Publications by authors named "Thomas A Neubert"

154 Publications

Mitovesicles are a novel population of extracellular vesicles of mitochondrial origin altered in Down syndrome.

Sci Adv 2021 Feb 12;7(7). Epub 2021 Feb 12.

Center for Dementia Research, Nathan S. Kline Institute for Psychiatric Research, Orangeburg, NY 10962, USA.

Mitochondrial dysfunction is an established hallmark of aging and neurodegenerative disorders such as Down syndrome (DS) and Alzheimer's disease (AD). Using a high-resolution density gradient separation of extracellular vesicles (EVs) isolated from murine and human DS and diploid control brains, we identify and characterize a previously unknown population of double-membraned EVs containing multiple mitochondrial proteins distinct from previously described EV subtypes, including microvesicles and exosomes. We term these newly identified mitochondria-derived EVs "mitovesicles." We demonstrate that brain-derived mitovesicles contain a specific subset of mitochondrial constituents and that their levels and cargo are altered during pathophysiological processes where mitochondrial dysfunction occurs, including in DS. The development of a method for the selective isolation of mitovesicles paves the way for the characterization in vivo of biological processes connecting EV biology and mitochondria dynamics and for innovative therapeutic and diagnostic strategies.
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http://dx.doi.org/10.1126/sciadv.abe5085DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7880603PMC
February 2021

Serine phosphorylation regulates the P-type potassium pump KdpFABC.

Elife 2020 09 21;9. Epub 2020 Sep 21.

Skirball Institute, Dept. of Cell Biology, New York University School of Medicine, New York, United States.

KdpFABC is an ATP-dependent K pump that ensures bacterial survival in K-deficient environments. Whereas transcriptional activation of kdpFABC expression is well studied, a mechanism for down-regulation when K levels are restored has not been described. Here, we show that KdpFABC is inhibited when cells return to a K-rich environment. The mechanism of inhibition involves phosphorylation of Ser162 on KdpB, which can be reversed in vitro by treatment with serine phosphatase. Mutating Ser162 to Alanine produces constitutive activity, whereas the phosphomimetic Ser162Asp mutation inactivates the pump. Analyses of the transport cycle show that serine phosphorylation abolishes the K-dependence of ATP hydrolysis and blocks the catalytic cycle after formation of the aspartyl phosphate intermediate (E1~P). This regulatory mechanism is unique amongst P-type pumps and this study furthers our understanding of how bacteria control potassium homeostasis to maintain cell volume and osmotic potential.
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http://dx.doi.org/10.7554/eLife.55480DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7535926PMC
September 2020

Zinc induced structural changes in the intrinsically disordered BDNF Met prodomain confer synaptic elimination.

Metallomics 2020 08;12(8):1208-1219

Weill Cornell Medicine, Department of Biochemistry, New York, NY, USA.

Human brain derived neurotrophic factor (BDNF) encodes a protein product consisting of a C-terminal mature domain (mature BDNF) and an N-terminal prodomain, which is an intrinsically disordered protein. A common single nucleotide polymorphism in humans results in a methionine substitution for valine at position 66 of the prodomain, and is associated with memory deficits, depression and anxiety disorders. The BDNF Met66 prodomain, but not the Val66 prodomain, promotes rapid structural remodeling of hippocampal neurons' growth cones and dendritic spines by interacting directly with the SorCS2 receptor. While it has been reported that the Met66 and Val66 prodomains exhibit only modest differences in structural propensities in the apo state, here we show that Val66 and Met66 prodomains differentially bind zinc (Zn). Zn2+ binds with higher affinity and more broadly impacts residues on the Met66 prodomain compared to the Val66 prodomain as shown by NMR and ITC. Zn2+ binding to the Met66 and Val66 prodomains results in distinct conformational and macroscopic differences observed by NMR, light scattering and cryoEM. To determine if Zn2+ mediated conformational change in the Met66 prodomain is required for biological effect, we mutated His40, a Zn2+ binding site, and observed a loss of Met66 prodomain bioactivity. As the His40 site is distant from the known region of the prodomain involved in receptor binding, we suggest that Met66 prodomain bioactivity involves His40 mediated stabilization of the multimeric structure. Our results point to the necessity of a Zn2+-mediated higher order molecular assembly of the Met66 prodomain to mediate neuronal remodeling.
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http://dx.doi.org/10.1039/d0mt00108bDOI Listing
August 2020

Molecular Stressors Engender Protein Connectivity Dysfunction through Aberrant N-Glycosylation of a Chaperone.

Cell Rep 2020 06;31(13):107840

Chemical Biology Program, Memorial Sloan Kettering Cancer Center, New York, NY 10065, USA; Department of Medicine, Memorial Sloan Kettering Cancer Center, New York, NY 10065, USA. Electronic address:

Stresses associated with disease may pathologically remodel the proteome by both increasing interaction strength and altering interaction partners, resulting in proteome-wide connectivity dysfunctions. Chaperones play an important role in these alterations, but how these changes are executed remains largely unknown. Our study unveils a specific N-glycosylation pattern used by a chaperone, Glucose-regulated protein 94 (GRP94), to alter its conformational fitness and stabilize a state most permissive for stable interactions with proteins at the plasma membrane. This "protein assembly mutation' remodels protein networks and properties of the cell. We show in cells, human specimens, and mouse xenografts that proteome connectivity is restorable by inhibition of the N-glycosylated GRP94 variant. In summary, we provide biochemical evidence for stressor-induced chaperone-mediated protein mis-assemblies and demonstrate how these alterations are actionable in disease.
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http://dx.doi.org/10.1016/j.celrep.2020.107840DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7372946PMC
June 2020

Neuronal Inactivity Co-opts LTP Machinery to Drive Potassium Channel Splicing and Homeostatic Spike Widening.

Cell 2020 06 2;181(7):1547-1565.e15. Epub 2020 Jun 2.

Department of Neuroscience and Physiology, Neuroscience Institute, NYU Grossman Medical Center, New York, NY 10016, USA; Center for Neural Science, New York University, New York, NY 10003, USA. Electronic address:

Homeostasis of neural firing properties is important in stabilizing neuronal circuitry, but how such plasticity might depend on alternative splicing is not known. Here we report that chronic inactivity homeostatically increases action potential duration by changing alternative splicing of BK channels; this requires nuclear export of the splicing factor Nova-2. Inactivity and Nova-2 relocation were connected by a novel synapto-nuclear signaling pathway that surprisingly invoked mechanisms akin to Hebbian plasticity: Ca-permeable AMPA receptor upregulation, L-type Ca channel activation, enhanced spine Ca transients, nuclear translocation of a CaM shuttle, and nuclear CaMKIV activation. These findings not only uncover commonalities between homeostatic and Hebbian plasticity but also connect homeostatic regulation of synaptic transmission and neuronal excitability. The signaling cascade provides a full-loop mechanism for a classic autoregulatory feedback loop proposed ∼25 years ago. Each element of the loop has been implicated previously in neuropsychiatric disease.
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http://dx.doi.org/10.1016/j.cell.2020.05.013DOI Listing
June 2020

Molecular basis for receptor tyrosine kinase A-loop tyrosine transphosphorylation.

Nat Chem Biol 2020 03 20;16(3):267-277. Epub 2020 Jan 20.

Department of Biochemistry and Molecular Pharmacology, New York University School of Medicine, New York, NY, USA.

A long-standing mystery shrouds the mechanism by which catalytically repressed receptor tyrosine kinase domains accomplish transphosphorylation of activation loop (A-loop) tyrosines. Here we show that this reaction proceeds via an asymmetric complex that is thermodynamically disadvantaged because of an electrostatic repulsion between enzyme and substrate kinases. Under physiological conditions, the energetic gain resulting from ligand-induced dimerization of extracellular domains overcomes this opposing clash, stabilizing the A-loop-transphosphorylating dimer. A unique pathogenic fibroblast growth factor receptor gain-of-function mutation promotes formation of the complex responsible for phosphorylation of A-loop tyrosines by eliminating this repulsive force. We show that asymmetric complex formation induces a more phosphorylatable A-loop conformation in the substrate kinase, which in turn promotes the active state of the enzyme kinase. This explains how quantitative differences in the stability of ligand-induced extracellular dimerization promotes formation of the intracellular A-loop-transphosphorylating asymmetric complex to varying extents, thereby modulating intracellular kinase activity and signaling intensity.
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http://dx.doi.org/10.1038/s41589-019-0455-7DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7040854PMC
March 2020

The epichaperome is a mediator of toxic hippocampal stress and leads to protein connectivity-based dysfunction.

Nat Commun 2020 01 16;11(1):319. Epub 2020 Jan 16.

Chemical Biology Program, Memorial Sloan Kettering Cancer Center, New York, NY, 10065, USA.

Optimal functioning of neuronal networks is critical to the complex cognitive processes of memory and executive function that deteriorate in Alzheimer's disease (AD). Here we use cellular and animal models as well as human biospecimens to show that AD-related stressors mediate global disturbances in dynamic intra- and inter-neuronal networks through pathologic rewiring of the chaperome system into epichaperomes. These structures provide the backbone upon which proteome-wide connectivity, and in turn, protein networks become disturbed and ultimately dysfunctional. We introduce the term protein connectivity-based dysfunction (PCBD) to define this mechanism. Among most sensitive to PCBD are pathways with key roles in synaptic plasticity. We show at cellular and target organ levels that network connectivity and functional imbalances revert to normal levels upon epichaperome inhibition. In conclusion, we provide proof-of-principle to propose AD is a PCBDopathy, a disease of proteome-wide connectivity defects mediated by maladaptive epichaperomes.
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http://dx.doi.org/10.1038/s41467-019-14082-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6965647PMC
January 2020

Sam68 Enables Metabotropic Glutamate Receptor-Dependent LTD in Distal Dendritic Regions of CA1 Hippocampal Neurons.

Cell Rep 2019 11;29(7):1789-1799.e6

Dominick P. Purpura Department of Neuroscience, Albert Einstein College of Medicine, Bronx, New York, NY 10461, USA; Department of Psychiatry and Behavioral Sciences, Albert Einstein College of Medicine, Bronx, New York, NY 10461, USA. Electronic address:

The transport and translation of dendritic mRNAs by RNA-binding proteins (RBPs) allows for spatially restricted gene expression in neuronal processes. Although local translation in neuronal dendrites is now well documented, there is little evidence for corresponding effects on local synaptic function. Here, we report that the RBP Sam68 promotes the localization and translation of Arc mRNA preferentially in distal dendrites of rodent hippocampal CA1 pyramidal neurons. Consistent with Arc function in translation-dependent synaptic plasticity, we find that Sam68 knockout (KO) mice display impaired metabotropic glutamate-receptor-dependent long-term depression (mGluR-LTD) and impaired structural plasticity exclusively at distal Schaffer-collateral synapses. Moreover, by using quantitative proteomics, we find that the Sam68 interactome contains numerous regulators of mRNA translation and synaptic function. This work identifies an important player in Arc expression, provides a general framework for Sam68 regulation of protein synthesis, and uncovers a mechanism that enables the precise spatiotemporal expression of long-term plasticity throughout neurons.
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http://dx.doi.org/10.1016/j.celrep.2019.10.030DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6871770PMC
November 2019

Lipidome-wide C flux analysis: a novel tool to estimate the turnover of lipids in organisms and cultures.

J Lipid Res 2020 01 11;61(1):95-104. Epub 2019 Nov 11.

Departments of Anesthesiology, New York University School of Medicine, New York, NY 10016; Cell Biology, New York University School of Medicine, New York, NY 10016.

Lipid metabolism plays an important role in the regulation of cellular homeostasis. However, because it is difficult to measure the actual rates of synthesis and degradation of individual lipid species, lipid compositions are often used as a surrogate to evaluate lipid metabolism even though they provide only static snapshots of the lipodome. Here, we designed a simple method to determine the turnover rate of phospholipid and acylglycerol species based on the incorporation of C-glucose combined with LC-MS/MS. We labeled adult with C-glucose that incorporates into the entire lipidome, derived kinetic parameters from mass spectra, and studied effects of deletion of CG6718, the fly homolog of the calcium-independent phospholipase A2β, on lipid metabolism. Although C-glucose gave rise to a complex pattern of C incorporation, we were able to identify discrete isotopomers in which C atoms were confined to the glycerol group. With these isotopomers, we calculated turnover rate constants, half-life times, and fluxes of the glycerol backbone of multiple lipid species. To perform these calculations, we estimated the fraction of labeled molecules in glycerol-3-phosphate, the lipid precursor, by mass isotopomer distribution analysis of the spectra of phosphatidylglycerol. When we applied this method to , we found a range of lipid half-lives from 2 to 200 days, demonstrated tissue-specific fluxes of individual lipid species, and identified a novel function of CG6718 in triacylglycerol metabolism. This method provides fluxomics-type data with significant potential to improve the understanding of complex lipid regulation in a variety of research models.
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http://dx.doi.org/10.1194/jlr.D119000318DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6939592PMC
January 2020

A glucose-sensing neuron pair regulates insulin and glucagon in Drosophila.

Nature 2019 10 23;574(7779):559-564. Epub 2019 Oct 23.

Skirball Institute of Biomolecular Medicine, Department of Cell Biology, Neuroscience Institute, New York University School of Medicine, New York, NY, USA.

Although glucose-sensing neurons were identified more than 50 years ago, the physiological role of glucose sensing in metazoans remains unclear. Here we identify a pair of glucose-sensing neurons with bifurcated axons in the brain of Drosophila. One axon branch projects to insulin-producing cells to trigger the release of Drosophila insulin-like peptide 2 (dilp2) and the other extends to adipokinetic hormone (AKH)-producing cells to inhibit secretion of AKH, the fly analogue of glucagon. These axonal branches undergo synaptic remodelling in response to changes in their internal energy status. Silencing of these glucose-sensing neurons largely disabled the response of insulin-producing cells to glucose and dilp2 secretion, disinhibited AKH secretion in corpora cardiaca and caused hyperglycaemia, a hallmark feature of diabetes mellitus. We propose that these glucose-sensing neurons maintain glucose homeostasis by promoting the secretion of dilp2 and suppressing the release of AKH when haemolymph glucose levels are high.
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http://dx.doi.org/10.1038/s41586-019-1675-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6857815PMC
October 2019

Vezatin is required for the maturation of the neuromuscular synapse.

Mol Biol Cell 2019 09 14;30(20):2571-2583. Epub 2019 Aug 14.

Helen L. and Martin S. Kimmel Center for Biology and Medicine, Skirball Institute of Biomolecular Medicine, New York University School of Medicine, New York, NY 10016.

Key genes, such as , , and , are required for the initial formation, subsequent maturation, and long-term stabilization of mammalian neuromuscular synapses. Additional molecules are thought to function selectively during the evolution and stabilization of these synapses, but these molecular players are largely unknown. Here, we used mass spectrometry to identify vezatin, a two-pass transmembrane protein, as an acetylcholine receptor (AChR)-associated protein, and we provide evidence that vezatin binds directly to AChRs. We show that vezatin is dispensable for the formation of synapses but plays a later role in the emergence of a topologically complex and branched shape of the synapse, as well as the stabilization of AChRs. In addition, neuromuscular synapses in mutant mice display premature signs of deterioration, normally found only during aging. Thus, vezatin has a selective role in the structural elaboration and postnatal maturation of murine neuromuscular synapses.
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http://dx.doi.org/10.1091/mbc.E19-06-0313DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6740198PMC
September 2019

Haploinsufficiency in the ANKS1B gene encoding AIDA-1 leads to a neurodevelopmental syndrome.

Nat Commun 2019 08 6;10(1):3529. Epub 2019 Aug 6.

Dominick P. Purpura Department of Neuroscience, Albert Einstein College of Medicine, Bronx, 10461, NY, USA.

Neurodevelopmental disorders, including autism spectrum disorder, have complex polygenic etiologies. Single-gene mutations in patients can help define genetic factors and molecular mechanisms underlying neurodevelopmental disorders. Here we describe individuals with monogenic heterozygous microdeletions in ANKS1B, a predicted risk gene for autism and neuropsychiatric diseases. Affected individuals present with a spectrum of neurodevelopmental phenotypes, including autism, attention-deficit hyperactivity disorder, and speech and motor deficits. Neurons generated from patient-derived induced pluripotent stem cells demonstrate loss of the ANKS1B-encoded protein AIDA-1, a brain-specific protein highly enriched at neuronal synapses. A transgenic mouse model of Anks1b haploinsufficiency recapitulates a range of patient phenotypes, including social deficits, hyperactivity, and sensorimotor dysfunction. Identification of the AIDA-1 interactome using quantitative proteomics reveals protein networks involved in synaptic function and the etiology of neurodevelopmental disorders. Our findings formalize a link between the synaptic protein AIDA-1 and a rare, previously undefined genetic disease we term ANKS1B haploinsufficiency syndrome.
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http://dx.doi.org/10.1038/s41467-019-11437-wDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6684583PMC
August 2019

Stable Isotope Labeling by Amino Acids in Cell Culture (SILAC) for Quantitative Proteomics.

Adv Exp Med Biol 2019 ;1140:531-539

Kimmel Center for Biology and Medicine at the Skirball Institute and Department of Cell Biology, New York University School of Medicine, New York, NY, USA.

Stable isotope labeling by amino acids in cell culture (SILAC) is a powerful approach for high-throughput quantitative proteomics. SILAC allows highly accurate protein quantitation through metabolic encoding of whole cell proteomes using stable isotope labeled amino acids. Since its introduction in 2002, SILAC has become increasingly popular. In this chapter we review the methodology and application of SILAC, with an emphasis on three research areas: dynamics of posttranslational modifications, protein-protein interactions, and protein turnover.
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http://dx.doi.org/10.1007/978-3-030-15950-4_31DOI Listing
September 2019

Combinatory microRNA serum signatures as classifiers of Parkinson's disease.

Parkinsonism Relat Disord 2019 07 11;64:202-210. Epub 2019 Apr 11.

Department of Biological Sciences, St. John's University, New York, NY, USA
. Electronic address:

Introduction: As current clinical diagnostic protocols for Parkinson's disease (PD) may be prone to inaccuracies there is a need to identify and validate molecular biomarkers, such as circulating microRNAs, which will complement current practices and increase diagnostic accuracy. This study identifies, verifies and validates combinatory serum microRNA signatures as diagnostic classifiers of PD across different patient cohorts.

Methods: 370 PD (drug naïve) and control serum samples from the Norwegian ParkWest study were used for identification and verification of differential microRNA levels in PD which were validated in a blind study using 64 NY Parkinsonism in UMeå (NYPUM) study serum samples and tested for specificity in 48 Dementia Study of Western Norway (DemWest) study Alzheimer's disease (AD) serum samples using miRNA-microarrays, and quantitative (q) RT-PCR. Proteomic approaches identified potential molecular targets for these microRNAs.

Results: Using Affymetrix GeneChip miRNA 4.0 arrays and qRT-PCR we comprehensively analyzed serum microRNA levels and found that the microRNA (PARKmiR)-combinations, hsa-miR-335-5p/hsa-miR-3613-3p (95% CI, 0.87-0.94), hsa-miR-335-5p/hsa-miR-6865-3p (95% CI, 0.87-0.93), and miR-335-5p/miR-3613-3p/miR-6865-3p (95% CI, 0.87-0.94) show a high degree of discriminatory accuracy (AUC 0.9-1.0). The PARKmiR signatures were validated in an independent PD cohort (AUC ≤ 0.71) and analysis in AD serum samples showed PARKmiR signature specificity to PD. Proteomic analyses showed that the PARKmiRs regulate key PD-associated proteins, including alpha-synuclein and Leucine Rich Repeat Kinase 2.

Conclusions: Our study has identified and validated unique miRNA serum signatures that represent PD classifiers, which may complement and increase the accuracy of current diagnostic protocols.
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http://dx.doi.org/10.1016/j.parkreldis.2019.04.010DOI Listing
July 2019

Altered steady state and activity-dependent de novo protein expression in fragile X syndrome.

Nat Commun 2019 04 12;10(1):1710. Epub 2019 Apr 12.

Center for Neural Science, New York University, New York, NY, 10003, USA.

Whether fragile X mental retardation protein (FMRP) target mRNAs and neuronal activity contributing to elevated basal neuronal protein synthesis in fragile X syndrome (FXS) is unclear. Our proteomic experiments reveal that the de novo translational profile in FXS model mice is altered at steady state and in response to metabotropic glutamate receptor (mGluR) stimulation, but the proteins expressed differ under these conditions. Several altered proteins, including Hexokinase 1 and Ras, also are expressed in the blood of FXS model mice and pharmacological treatments previously reported to ameliorate phenotypes modify their abundance in blood. In addition, plasma levels of Hexokinase 1 and Ras differ between FXS patients and healthy volunteers. Our data suggest that brain-based de novo proteomics in FXS model mice can be used to find altered expression of proteins in blood that could serve as disease-state biomarkers in individuals with FXS.
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http://dx.doi.org/10.1038/s41467-019-09553-8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6461708PMC
April 2019

Extramitochondrial cardiolipin suggests a novel function of mitochondria in spermatogenesis.

J Cell Biol 2019 05 26;218(5):1491-1502. Epub 2019 Mar 26.

Department of Anesthesiology, New York University School of Medicine, New York, NY

Mitochondria contain cardiolipin (CL), an organelle-specific phospholipid that carries four fatty acids with a strong preference for unsaturated chains. Unsaturation is essential for the stability and for the function of mitochondrial CL. Surprisingly, we found tetrapalmitoyl-CL (TPCL), a fully saturated species, in the testes of humans and mice. TPCL was absent from other mouse tissues but was the most abundant CL species in testicular germ cells. Most intriguingly, TPCL was not localized in mitochondria but was in other cellular membranes even though mitochondrial CL was the substrate from which TPCL was synthesized. During spermiogenesis, TPCL became associated with the acrosome, a sperm-specific organelle, along with a subset of authentic mitochondrial proteins, including Ant4, Suox, and Spata18. Our data suggest that mitochondria-derived membranes are assembled into the acrosome, challenging the concept that this organelle is strictly derived from the Golgi apparatus and revealing a novel function of mitochondria.
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http://dx.doi.org/10.1083/jcb.201808131DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6504895PMC
May 2019

Tau antibody chimerization alters its charge and binding, thereby reducing its cellular uptake and efficacy.

EBioMedicine 2019 Apr 22;42:157-173. Epub 2019 Mar 22.

New York University School of Medicine, Department of Neuroscience and Physiology, and The Neuroscience Institute, 435 E 30th St. SB1123, New York, NY 10016, United States of America; New York University School of Medicine, Department of Psychiatry, 435 E 30th St. Science Building SB1115, New York, NY 10016, United States of America. Electronic address:

Background: Bringing antibodies from pre-clinical studies to human trials requires humanization, but this process may alter properties that are crucial for efficacy. Since pathological tau protein is primarily intraneuronal in Alzheimer's disease, the most efficacious antibodies should work both intra- and extracellularly. Thus, changes which impact uptake or antibody binding will affect antibody efficacy.

Methods: Initially, we examined four tau mouse monoclonal antibodies with naturally differing charges. We quantified their neuronal uptake, and efficacy in preventing toxicity and pathological seeding induced by human-derived pathological tau. Later, we generated a human chimeric 4E6 (h4E6), an antibody with well documented efficacy in multiple tauopathy models. We compared the uptake and efficacy of unmodified and chimeric antibodies in neuronal and differentiated neuroblastoma cultures. Further, we analyzed tau binding using ELISA assays.

Findings: Neuronal uptake of tau antibodies and their efficacy strongly depends on antibody charge. Additionally, their ability to prevent tau toxicity and seeding of tau pathology does not necessarily go together. Particularly, chimerization of 4E6 increased its charge from 6.5 to 9.6, which blocked its uptake into human and mouse cells. Furthermore, h4E6 had altered binding characteristics despite intact binding sites, compared to the mouse antibody. Importantly, these changes in uptake and binding substantially decreased its efficacy in preventing tau toxicity, although under certain conditions it did prevent pathological seeding of tau.

Conclusions: These results indicate that efficacy of chimeric/humanized tau antibodies should be thoroughly characterized prior to clinical trials, which may require further engineering to maintain or improve their therapeutic potential. FUND: National Institutes of Health (NS077239, AG032611, R24OD18340, R24OD018339 and RR027990, Alzheimer's Association (2016-NIRG-397228) and Blas Frangione Foundation.
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http://dx.doi.org/10.1016/j.ebiom.2019.03.033DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6492224PMC
April 2019

MACF1 links Rapsyn to microtubule- and actin-binding proteins to maintain neuromuscular synapses.

J Cell Biol 2019 05 6;218(5):1686-1705. Epub 2019 Mar 6.

Helen L. and Martin S. Kimmel Center for Biology and Medicine at the Skirball Institute of Biomolecular Medicine, New York University Medical School, New York, NY

Complex mechanisms are required to form neuromuscular synapses, direct their subsequent maturation, and maintain the synapse throughout life. Transcriptional and post-translational pathways play important roles in synaptic differentiation and direct the accumulation of the neurotransmitter receptors, acetylcholine receptors (AChRs), to the postsynaptic membrane, ensuring for reliable synaptic transmission. Rapsyn, an intracellular peripheral membrane protein that binds AChRs, is essential for synaptic differentiation, but how Rapsyn acts is poorly understood. We screened for proteins that coisolate with AChRs in a Rapsyn-dependent manner and show that microtubule actin cross linking factor 1 (MACF1), a scaffolding protein with binding sites for microtubules (MT) and actin, is concentrated at neuromuscular synapses, where it binds Rapsyn and serves as a synaptic organizer for MT-associated proteins, EB1 and MAP1b, and the actin-associated protein, Vinculin. MACF1 plays an important role in maintaining synaptic differentiation and efficient synaptic transmission in mice, and variants in are associated with congenital myasthenia in humans.
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http://dx.doi.org/10.1083/jcb.201810023DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6504910PMC
May 2019

HSP90-incorporating chaperome networks as biosensor for disease-related pathways in patient-specific midbrain dopamine neurons.

Nat Commun 2018 10 19;9(1):4345. Epub 2018 Oct 19.

The Center for Stem Cell Biology, Memorial Sloan Kettering Cancer Center, 1275 York Avenue, Box 256, New York, NY, 10065, USA.

Environmental and genetic risk factors contribute to Parkinson's Disease (PD) pathogenesis and the associated midbrain dopamine (mDA) neuron loss. Here, we identify early PD pathogenic events by developing methodology that utilizes recent innovations in human pluripotent stem cells (hPSC) and chemical sensors of HSP90-incorporating chaperome networks. We show that events triggered by PD-related genetic or toxic stimuli alter the neuronal proteome, thereby altering the stress-specific chaperome networks, which produce changes detected by chemical sensors. Through this method we identify STAT3 and NF-κB signaling activation as examples of genetic stress, and phospho-tyrosine hydroxylase (TH) activation as an example of toxic stress-induced pathways in PD neurons. Importantly, pharmacological inhibition of the stress chaperome network reversed abnormal phospho-STAT3 signaling and phospho-TH-related dopamine levels and rescued PD neuron viability. The use of chemical sensors of chaperome networks on hPSC-derived lineages may present a general strategy to identify molecular events associated with neurodegenerative diseases.
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http://dx.doi.org/10.1038/s41467-018-06486-6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6195591PMC
October 2018

Quantitative Comparison of Proteomes Using SILAC.

Curr Protoc Protein Sci 2019 02 20;95(1):e74. Epub 2018 Sep 20.

Kimmel Center for Biology and Medicine at the Skirball Institute and Department of Cell Biology, New York University School of Medicine, New York, New York.

Stable isotope labeling by amino acids in cell culture (SILAC) has become very popular as a quantitative proteomic method since it was firstly introduced by Matthias Mann's group in 2002. It is a metabolic labeling strategy in which isotope-labeled amino acids are metabolically incorporated in vivo into proteins during translation. After natural (light) or heavy amino acid incorporation, differentially labeled samples are mixed immediately after cell lysis and before any further processing, which minimizes quantitative errors caused by handling different samples in parallel. In this unit, we describe protocols for basic duplex SILAC, triplex SILAC for use in nondividing cells such as neurons, and for measuring amounts of newly synthesized proteins. © 2018 by John Wiley & Sons, Inc.
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http://dx.doi.org/10.1002/cpps.74DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6342620PMC
February 2019

Dppa2/4 Facilitate Epigenetic Remodeling during Reprogramming to Pluripotency.

Cell Stem Cell 2018 09 23;23(3):396-411.e8. Epub 2018 Aug 23.

Department of Genetics, Yale University, New Haven, CT, USA; Yale Stem Cell Center, Yale University, New Haven, CT, USA. Electronic address:

As somatic cells are converted into induced pluripotent stem cells (iPSCs), their chromatin is remodeled to a pluripotent configuration with unique euchromatin-to-heterochromatin ratios, DNA methylation patterns, and enhancer and promoter status. The molecular machinery underlying this process is largely unknown. Here, we show that embryonic stem cell (ESC)-specific factors Dppa2 and Dppa4 play a key role in resetting the epigenome to a pluripotent state. They are induced in reprogramming intermediates, function as a heterodimer, and are required for efficient reprogramming of mouse and human cells. When co-expressed with Oct4, Klf4, Sox2, and Myc (OKSM) factors, Dppa2/4 yield reprogramming efficiencies that exceed 80% and accelerate reprogramming kinetics, generating iPSCs in 2 to 4 days. When bound to chromatin, Dppa2/4 initiate global chromatin decompaction via the DNA damage response pathway and contribute to downregulation of somatic genes and activation of ESC enhancers, all of which enables an efficient transition to pluripotency. Our work provides critical insights into how the epigenome is remodeled during acquisition of pluripotency.
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http://dx.doi.org/10.1016/j.stem.2018.08.001DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6128737PMC
September 2018

The vimentin intermediate filament network restrains regulatory T cell suppression of graft-versus-host disease.

J Clin Invest 2018 10 14;128(10):4604-4621. Epub 2018 Aug 14.

Division of Blood and Marrow Transplantation, Department of Pediatrics, Masonic Cancer Center, University of Minnesota, Minneapolis, Minnesota, USA.

Regulatory T cells (Tregs) are critical for maintaining immune homeostasis. However, current Treg immunotherapies do not optimally treat inflammatory diseases in patients. Understanding the cellular processes that control Treg function may allow for the augmentation of therapeutic efficacy. In contrast to activated conventional T cells, in which protein kinase C-θ (PKC-θ) localizes to the contact point between T cells and antigen-presenting cells, in human and mouse Tregs, PKC-θ localizes to the opposite end of the cell in the distal pole complex (DPC). Here, using a phosphoproteomic screen, we identified the intermediate filament vimentin as a PKC-θ phospho target and show that vimentin forms a DPC superstructure on which PKC-θ accumulates. Treatment of mouse Tregs with either a clinically relevant PKC-θ inhibitor or vimentin siRNA disrupted vimentin and enhanced Treg metabolic and suppressive activity. Moreover, vimentin-disrupted mouse Tregs were significantly better than controls at suppressing alloreactive T cell priming in graft-versus-host disease (GVHD) and GVHD lethality, using a complete MHC-mismatch mouse model of acute GVHD (C57BL/6 donor into BALB/c host). Interestingly, vimentin disruption augmented the suppressor function of PKC-θ-deficient mouse Tregs. This suggests that enhanced Treg activity after PKC-θ inhibition is secondary to effects on vimentin, not just PKC-θ kinase activity inhibition. Our data demonstrate that vimentin is a key metabolic and functional controller of Treg activity and provide proof of principle that disruption of vimentin is a feasible, translationally relevant method to enhance Treg potency.
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http://dx.doi.org/10.1172/JCI95713DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6159973PMC
October 2018

Unveiling Brain Aβ Heterogeneity Through Targeted Proteomic Analysis.

Methods Mol Biol 2018 ;1779:23-43

Department of Pathology, New York University School of Medicine, New York, NY, USA.

Amyloid β (Aβ) is the major constituent of the brain deposits found in parenchymal plaques and cerebral blood vessels of patients with Alzheimer's disease (AD). Besides classic full-length peptides, biochemical analyses of brain deposits have revealed high degree of Aβ heterogeneity likely resulting from the action of multiple proteolytic enzymes. This chapter describes a sequential extraction protocol allowing the differential fractionation of soluble and deposited Aβ species taking advantage of their differential solubility properties. Soluble Aβ is extracted by water-based buffers like phosphate-buffered saline-PBS-whereas pre-fibrillar and fibrillar deposits, usually poorly soluble in PBS, are extractable in detergent containing solutions or more stringent conditions as formic acid. The extraction procedure is followed by the biochemical identification of the extracted Aβ species using Western blot and a targeted proteomic analysis which combines immunoprecipitation with MALDI-ToF mass spectrometry. This approach revealed the presence of numerous C- and N-terminal truncated Aβ species in addition to Aβ1-40/42. Notably, the more soluble C-terminal cleaved fragments constitute a main part of PBS homogenates. On the contrary, N-terminal truncated species typically require more stringent conditions for the extraction in agreement with their lower solubility and enhanced aggregability. Detailed assessment of the molecular diversity of Aβ species composing interstitial fluid and amyloid deposits at different disease stages, as well as the evaluation of the truncation profile during various pharmacologic approaches will provide a comprehensive understanding of the still undefined contribution of Aβ truncations to AD pathogenesis and their potential as novel therapeutic targets.
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http://dx.doi.org/10.1007/978-1-4939-7816-8_3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6746306PMC
July 2019

Communicating the nutritional value of sugar in .

Proc Natl Acad Sci U S A 2018 03 5;115(12):E2829-E2838. Epub 2018 Mar 5.

Skirball Institute of Biomolecular Medicine, New York University School of Medicine, New York, NY 10016;

Sweet-insensitive mutants are unable to readily identify sugar. In presence of wild-type (WT) flies, however, these mutant flies demonstrated a marked increase in their preference for nutritive sugar. Real-time recordings of starved WT flies revealed that these flies discharge a drop from their gut end after consuming nutritive sugars, but not nonnutritive sugars. We proposed that the drop may contain a molecule(s) named calorie-induced secreted factor (CIF), which serves as a signal to inform other flies about its nutritional value. Consistent with this, we observed a robust preference of flies for nutritive sugar containing CIF over nutritive sugar without CIF. Feeding appears to be a prerequisite for the release of CIF, given that fed flies did not produce it. Additionally, correlation analyses and pharmacological approaches suggest that the nutritional value, rather than the taste, of the consumed sugar correlates strongly with the amount (or intensity) of the released CIF. We observed that the release of this attractant signal requires the consumption of macronutrients, specifically nutritive sugars and l-enantiomer essential amino acids (l-eAAs), but it is negligibly released when flies are fed nonnutritive sugars, unnatural d-enantiomer essential amino acids (d-eAAs), fatty acids, alcohol, or salts. Finally, CIF () is not detected by the olfactory system, () is not influenced by the sex of the fly, and () is not limited to one species of .
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http://dx.doi.org/10.1073/pnas.1719827115DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5866586PMC
March 2018

Sample Preparation for Relative Quantitation of Proteins Using Tandem Mass Tags (TMT) and Mass Spectrometry (MS).

Methods Mol Biol 2018 ;1741:135-149

Department of Cell Biology, Kimmel Center for Biology and Medicine at the Skirball Institute, New York University School of Medicine, New York, NY 10016, USA.

Quantitative proteome analysis allows comparisons of protein or phosphoprotein levels across multiple cell types or conditions. A number of experimental approaches have been described toward quantitative proteomics. In this chapter, we focus on Tandem Mass Tag (TMT) isobaric labeling of peptides for global, relative quantitation of proteins and phosphopeptides. To date, there has been no published protocol describing chemical labeling of small amounts of peptides specifically extracted from small tumor samples, for which rigorous sample preparation is necessary to ensure reproducible TMT labeling.
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http://dx.doi.org/10.1007/978-1-4939-7659-1_11DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6472261PMC
December 2018

Metabolomic Analysis of Glioma Cells Using Nanoflow Liquid Chromatography-Tandem Mass Spectrometry.

Methods Mol Biol 2018 ;1741:125-134

Department of Cell Biology, Kimmel Center for Biology and Medicine at the Skirball Institute, New York University School of Medicine, New York, NY, USA.

Mass spectrometry (MS)-based techniques have been finding utility as sensitive, high throughput metabolite analysis tools for complex biological samples. We describe here a nanoflow liquid chromatography-tandem mass spectrometry (nano-LC-MS/MS) system we developed and applied to metabolic profiling of human cells. Metabolites are extracted from cells using methanol, and filtered through a C18 StageTip to remove large particles. Metabolite samples are separated by HPLC at a flow rate of 400-500 nl/min, then analyzed in both positive and negative ion modes in an LTQ-Orbitrap MS. Metabolite identification and differential analysis are performed using commercial or open source software. Protocols outlined in this chapter describe how nano-LC-MS can be applied to investigate metabolic profiling with limited biomass amount.
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http://dx.doi.org/10.1007/978-1-4939-7659-1_10DOI Listing
December 2018

A Non-canonical BCOR-PRC1.1 Complex Represses Differentiation Programs in Human ESCs.

Cell Stem Cell 2018 02 11;22(2):235-251.e9. Epub 2018 Jan 11.

Department of Genetics, Yale University, New Haven, CT 06520, USA; Yale Stem Cell Center, Yale University, New Haven, CT 06520, USA. Electronic address:

Polycomb group proteins regulate self-renewal and differentiation in many stem cell systems. When assembled into two canonical complexes, PRC1 and PRC2, they sequentially deposit H3K27me3 and H2AK119ub histone marks and establish repressive chromatin, referred to as Polycomb domains. Non-canonical PRC1 complexes retain RING1/RNF2 E3-ubiquitin ligases but have unique sets of accessory subunits. How these non-canonical complexes recognize and regulate their gene targets remains poorly understood. Here, we show that the BCL6 co-repressor (BCOR), a member of the PRC1.1 complex, is critical for maintaining primed pluripotency in human embryonic stem cells (ESCs). BCOR depletion leads to the erosion of Polycomb domains at key developmental loci and the initiation of differentiation along endoderm and mesoderm lineages. The C terminus of BCOR regulates the assembly and targeting of the PRC1.1 complex, while the N terminus contributes to BCOR-PRC1.1 repressor function. Our findings advance understanding of Polycomb targeting and repression in ESCs and could apply broadly across developmental systems.
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http://dx.doi.org/10.1016/j.stem.2017.12.002DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5797497PMC
February 2018

Sex-Specific Differences in Oxytocin Receptor Expression and Function for Parental Behavior.

Gend Genome 2017 Dec;1(4):142-166

Skirball Institute for Biomolecular Medicine, New York University School of Medicine, New York, New York.

Parental care is among the most profound behavior expressed by humans and other animals. Despite intense interest in understanding the biological basis of parental behaviors, it remains unknown how much of parenting is encoded by the genome and which abilities instead are learned or can be refined by experience. One critical factor at the intersection between innate behaviors and experience-dependent learning is oxytocin, a neurohormone important for maternal physiology and neuroplasticity. Oxytocin acts throughout the body and brain to promote prosocial and maternal behaviors and modulates synaptic transmission to affect neural circuit dynamics. Recently we developed specific antibodies to mouse oxytocin receptors, found that oxytocin receptors are left lateralized in female auditory cortex, and examined how oxytocin enables maternal behavior by sensitizing the cortex to infant distress sounds. In this study we compare oxytocin receptor expression and function in male and female mice. Receptor expression is higher in adult female left auditory cortex than in right auditory cortex or males. Developmental profiles and mRNA expression were comparable between males and females. Behaviorally, male and female mice began expressing parental behavior similarly after cohousing with experienced females; however, oxytocin enhanced parental behavior onset in females but not males. This suggests that left lateralization of oxytocin receptor expression in females provides a mechanism for accelerating maternal behavior onset, although male mice can also effectively co-parent after experience with infants. The sex-specific pattern of oxytocin receptor expression might genetically predispose female cortex to respond to infant cues, which both males and females can also rapidly learn.
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http://dx.doi.org/10.1089/gg.2017.0017DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7500123PMC
December 2017

Low-Grade Astrocytoma Mutations in IDH1, P53, and ATRX Cooperate to Block Differentiation of Human Neural Stem Cells via Repression of SOX2.

Cell Rep 2017 Oct;21(5):1267-1280

Department of Neurosurgery, NYU School of Medicine, New York, NY 10016, USA; Kimmel Center for Stem Cell Biology, NYU School of Medicine, New York, NY 10016, USA; Laura and Isaac Perlmutter Cancer Center, NYU School of Medicine, New York, NY 10016, USA; Brain Tumor Center, NYU School of Medicine, New York, NY 10016, USA; Neuroscience Institute, NYU School of Medicine, New York, NY 10016, USA. Electronic address:

Low-grade astrocytomas (LGAs) carry neomorphic mutations in isocitrate dehydrogenase (IDH) concurrently with P53 and ATRX loss. To model LGA formation, we introduced R132H IDH1, P53 shRNA, and ATRX shRNA into human neural stem cells (NSCs). These oncogenic hits blocked NSC differentiation, increased invasiveness in vivo, and led to a DNA methylation and transcriptional profile resembling IDH1 mutant human LGAs. The differentiation block was caused by transcriptional silencing of the transcription factor SOX2 secondary to disassociation of its promoter from a putative enhancer. This occurred because of reduced binding of the chromatin organizer CTCF to its DNA motifs and disrupted chromatin looping. Our human model of IDH mutant LGA formation implicates impaired NSC differentiation because of repression of SOX2 as an early driver of gliomagenesis.
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http://dx.doi.org/10.1016/j.celrep.2017.10.009DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5687844PMC
October 2017