Protocol for the longitudinal study of neuroinflammation and reactive astrocytes in Lcn2CreERT2 mice.

During brain disease, astrocytes can reprogram into a reactive state that alters many of their functions. Here, we present a protocol for studying neuroinflammation and reactive astrogliosis in mice using lipopolysaccharide (LPS) from E. coli. We describe steps for employing the Lcn2CreERT2 mouse crossed into a fluorescent Cre reporter line to label a subset of reactive astrocytes during and after inflammation. We then detail procedures for the longitudinal study of reactive astrocytes during the induction, progression, and/or resolution of astrogliosis. For complete details on the use and execution of this protocol, please refer to Agnew-Svoboda et al.1.

An adhesion signaling axis involving Dystroglycan, ß1-Integrin, and Cas adaptor proteins regulates the establishment of the cortical glial scaffold.

The mature mammalian cortex is composed of 6 architecturally and functionally distinct layers. Two key steps in the assembly of this layered structure are the initial establishment of the glial scaffold and the subsequent migration of postmitotic neurons to their final position. These processes involve the precise and timely regulation of adhesion and detachment of neural cells from their substrates. Although much is known about the roles of adhesive substrates during neuronal migration and the formation of the glial scaffold, less is understood about how these signals are interpreted and integrated within these neural cells. Here, we provide in vivo evidence that Cas proteins, a family of cytoplasmic adaptors, serve a functional and redundant role during cortical lamination. Cas triple conditional knock-out (Cas TcKO) mice display severe cortical phenotypes that feature cobblestone malformations. Molecular epistasis and genetic experiments suggest that Cas proteins act downstream of transmembrane Dystroglycan and ß1-Integrin in a radial glial cell-autonomous manner. Overall, these data establish a new and essential role for Cas adaptor proteins during the formation of cortical circuits and reveal a signaling axis controlling cortical scaffold formation.

A genetic tool for the longitudinal study of a subset of post-inflammatory reactive astrocytes.

Astrocytes are vital support cells that ensure proper brain function. In brain disease, astrocytes reprogram into a reactive state that alters many of their cellular roles. A long-standing question in the field is whether downregulation of reactive astrocyte (RA) markers during resolution of inflammation is because these astrocytes revert back to a non-reactive state or die and are replaced. This has proven difficult to answer mainly because existing genetic tools cannot distinguish between healthy versus RAs. Here we describe the generation of an inducible genetic tool that can be used to specifically target and label a subset of RAs. Longitudinal analysis of an acute inflammation model using this tool revealed that the previously observed downregulation of RA markers after inflammation is likely due to changes in gene expression and not because of cell death. Our findings suggest that cellular changes associated with astrogliosis after acute inflammation are largely reversible.

ExBoX - a simple Boolean exclusion strategy to drive expression in neurons.

The advent of modern single-cell biology has revealed the striking molecular diversity of cell populations once thought to be more homogeneous. This newly appreciated complexity has made intersectional genetic approaches essential to understanding and probing cellular heterogeneity at the functional level. Here, we build on previous knowledge to develop a simple adeno-associated virus (AAV)-based approach to define specific subpopulations of cells by Boolean exclusion logic (AND NOT). This expression by Boolean exclusion (ExBoX) system encodes for a gene of interest that is turned on by a particular recombinase (Cre or FlpO) and turned off by another. ExBoX allows for the specific transcription of a gene of interest in cells expressing only the activating recombinase, but not in cells expressing both. We show the ability of the ExBoX system to tightly regulate expression of fluorescent reporters in vitro and in vivo, and further demonstrate the adaptability of the system by achieving expression of a variety of virally delivered coding sequences in the mouse brain. This simple strategy will expand the molecular toolkit available for cell- and time-specific gene expression in a variety of systems.

A Human Embryonic Stem Cell Model of Aß-Dependent Chronic Progressive Neurodegeneration.

We describe the construction and phenotypic analysis of a human embryonic stem cell model of progressive Aß-dependent neurodegeneration (ND) with potential relevance to Alzheimer's disease (AD). We modified one allele of the normal APP locus to directly express a secretory form of Aß40 or Aß42, enabling expression from this edited allele to bypass the normal amyloidogenic APP processing pathway. Following neuronal differentiation, edited cell lines specifically accumulate intracellular aggregated/oligomeric Aß, exhibit a synaptic deficit, and have an abnormal accumulation of endolysosomal vesicles. Edited cultures progress to a stage of overt ND. All phenotypes appear at earlier culture times for Aß42 relative to Aß40. Whole transcriptome RNA-Seq analysis identified 23 up and 70 down regulated genes (differentially expressed genes) with similar directional fold change but larger absolute values in the Aß42 samples suggesting common underlying pathogenic mechanisms. Pathway/annotation analysis suggested that down regulation of extracellular matrix and cilia functions is significantly overrepresented. This cellular model could be useful for uncovering mechanisms directly linking Aß to neuronal death and as a tool to screen for new therapeutic agents that slow or prevent human ND.