Astrocytes Transplanted during Early Postnatal Development Integrate, Mature, and Survive Long Term in Mouse Cortex.

Astrocytes have complex structural, molecular, and physiological properties and form specialized microenvironments that support circuit-specific functions in the CNS. To better understand how astrocytes acquire their unique features, we transplanted immature mouse cortical astrocytes into the developing cortex of male and female mice and assessed their integration, maturation, and survival. Within days, transplanted astrocytes developed morphologies and acquired territories and tiling behavior typical of cortical astrocytes. At 35-47 d post-transplantation, astrocytes appeared morphologically mature and expressed levels of EAAT2/GLT1 similar to nontransplanted astrocytes. Transplanted astrocytes also supported excitatory/inhibitory (E/I) presynaptic terminals within their territories, and displayed normal Ca2+ events. Transplanted astrocytes showed initially reduced expression of aquaporin 4 (AQP4) at endfeet and elevated expression of EAAT1/GLAST, with both proteins showing normalized expression by 110 d and one year post-transplantation, respectively. To understand how specific brain regions support astrocytic integration and maturation, we transplanted cortical astrocytes into the developing cerebellum. Cortical astrocytes interlaced with Bergmann glia (BG) in the cerebellar molecular layer to establish discrete territories. However, transplanted astrocytes retained many cortical astrocytic features including higher levels of EAAT2/GLT1, lower levels of EAAT1/GLAST, and the absence of expression of the AMPAR subunit GluA1. Collectively, our findings demonstrate that immature cortical astrocytes integrate, mature, and survive (more than one year) following transplantation and retain cortical astrocytic properties. Astrocytic transplantation can be useful for investigating cell-autonomous (intrinsic) and non-cell-autonomous (environmental) mechanisms contributing to astrocytic development/diversity, and for determining the optimal timing for transplanting astrocytes for cellular delivery or replacement in regenerative medicine.SIGNIFICANCE STATEMENT The mechanisms that enable astrocytes to acquire diverse molecular and structural properties remain to be better understood. In this study, we systematically analyzed the properties of cortical astrocytes following their transplantation to the early postnatal brain. We found that immature cortical astrocytes transplanted into cerebral cortex during early postnatal mouse development integrate and establish normal astrocytic properties, and show long-term survival in vivo (more than one year). In contrast, transplanted cortical astrocytes display reduced or altered ability to integrate into the more mature cerebral cortex or developing cerebellum, respectively. This study demonstrates the developmental potential of transplanted cortical astrocytes and provides an approach to tease apart cell-autonomous (intrinsic) and non-cell-autonomous (environmental) mechanisms that determine the structural, molecular, and physiological phenotype of astrocytes.

Plant-derived virus-like particle vaccines drive cross-presentation of influenza A hemagglutinin peptides by human monocyte-derived macrophages.

A growing body of evidence supports the importance of T cell responses to protect against severe influenza, promote viral clearance, and ensure long-term immunity. Plant-derived virus-like particle (VLP) vaccines bearing influenza hemagglutinin (HA) have been shown to elicit strong humoral and CD4+ T cell responses in both pre-clinical and clinical studies. To better understand the immunogenicity of these vaccines, we tracked the intracellular fate of a model HA (A/California/07/2009 H1N1) in human monocyte-derived macrophages (MDMs) following delivery either as VLPs (H1-VLP) or in soluble form. Compared to exposure to soluble HA, pulsing with VLPs resulted in ~3-fold greater intracellular accumulation of HA at 15 min that was driven by clathrin-mediated and clathrin-independent endocytosis as well as macropinocytosis/phagocytosis. At 45 min, soluble HA had largely disappeared suggesting its handling primarily by high-degradative endosomal pathways. Although the overall fluorescence intensity/cell had declined 25% at 45 min after H1-VLP exposure, the endosomal distribution pattern and degree of aggregation suggested that HA delivered by VLP had entered both high-degradative late and low-degradative static early and/or recycling endosomal pathways. At 45 min in the cells pulsed with VLPs, HA was strongly co-localized with Rab5, Rab7, Rab11, MHC II, and MHC I. High-resolution tandem mass spectrometry identified 115 HA-derived peptides associated with MHC I in the H1-VLP-treated MDMs. These data suggest that HA delivery to antigen-presenting cells on plant-derived VLPs facilitates antigen uptake, endosomal processing, and cross-presentation. These observations may help to explain the broad and cross-reactive immune responses generated by these vaccines.

An Optogenetic Kindling Model of Neocortical Epilepsy.

Epileptogenesis is the gradual process by which the healthy brain develops epilepsy. However, the neuronal circuit changes that underlie epileptogenesis are not well understood. Unfortunately, current chemically or electrically induced epilepsy models suffer from lack of cell specificity, so it is seldom known which cells were activated during epileptogenesis. We therefore sought to develop an optogenetic variant of the classical kindling model of epilepsy in which activatable cells are both genetically defined and fluorescently tagged. We briefly optogenetically activated pyramidal cells (PCs) in awake behaving mice every two days and conducted a series of experiments to validate the effectiveness of the model. Although initially inert, brief optogenetic stimuli eventually elicited seizures that increased in number and severity with additional stimulation sessions. Seizures were associated with long-lasting plasticity, but not with tissue damage or astrocyte reactivity. Once optokindled, mice retained an elevated seizure susceptibility for several weeks in the absence of additional stimulation, indicating a form of long-term sensitization. We conclude that optokindling shares many features with classical kindling, with the added benefit that the role of specific neuronal populations in epileptogenesis can be studied. Links between long-term plasticity and epilepsy can thus be elucidated.

The Nogo Receptor Ligand LGI1 Regulates Synapse Number and Synaptic Activity in Hippocampal and Cortical Neurons.

Leucine-rich glioma-inactivated protein 1 (LGI1) is a secreted neuronal protein and a Nogo receptor 1 (NgR1) ligand. Mutations in LGI1 in humans causes autosomal dominant lateral temporal lobe epilepsy and homozygous deletion of LGI1 in mice results in severe epileptic seizures that cause early postnatal death. NgR1 plays an important role in the development of CNS synapses and circuitry by limiting plasticity in the adult cortex via the activation of RhoA. These relationships and functions prompted us to examine the effect of LGI1 on synapse formation in vitro and in vivo. We report that application of LGI1 increases synaptic density in neuronal culture and that LGI1 null hippocampus has fewer dendritic mushroom spines than in wild-type (WT) littermates. Further, our electrophysiological investigations demonstrate that LGI1 null hippocampal neurons possess fewer and weaker synapses. RhoA activity is significantly increased in cortical cultures derived from LGI1 null mice and using a reconstituted system; we show directly that LGI1 antagonizes NgR1-tumor necrosis factor receptor orphan Y (TROY) signaling. Our data suggests that LGI1 enhances synapse formation in cortical and hippocampal neurons by reducing NgR1 signaling.

Morphological characterization of a plant-made virus-like particle vaccine bearing influenza virus hemagglutinins by electron microscopy.

Plant-made virus-like particle (VLP) vaccines that display wild-type influenza hemagglutinin (HA) are rapidly advancing through clinical trials. Produced by transient transfection of Nicotiana benthamiana, these novel vaccines are unusually immunogenic, eliciting both humoral and cellular responses. Here, we directly visualized VLPs bearing either HA trimers derived from strains A/California/7/2009 or A/Indonesia/5/05 using cryo-electron microscopy and determined the 3D organization of the VLPs using cryo-electron tomography. More than 99.9% of the HA trimers in the vaccine preparations were found on discoid and ovoid-shaped particles. The discoid-shaped VLPs presented HA trimers on their outer diameter. The ovoid-shaped VLPs contained HA trimers evenly distributed at their surface. The VLPs were stable for 12 months at 4 °C. Early interactions of the VLPs with mouse dendritic and human monocytoid (U-937) cells were visualized by electron microscopy after resin-embedding and sectioning. The VLP particles were observed bound to plasma membranes as well as inside vesicles. Mouse dendritic cells exposed to VLPs displayed classic morphological changes associated with activation including the extensive formation of dendrites. Our findings demonstrate that plant-made VLPs bearing influenza HA trimers are morphologically stable over time and raise the possibility that these VLPs may interact with and activate antigen-presenting cells in a manner similar to the intact virus.

SPARC and GluA1-Containing AMPA Receptors Promote Neuronal Health Following CNS Injury.

The proper formation and maintenance of functional synapses in the central nervous system (CNS) requires communication between neurons and astrocytes and the ability of astrocytes to release neuromodulatory molecules. Previously, we described a novel role for the astrocyte-secreted matricellular protein SPARC (Secreted Protein, Acidic and Rich in Cysteine) in regulating α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors (AMPARs) and plasticity at developing synapses. SPARC is highly expressed by astrocytes and microglia during CNS development but its level is reduced in adulthood. Interestingly, SPARC has been shown to be upregulated in CNS injury and disease. However, the role of SPARC upregulation in these contexts is not fully understood. In this study, we investigated the effect of chronic SPARC administration on glutamate receptors on mature hippocampal neuron cultures and following CNS injury. We found that SPARC treatment increased the number of GluA1-containing AMPARs at synapses and enhanced synaptic function. Furthermore, we determined that the increase in synaptic strength induced by SPARC could be inhibited by Philanthotoxin-433, a blocker of homomeric GluA1-containing AMPARs. We then investigated the effect of SPARC treatment on neuronal health in an injury context where SPARC expression is upregulated. We found that SPARC levels are increased in astrocytes and microglia following middle cerebral artery occlusion (MCAO) in vivo and oxygen-glucose deprivation (OGD) in vitro. Remarkably, chronic pre-treatment with SPARC prevented OGD-induced loss of synaptic GluA1. Furthermore, SPARC treatment reduced neuronal death through Philanthotoxin-433 sensitive GluA1 receptors. Taken together, this study suggests a novel role for SPARC and GluA1 in promoting neuronal health and recovery following CNS damage.

Plant-made virus-like particles bearing influenza hemagglutinin (HA) recapitulate early interactions of native influenza virions with human monocytes/macrophages.

INTRODUCTION: Plant-made virus-like particles (VLP) bearing influenza virus hemagglutinins (HA) are novel vaccine candidates that induce cross-reactive humoral and poly-functional T cell responses. To better understand the mechanisms that underlie this broad immunogenicity we studied early interactions of VLPs bearing either H1 (A/California/07/2009 (H1N1)) or H5 (A/Indonesia/05/2005 (H5N1)) with a human monocytoid cell line (U-937 cells) and human monocyte-derived macrophages (MDMs) as model antigen-presenting cells (APC). METHODS AND RESULTS: Using Vibrio cholerae sialidase and lectins that target α2,6- (Sambucus nigra lectin) or α2,3-linked sialic acids (Maackia amurensis lectin I), we demonstrated that VLPs bind to these APCs in a sialic acid-dependent manner. Using lysosomal markers and DiD-labelled VLPs, we found that attachment to the cell surface leads to internalization, trafficking to acidic cell compartments and fusion of the VLP lipid envelope with endosomal membranes. Incubation of MDMs with H1- but not H5-VLPs induced proliferation of autologous peripheral blood mononuclear cells suggesting antigen processing and stimulation of a memory T cell response. CONCLUSIONS: Plant-made VLPs bearing influenza HA not only mimic the structure of influenza virions to some degree but also recapitulate key features of the initial virus-APC interaction. These observations may help to explain the balanced humoral and cellular responses to plant-made VLP vaccines.

Neurons diversify astrocytes in the adult brain through sonic hedgehog signaling.

Astrocytes are specialized and heterogeneous cells that contribute to central nervous system function and homeostasis. However, the mechanisms that create and maintain differences among astrocytes and allow them to fulfill particular physiological roles remain poorly defined. We reveal that neurons actively determine the features of astrocytes in the healthy adult brain and define a role for neuron-derived sonic hedgehog (Shh) in regulating the molecular and functional profile of astrocytes. Thus, the molecular and physiological program of astrocytes is not hardwired during development but, rather, depends on cues from neurons that drive and sustain their specialized properties.

Activity maintains structural plasticity of mossy fiber terminals in the hippocampus.

Neural activity plays an important role in organizing and optimizing neural circuits during development and in the mature nervous system. However, the cellular events that underlie this process still remain to be fully understood. In this study, we investigated the role of neural activity in regulating the structural plasticity of presynaptic terminals in the hippocampal formation. We designed a virus to drive the Drosophila Allatostatin receptor in individual dentate granule neurons to suppress activity of complex mossy fiber terminals 'on-demand' in organotypic slices and used time-lapse confocal imaging to determine the impact on presynaptic remodeling. We found that activity played an important role in maintaining the structural plasticity of the core region of the mossy fiber terminal (MFT) that synapses onto CA3 pyramidal cell thorny excrescences but was not essential for the motility of terminal filopodial extensions that contact local inhibitory neurons. Short-term suppression of activity did not have an impact on the size of the MFT, however, longer-term suppression reduced the overall size of the MFT. Remarkably, global blockade of activity with tetrodotoxin (TTX) interfered with the ability of single cell activity deprivation to slow down terminal dynamics suggesting that differences in activity levels among neighboring synapses promote synaptic remodeling events. The results from our studies indicate that neural activity plays an important role in maintaining structural plasticity of presynaptic compartments in the central nervous system and provide new insight into the time-frame during which activity can affect the morphology of synaptic connections.

Lipocalin 2 plays an immunomodulatory role and has detrimental effects after spinal cord injury.

Lipocalin 2 (Lcn2) plays an important role in defense against bacterial infection by interfering with bacterial iron acquisition. Although Lcn2 is expressed in a number of aseptic inflammatory conditions, its role in these conditions remains unclear. We examined the expression and role of Lcn2 after spinal cord injury (SCI) in adult mice by using a contusion injury model. Lcn2 expression at the protein level is rapidly increased 12-fold at 1 d after SCI and decreases gradually thereafter, being three times as high as control levels at 21 d after injury. Lcn2 expression is strongly induced after contusion injury in astrocytes, neurons, and neutrophils. The Lcn2 receptor (Lcn2R), which has been shown to influence cell survival, is also expressed after SCI in the same cell types. Lcn2-deficient (Lcn2⁻/⁻) mice showed significantly better locomotor recovery after spinal cord contusion injury than wild-type (Lcn2⁺/⁺) mice. Histological assessments indicate improved neuronal and tissue survival and greater sparing of myelin in Lcn2⁻/⁻ mice after contusion injury. Flow cytometry showed a decrease in neutrophil influx and a small increase in the monocyte population in Lcn2⁻/⁻ injured spinal cords. This change was accompanied by a reduction in the expression of several pro-inflammatory chemokines and cytokines as well as inducible nitric oxide synthase early after SCI in Lcn2⁻/⁻ mice compared with wild-type animals. Our results, therefore, suggest a role for Lcn2 in regulating inflammation in the injured spinal cord and that lack of Lcn2 reduces secondary damage and improves locomotor recovery after spinal cord contusion injury.

Astrocytes control glutamate receptor levels at developing synapses through SPARC-beta-integrin interactions.

Neurons recruit numerous mechanisms to facilitate the development of synaptic connections. However, little is known about activity-dependent mechanisms that control the timing and fidelity of this process. Here we describe a novel pathway used by neurons to regulate glutamate receptors at maturing central synapses. This pathway relies on communication between neurons and astrocytes and the ability of astrocytes to release the factor SPARC (secreted protein, acidic and rich in cysteine). SPARC expression is dynamically regulated and plays a critical role in determining the level of synaptic AMPARs. SPARC ablation in mice increases excitatory synapse function, causes an abnormal accumulation of surface AMPARs at synapses, and impairs synaptic plasticity during development. We further demonstrate that SPARC inhibits the properties of neuronal ß3-integrin complexes, which are intimately coupled to AMPAR stabilization at synapses. Thus neuron-glial signals control glutamate receptor levels at developing synapses to enable activity-driven modifications of synaptic strength.

Developmental course of EphA4 cellular and subcellular localization in the postnatal rat hippocampus.

From embryonic development to adulthood, the EphA4 receptor and several of its ephrin-A or -B ligands are expressed in the hippocampus, where they presumably play distinct roles at different developmental stages. To help clarify these diverse roles in the assembly and function of the hippocampus, we examined the cellular and subcellular localization of EphA4 in postnatal rat hippocampus by light and electron microscopic immunocytochemistry. On postnatal day (P) 1, the EphA4 immunostaining was robust in most layers of CA1, CA3, and dentate gyrus and then decreased gradually, until P21, especially in the cell body layers. At the ultrastructural level, focal spots of EphA4 immunoreactivity were detected all over the plasma membrane of pyramidal and granule cells, between P1 and P14, from the perikarya to the dendritic and axonal extremities, including growth cones and filopodia. This cell surface immunoreactivity then became restricted to the synapse-associated dendritic spines and axon terminals by P21. In astrocytes, the EphA4 immunolabeling showed a similar cell surface redistribution, from the perikarya and large processes at P1-P7, to small perisynaptic processes at P14-P21. In both cell types, spots of EphA4 immunoreactivity were also detected, with an incidence decreasing with maturation, on the endoplasmic reticulum, Golgi apparatus, and vesicles, organelles involved in protein synthesis, posttranslational modifications, and transport. The cell surface evolution of EphA4 localization in neuronal and glial cells is consistent with successive involvements in the developmental movements of cell bodies first, followed by process outgrowth and guidance, synaptogenesis, and finally synaptic maintenance and plasticity.

Maternal enrichment during pregnancy accelerates retinal development of the fetus.

The influence of maternal environment on fetal development is largely unexplored, the available evidence concerns only the deleterious effects elicited by prenatal stress. Here we investigated the influence of prenatal enrichment on the early development of the visual system in the fetus. We studied the anatomical development of the rat retina, by analyzing the migration of neural progenitors and the process of retinal ganglion cell death, which exerts a key role in sculpturing the developing retinal system at perinatal ages. The number of apoptotic cells in the retinal ganglion cell layer was analyzed using two distinct methods: the presence of pyknotic nuclei stained for cresyl violet and the appearance of DNA fragmentation (Tunel method). We report that environmental enrichment of the mother during pregnancy affects the structural maturation of the retina, accelerating the migration of neural progenitors and the dynamics of natural cell death. These effects seem to be under the control of insulin-like growth factor-I: its levels, higher in enriched pregnant rats and in their milk, are increased also in their offspring, its neutralization abolishes the action of maternal enrichment on retinal development and chronic insulin-like growth factor-I injection to standard-reared females mimics the effects of enrichment in the fetuses. Thus, the development of the visual system is sensitive to environmental stimulation during prenatal life. These findings could have a bearing in orienting clinical research in the field of prenatal therapy.

The ability of axons to regenerate their growth cones depends on axonal type and age, and is regulated by calcium, cAMP and ERK.

The processes activated at the time of axotomy and leading to the formation of a new growth cone are the first step in regeneration, but are still poorly characterized. We investigated this event in an in vitro model of axotomy performed on dorsal root ganglia and retinal explants. We observed that the dorsal root ganglion axons and retinal ganglion cell axons, which had grown out on a poly d-lysine/laminin substrate at the time of culture preparation greatly differed in their regenerative response after a subsequent in vitro lesion made far from the cell body. The majority of axons of adult dorsal root ganglia but only a small percentage of axons of adult retinal ganglion cells regenerated new growth cones within four hours after in vitro axotomy, though both kinds of axons were growing before the lesion. The depletion of extracellular calcium and the inhibition of extracellular-signal regulated kinase 1,2 (ERK) and protein kinase A (PKA) at the time of injury significantly impaired the capacity of dorsal root ganglia axons to re-initiate growth cones without affecting growth cone motility. Pharmacological treatments directed at increasing the level of cAMP promoted growth cone regeneration in adult retinal ganglion cell axons in spite of the low regenerative potential exhibited in normal conditions. Understanding the cellular mechanisms activated at the time of lesion and leading to the formation of a new growth cone is necessary for devising treatments aimed at enhancing the regenerative response of injured axons.

Axonal protein synthesis and degradation are necessary for efficient growth cone regeneration.

Axonal regeneration can occur within hours of injury, the first step being the formation of a new growth cone. For sensory and retinal axons, regenerative ability in vivo correlates with the potential to form a new growth cone after axotomy in vitro. We show that this ability to regenerate a new growth cone depends on local protein synthesis and degradation within the axon. Axotomy in vitro leads to a fourfold to sixfold increase in 3H-leucine incorporation in both neurones and axons, starting within 10 min and peaking 1 h after axotomy. Application of protein synthesis inhibitors (cycloheximide and anisomycin) to cut axons, including axons whose cell bodies were removed, or proteasome inhibitors (lactacystin and N-acetyl-Nor-Leu-Leu-Al) all result in a reduction in the proportion of transected axons able to reform growth cones. Similar inhibition of growth cone formation was observed on addition of target of rapamycin (TOR), p38 MAPK (mitogen-activated protein kinase), and caspase-3 inhibitors. Comparing retinal and sensory axons of different developmental stages, levels of ribosomal protein P0 and phosphorylated translation initiation factor are high in sensory axons, lower in embryonic axons, and absent in adult retinal axons. Conditioning lesions, which increase the regenerative ability of sensory axons, lead to increases in intra-axonal protein synthetic and degradative machinery both in vitro and in vivo. Collectively, these findings suggest that local protein synthesis and degradation, controlled by various TOR-, p38 MAPK-, and caspase-dependent pathways, underlie growth cone initiation after axotomy.

Reactivation of ocular dominance plasticity in the adult visual cortex.

In young animals, monocular deprivation leads to an ocular dominance shift, whereas in adults after the critical period there is no such shift. Chondroitin sulphate proteoglycans (CSPGs) are components of the extracellular matrix (ECM) inhibitory for axonal sprouting. We tested whether the developmental maturation of the ECM is inhibitory for experience-dependent plasticity in the visual cortex. The organization of CSPGs into perineuronal nets coincided with the end of the critical period and was delayed by dark rearing. After CSPG degradation with chondroitinase-ABC in adult rats, monocular deprivation caused an ocular dominance shift toward the nondeprived eye. The mature ECM is thus inhibitory for experience-dependent plasticity, and degradation of CSPGs reactivates cortical plasticity.