O-GlcNAc glycosylation (or O-GlcNAcylation) is a dynamic, inducible posttranslational modification found on proteins associated with neurodegenerative diseases such as α-synuclein, amyloid precursor protein, and tau. Deletion of the O-GlcNAc transferase (ogt) gene responsible for the modification causes early postnatal lethality in mice, complicating efforts to study O-GlcNAcylation in mature neurons and to understand its roles in disease. Here, we report that forebrain-specific loss of OGT in adult mice leads to progressive neurodegeneration, including widespread neuronal cell death, neuroinflammation, increased production of hyperphosphorylated tau and amyloidogenic Aβ-peptides, and memory deficits. Furthermore, we show that human cortical brain tissue from Alzheimer’s disease patients has significantly reduced levels of OGT protein expression compared with cortical tissue from control individuals. Together, these studies indicate that O-GlcNAcylation regulates pathways critical for the maintenance of neuronal health and suggest that dysfunctional O-GlcNAc signaling may be an important contributor to neurodegenerative diseases.

Iron dysregulation has been implicated in multiple neurodegenerative diseases, including Parkinson’s disease (PD). Iron-loaded microglia are frequently found in affected brain regions, but how iron accumulation influences microglia physiology and contributes to neurodegeneration is poorly understood. Here we show that human induced pluripotent stem cell-derived microglia grown in a tri-culture system are highly responsive to iron and susceptible to ferroptosis, an iron-dependent form of cell death. Furthermore, iron overload causes a marked shift in the microglial transcriptional state that overlaps with a transcriptomic signature found in PD postmortem brain microglia. Our data also show that this microglial response contributes to neurodegeneration, as removal of microglia from the tri-culture system substantially delayed iron-induced neurotoxicity. To elucidate the mechanisms regulating iron response in microglia, we performed a genome-wide CRISPR screen and identified novel regulators of ferroptosis, including the vesicle trafficking gene SEC24B. These data suggest a critical role for microglia iron overload and ferroptosis in neurodegeneration.

Amyotrophic lateral sclerosis (ALS) is a fatal neuromuscular disease characterized by motor neuron (MN) death. Lipid dysregulation manifests during disease; however, it is unclear whether lipid homeostasis is adversely affected in the in the spinal cord gray matter (GM), and if so, whether it is because of an aberrant increase in lipid synthesis. Moreover, it is unknown whether lipid dysregulation contributes to MN death. Here, we show that cholesterol ester (CE) and triacylglycerol levels are elevated several-fold in the spinal cord GM of male sporadic ALS patients. Interestingly, HMG-CoA reductase, the rate-limiting enzyme in cholesterol synthesis, was reduced in the spinal cord GM of ALS patients. Increased cytosolic phospholipase A2 activity and lyso-phosphatidylcholine (Lyso-PC) levels in ALS patients suggest that CE accumulation was driven by acyl group transfer from PC to cholesterol. Notably, Lyso-PC, a byproduct of CE synthesis, was toxic to human MNs in vitro. Elevations in CE, triacylglycerol, and Lyso-PC were also found in the spinal cord of SOD1 G93A mice, a model of ALS. Similar to ALS patients, a compensatory downregulation of cholesterol synthesis occurred in the spinal cord of SOD1 G93A mice; levels of sterol regulatory element binding protein 2, a transcriptional regulator of cholesterol synthesis, progressively declined. Remarkably, overexpressing sterol regulatory element binding protein 2 in the spinal cord of normal mice to model CE accumulation led to ALS-like lipid pathology, MN death, astrogliosis, paralysis, and reduced survival. Thus, spinal cord lipid dysregulation in ALS likely contributes to neurodegeneration and developing therapies to restore lipid homeostasis may lead to a treatment for ALS.

The post-translational modification of serine or threonine residues of proteins with a single N-acetylglucosamine monosaccharide (O-GlcNAcylation) is essential for cell survival and function. However, relatively few O-GlcNAc modification sites have been mapped due to the difficulty of enriching and detecting O-GlcNAcylated peptides from complex samples. Here we describe an improved approach to quantitatively label and enrich O-GlcNAcylated proteins for site identification. Chemoenzymatic labelling followed by copper(I)-catalysed azide-alkyne cycloaddition (CuAAC) installs a new mass spectrometry (MS)-compatible linker designed for facile purification of O-GlcNAcylated proteins from cell lysates. The linker also allows quantitative release of O-GlcNAcylated proteins for downstream MS analysis. We validate the approach by unambiguously identifying several established O-GlcNAc sites on the proteins α-crystallin and O-GlcNAc transferase (OGT) as well as discovering new, previously unreported sites on OGT. Notably, these novel sites on OGT lie in key functional domains of the protein, underscoring how this site identification method may reveal important biological insights into protein activity and regulation.

Amyotrophic Lateral Sclerosis (ALS) is a neurodegenerative disease with a complex, multifactorial pathophysiology, most commonly manifest as loss of motor neurons. We introduce a new mechanism of ALS pathogenesis via a novel drug-like small molecule series that targets protein disulfide isomerase (PDI) within a previously unappreciated transient and energy-dependent multi-protein complex. This novel drug was found to have activity in cellular models for both familial and sporadic ALS, as well as in transgenic worms, flies, and mice bearing a diversity of human genes with ALS-associated mutations. These compounds were initially identified as modulators of human immunodeficiency virus (HIV) capsid assembly in cell-free protein synthesis and assembly (CFPSA) systems, with demonstrated antiviral activity in cell culture. Their advancement as ALS-therapeutics, and the subsequent separation of activity against HIV and ALS in chemical subseries through structure-activity-relationship optimization, may provide insights into the molecular mechanisms governing pathophysiology of disordered homeostasis relevant to ALS.

Iron dysregulation has been implicated in multiple neurodegenerative diseases, including Parkinson’s Disease (PD), Amyotrophic Lateral Sclerosis (ALS), and Multiple Sclerosis (MS). One prominent feature of affected brain regions are iron-loaded microglia, but how iron overload influences microglia physiology and disease response is poorly understood. Here we show that microglia are highly susceptible to ferroptosis, an iron-dependent form of cell death. In a tri-culture of human iPSC-derived neurons, astrocytes, and microglia, under ferroptosis-inducing conditions, microglia undergo a drastic shift in cell state, with increased ferritin levels, disrupted glutathione homeostasis, and altered cytokine signaling. Similar ferroptosis-associated signature (FAS) microglia were uncovered in PD, and the signature was also found in a large cohort of PD patient blood samples, raising the possibility that ferroptosis can be identified clinically. We performed a genome-wide CRISPR screen which revealed a novel regulator of ferroptosis, the vesicle trafficking gene SEC24B. A small molecule screen also nominated several candidates which blocked ferroptosis, some of which are already in clinical use. These data suggest that ferroptosis sits at the interface of cell death and inflammation, and inhibition of this process in microglia and other brain cells may provide new ways for treating neurodegenerative disease.

The post-translational modification (PTM) of proteins by O-linked β-N-acetyl-D-glucosamine (O-GlcNAcylation) is widespread across the proteome during the lifespan of all multicellular organisms. However, nearly all functional studies have focused on individual protein modifications, overlooking the multitude of simultaneous O-GlcNAcylation events that work together to coordinate cellular activities. Here, we describe Networking of Interactors and SubstratEs (NISE), a novel, systems-level approach to rapidly and comprehensively monitor O-GlcNAcylation across the proteome. Our method integrates affinity purification-mass spectrometry (AP-MS) and site-specific chemoproteomic technologies with network generation and unsupervised partitioning to connect potential upstream regulators with downstream targets of O-GlcNAcylation. The resulting network provides a data-rich framework that reveals both conserved activities of O-GlcNAcylation such as epigenetic regulation as well as tissue-specific functions like synaptic morphology. Beyond O-GlcNAc, this holistic and unbiased systems-level approach provides a broadly applicable framework to study PTMs and discover their diverse roles in specific cell types and biological states.

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