The laminar position, morphology, and gene expression profiles of cortical astrocytes are influenced by time of birth from ventricular/subventricular progenitors.

Astrocytes that reside in superficial (SL) and deep cortical layers have distinct molecular profiles and morphologies, which may underlie specific functions. Here, we demonstrate that the production of SL and deep layer (DL) astrocyte populations from neural progenitor cells in the mouse is temporally regulated. Lineage tracking following in utero and postnatal electroporation with PiggyBac (PB) EGFP and birth dating with EdU and FlashTag, showed that apical progenitors produce astrocytes during late embryogenesis (E16.5) that are biased to the SL, while postnatally labeled (P0) astrocytes are biased to the DL. In contrast, astrocytes born during the predominantly neurogenic window (E14.5) showed a random distribution in the SL and DL. Of interest, E13.5 astrocytes birth dated at E13.5 with EdU showed a lower layer bias, while FT labeling of apical progenitors showed no bias. Finally, examination of the morphologies of "biased" E16.5- and P0-labeled astrocytes demonstrated that E16.5-labeled astrocytes exhibit different morphologies in different layers, while P0-labeled astrocytes do not. Differences based on time of birth are also observed in the molecular profiles of E16.5 versus P0-labeled astrocytes. Altogether, these results suggest that the morphological, molecular, and positional diversity of cortical astrocytes is related to their time of birth from ventricular/subventricular zone progenitors.

Homozygous ARHGEF2 mutation causes intellectual disability and midbrain-hindbrain malformation.

Mid-hindbrain malformations can occur during embryogenesis through a disturbance of transient and localized gene expression patterns within these distinct brain structures. Rho guanine nucleotide exchange factor (ARHGEF) family members are key for controlling the spatiotemporal activation of Rho GTPase, to modulate cytoskeleton dynamics, cell division, and cell migration. We identified, by means of whole exome sequencing, a homozygous frameshift mutation in the ARHGEF2 as a cause of intellectual disability, a midbrain-hindbrain malformation, and mild microcephaly in a consanguineous pedigree of Kurdish-Turkish descent. We show that loss of ARHGEF2 perturbs progenitor cell differentiation and that this is associated with a shift of mitotic spindle plane orientation, putatively favoring more symmetric divisions. The ARHGEF2 mutation leads to reduction in the activation of the RhoA/ROCK/MLC pathway crucial for cell migration. We demonstrate that the human brain malformation is recapitulated in Arhgef2 mutant mice and identify an aberrant migration of distinct components of the precerebellar system as a pathomechanism underlying the midbrain-hindbrain phenotype. Our results highlight the crucial function of ARHGEF2 in human brain development and identify a mutation in ARHGEF2 as novel cause of a neurodevelopmental disorder.

The emerging link between O-GlcNAc and Alzheimer disease.

Regional glucose hypometabolism is a defining feature of Alzheimer disease (AD). One emerging link between glucose hypometabolism and progression of AD is the nutrient-responsive post-translational O-GlcNAcylation of nucleocytoplasmic proteins. O-GlcNAc is abundant in neurons and occurs on both tau and amyloid precursor protein. Increased brain O-GlcNAcylation protects against tau and amyloid-ß peptide toxicity. Decreased O-GlcNAcylation occurs in AD, suggesting that glucose hypometabolism may impair the protective roles of O-GlcNAc within neurons and enable neurodegeneration. Here, we review how O-GlcNAc may link cerebral glucose hypometabolism to progression of AD and summarize data regarding the protective role of O-GlcNAc in AD models.

O-GlcNAc and neurodegeneration: biochemical mechanisms and potential roles in Alzheimer's disease and beyond.

Alzheimer disease (AD) is a growing problem for aging populations worldwide. Despite significant efforts, no therapeutics are available that stop or slow progression of AD, which has driven interest in the basic causes of AD and the search for new therapeutic strategies. Longitudinal studies have clarified that defects in glucose metabolism occur in patients exhibiting Mild Cognitive Impairment (MCI) and glucose hypometabolism is an early pathological change within AD brain. Further, type 2 diabetes mellitus (T2DM) is a strong risk factor for the development of AD. These findings have stimulated interest in the possibility that disrupted glucose regulated signaling within the brain could contribute to the progression of AD. One such process of interest is the addition of O-linked N-acetylglucosamine (O-GlcNAc) residues onto nuclear and cytoplasmic proteins within mammals. O-GlcNAc is notably abundant within brain and is present on hundreds of proteins including several, such as tau and the amyloid precursor protein, which are involved in the pathophysiology AD. The cellular levels of O-GlcNAc are coupled to nutrient availability through the action of just two enzymes. O-GlcNAc transferase (OGT) is the glycosyltransferase that acts to install O-GlcNAc onto proteins and O-GlcNAcase (OGA) is the glycoside hydrolase that acts to remove O-GlcNAc from proteins. Uridine 5'-diphosphate-N-acetylglucosamine (UDP-GlcNAc) is the donor sugar substrate for OGT and its levels vary with cellular glucose availability because it is generated from glucose through the hexosamine biosynthetic pathway (HBSP). Within the brains of AD patients O-GlcNAc levels have been found to be decreased and aggregates of tau appear to lack O-GlcNAc entirely. Accordingly, glucose hypometabolism within the brain may result in disruption of the normal functions of O-GlcNAc within the brain and thereby contribute to downstream neurodegeneration. While this hypothesis remains largely speculative, recent studies using different mouse models of AD have demonstrated the protective benefit of pharmacologically increased brain O-GlcNAc levels. In this review we summarize the state of knowledge in the area of O-GlcNAc as it pertains to AD while also addressing some of the basic biochemical roles of O-GlcNAc and how these might contribute to protecting against AD and other neurodegenerative diseases.

Increasing O-GlcNAc slows neurodegeneration and stabilizes tau against aggregation.

Oligomerization of tau is a key process contributing to the progressive death of neurons in Alzheimer's disease. Tau is modified by O-linked N-acetylglucosamine (O-GlcNAc), and O-GlcNAc can influence tau phosphorylation in certain cases. We therefore speculated that increasing tau O-GlcNAc could be a strategy to hinder pathological tau-induced neurodegeneration. Here we found that treatment of hemizygous JNPL3 tau transgenic mice with an O-GlcNAcase inhibitor increased tau O-GlcNAc, hindered formation of tau aggregates and decreased neuronal cell loss. Notably, increases in tau O-GlcNAc did not alter tau phosphorylation in vivo. Using in vitro biochemical aggregation studies, we found that O-GlcNAc modification, on its own, hinders tau oligomerization. O-GlcNAc also inhibits thermally induced aggregation of an unrelated protein, TAK-1 binding protein, suggesting that a basic biochemical function of O-GlcNAc may be to prevent protein aggregation. These results also suggest O-GlcNAcase as a potential therapeutic target that could hinder progression of Alzheimer's disease.

LRIG1 controls proliferation of adult neural stem cells by facilitating TGFß and BMP signalling pathways.

Adult Neural Stem Cells (aNSCs) in the ventricular-subventricular zone (V-SVZ) are largely quiescent. Here, we characterize the mechanism underlying the functional role of a cell-signalling inhibitory protein, LRIG1, in the control of aNSCs proliferation. Using Lrig1 knockout models, we show that Lrig1 ablation results in increased aNSCs proliferation with no change in neuronal progeny and that this hyperproliferation likely does not result solely from activation of the epidermal growth factor receptor (EGFR). Loss of LRIG1, however, also leads to impaired activation of transforming growth factor beta (TGFß) and bone morphogenic protein (BMP) signalling. Biochemically, we show that LRIG1 binds TGFß/BMP receptors and the TGFß1 ligand. Finally, we show that the consequences of these interactions are to facilitate SMAD phosphorylation. Collectively, these data suggest that unlike in embryonic NSCs where EGFR may be the primary mechanism of action, in aNSCs, LRIG1 and TGFß pathways function together to fulfill their inhibitory roles.

Integrating single-cell and spatially resolved transcriptomic strategies to survey the astrocyte response to stroke in male mice.

Astrocytes, a type of glial cell in the central nervous system (CNS), adopt diverse states in response to injury that are influenced by their location relative to the insult. Here, we describe a platform for spatially resolved, single-cell transcriptomics and proteomics, called tDISCO (tissue-digital microfluidic isolation of single cells for -Omics). We use tDISCO alongside two high-throughput platforms for spatial (Visium) and single-cell transcriptomics (10X Chromium) to examine the heterogeneity of the astrocyte response to a cortical ischemic stroke in male mice. We show that integration of Visium and 10X Chromium datasets infers two astrocyte populations, proximal or distal to the injury site, while tDISCO determines the spatial boundaries and molecular profiles that define these populations. We find that proximal astrocytes show differences in lipid shuttling, with enriched expression of Apoe and Fabp5. Our datasets provide a resource for understanding the roles of astrocytes in stroke and showcase the utility of tDISCO for hypothesis-driven, spatially resolved single-cell experiments.

Insights into O-linked N-acetylglucosamine ([0-9]O-GlcNAc) processing and dynamics through kinetic analysis of O-GlcNAc transferase and O-GlcNAcase activity on protein substrates.

Cellular O-linked N-acetylglucosamine (O-GlcNAc) levels are modulated by two enzymes: uridine diphosphate-N-acetyl-D-glucosamine:polypeptidyltransferase (OGT) and O-GlcNAcase (OGA). To quantitatively address the activity of these enzymes on protein substrates, we generated five structurally diverse proteins in both unmodified and O-GlcNAc-modified states. We found a remarkably invariant upper limit for k(cat)/K(m) values for human OGA (hOGA)-catalyzed processing of these modified proteins, which suggests that hOGA processing is driven by the GlcNAc moiety and is independent of the protein. Human OGT (hOGT) activity ranged more widely, by up to 15-fold, suggesting that hOGT is the senior partner in fine tuning protein O-GlcNAc levels. This was supported by the observation that K(m,app) values for UDP-GlcNAc varied considerably (from 1 µM to over 20 µM), depending on the protein substrate, suggesting that some OGT substrates will be nutrient-responsive, whereas others are constitutively modified. The ratios of k(cat)/K(m) values obtained from hOGT and hOGA kinetic studies enable a prediction of the dynamic equilibrium position of O-GlcNAc levels that can be recapitulated in vitro and suggest the relative O-GlcNAc stoichiometries of target proteins in the absence of other factors. We show that changes in the specific activities of hOGT and hOGA measured in vitro on calcium/calmodulin-dependent kinase IV (CaMKIV) and its pseudophosphorylated form can account for previously reported changes in CaMKIV O-GlcNAc levels observed in cells. These studies provide kinetic evidence for the interplay between O-GlcNAc and phosphorylation on proteins and indicate that these effects can be mediated by changes in hOGT and hOGA kinetic activity.

Metabolism of vertebrate amino sugars with N-glycolyl groups: intracellular ß-O-linked N-glycolylglucosamine (GlcNGc), UDP-GlcNGc, and the biochemical and structural rationale for the substrate tolerance of ß-O-linked ß-N-acetylglucosaminidase.

The O-GlcNAc modification involves the attachment of single ß-O-linked N-acetylglucosamine residues to serine and threonine residues of nucleocytoplasmic proteins. Interestingly, previous biochemical and structural studies have shown that O-GlcNAcase (OGA), the enzyme that removes O-GlcNAc from proteins, has an active site pocket that tolerates various N-acyl groups in addition to the N-acetyl group of GlcNAc. The remarkable sequence and structural conservation of residues comprising this pocket suggest functional importance. We hypothesized this pocket enables processing of metabolic variants of O-GlcNAc that could be formed due to inaccuracy within the metabolic machinery of the hexosamine biosynthetic pathway. In the accompanying paper (Bergfeld, A. K., Pearce, O. M., Diaz, S. L., Pham, T., and Varki, A. (2012) J. Biol. Chem. 287, 28865-28881), N-glycolylglucosamine (GlcNGc) was shown to be a catabolite of NeuNGc. Here, we show that the hexosamine salvage pathway can convert GlcNGc to UDP-GlcNGc, which is then used to modify proteins with O-GlcNGc. The kinetics of incorporation and removal of O-GlcNGc in cells occur in a dynamic manner on a time frame similar to that of O-GlcNAc. Enzymatic activity of O-GlcNAcase (OGA) toward a GlcNGc glycoside reveals OGA can process glycolyl-containing substrates fairly efficiently. A bacterial homolog (BtGH84) of OGA, from a human gut symbiont, also processes O-GlcNGc substrates, and the structure of this enzyme bound to a GlcNGc-derived species reveals the molecular basis for tolerance and binding of GlcNGc. Together, these results demonstrate that analogs of GlcNAc, such as GlcNGc, are metabolically viable species and that the conserved active site pocket of OGA likely evolved to enable processing of mis-incorporated analogs of O-GlcNAc and thereby prevent their accumulation. Such plasticity in carbohydrate processing enzymes may be a general feature arising from inaccuracy in hexosamine metabolic pathways.

Culture Protocol and Transcriptomic Analysis of Murine SVZ NPCs and OPCs.

The mammalian adult brain contains two neural stem and precursor (NPC) niches: the subventricular zone [SVZ] lining the lateral ventricles and the subgranular zone [SGZ] in the hippocampus. From these, SVZ NPCs represent the largest NPC pool. While SGZ NPCs typically only produce neurons and astrocytes, SVZ NPCs produce neurons, astrocytes and oligodendrocytes throughout life. Of particular importance is the generation and replacement of oligodendrocytes, the only myelinating cells of the central nervous system (CNS). SVZ NPCs contribute to myelination by regenerating the parenchymal oligodendrocyte precursor cell (OPC) pool and by differentiating into oligodendrocytes in the developing and demyelinated brain. The neurosphere assay has been widely adopted by the scientific community to facilitate the study of NPCs in vitro. Here, we present a streamlined protocol for culturing postnatal and adult SVZ NPCs and OPCs from primary neurosphere cells. We characterize the purity and differentiation potential as well as provide RNA-sequencing profiles of postnatal SVZ NPCs, postnatal SVZ OPCs and adult SVZ NPCs. We show that primary neurospheres cells generated from postnatal and adult SVZ differentiate into neurons, astrocytes and oligodendrocytes concurrently and at comparable levels. SVZ OPCs are generated by subjecting primary neurosphere cells to OPC growth factors fibroblast growth factor (FGF) and platelet-derived growth factor-AA (PDGF-AA). We further show SVZ OPCs can differentiate into oligodendrocytes in the absence and presence of thyroid hormone T3. Transcriptomic analysis confirmed the identities of each cell population and revealed novel immune and signalling pathways expressed in an age and cell type specific manner.

Mapping O-GlcNAc modification sites on tau and generation of a site-specific O-GlcNAc tau antibody.

The microtubule-associated protein tau is known to be post-translationally modified by the addition of N-acetyl-D: -glucosamine monosaccharides to certain serine and threonine residues. These O-GlcNAc modification sites on tau have been challenging to identify due to the inherent complexity of tau from mammalian brains and the fact that the O-GlcNAc modification typically has substoichiometric occupancy. Here, we describe a method for the production of recombinant O-GlcNAc modified tau and, using this tau, we have mapped sites of O-GlcNAc on tau at Thr-123 and Ser-400 using mass spectrometry. We have also detected the presence of a third O-GlcNAc site on either Ser-409, Ser-412, or Ser-413. Using this information we have raised a rabbit polyclonal IgG antibody (3925) that detects tau O-GlcNAc modified at Ser-400. Further, using this antibody we have detected the Ser-400 tau O-GlcNAc modification in rat brain, which confirms the validity of this in vitro mapping approach. The identification of these O-GlcNAc sites on tau and this antibody will enable both in vivo and in vitro experiments designed to understand the possible functional roles of O-GlcNAc on tau.

A potent mechanism-inspired O-GlcNAcase inhibitor that blocks phosphorylation of tau in vivo.

Pathological hyperphosphorylation of the microtubule-associated protein tau is characteristic of Alzheimer's disease (AD) and the associated tauopathies. The reciprocal relationship between phosphorylation and O-GlcNAc modification of tau and reductions in O-GlcNAc levels on tau in AD brain offers motivation for the generation of potent and selective inhibitors that can effectively enhance O-GlcNAc in vertebrate brain. We describe the rational design and synthesis of such an inhibitor (thiamet-G, K(i) = 21 nM; 1) of human O-GlcNAcase. Thiamet-G decreased phosphorylation of tau in PC-12 cells at pathologically relevant sites including Thr231 and Ser396. Thiamet-G also efficiently reduced phosphorylation of tau at Thr231, Ser396 and Ser422 in both rat cortex and hippocampus, which reveals the rapid and dynamic relationship between O-GlcNAc and phosphorylation of tau in vivo. We anticipate that thiamet-G will find wide use in probing the functional role of O-GlcNAc in vertebrate brain, and it may also offer a route to blocking pathological hyperphosphorylation of tau in AD.

LRIG1-Mediated Inhibition of EGF Receptor Signaling Regulates Neural Precursor Cell Proliferation in the Neocortex.

Here, we ask how neural stem cells (NSCs) transition in the developing neocortex from a rapidly to a slowly proliferating state, a process required to maintain lifelong stem cell pools. We identify LRIG1, known to regulate receptor tyrosine kinase signaling in other cell types, as a negative regulator of cortical NSC proliferation. LRIG1 is expressed in murine cortical NSCs as they start to proliferate more slowly during embryogenesis and then peaks postnatally when they transition to give rise to a portion of adult NSCs. Constitutive or acute loss of Lrig1 in NSCs over this developmental time frame causes stem cell expansion due to increased proliferation. LRIG1 controls NSC proliferation by associating with and negatively regulating the epidermal growth factor receptor (EGFR). These data support a model in which LRIG1 dampens the stem cell response to EGFR ligands within the cortical environment to slow their proliferation as they transition to postnatal adult NSCs.

Peripheral Nerve Single-Cell Analysis Identifies Mesenchymal Ligands that Promote Axonal Growth.

Peripheral nerves provide a supportive growth environment for developing and regenerating axons and are essential for maintenance and repair of many non-neural tissues. This capacity has largely been ascribed to paracrine factors secreted by nerve-resident Schwann cells. Here, we used single-cell transcriptional profiling to identify ligands made by different injured rodent nerve cell types and have combined this with cell-surface mass spectrometry to computationally model potential paracrine interactions with peripheral neurons. These analyses show that peripheral nerves make many ligands predicted to act on peripheral and CNS neurons, including known and previously uncharacterized ligands. While Schwann cells are an important ligand source within injured nerves, more than half of the predicted ligands are made by nerve-resident mesenchymal cells, including the endoneurial cells most closely associated with peripheral axons. At least three of these mesenchymal ligands, ANGPT1, CCL11, and VEGFC, promote growth when locally applied on sympathetic axons. These data therefore identify an unexpected paracrine role for nerve mesenchymal cells and suggest that multiple cell types contribute to creating a highly pro-growth environment for peripheral axons.

Acquisition of a Unique Mesenchymal Precursor-like Blastema State Underlies Successful Adult Mammalian Digit Tip Regeneration.

Here, we investigate the origin and nature of blastema cells that regenerate the adult murine digit tip. We show that Pdgfra-expressing mesenchymal cells in uninjured digits establish the regenerative blastema and are essential for regeneration. Single-cell profiling shows that the mesenchymal blastema cells are distinct from both uninjured digit and embryonic limb or digit Pdgfra-positive cells. This unique blastema state is environmentally determined; dermal fibroblasts transplanted into the regenerative, but not non-regenerative, digit express blastema-state genes and contribute to bone regeneration. Moreover, lineage tracing with single-cell profiling indicates that endogenous osteoblasts or osteocytes acquire a blastema mesenchymal transcriptional state and contribute to both dermis and bone regeneration. Thus, mammalian digit tip regeneration occurs via a distinct adult mechanism where the regenerative environment promotes acquisition of a blastema state that enables cells from tissues such as bone to contribute to the regeneration of other mesenchymal tissues such as the dermis.

Molecular basis for inhibition of GH84 glycoside hydrolases by substituted azepanes: conformational flexibility enables probing of substrate distortion.

Here we report the synthesis of a series of polyhydroxylated 3- and 5-acetamido azepanes and detail the molecular basis of their inhibition of family 84 glycoside hydrolases. These family 84 enzymes include human O-GlcNAcase, an enzyme involved in post-translational processing of intracellular proteins modified by O-linked beta-N-acetylglucosamine residues. Detailed structural analysis of the binding of these azepanes to BtGH84, a bacterial homologue of O-GlcNAcase, highlights their conformational flexibility. Molecular mechanics and molecular dynamics calculations reveal that binding to the enzyme involves significant conformational distortion of these inhibitors from their preferred solution conformations. The binding of these azepanes provides structural insight into substrate distortion that likely occurs along the reaction coordinate followed by O-GlcNAcase during glycoside hydrolysis. This class of inhibitors may prove to be useful probes for evaluating the conformational itineraries of glycosidases and aid the development of more potent and specific glycosidase inhibitors.

Mesenchymal Precursor Cells in Adult Nerves Contribute to Mammalian Tissue Repair and Regeneration.

Peripheral innervation plays an important role in regulating tissue repair and regeneration. Here we provide evidence that injured peripheral nerves provide a reservoir of mesenchymal precursor cells that can directly contribute to murine digit tip regeneration and skin repair. In particular, using single-cell RNA sequencing and lineage tracing, we identify transcriptionally distinct mesenchymal cell populations within the control and injured adult nerve, including neural crest-derived cells in the endoneurium with characteristics of mesenchymal precursor cells. Culture and transplantation studies show that these nerve-derived mesenchymal cells have the potential to differentiate into non-nerve lineages. Moreover, following digit tip amputation, neural crest-derived nerve mesenchymal cells contribute to the regenerative blastema and, ultimately, to the regenerated bone. Similarly, neural crest-derived nerve mesenchymal cells contribute to the dermis during skin wound healing. These findings support a model where peripheral nerves directly contribute mesenchymal precursor cells to promote repair and regeneration of injured mammalian tissues.

A Translational Repression Complex in Developing Mammalian Neural Stem Cells that Regulates Neuronal Specification.

The mechanisms instructing genesis of neuronal subtypes from mammalian neural precursors are not well understood. To address this issue, we have characterized the transcriptional landscape of radial glial precursors (RPs) in the embryonic murine cortex. We show that individual RPs express mRNA, but not protein, for transcriptional specifiers of both deep and superficial layer cortical neurons. Some of these mRNAs, including the superficial versus deep layer neuron transcriptional regulators Brn1 and Tle4, are translationally repressed by their association with the RNA-binding protein Pumilio2 (Pum2) and the 4E-T protein. Disruption of these repressive complexes in RPs mid-neurogenesis by knocking down 4E-T or Pum2 causes aberrant co-expression of deep layer neuron specification proteins in newborn superficial layer neurons. Thus, cortical RPs are transcriptionally primed to generate diverse types of neurons, and a Pum2/4E-T complex represses translation of some of these neuronal identity mRNAs to ensure appropriate temporal specification of daughter neurons.

Production of O-GlcNAc Modified Recombinant Tau in E. coli and Detection of Ser400 O-GlcNAc Tau In Vivo.

Assembly of the microtubule-associated protein tau (tau) into paired helical filaments that ultimately give rise to neurofibrillary tangles (NFTs) makes up one half of the two hallmark pathologies of Alzheimer's disease (AD). Tau has been shown to be modified with O-linked N-acetylglucosamine residues (O-GlcNAc), which is the modification of serine and threonine residues of nucleocytoplasmic proteins with N-acetyl-D-glucosamine (GlcNAc) moieties. Increasing O-GlcNAc in mouse models of tauopathy has been shown to hinder the progression of symptoms in these mice and impair the aggregation of tau into NFTs. In order to study how O-GlcNAc on tau may contribute to the protective effects observed in tauopathy mouse models, it is beneficial to study O-GlcNAc modified tau in vitro. Here we describe a method for producing, purifying and enriching recombinant tau that is O-GlcNAc modified. These methods have enabled the identification of O-GlcNAc modification sites on tau including Ser400. We further describe the detection of Ser400 O-GlcNAc on tau from brain lysates.

Deciphering cell-cell communication in the developing mammalian brain.

The diverse subtypes of neurons that comprise the mammalian cerebral cortex are produced from a single population of cortical neural precursor cells during the period of embryonic neurogenesis. While this process of neurogenesis is tightly controlled at the transcriptional and translational levels, substantial opportunity exists for extrinsic or niche control of the process of neurogenesis. In our recently published work we made use of a combination of computational and biologic approaches to characterize cell-cell communication between cortical neurons and cortical precursor cells and thereby reveal an unexpectedly complex growth factor communication network that accurately predicted new regulators of cortical neurogenesis.