Cell-specific and region-specific transcriptomics in the multiple sclerosis model: Focus on astrocytes.

Changes in gene expression that occur across the central nervous system (CNS) during neurological diseases do not address the heterogeneity of cell types from one CNS region to another and are complicated by alterations in cellular composition during disease. Multiple sclerosis (MS) is multifocal by definition. Here, a cell-specific and region-specific transcriptomics approach was used to determine gene expression changes in astrocytes in the most widely used MS model, experimental autoimmune encephalomyelitis (EAE). Astrocyte-specific RNAs from various neuroanatomic regions were attained using RiboTag technology. Sequencing and bioinformatics analyses showed that EAE-induced gene expression changes differed between neuroanatomic regions when comparing astrocytes from spinal cord, cerebellum, cerebral cortex, and hippocampus. The top gene pathways that were changed in astrocytes from spinal cord during chronic EAE involved decreases in expression of cholesterol synthesis genes while immune pathway gene expression in astrocytes was increased. Optic nerve from EAE and optic chiasm from MS also showed decreased cholesterol synthesis gene expression. The potential role of cholesterol synthesized by astrocytes during EAE and MS is discussed. Together, this provides proof-of-concept that a cell-specific and region-specific gene expression approach can provide potential treatment targets in distinct neuroanatomic regions during multifocal neurological diseases

Imaging calcium microdomains within entire astrocyte territories and endfeet with GCaMPs expressed using adeno-associated viruses.

Intracellular Ca(2+) transients are considered a primary signal by which astrocytes interact with neurons and blood vessels. With existing commonly used methods, Ca(2+) has been studied only within astrocyte somata and thick branches, leaving the distal fine branchlets and endfeet that are most proximate to neuronal synapses and blood vessels largely unexplored. Here, using cytosolic and membrane-tethered forms of genetically encoded Ca(2+) indicators (GECIs; cyto-GCaMP3 and Lck-GCaMP3), we report well-characterized approaches that overcome these limitations. We used in vivo microinjections of adeno-associated viruses to express GECIs in astrocytes and studied Ca(2+) signals in acute hippocampal slices in vitro from adult mice (aged ∼P80) two weeks after infection. Our data reveal a sparkling panorama of unexpectedly numerous, frequent, equivalently scaled, and highly localized Ca(2+) microdomains within entire astrocyte territories in situ within acute hippocampal slices, consistent with the distribution of perisynaptic branchlets described using electron microscopy. Signals from endfeet were revealed with particular clarity. The tools and experimental approaches we describe in detail allow for the systematic study of Ca(2+) signals within entire astrocytes, including within fine perisynaptic branchlets and vessel-associated endfeet, permitting rigorous evaluation of how astrocytes contribute to brain function

Juvenile neurogenesis makes essential contributions to adult brain structure and plays a sex-dependent role in fear memories

Postnatal neurogenesis (PNN) contributes neurons to olfactory bulb (OB) and dentate gyrus (DG) throughout juvenile development, but the quantitative amount, temporal dynamics and functional roles of this contribution have not been defined. By using transgenic mouse models for cell lineage tracing and conditional cell ablation, we found that juvenile neurogenesis gradually increased the total number of granule neurons by approximately 40% in OB, and by 25% in DG, between 2 weeks and 2 months of age, and that total numbers remained stable thereafter. These findings indicate that the overwhelming majority of net postnatal neuronal addition in these regions occurs during the juvenile period and that adult neurogenesis contributes primarily to replacement of granule cells in both regions. Behavioral analysis in our conditional cell ablation mouse model showed that complete loss of PNN throughout both the juvenile and young adult period produced a specific set of sex-dependent cognitive changes. We observed normal hippocampus-independent delay fear conditioning, but excessive generalization of fear to a novel auditory stimulus, which is consistent with a role for PNN in psychopathology. Standard contextual fear conditioning was intact, however, pre-exposure dependent contextual fear was impaired suggesting a specific role for PNN in incidental contextual learning. Contextual discrimination between two highly similar contexts was enhanced; suggesting either enhanced contextual pattern separation or impaired temporal integration. We also observed a reduced reliance on olfactory cues, consistent with a role for OB PNN in the efficient processing of olfactory information. Thus, juvenile neurogenesis adds substantively to the total numbers of granule neurons in OB and DG during periods of critical juvenile behavioral development, including weaning, early social interactions and sexual maturation, and plays a sex-dependent role in fear memories

Disruption of Astrocyte STAT3 Signaling Decreases Mitochondrial Function and Increases Oxidative Stress In Vitro

Astrocytes exert a wide variety of functions in health and disease and respond to a wide range of signaling pathways, including members of the Janus-kinase signal transducers and activators of transcription (Jak-STAT) family. We have recently shown that STAT3 is an important regulator of astrocyte reactivity after spinal cord injury in vivo[1].Here, we used both a conditional gene deletion strategy that targets the deletion of STAT3 selectively to astrocytes (STAT3-CKO), and a pharmacological inhibitor of JAK-2, AG490, in cultured astrocytes in vitro, to investigate potential functions and molecules influenced by STAT3 signaling in relation to mitochondrial function and oxidative stress. Our findings show that the absence of STAT3 signaling in astrocytes leads to (i) increased production of superoxide anion and other reactive oxygen species and decreased level of glutathione, (ii) decreased mitochondrial membrane potential and decreased ATP production, and (iii) decreased rate of cell proliferation. Many of the differences observed in STAT3-CKO astrocytes were distinctly altered by exposure to rotenone, suggesting a role for complex I of the mitochondrial electron transport chain. Gene expression microarray studies identified numerous changes in STAT3-CKO cells that may have contributed to the identified deficits in cell function.Taken together, these STAT3-dependent alterations in cell function and gene expression have relevance to both reactive gliosis and to the support and protection of surrounding cells in neural tissue

Maternal embryonic leucine zipper kinase (MELK) regulates multipotent neural progenitor proliferation.

Maternal embryonic leucine zipper kinase (MELK) was previously identified in a screen for genes enriched in neural progenitors. Here, we demonstrate expression of MELK by progenitors in developing and adult brain and that MELK serves as a marker for self-renewing multipotent neural progenitors (MNPs) in cultures derived from the developing forebrain and in transgenic mice. Overexpression of MELK enhances (whereas knockdown diminishes) the ability to generate neurospheres from MNPs, indicating a function in self-renewal. MELK down-regulation disrupts the production of neurogenic MNP from glial fibrillary acidic protein (GFAP)-positive progenitors in vitro. MELK expression in MNP is cell cycle regulated and inhibition of MELK expression down-regulates the expression of B-myb, which is shown to also mediate MNP proliferation. These findings indicate that MELK is necessary for proliferation of embryonic and postnatal MNP and suggest that it regulates the transition from GFAP-expressing progenitors to rapid amplifying progenitors in the postnatal brain

Neurological diseases as primary gliopathies: a reassessment of neurocentrism

Diseases of the human brain are almost universally attributed to malfunction or loss of nerve cells. However, a considerable amount of work has, during the last decade, expanded our view on the role of astrocytes in CNS (central nervous system), and this analysis suggests that astrocytes contribute to both initiation and propagation of many (if not all) neurological diseases. Astrocytes provide metabolic and trophic support to neurons and oligodendrocytes. Here, we shall endeavour a broad overviewing of the progress in the field and forward the idea that loss of homoeostatic astroglial function leads to an acute loss of neurons in the setting of acute insults such as ischaemia, whereas more subtle dysfunction of astrocytes over periods of months to years contributes to epilepsy and to progressive loss of neurons in neurodegenerative diseases. The majority of therapeutic drugs currently in clinical use target neuronal receptors, channels or transporters. Future therapeutic efforts may benefit by a stronger focus on the supportive homoeostatic functions of astrocytes

Oestrogen receptor β ligand: a novel treatment to enhance endogenous functional remyelination

Demyelinating diseases, such as multiple sclerosis, are characterized by inflammatory demyelination and neurodegeneration of the central nervous system. Therapeutic strategies that induce effective neuroprotection and enhance intrinsic repair mechanisms are central goals for future therapy of multiple sclerosis. Oestrogens and oestrogen receptor ligands are promising treatments to prevent multiple sclerosis-induced neurodegeneration. In the present study we investigated the capacity of oestrogen receptor β ligand treatment to affect callosal axon demyelination and stimulate endogenous myelination in chronic experimental autoimmune encephalomyelitis using electrophysiology, electron microscopy, immunohistochemistry and tract-tracing methods. Oestrogen receptor β ligand treatment of experimental autoimmune encephalomyelitis mice prevented both histopathological and functional abnormalities of callosal axons despite the presence of inflammation. Specifically, there were fewer demyelinated, damaged axons and more myelinated axons with intact nodes of Ranvier in oestrogen receptor β ligand-treated mice. In addition, oestrogen receptor β ligand treatment caused an increase in mature oligodendrocyte numbers, a significant increase in myelin sheath thickness and axon transport. Functional analysis of callosal axon conduction showed a significant improvement in compound action potential amplitudes, latency and in axon refractoriness. These findings show a direct neuroprotective effect of oestrogen receptor β ligand treatment on oligodendrocyte differentiation, myelination and axon conduction during experimental autoimmune encephalomyelitis

The Neurotoxicity of DOPAL: Behavioral and Stereological Evidence for Its Role in Parkinson Disease Pathogenesis

BACKGROUND: The etiology of Parkinson disease (PD) has yet to be fully elucidated. We examined the consequences of injections of 3,4-dihydroxyphenylacetaldehyde (DOPAL), a toxic metabolite of dopamine, into the substantia nigra of rats on motor behavior and neuronal survival. METHODS/PRINCIPAL FINDINGS: A total of 800 nl/rat of DOPAL (1 µg/200 nl) was injected stereotaxically into the substantia nigra over three sites while control animals received similar injections of phosphate buffered saline. Rotational behavior of these rats was analyzed, optical density of striatal tyrosine hydroxylase was calculated, and unbiased stereological counts of the substantia nigra were made. The rats showed significant rotational asymmetry ipsilateral to the lesion, supporting disruption of dopaminergic nigrostriatal projections. Such disruption was verified since the density of striatal tyrosine hydroxylase decreased significantly (p<0.001) on the side ipsilateral to the DOPAL injections when compared to the non-injected side. Stereological counts of neurons stained for Nissl in pars compacta of the substantia nigra significantly decreased (p<0.001) from control values, while counts of those in pars reticulata were unchanged after DOPAL injections. Counts of neurons immunostained for tyrosine hydroxylase also showed a significant (p=0.032) loss of dopaminergic neurons. In spite of significant loss of dopaminergic neurons, DOPAL injections did not induce significant glial reaction in the substantia nigra. CONCLUSIONS: The present study provides the first in vivo quantification of substantia nigra pars compacta neuronal loss after injection of the endogenous toxin DOPAL. The results demonstrate that injections of DOPAL selectively kills SN DA neurons, suggests loss of striatal DA terminals, spares non-dopaminergic neurons of the pars reticulata, and triggers a behavioral phenotype (rotational asymmetry) consistent with other PD animal models. This study supports the "catecholaldehyde hypothesis" as an important link for the etiology of sporadic PD