O-GlcNAc Modification of tau Directly Inhibits Its Aggregation without Perturbing the Conformational Properties of tau Monomers

Abstract The aggregation of the microtubule-associated protein tau into paired helical filaments to form neurofibrillary tangles constitutes one of the pathological hallmarks of Alzheimer's disease. Tau is post-translationally modified by the addition of N-acetyl-D-glucosamine O-linked to several serine and threonine residues (O-GlcNAc). Previously, increased O-GlcNAcylation of tau has been shown to block the accumulation of tau aggregates within a tauopathy mouse model. Here we show that O-GlcNAc modification of full-length human tau impairs the rate and extent of its heparin-induced aggregation without perturbing its activity toward microtubule polymerization. O-GlcNAcylation, however, does not impact the "global-fold" of tau as measured by a Förster resonance energy transfer assay. Similarly, nuclear magnetic resonance studies demonstrated that O-GlcNAcylation only minimally perturbs the local structural and dynamic features of a tau fragment (residues 353-408) spanning the last microtubule binding repeat to the major GlcNAc-acceptor Ser400. These data indicate that the inhibitory effects of O-GlcNAc on tau aggregation may result from enhanced monomer solubility or the destabilization of fibrils or soluble aggregates, rather than by altering the conformational properties of the monomeric protein. This work further underscores the potential of targeting the O-GlcNAc pathway for potential Alzheimer's disease therapeutics

O-GlcNAc Modification of tau Directly Inhibits Its Aggregation without Perturbing the Conformational Properties of tau Monomers

The aggregation of the microtubule-associated protein tau into paired helical filaments to form neurofibrillary tangles constitutes one of the pathological hallmarks of Alzheimer\u27s disease. Tau is post-translationally modified by the addition of N-acetyl-d-glucosamine O-linked to several serine and threonine residues (O-GlcNAc). Previously, increased O-GlcNAcylation of tau has been shown to block the accumulation of tau aggregates within a tauopathy mouse model. Here we show that O-GlcNAc modification of full-length human tau impairs the rate and extent of its heparin-induced aggregation without perturbing its activity toward microtubule polymerization. O-GlcNAcylation, however, does not impact the “global-fold” of tau as measured by a Förster resonance energy transfer assay. Similarly, nuclear magnetic resonance studies demonstrated that O-GlcNAcylation only minimally perturbs the local structural and dynamic features of a tau fragment (residues 353–408) spanning the last microtubule binding repeat to the major GlcNAc-acceptor Ser400. These data indicate that the inhibitory effects of O-GlcNAc on tau aggregation may result from enhanced monomer solubility or the destabilization of fibrils or soluble aggregates, rather than by altering the conformational properties of the monomeric protein. This work further underscores the potential of targeting the O-GlcNAc pathway for potential Alzheimer\u27s disease therapeutics

Homozygous ARHGEF2 mutation causes intellectual disability and midbrain- hindbrain malformation

Abstract 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. Author summary During brain development, localized gene expression is crucial for the formation and function of specific brain regions. Various groups of proteins are known to regulate segmentation through controlled gene expression, among them, the Rho GTPase regulator family. In this study, we identified a frameshift mutation in the Rho guanine nucleotide exchange factor 2 gene (ARHGEF2) in two children presenting with intellectual disability, mild microcephaly, and a midbrain- hindbrain malformation. This phenotype is also observed in Arhgef2 mutant mice, highlighting the importance of ARHGEF2 across development of distinct mammalian species. We show that loss of Arhgef2 affects neurogenesis and also cell migration. In addition, we extended the current knowledge of ARHGEF2 expression and its role in early central nervous system development, with special reference to the formation of the precerebellar system. In addition to extensive literature on ARHGEF2, we now provide evidence for its significant role in neuronal migration in brain development and link the gene to human neurodevelopmental disease

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 β-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

O -GlcNAc and Neurodegeneration: Biochemical Mechanisms and Potential Roles in Alzheimer\u27s 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

Elevation of Global <em>O</em>-GlcNAc in Rodents Using a Selective <em>O</em>-GlcNAcase Inhibitor Does Not Cause Insulin Resistance or Perturb Glucohomeostasis

The O-GlcNAc modification is proposed to be a nutrient sensor with studies suggesting that global increases in O-GlcNAc levels cause insulin resistance and impaired glucohomeostasis. We address this hypothesis by using a potent and selective inhibitor of O-GlcNAcase, known as NButGT, in a series of in vivo studies. Treatment of rats and mice with NButGT, for various time regimens and doses, dramatically increases O-GlcNAc levels throughout all tissues but does not perturb insulin sensitivity or alter glucohomeostasis. NButGT also does not affect the severity or onset of insulin resistance induced by a high-fat diet. These results suggest that pharmacological increases in global O-GlcNAc levels do not cause insulin resistance nor do they appear to disrupt glucohomeostasis. Therefore, the protective benefits of elevated O-GlcNAc levels may be achieved without deleteriously affecting glucohomeostasis.</p

Developmental Emergence of Adult Neural Stem Cells as Revealed by Single-Cell Transcriptional Profiling

Adult neural stem cells (NSCs) derive from embryonic precursors, but little is known about how or when this occurs. We have addressed this issue using single-cell RNA sequencing at multiple developmental time points to analyze the embryonic murine cortex, one source of adult forebrain NSCs. We computationally identify all major cortical cell types, including the embryonic radial precursors (RPs) that generate adult NSCs. We define the initial emergence of RPs from neuroepithelial stem cells at E11.5. We show that, by E13.5, RPs express a transcriptional identity that is maintained and reinforced throughout their transition to a non-proliferative state between E15.5 and E17.5. These slowly proliferating late embryonic RPs share a core transcriptional phenotype with quiescent adult forebrain NSCs. Together, these findings support a model wherein cortical RPs maintain a core transcriptional identity from embryogenesis through to adulthood and wherein the transition to a quiescent adult NSC occurs during late neurogenesis