Cortical Cells Research Articles

Single-cell dissection of the human motor and prefrontal cortices in ALS and FTLD

Amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration (FTLD) share many clinical, pathological, and genetic features, but a detailed understanding of their associated transcriptional alterations across vulnerable cortical cell types is lacking. Here, we report a high-resolution, comparative single-cell molecular atlas of the human primary motor and dorsolateral prefrontal cortices and their transcriptional alterations in sporadic and familial ALS and FTLD. By integrating transcriptional and genetic information, we identify known and previously unidentified vulnerable populations in cortical layer 5 and show that ALS- and FTLD-implicated motor and spindle neurons possess a virtually indistinguishable molecular identity. We implicate potential disease mechanisms affecting these cell types as well as non-neuronal drivers of pathogenesis. Finally, we show that neuron loss in cortical layer 5 tracks more closely with transcriptional identity rather than cellular morphology and extends beyond previously reported vulnerable cell types.

LPS-induced inflammation reduces GABAergic interneuron markers and brain-derived neurotrophic factor in mouse prefrontal cortex and hippocampus

Inflammation, reduced gamma-aminobutyric acidergic (GABAergic) function and altered neuroplasticity are co-occurring pathophysiologies in major depressive disorder (MDD). However, the link between these biological changes remains unclear. We hypothesized that inflammation induces deficits in GABAergic interneuron markers and that this effect is mediated by brain-derived neurotrophic factor (BDNF). We report here that systemic inflammation induced by intraperitoneal injection of lipopolysaccharide (LPS) (0.125, 0.25, 0.5, 1, 2 mg/kg) in the first cohort of C57BL/6 mice (n = 72; 10–11 weeks; 50% female) resulted in increased interleukin 1-beta and interleukin-6 in prefrontal cortex (PFC) and hippocampus (HPC), as measured using enzyme-linked immunosorbent assay (ELISA). Quantitative real-time polymerase reaction (qPCR) was used to explore the effect of LPS on the expression of GABAergic interneuron markers. In the PFC of the second cohort (n = 39; 10–11 weeks; 50% female), 2 mg/kg of LPS decreased the expression of somatostatin (Sst) (p = 0.0014), parvalbumin (Pv) (p = 0.0257), cortistatin (Cort) (p = 0.0003), neuropeptide Y (Npy) (p = 0.0033) and cholecystokinin (Cck) (p = 0.0041), and did not affect corticotropin-releasing hormone (Crh) and vasoactive intestinal peptide (Vip) expression. In the HPC, 2 mg/kg of LPS decreased the expression of Sst (p = 0.0543), Cort (p = 0.0011), Npy (p = 0.0001), and Cck (p < 0.0001), and did not affect Crh, Pv, and Vip expression. LPS decreased the expression of Bdnf in the PFC (p < 0.0001) and HPC (p = 0.0003), which significantly correlated with affected markers (Sst, Pv, Cort, Cck, and Npy). Collectively, these results suggest that inflammation may causally contribute to cortical cell microcircuit GABAergic deficits observed in MDD.

Characterization of the fiber-like cortical cells in moss gametophytes.

Fiber-like cells with thickened cell walls of specific structure and polymer composition that includes (1 → 4)-β-galactans develop in the outer stem cortex of several moss species gametophytes. The early land plants evolved several specialized cell types and tissues that did not exist in their aquatic ancestors. Of these, water-conducting elements and reproductive organs have received most of the research attention. The evolution of tissues specialized to fulfill a mechanical function is by far less studied despite their wide distribution in land plants. For vascular plants following a homoiohydric trajectory, the evolutionary emergence of mechanical tissues is mainly discussed starting with the fern-like plants with their hypodermal sterome or sclerified fibers that have xylan and lignin-based cell walls. However, mechanical challenges were also faced by bryophytes, which lack lignified cell-walls. To characterize mechanical tissues in the bryophyte lineage, following a poikilohydric trajectory, we used six wild moss species (Polytrichum juniperinum, Dicranum sp., Rhodobryum roseum, Eurhynchiadelphus sp., Climacium dendroides, and Hylocomium splendens) and analyzed the structure and composition of their cell walls. In all of them, the outer stem cortex of the leafy gametophytic generation had fiber-like cells with a thickened but non-lignified cell wall. Such cells have a spindle-like shape with pointed tips. The additional thick cell wall layer in those fiber-like cells is composed of sublayers with structural evidence for different cellulose microfibril orientation, and with specific polymer composition that includes (1 → 4)-β-galactans. Thus, the basic cellular characters of the cells that provide mechanical support in vascular plant taxa (elongated cell shape, location at the periphery of a primary organ, the thickened cell wall and its peculiar composition and structure) also exist in mosses.

Induction of SK-MEL-28 Invasion by Brain Cortical Cell-Conditioned Medium Through CXCL10 Signaling.

Melanoma, an infrequent yet significant variant of skin cancer, emerges as a primary cause of brain metastasis among various malignancies. Despite recognizing the involvement of inflammatory molecules, particularly chemokines, in shaping the metastatic microenvironment, the intricate cellular signaling mechanisms underlying cerebral metastasis remain elusive. In our pursuit to unravel the role of cytokines in melanoma metastasis, we devised a protocol utilizing mixed cerebral cortical cells and SK-MEL-28 melanoma cell lines. Contrary to expectations, we observed no discernible morphological change in melanoma cells exposed to a cerebral conditioned medium (CM). However, a substantial increase in both migration and proliferation was quantitatively noted. Profiling the chemokine secretion by melanoma in response to the cerebral CM unveiled the pivotal role of interferon gamma-induced protein 10 (CXCL10), inhibiting the secretion of interleukin 8 (CXCL8). Furthermore, through a transwell assay, we demonstrated that knockdown CXCL10 led to a significant decrease in the migration of the SK-MEL-28 cell line. In conclusion, our findings suggest that a cerebral CM induces melanoma cell migration, while modulating the secretion of CXCL10 and CXCL8 in the context of brain metastases. These insights advance our understanding of the underlying mechanisms in melanoma cerebral metastasis, paving the way for further exploration and targeted therapeutic interventions.

ERK signaling expands mammalian cortical radial glial cells and extends the neurogenic period

The molecular basis for cortical expansion during evolution remains largely unknown. Here, we report that fibroblast growth factor (FGF)-extracellular signal-regulated kinase (ERK) signaling promotes the self-renewal and expansion of cortical radial glial (RG) cells. Furthermore, FGF-ERK signaling induces bone morphogenic protein 7 (Bmp7) expression in cortical RG cells, which increases the length of the neurogenic period. We demonstrate that ERK signaling and Sonic Hedgehog (SHH) signaling mutually inhibit each other in cortical RG cells. We provide evidence that ERK signaling is elevated in cortical RG cells during development and evolution. We propose that the expansion of the mammalian cortex, notably in human, is driven by the ERK-BMP7-GLI3R signaling pathway in cortical RG cells, which participates in a positive feedback loop through antagonizing SHH signaling. We also propose that the relatively short cortical neurogenic period in mice is partly due to mouse cortical RG cells receiving higher SHH signaling that antagonizes ERK signaling.

Rethinking superpixel segmentation from biologically inspired mechanisms

Recently, advancements in deep learning-based superpixel segmentation methods have brought about improvements in both the efficiency and the performance of segmentation. However, a significant challenge remains in generating superpixels that strictly adhere to object boundaries while conveying rich visual significance, especially with limited training data, leading to the generation of blunt superpixel that encompass different semantics. To address these challenges, we propose a novel bio-inspired superpixel segmentation network (BINet), drawing inspiration from neural structures and visual mechanisms. Specifically, the BINet includes an Enhanced Screening Module (ESM) and a novel Boundary-Aware Label (BAL) for superpixel segmentation. The ESM enhances semantic information by simulating visual cortex interactive projection mechanisms, while the BAL emulates spatial frequency characteristics of visual cortical cells to generate superpixels with strong boundary adherence. Extensive experiments on BSDS500, NYUv2 and KITTI datasets show that our method achieves state-of-the-art performances but maintain satisfactory inference efficiency. Our code is available at https://github.com/zhaotingyu-ss/BINet.

Cortical cell assemblies and their underlying connectivity: An in silico study.

Recent developments in experimental techniques have enabled simultaneous recordings from thousands of neurons, enabling the study of functional cell assemblies. However, determining the patterns of synaptic connectivity giving rise to these assemblies remains challenging. To address this, we developed a complementary, simulation-based approach, using a detailed, large-scale cortical network model. Using a combination of established methods we detected functional cell assemblies from the stimulus-evoked spiking activity of 186,665 neurons. We studied how the structure of synaptic connectivity underlies assembly composition, quantifying the effects of thalamic innervation, recurrent connectivity, and the spatial arrangement of synapses on dendrites. We determined that these features reduce up to 30%, 22%, and 10% of the uncertainty of a neuron belonging to an assembly. The detected assemblies were activated in a stimulus-specific sequence and were grouped based on their position in the sequence. We found that the different groups were affected to different degrees by the structural features we considered. Additionally, connectivity was more predictive of assembly membership if its direction aligned with the temporal order of assembly activation, if it originated from strongly interconnected populations, and if synapses clustered on dendritic branches. In summary, reversing Hebb's postulate, we showed how cells that are wired together, fire together, quantifying how connectivity patterns interact to shape the emergence of assemblies. This includes a qualitative aspect of connectivity: not just the amount, but also the local structure matters; from the subcellular level in the form of dendritic clustering to the presence of specific network motifs.

Interplay between developmental cues and rhizosphere signals from mycorrhizal fungi shape root anatomy, impacting crop productivity

Abstract Background The rhizosphere is the interface between roots and the soil and the site of nutrient and water uptake for plant growth. Root anatomy and the physical, chemical, and biological components of the rhizosphere interact to influence plant growth. Several root developmental and rhizosphere signals combine in the patterning of root cortical anatomy and have implications for the plant’s hydro-mineral nutrition and carbon partitioning and therefore crop productivity, especially in edaphic stress. Scope Here, we highlight how mutualistic mycorrhizal fungi from the rhizosphere mobilize plant molecular actors controlling root anatomical traits, including cortical cell size, to facilitate their establishment and accommodation within the cortex. We explore the effects on plant growth and stress tolerance that may result from the changes in root anatomy driven by interactions with arbuscular mycorrhizal fungi, including altering the metabolic efficiency required for nutrient exploitation. We also discuss opportunities for understanding the genetic control of root anatomy and rhizosphere interactions to enable a comprehensive understanding of the benefits and trade-offs of root-rhizosphere interactions for more productive crops.

Thromboxane A2 Modulates de novo Synthesis of Adrenal Corticosterone in Mice via p38/14-3-3γ/StAR Signaling.

Prostanoids are endogenous lipid bioactive mediators that play essential roles in physiological processes such as glucocorticoid secretion. Here, it is found that the thromboxane (Tx)A2 receptor (TP) is highly expressed in the adrenal cortex of mice. Both global and adrenocortical-specific deletion of the TP receptor lead to increased adiposity in mice by elevating corticosterone synthesis. Mechanistically, the TP receptor deletion increases the phosphorylation of steroidogenic acute regulatory protein (StAR) and corticosterone synthesis in adrenal cortical cells by suppressing p-p38-mediated phosphorylation of 14-3-3γ adapter protein at S71. The activation of the p38 in the adrenal cortical cells by forced expression of the MKK6EE gene attenuates hypercortisolism in TP-deficient mice. These observations suggest that the TxA2/TP signaling regulates adrenal corticosterone homeostasis independent of the hypothalamic-pituitary-adrenal axis and the TP receptor may serve as a promising therapeutic target for hypercortisolism.

Model biological systems demonstrate the inducibility of pathways that strongly reduce cryoprotectant toxicity

Cryoprotectant toxicity is a limiting factor for the cryopreservation of many living systems. We were moved to address this problem by the potential of organ vitrification to relieve the severe shortage of viable donor organs available for human transplantation. The M22 vitrification solution is presently the only solution that has enabled the vitrification and subsequent transplantation with survival of large mammalian organs, but its toxicity remains an obstacle to organ stockpiling for transplantation. We therefore undertook a series of exploratory studies to identify potential pretreatment interventions that might reduce the toxic effects of M22. Hormesis, in which a living system becomes more resistant to toxic stress after prior subtoxic exposure to a related stress, was investigated as a potential remedy for M22 toxicity in yeast, in the nematode worm C. elegans, and in mouse kidney slices. In yeast, heat shock pretreatment increased survival by 18-fold after exposure to formamide and by over 9-fold after exposure to M22 at 30 °C; at 0 °C and with two-step addition, treatment with 90% M22 resulted in 100% yeast survival. In nematodes, surveying a panel of pretreatment interventions revealed 3 that conferred nearly total protection from acute whole-worm M22-induced damage. One of these protective pretreatments (exposure to hydrogen peroxide) was applied to mouse kidney slices in vitro and was found to strongly protect nuclear and plasma membrane integrity in both cortical and medullary renal cells exposed to 75–100% M22 at room temperature for 40 min. These studies demonstrate for the first time that endogenous cellular defenses, conserved from yeast to mammals, can be marshalled to substantially ameliorate the toxic effects of one of the most toxic single cryoprotectants and the toxicity of the most concentrated vitrification solution so far described for whole organs.

Elevation-driven modifications in tissue architecture and physiobiochemical traits of Panicum antidotale Retz. in the Pothohar Plateau, Pakistan

Mountainous ecosystems are experiencing rapid shifts in distribution patterns and community structure due to the impacts of climate change and vegetation dynamics. This study offers a comprehensive insight into the adaptive mechanisms of Panicum antidotale to varying elevations within the Pothohar Plateau, shedding light on its survival strategies in climate-affected regions. The findings reveal that populations at lower elevations exhibit maximum height, leaf area, shoot length, and fresh weight, whereas those at higher elevations display significant decrease in these parameters. Moreover, a significant decline in species density, frequency and cover is evident at high elevation. High-elevation populations demonstrate decreased photosynthetic traits, including chlorophyll, carotenoid, and Na and Ca content. Furthermore, there are notable variations in the antioxidant enzyme activity across elevation gradients. Populations at lower elevations exhibit higher activities of superoxide dismutase and peroxidase. Across the gradient, there were significant reductions in root and stem radius, sclerenchyma thickness, bulliform cell size, metaxylem vessels, and phloem area. Populations at moderate elevations displayed thicker lamina and epidermis, larger cortical cells, wider xylem vessels, and increased phloem area; however, these traits declined at higher elevations. High-elevation populations showed thicker lamina and epidermis, extensive sclerenchyma, greater cortical cell area, and higher stomatal density. Photosynthetic traits and enzymatic activity were major contributors at moderate elevations while stem and leaf traits were significantly changed at high elevations. Overall, this research contributes significantly to the broader knowledge of plant adaptation in mountainous ecosystems and stresses the importance of considering elevation as a critical factor in ecological studies.

Preconditioning exercise reduces brain damage of ischemic stroke in rats via PI3K-AKT pathway by bioinformatic analysis.

Ischemic stroke is one of the most vital causes of high neurological morbidity and mortality in the world. Preconditioning exercise is considered as the primary prevention of stroke to resistance to subsequent injury. We tried to research the underlying biological mechanisms of this exercise. Forty-two SD rats were randomly divided into three groups: middle cerebral artery occlusion (MCAO) group, exercise group with MCAO (EX + MCAO) group, and sham group, with 14 rats in each group. The EX + MCAO group underwent exercise preconditioning for 3weeks before occlusion, and the other two groups were fed and exercised normally. After 3weeks, MCAO model was made by thread plug method in the EX + MCAO group and MCAO group. After successful modeling, the Longa scale was used to evaluate the neurological impairment of rats at day 0, day 1, and day 2. The rats in each group were killed on the third day after modeling. TTC staining measured the infarct volume of each group. The morphology and apoptosis of cortical cells were observed by HE and Tunel staining. Three rats in each group underwent high-throughput sequencing. Bioinformatic analysis was used to find the deferentially expressed genes (DEGs) and predict the transcription factor binding sites (TFBS) of the next-generation sequencing results. Gene enrichment (GSEA) was used to analyze potential functional genes and their corresponding signaling pathways. The Longa scale showed EX + MCAO group had the neurological function better than the modeling group (P < 0.001). TTC staining showed that the infarct size of EX + MCAO group was less than MCAO group (P < 0.05). HE and Tunel staining showed that the cells in the EX + MCAO group and the sham group had normal morphology and fewer apoptotic cells than MCAO group. A new gene named 7994 was discovered and TFBS of this gene was predicted, which could interact with key genes such as Foxd3, Foxa2, NR4A2, SP1, CEBPA, and SOX10. GSEA showed that EX + MCAO group could promote and regulate angiogenesis and apoptosis through PI3K-AKT pathway. Preconditioning exercise could improve nerve function and reduce infarct size in rats. The underlying mechanism is to regulate the PI3K-AKT pathway through several key genes, promote cerebral angiogenesis, and reduce apoptosis.

Phase contrast reflectance confocal brain imaging at 1650nm.

The imaging depth of microscopy techniques is limited by the ability of light to penetrate biological tissue. Recent research has addressed this limitation by combining a reflectance confocal microscope with the NIR-II (or shortwave infrared) spectrum. This approach offers significant imaging depth, is straightforward in design, and remains cost-effective. However, the imaging system, which relies on intrinsic signals, could benefit from adjustments in its optical design and post-processing methods to differentiate cortical cells, such as neurons and small blood vessels. We implemented a phase contrast detection scheme to a reflectance confocal microscope using NIR-II spectral range as illumination. We analyzed the features retrieved in the images while testing the imaging depth. Moreover, we introduce an acquisition method for distinguishing dynamic signals from the background, allowing the creation of vascular maps similar to those produced by optical coherence tomography. The phase contrast implementation is successful to retrieve deep images in the cortex up to using a cranial window. Vascular maps were retrieved at similar cortical depth and the possibility of combining multiple images can provide a vessel network. Phase contrast reflectance confocal microscopy can improve the outlining of cortical cell bodies. With the presented framework, angiograms can be retrieved from the dynamic signal in the biological tissue. Our work presents an optical implementation and analysis techniques from a former microscope design.

In Vitro Pharmacological Modulation of PIEZO1 Channels in Frontal Cortex Neuronal Networks.

PIEZO1 is a mechanosensitive ion channel expressed in various organs, including but not limited to the brain, heart, lungs, kidneys, bone, and skin. PIEZO1 has been implicated in astrocyte, microglia, capillary, and oligodendrocyte signaling in the mammalian cortex. Using murine embryonic frontal cortex tissue, we examined the protein expression and functionality of PIEZO1 channels in cultured networks leveraging substrate-integrated microelectrode arrays (MEAs) with additional quantitative results from calcium imaging and whole-cell patch-clamp electrophysiology. MEA data show that the PIEZO1 agonist Yoda1 transiently enhances the mean firing rate (MFR) of single units, while the PIEZO1 antagonist GsMTx4 inhibits both spontaneous activity and Yoda1-induced increase in MFR in cortical networks. Furthermore, calcium imaging experiments revealed that Yoda1 significantly increased the frequency of calcium transients in cortical cells. Additionally, in voltage clamp experiments, Yoda1 exposure shifted the cellular reversal potential towards depolarized potentials consistent with the behavior of PIEZO1 as a non-specific cation-permeable channel. Our work demonstrates that murine frontal cortical neurons express functional PIEZO1 channels and quantifies the electrophysiological effects of channel activation in vitro. By quantifying the electrophysiological effects of PIEZO1 activation in vitro, our study establishes a foundation for future investigations into the role of PIEZO1 in neurological processes and potential therapeutic applications targeting mechanosensitive channels in various physiological contexts.

Therapeutic Polymer-Based Cannabidiol Formulation: Tackling Neuroinflammation Associated with Ischemic Events in the Brain.

Cannabidiol (CBD) is the most relevant nonpsychostimulant phytocompound found in Cannabis sativa. CBD has been extensively studied and has been proposed as a therapeutic candidate for neuroinflammation-related conditions. However, being a highly lipophilic drug, it has several drawbacks for pharmaceutical use, including low solubility and high permeability. Synthetic polymers can be used as drug delivery systems to improve CBD's stability, half-life, and biodistribution. Here, we propose using a synthetic polymer as a nanoparticulate vehicle for CBD (NPCBD) to overcome the pharmacological drawbacks of free drugs. We tested the NPCBD-engineered system in the context of ischemic events in a relevant oxygen and glucose deprivation (OGD) model in primary cortical cells (PCC). Moreover, we have characterized the inflammatory response of relevant cell types, such as THP-1 (human monocytes), HMC3 (human microglia), and PCC, to NPCBD and observed a shift in the inflammatory state of the treated cells after the ischemic event. In addition, NPCBD exhibited a promising ability to restore mitochondrial function after OGD insult in both HMC3 and PCC cells at low doses of 1 and 0.2 μM CBD. Taken together, these results suggest the potential for preclinical use.

Comparative light and scanning electron microscopic studies of the lenses in the insectivorous bat (Pipistrellus kuhlii) and Egyptian fruit bat (Rousettus aegyptiacus).

Bats have the ability to fly without eye application in the darkness. In this study, we aimed to characterize the functional and structural acclimations of the lenses of two common bats with a various lifestyle in the Egyptian environment: the insectivorous bat (IB) (Pipistrellus kuhlii) and Egyptian fruit bat (FB) (Rousettus aegyptiacus). From each species, seven lenses were extracted from adult eyes. The scanning electron microscopic (SEM) and light microscopic examination of the lens were carried out. FB lenses were made up primarily of fiber cells and sheets, which were encapsulated by a thin collagenous capsule and covered by single epithelial layer anteriorly. On the other hand, the IB lens had two poles and was visibly oval shaped. Both lenses had epithelial cells of the same cuboidal form that were subjected to continuous division and differentiation into new fiber cells at the center. SEM revealed that the normal FB lens had regularly organized shells of fiber cells of intact lens fibers which were connected by membrane interdigitations with different shapes mainly ball-and-socket junctions through the superficial cortical fiber cells. The IB lens was composedof parallel, evenly spaced fibers with various types of interdigitations between fibers that can be seen and increased close to the middle region revealing tiny bumps along the scrubby portions and sockets and balls in the center of the wide portions. Near the center of both lenses, there were large interlocking paddles with little and lengthy protrusions along their short sides. In conclusion, our study discovered several ultrastructural and structural variations among the investigated species. The detection of specialized membrane interdigitations with different shapes protruding from the lens fiber sheets is considered the most characteristic of the FB lens. RESEARCH HIGHLIGHTS: FB lens has more organized sheets of fibers parallel to each other than IB lens. Different shapes of interdigitations protruded from the FB lens have been detected. Interlocking paddles, balls, and sockets with tongue-like fiber flabs are characteristic to FB lens.

Integration of Microfluidic Devices with Microelectrode Arrays to Functionally Assay Amyloid-β-Induced Synaptotoxicity.

Alzheimer's disease (AD) is a neurodegenerative disease and the most frequent cause of dementia. It is characterized by the accumulation in the brain of two pathological protein aggregates: amyloid-β peptides (Aβ) and abnormally phosphorylated tau. The progressive cognitive decline observed in patients strongly correlates with the synaptic loss. Many lines of evidence suggest that soluble forms of Aβ accumulate into the brain where they cause synapse degeneration. Stopping their spreading and/or targeting the pathophysiological mechanisms leading to synaptic loss would logically be beneficial for the patients. However, we are still far from understanding these processes. Our objective was therefore to develop a versatile model to assay and study Aβ-induced synaptotoxicity. We integrated a microfluidic device that physically isolates synapses from presynaptic and postsynaptic neurons with a microelectrode array. We seeded mouse primary cortical cells in the presynaptic and postsynaptic chambers. After functional synapses have formed in the synaptic chamber, we exposed them to concentrated conditioned media from cell lines overexpressing the wild-type or mutated amyloid precursor protein and thus secreting different levels of Aβ. We recorded the neuronal activity before and after exposition to Aβ and quantified Aβ's effects on the connectivity between presynaptic and postsynaptic neurons. We observed that the application of Aβ on the synapses for 48 h strongly decreased the interchamber connectivity without significantly affecting the neuronal activity in the presynaptic or postsynaptic chambers. Thus, through this model, we are able to functionally assay the impact of Aβ peptides (or other molecules) on synaptic connectivity and to use the latter as a proxy to study Aβ-induced synaptotoxicity. Moreover, since the presynaptic, postsynaptic, and synaptic chambers can be individually targeted, our assay provides a powerful tool to evaluate the involvement of candidate genes in synaptic vulnerability and/or test therapeutic strategies for AD.

Nfe2l3 promotes neuroprotection and long-distance axon regeneration after injury in vivo

Nuclear factor erythroid 2 like (Nfe2l) gene family members 1–3 mediate cellular response to oxidative stress, including in the central nervous system (CNS). However, neuronal functions of Nfe2l3 are unknown. Here, we comparatively evaluated expression of Nfe2l1, Nfe2l2, and Nfe2l3 in singe cell RNA-seq (scRNA-seq)-profiled cortical and retinal ganglion cell (RGC) CNS projection neurons, investigated whether Nfe2l3 regulates neuroprotection and axon regeneration after CNS injury in vivo, and characterized a gene network associated with Nfe2l3 in neurons. We showed that, Nfe2l3 expression transiently peaks in developing immature cortical and RGC projection neurons, but is nearly abolished in adult neurons and is not upregulated after injury. Furthermore, within the retina, Nfe2l3 is enriched in RGCs, primarily neonatally, and not upregulated in injured RGCs, whereas Nfe2l1 and Nfe2l2 are expressed robustly in other retinal cell types as well and are upregulated in injured RGCs. We also found that, expressing Nfe2l3 in injured RGCs through localized intralocular viral vector delivery promotes neuroprotection and long-distance axon regeneration after optic nerve injury in vivo. Moreover, Nfe2l3 provided a similar extent of neuroprotection and axon regeneration as viral vector-targeting of Pten and Klf9, which are prominent regulators of neuroprotection and long-distance axon regeneration. Finally, we bioinformatically characterized a gene network associated with Nfe2l3 in neurons, which predicted the association of Nfe2l3 with established mechanisms of neuroprotection and axon regeneration. Thus, Nfe2l3 is a novel neuroprotection and axon regeneration-promoting factor with a therapeutic potential for treating CNS injury and disease.

A simple and reliable method for claustrum localization across age in mice

The anatomical organization of the rodent claustrum remains obscure due to lack of clear borders that distinguish it from neighboring forebrain structures. Defining what constitutes the claustrum is imperative for elucidating its functions. Methods based on gene/protein expression or transgenic mice have been used to spatially outline the claustrum but often report incomplete labeling and/or lack of specificity during certain neurodevelopmental timepoints. To reliably identify claustrum projection cells in mice, we propose a simple immunolabelling method that juxtaposes the expression pattern of claustrum-enriched and cortical-enriched markers. We determined that claustrum cells immunoreactive for the claustrum-enriched markers Nurr1 and Nr2f2 are devoid of the cortical marker Tle4, which allowed us to differentiate the claustrum from adjoining cortical cells. Using retrograde tracing, we verified that nearly all claustrum projection neurons lack Tle4 but expressed Nurr1/Nr2f2 markers to different degrees. At neonatal stages between 7 and 21 days, claustrum projection neurons were identified by their Nurr1-postive/Tle4-negative expression profile, a time-period when other immunolabelling techniques used to localize the claustrum in adult mice are ineffective. Finally, exposure to environmental novelty enhanced the expression of the neuronal activation marker c-Fos in the claustrum region. Notably, c-Fos labeling was mainly restricted to Nurr1-positive cells and nearly absent from Tle4-positive cells, thus corroborating previous work reporting novelty-induced claustrum activation. Taken together, this method will aid in studying the claustrum during postnatal development and may improve histological and functional studies where other approaches are not amenable.

STING-Dependent Signaling in Microglia or Peripheral Immune Cells Orchestrates the Early Inflammatory Response and Influences Brain Injury Outcome.

While originally identified as an antiviral pathway, recent work has implicated that cyclic GMP-AMP-synthase-Stimulator of Interferon Genes (cGAS-STING) signaling is playing a critical role in the neuroinflammatory response to traumatic brain injury (TBI). STING activation results in a robust inflammatory response characterized by the production of inflammatory cytokines called interferons, as well as hundreds of interferon stimulated genes (ISGs). Global knock-out (KO) mice inhibiting this pathway display neuroprotection with evidence that this pathway is active days after injury; yet, the early neuroinflammatory events stimulated by STING signaling remain understudied. Furthermore, the source of STING signaling during brain injury is unknown. Using a murine controlled cortical impact (CCI) model of TBI, we investigated the peripheral immune and microglial response to injury utilizing male chimeric and conditional STING KO animals, respectively. We demonstrate that peripheral and microglial STING signaling contribute to negative outcomes in cortical lesion volume, cell death, and functional outcomes postinjury. A reduction in overall peripheral immune cell and neutrophil infiltration at the injury site is STING dependent in these models at 24 h. Transcriptomic analysis at 2 h, when STING is active, reveals that microglia drive an early, distinct transcriptional program to elicit proinflammatory genes including interleukin 1-β (IL-1β), which is lost in conditional knock-out mice. The upregulation of alternative innate immune pathways also occurs after injury in these animals, which supports a complex relationship between brain-resident and peripheral immune cells to coordinate the proinflammatory response and immune cell influx to damaged tissue after injury.