Single-cell peripheral immunoprofiling of lewy body and Parkinson's disease in a multi-site cohort.

Background: Multiple lines of evidence support peripheral organs in the initiation or progression of Lewy body disease (LBD), a spectrum of neurodegenerative diagnoses that include Parkinson's Disease (PD) without or with dementia (PDD) and dementia with Lewy bodies (DLB). However, the potential contribution of the peripheral immune response to LBD remains unclear. This study aims to characterize peripheral immune responses unique to participants with LBD at single-cell resolution to highlight potential biomarkers and increase mechanistic understanding of LBD pathogenesis in humans.

Cross-species comparative analysis of single presynapses.

Comparing brain structure across species and regions enables key functional insights. Leveraging publicly available data from a novel mass cytometry-based method, synaptometry by time of flight (SynTOF), we applied an unsupervised machine learning approach to conduct a comparative study of presynapse molecular abundance across three species and three brain regions. We used neural networks and their attractive properties to model complex relationships among high dimensional data to develop a unified, unsupervised framework for comparing the profile of more than 4.

Discovery of azaspirocyclic 1H-3,4,5-Trisubstitued pyrazoles as novel G2019S-LRRK2 selective kinase inhibitors.

Mutations in the Leucine Rich Repeat Protein Kinase 2 gene (LRRK2) are genetic predispositions for Parkinson's Disease, of which the G2019S (GS) missense mutation is the most common. GS-LRRK2 has a hyperactive kinase, and although numerous drug discovery programs have targeted the LRRK2 kinase, few have reached clinical trials. We recently reported on the discovery of a novel LRRK2 kinase inhibitor chemotype, 1H-pyrazole biaryl sulfonamides.

Discovery of 1Pyrazole Biaryl Sulfonamides as Novel G2019S-LRRK2 Kinase Inhibitors.

G2019S (GS) is the most prevalent mutation in the leucine rich repeat protein kinase 2 gene (), a genetic predisposition that is common for Parkinson's disease, as well as for some forms of cancer, and is a shared risk allele for Crohn's disease. GS-LRRK2 has a hyperactive kinase, and although numerous drug discovery programs have targeted LRRK2 kinase, few have reached clinical development. We report the discovery and preliminary development of an entirely novel structural class of potent and selective GS-LRRK2 kinase inhibitors: biaryl-1-pyrazoles.

Mass Synaptometry: Applying Mass Cytometry to Single Synapse Analysis.

Synaptic degeneration is one of the earliest and phenotypically most significant features associated with numerous neurodegenerative conditions, including Alzheimer's and Parkinson's diseases. Synaptic changes are also known to be important in neurocognitive disorders such as schizophrenia and autism spectrum disorders. Several labs, including ours, have demonstrated that conventional (fluorescence-based) flow cytometry of individual synaptosomes is a robust and reproducible method.

Discovery of G2019S-Selective Leucine Rich Repeat Protein Kinase 2 inhibitors with in vivo efficacy.

Mutations in the Leucine Rich Repeat Protein Kinase 2 gene (LRRK2) are the most common genetic causes of Parkinson's Disease (PD). The G2019S mutation is the most common inherited LRRK2 mutation, occurs in the kinase domain, and results in increased kinase activity. We report the discovery and development of compound 38, an indazole-based, G2019S-selective (>2000-fold vs.

Single-synapse analyses of Alzheimer's disease implicate pathologic tau, DJ1, CD47, and ApoE.

Synaptic molecular characterization is limited for Alzheimer’s disease (AD). Our newly invented mass cytometry–based method, synaptometry by time of flight (SynTOF), was used to measure 38 antibody probes in approximately 17 million single-synapse events from human brains without pathologic change or with pure AD or Lewy body disease (LBD), nonhuman primates (NHPs), and PS/APP mice. Synaptic molecular integrity in humans and NHP was similar.

GATM and GAMT synthesize creatine locally throughout the mammalian body and within oligodendrocytes of the brain.

The enzymes glycine amidinotransferase, mitochondrial (GATM also known as AGAT) and guanidinoacetate N-methyltransferase (GAMT) function together to synthesize creatine from arginine, glycine, and S-Adenosyl methionine. Deficiency in either enzyme or the creatine transporter, CT1, results in a devastating neurological disorder, Cerebral Creatine Deficiency Syndrome (CCDS). To better understand the pathophysiology of CCDS, we mapped the distribution of GATM and GAMT at single cell resolution, leveraging RNA sequencing analysis combined with in vivo immunofluorescence (IF).

Enantiomers of 4-aminopentanoic acid act as false GABAergic neurotransmitters and impact mouse behavior.

Imbalance in the metabolic pathway linking excitatory and inhibitory neurotransmission has been implicated in multiple psychiatric and neurologic disorders. Recently, we described enantiomer-specific effects of 2-methylglutamate, which is not decarboxylated to the corresponding methyl analogue of gamma-aminobutyric acid (GABA): 4-aminopentanoic acid (4APA). Here, we tested the hypothesis that 4APA also has enantiomer-specific actions in brain.

Enantiomers of 2-methylglutamate and 2-methylglutamine selectively impact mouse brain metabolism and behavior.

Imbalance of excitatory and inhibitory neurotransmission is implicated in a wide range of psychiatric and neurologic disorders. Here we tested the hypothesis that insertion of a methyl group on the stereogenic alpha carbon of L-Glu or L-Gln would impact the γ-aminobutyric acid (GABA) shunt and the glutamate-glutamine cycle. (S)-2-methylglutamate, or (S)-2MeGlu, was efficiently transported into brain and synaptosomes where it was released by membrane depolarization in a manner equivalent to endogenous L-Glu.

Mass-tag barcoding for multiplexed analysis of human synaptosomes and other anuclear events.

Mass-tag cell barcoding has increased the throughput, multiplexing, and robustness of multiple cytometry approaches. Previously, we adapted mass cytometry for cells to analyze synaptosome preparations (mass synaptometry or SynTOF), extending mass cytometry to these smaller, anuclear particles. To improve throughput and individual event resolution, we report here the application of palladium-based barcoding in human synaptosomes.

Creatine transport and pathological changes in creatine transporter deficient mice.

The severe impact on brain function and lack of effective therapy for patients with creatine (Cr) transporter deficiency motivated the generation of three ubiquitous Slc6a8 deficient mice (-/y). While each mouse knock-out line has similar behavioral effects at 2 to 3 months of age, other features critical to the efficient use of these mice in drug discovery are unclear or lacking: the concentration of Cr in brain and heart differ widely between mouse lines, there are limited data on histopathologic changes, and no data on Cr uptake. Here, we determined survival, measured endogenous Cr and uptake of its deuterium-labeled analogue Cr-d3 using a liquid chromatography coupled with tandem mass spectrometry assay, and performed comprehensive histopathologic examination on the Slc6a8 mouse developed by Skelton et al.

Single-cell peripheral immunoprofiling of Alzheimer's and Parkinson's diseases.

Peripheral blood mononuclear cells (PBMCs) may provide insight into the pathogenesis of Alzheimer's disease (AD) or Parkinson's disease (PD). We investigated PBMC samples from 132 well-characterized research participants using seven canonical immune stimulants, mass cytometric identification of 35 PBMC subsets, and single-cell quantification of 15 intracellular signaling markers, followed by machine learning model development to increase predictive power. From these, three main intracellular signaling pathways were identified specifically in PBMC subsets from people with AD versus controls: reduced activation of PLCγ2 across many cell types and stimulations and selectively variable activation of STAT1 and STAT5, depending on stimulant and cell type.

Mass synaptometry: High-dimensional multi parametric assay for single synapses.

Background: Synaptic alterations, especially presynaptic changes, are cardinal features of neurodegenerative diseases and strongly correlate with cognitive decline.

New Method: We report "Mass Synaptometry" for the high-dimensional analysis of individual human synaptosomes, enriched nerve terminals from brain. This method was adapted from cytometry by time-of-flight mass spectrometry (CyTOF), which is commonly used for single-cell analysis of immune and blood cells.

An Activity Switch in Human Telomerase Based on RNA Conformation and Shaped by TCAB1.

Ribonucleoprotein enzymes require dynamic conformations of their RNA constituents for regulated catalysis. Human telomerase employs a non-coding RNA (hTR) with a bipartite arrangement of domains-a template-containing core and a distal three-way junction (CR4/5) that stimulates catalysis through unknown means. Here, we show that telomerase activity unexpectedly depends upon the holoenzyme protein TCAB1, which in turn controls conformation of CR4/5.

Distributed hepatocytes expressing telomerase repopulate the liver in homeostasis and injury.

Hepatocytes are replenished gradually during homeostasis and robustly after liver injury. In adults, new hepatocytes originate from the existing hepatocyte pool, but the cellular source of renewing hepatocytes remains unclear. Telomerase is expressed in many stem cell populations, and mutations in telomerase pathway genes have been linked to liver diseases.

Loss of Lrp2 in zebrafish disrupts pronephric tubular clearance but not forebrain development.

Low-density lipoprotein receptor-related protein 2 (LRP2) is a multifunctional cell surface receptor conserved from nematodes to humans. In mammals, it acts as regulator of sonic hedgehog and bone morphogenetic protein pathways in patterning of the embryonic forebrain and as a clearance receptor in the adult kidney. Little is known about activities of this LRP in other phyla.

LRP2 in ependymal cells regulates BMP signaling in the adult neurogenic niche.

The microenvironment of growth factors in the subependymal zone (SEZ) of the adult brain provides the instructive milieu for neurogenesis to proceed in this germinal niche. In particular, tight regulation of bone morphogenetic protein (BMP) signaling is essential to balance proliferative and non-proliferative cell fate specification. However, the regulatory pathways that control BMP signaling in the SEZ are still poorly defined.