick Type C1 (NPC1) cholesterol transporter, which leads to neurodegenerative NPC disease, initiates a signaling cascade that alters the cholesterol/phosphatidylinositol 4-phosphate (PtdIns4P) countertransport cycle between Golgi-endoplasmic reticulum (ER), as well as lysosome-ER membrane contact sites (MCS). Central to these disruptions is increased recruitment of phosphatidylinositol 4-kinases-PI4KIIα and PI4KIIIß-which boosts PtdIns4P metabolism at Golgi and lysosomal membranes. Aberrantly increased PtdIns4P levels elevate constitutive anterograde secretion from the Golgi complex, and mTORC1 recruitment to lysosomes. NPC1 disease mutations phenocopy the transporter loss of function and can be rescued by inhibition or knockdown of either key phosphoinositide enzymes or their recruiting partners. In summary, we show that the lysosomal NPC1 cholesterol transporter tunes the molecular content of Golgi and lysosome MCS to regulate intracellular trafficking and growth signaling in health and disease.

MicroRNA Signatures of the Developing Primate Fovea.

Rod and cone photoreceptors differ in their shape, photopigment expression, synaptic connection patterns, light sensitivity, and distribution across the retina. Although rods greatly outnumber cones, human vision is mostly dependent on cone photoreceptors since cones are essential for our sharp visual acuity and color discrimination. In humans and other primates, the fovea centralis (fovea), a specialized region of the central retina, contains the highest density of cones. Despite the vast importance of the fovea for human vision, the molecular mechanisms guiding the development of this region are largely unknown. MicroRNAs (miRNAs) are small post-transcriptional regulators known to orchestrate developmental transitions and cell fate specification in the retina. Here, we have characterized the transcriptional landscape of the developing rhesus monkey retina. Our data indicates that non-human primate fovea development is significantly accelerated compared to the equivalent retinal region at the other side of the optic nerve head, as described previously. Notably, we also identify several miRNAs differentially expressed in the presumptive fovea, including miR-15b-5p, miR-342-5p, miR-30b-5p, miR-103-3p, miR-93-5p as well as the miRNA cluster miR-183/-96/-182. Interestingly, miR-342-5p is enriched in the nasal primate retina and in the peripheral developing mouse retina, while miR-15b is enriched in the temporal primate retina and increases over time in the mouse retina in a central-to-periphery gradient. Together our data constitutes the first characterization of the developing rhesus monkey retinal miRNome and provides novel datasets to attain a more comprehensive understanding of foveal development.

CRL5-dependent regulation of the small GTPases ARL4C and ARF6 controls hippocampal morphogenesis.

The small GTPase ARL4C participates in the regulation of cell migration, cytoskeletal rearrangements, and vesicular trafficking in epithelial cells. The ARL4C signaling cascade starts by the recruitment of the ARF-GEF cytohesins to the plasma membrane, which, in turn, bind and activate the small GTPase ARF6. However, the role of ARL4C-cytohesin-ARF6 signaling during hippocampal development remains elusive. Here, we report that the E3 ubiquitin ligase Cullin 5/RBX2 (CRL5) controls the stability of ARL4C and its signaling effectors to regulate hippocampal morphogenesis. Both RBX2 knockout and Cullin 5 knockdown cause hippocampal pyramidal neuron mislocalization and development of multiple apical dendrites. We used quantitative mass spectrometry to show that ARL4C, Cytohesin-1/3, and ARF6 accumulate in the RBX2 mutant telencephalon. Furthermore, we show that depletion of ARL4C rescues the phenotypes caused by Cullin 5 knockdown, whereas depletion of CYTH1 or ARF6 exacerbates overmigration. Finally, we show that ARL4C, CYTH1, and ARF6 are necessary for the dendritic outgrowth of pyramidal neurons to the superficial strata of the hippocampus. Overall, we identified CRL5 as a key regulator of hippocampal development and uncovered ARL4C, CYTH1, and ARF6 as CRL5-regulated signaling effectors that control pyramidal neuron migration and dendritogenesis.

A Combinatorial MAP Code Dictates Polarized Microtubule Transport.

Many eukaryotic cells distribute their intracellular components asymmetrically through regulated active transport driven by molecular motors along microtubule tracks. While intrinsic and extrinsic regulation of motor activity exists, what governs the overall distribution of activated motor-cargo complexes within cells remains unclear. Here, we utilize in vitro reconstitution of purified motor proteins and non-enzymatic microtubule-associated proteins (MAPs) to demonstrate that MAPs exhibit distinct influences on the motility of the three main classes of transport motors: kinesin-1, kinesin-3, and cytoplasmic dynein. Further, we dissect how combinations of MAPs affect motors and unveil MAP9 as a positive modulator of kinesin-3 motility. From these data, we propose a general "MAP code" that has the capacity to strongly bias directed movement along microtubules and helps elucidate the intricate intracellular sorting observed in highly polarized cells such as neurons.

NCAM2 Regulates Dendritic and Axonal Differentiation through the Cytoskeletal Proteins MAP2 and 14-3-3.

Neural cell adhesion molecule 2 (NCAM2) is involved in the development and plasticity of the olfactory system. Genetic data have implicated the NCAM2 gene in neurodevelopmental disorders including Down syndrome and autism, although its role in cortical development is unknown. Here, we show that while overexpression of NCAM2 in hippocampal neurons leads to minor alterations, its downregulation severely compromises dendritic architecture, leading to an aberrant phenotype including shorter dendritic trees, retraction of dendrites, and emergence of numerous somatic neurites. Further, our data reveal alterations in the axonal tree and deficits in neuronal polarization. In vivo studies confirm the phenotype and reveal an unexpected role for NCAM2 in cortical migration. Proteomic and cell biology experiments show that NCAM2 molecules exert their functions through a protein complex with the cytoskeletal-associated proteins MAP2 and 14-3-3γ and ζ. We provide evidence that NCAM2 depletion results in destabilization of the microtubular network and reduced MAP2 signal. Our results demonstrate a role for NCAM2 in dendritic formation and maintenance, and in neural polarization and migration, through interaction of NCAM2 with microtubule-associated proteins.

Let-7 regulates cell cycle dynamics in the developing cerebral cortex and retina.

In the neural progenitors of the developing central nervous system (CNS), cell proliferation is tightly controlled and coordinated with cell fate decisions. Progenitors divide rapidly during early development and their cell cycle lengthens progressively as development advances to eventually give rise to a tissue of the correct size and cellular composition. However, our understanding of the molecules linking cell cycle progression to developmental time is incomplete. Here, we show that the microRNA (miRNA) let-7 accumulates in neural progenitors over time throughout the developing CNS. Intriguingly, we find that the level and activity of let-7 oscillate as neural progenitors progress through the cell cycle by in situ hybridization and fluorescent miRNA sensor analyses. We also show that let-7 mediates cell cycle dynamics: increasing the level of let-7 promotes cell cycle exit and lengthens the S/G2 phase of the cell cycle, while let-7 knock down shortens the cell cycle in neural progenitors. Together, our findings suggest that let-7 may link cell proliferation to developmental time and regulate the progressive cell cycle lengthening that occurs during development.

Microtubules gate tau condensation to spatially regulate microtubule functions.

Tau is an abundant microtubule-associated protein in neurons. Tau aggregation into insoluble fibrils is a hallmark of Alzheimer's disease and other types of dementia1, yet the physiological state of tau molecules within cells remains unclear. Using single-molecule imaging, we directly observe that the microtubule lattice regulates reversible tau self-association, leading to localized, dynamic condensation of tau molecules on the microtubule surface. Tau condensates form selectively permissible barriers, spatially regulating the activity of microtubule-severing enzymes and the movement of molecular motors through their boundaries. We propose that reversible self-association of tau molecules, gated by the microtubule lattice, is an important mechanism of the biological functions of tau, and that oligomerization of tau is a common property shared between the physiological and disease-associated forms of the molecule.

A Gs-coupled purinergic receptor boosts Ca2+ influx and vascular contractility during diabetic hyperglycemia.

Elevated glucose increases vascular reactivity by promoting L-type CaV1.2 channel (LTCC) activity by protein kinase A (PKA). Yet, how glucose activates PKA is unknown. We hypothesized that a Gs-coupled P2Y receptor is an upstream activator of PKA mediating LTCC potentiation during diabetic hyperglycemia. Experiments in apyrase-treated cells suggested involvement of a P2Y receptor underlying the glucose effects on LTTCs. Using human tissue, expression for P2Y11, the only Gs-coupled P2Y receptor, was detected in nanometer proximity to CaV1.2 and PKA. FRET-based experiments revealed that the selective P2Y11 agonist NF546 and elevated glucose stimulate cAMP production resulting in enhanced PKA-dependent LTCC activity. These changes were blocked by the selective P2Y11 inhibitor NF340. Comparable results were observed in mouse tissue, suggesting that a P2Y11-like receptor is mediating the glucose response in these cells. These findings established a key role for P2Y11 in regulating PKA-dependent LTCC function and vascular reactivity during diabetic hyperglycemia.

Health and Safety at Work in the Transport Industry (TRANS-12): Factorial Structure, Reliability and Validity / Salud y seguridad laboral en la industria del transporte (TRANS-12): estructura factorial, fiabilidad y validez

Background: This study sets out the psychometric properties of the TRANS-18 scale and of a shorter version, the TRANS-12, both designed to detect safe behaviors (personal and vehicle-related) and psychophysiological disorders among professional drivers. Method: The investigation was divided into Study 1, into the factorial structure, reliability and validity of the TRANS-18, and Study 2, looking into the same aspects of the TRANS-12. The participants in both studies were resident in Spain. 272 professional drivers took part in Study 1, while Study 2 had 326 participants. Results: A confirmatory factor analysis was carried out for both studies. The results for Study 1 confirm an internal structure of three factors related to psychophysiological disorders and personal and vehicle-related safety behaviors, but the original TRANS-18 is discarded because it does not fit the model. With regard to Study 2, the results show a good fit of the three-factor model, appropriate reliability and evidence of validity. Conclusions: We conclude by considering the suitability of the psychometric properties of the TRANS-12 and its utility for identifying safe behaviors in work in the transport industry

Antecedentes: Este estudio expone las propiedades psicométricas de la escala TRANS-18 y de una versión más corta, el TRANS-12, ambos diseñados para detectar conductas seguras (personales y relacionadas con el vehículo) y trastornos psicofisiológicos entre conductores profesionales. Método: La investigación se divide en dos. Estudio 1, estructura factorial, fiabilidad y validez del TRANS-18, y Estudio 2, se estudian los mismos aspectos en el TRANS-12. Los participantes en ambos estudios fueron residentes en España. 272 conductores profesionales participaron en el Estudio 1, mientras que el Estudio 2 participaron 326 conductores. Resultados: Se realizó un análisis factorial confirmatorio para ambos estudios. Los resultados del Estudio 1 confirman una estructura interna de tres factores relacionados con los trastornos psicofisiológicos y las conductas de seguridad personales y con el vehículo, pero el TRANS-18 original se descarta por no ajustarse al modelo. Con respecto al Estudio 2, los resultados muestran un buen ajuste del modelo de tres factores, la fiabilidad apropiada y la evidencia de validez. Conclusiones: Concluimos considerando la idoneidad de las propiedades psicométricas del TRANS-12 y su utilidad para identificar comportamientos seguros en el trabajo en la industria del transporte

Comparative Analysis of cul5 and rbx2 Expression in the Developing and Adult Murine Brain and Their Essentiality During Mouse Embryogenesis.

BACKGROUND: The E3 Cullin 5-RING ubiquitin ligase (CRL5) is a multiprotein complex that has recently been highlighted as a major regulator of central nervous system development. Cullin 5 (Cul5) and the RING finger protein Rbx2 are two CRL5 core components required for CRL5 function in the brain, but their full expression patterns and developmental functions have not been described in detail. RESULTS: Using a gene-trap mouse model for Cul5 and a knock-in-knockout mouse model for Rbx2, we show that lack of Cul5, but not Rbx2, disrupts blastocyst formation. However, Rbx2 is required for embryo survival at later embryonic stages. We also show that cul5 is expressed in the embryo proper as early as E7.5 and its expression is mostly restricted to the central nervous system and limbs at later time points. Finally, we show that rbx2 and cul5 are co-expressed in most areas of the brain during development and in the adult. CONCLUSIONS: Our results show that Cul5, but not Rbx2, is required during early embryogenesis and suggests that Cul5 has Rbx2-independent functions in early development. In the brain, Cul5 and Rbx2 are expressed in a similar fashion, allowing the nucleation of an active CRL5 complex. Developmental Dynamics 247:1227-1236, 2018. © 2018 Wiley Periodicals, Inc.

RBX2 maintains final retinal cell position in a DAB1-dependent and -independent fashion.

The laminated structure of the retina is fundamental for the organization of the synaptic circuitry that translates light input into patterns of action potentials. However, the molecular mechanisms underlying cell migration and layering of the retina are poorly understood. Here, we show that RBX2, a core component of the E3 ubiquitin ligase CRL5, is essential for retinal layering and function. RBX2 regulates the final cell position of rod bipolar cells, cone photoreceptors and Muller glia. Our data indicate that sustained RELN/DAB1 signaling, triggered by depletion of RBX2 or SOCS7 - a CRL5 substrate adaptor known to recruit DAB1 - causes rod bipolar cell misposition. Moreover, whereas SOCS7 also controls Muller glia cell lamination, it is not responsible for cone photoreceptor positioning, suggesting that RBX2, most likely through CRL5 activity, controls other signaling pathways required for proper cone localization. Furthermore, RBX2 depletion reduces the number of ribbon synapses and disrupts cone photoreceptor function. Together, these results uncover RBX2 as a crucial molecular regulator of retina morphogenesis and cone photoreceptor function.