Molecular signatures underlying neurofibrillary tangle susceptibility in Alzheimer's disease

Neuron

2022 Jul 21

Otero-Garcia, M;Mahajani, SU;Wakhloo, D;Tang, W;Xue, YQ;Morabito, S;Pan, J;Oberhauser, J;Madira, AE;Shakouri, T;Deng, Y;Allison, T;He, Z;Lowry, WE;Kawaguchi, R;Swarup, V;Cobos, I;
PMID: 35882228 | DOI: 10.1016/j.neuron.2022.06.021

Tau aggregation in neurofibrillary tangles (NFTs) is closely associated with neurodegeneration and cognitive decline in Alzheimer's disease (AD). However, the molecular signatures that distinguish between aggregation-prone and aggregation-resistant cell states are unknown. We developed methods for the high-throughput isolation and transcriptome profiling of single somas with NFTs from the human AD brain, quantified the susceptibility of 20 neocortical subtypes for NFT formation and death, and identified both shared and cell-type-specific signatures. NFT-bearing neurons shared a marked upregulation of synaptic transmission-related genes, including a core set of 63 genes enriched for synaptic vesicle cycling. Oxidative phosphorylation and mitochondrial dysfunction were highly cell-type dependent. Apoptosis was only modestly enriched, and the susceptibilities of NFT-bearing and NFT-free neurons for death were highly similar. Our analysis suggests that NFTs represent cell-type-specific responses to stress and synaptic dysfunction. We provide a resource for biomarker discovery and the investigation of tau-dependent and tau-independent mechanisms of neurodegeneration.

NIH SenNet Consortium: Mapping Senescent Cells in the Human Body to Understand Health and Disease

Preprint

2022 Jul 12

Lee, P;Blood, P;Börner, K;Campisi, J;Chen, F;Daldrup-Link, H;De Jager, P;Ding, L;Duncan, F;Eickelberg, O;Fan, R;Finkel, T;Garovic, V;Gehlenborg, N;Glass, C;Bar-Joseph, Z;Katiyar, P;Kim, S;Königshoff, M;Kuchel, G;Lee, H;Lee, J;Ma, J;Ma, Q;Melov, S;Metis, K;Mora, A;Musi, N;Neretti, N;Passos, J;Rahman, I;Rivera-Mulia, J;Robson, P;Rojas, M;Roy, A;Schilling, B;Shi, P;Silverstein, J;Suryadevera, V;Xie, J;Wang, J;Wong, A;Niedernhofer, L;
| DOI: 10.20944/preprints202207.0160.v1

Cells respond to a myriad of stressors by senescing, acquiring stable growth arrest, morphologic and metabolic changes, and a senescence-associated-secretory-phenotype (SASP). The heterogeneity of senescent cells (SnCs) and their SASP is vast, yet poorly characterized. SnCs have diverse roles in health and disease and are therapeutically targetable, making characterization of SnCs and harmonization of their nomenclature a priority. The Cellular Senescence Network (SenNet), a NIH Common Fund initiative, will leverage emerging single cell and spatial-omics to identify and map SnCs in numerous organs across the lifespan of humans and mice. A common coordinate framework will integrate the data, using validated, standardized methods, creating public 4-dimensional SnC atlases. Key SenNet deliverables include development of innovative tools/technologies to detect SnCs, biomarker discovery, common annotations to describe SnCs and extensive public data sets. The goal is to comprehensively understand and map SnCs for diagnostic and therapeutic purposes to improve human health.

Recombinant Human Collagen Hydrogel Rapidly Reduces Methylglyoxal Adducts within Cardiomyocytes and Improves Borderzone Contractility after Myocardial Infarction in Mice

Advanced Functional Materials

2022 May 24

McLaughlin, S;Sedlakova, V;Zhang, Q;McNeill, B;Smyth, D;Seymour, R;Davis, D;Ruel, M;Brand, M;Alarcon, E;Suuronen, E;
| DOI: 10.1002/adfm.202204076

Methylglyoxal (MG) production after myocardial infarction (MI) leads to advanced glycation end-product formation, adverse remodeling, and loss of cardiac function. The extracellular matrix (ECM) is a main target for MG glycation. This suggests that ECM-mimicking biomaterial therapies may protect the post-MI environment by removing MG. In this study, mechanisms by which a recombinant human collagen type I hydrogel therapy confers cardioprotection are investigated. One-week post-MI, mice receive intramyocardial injection of hydrogel or PBS. The hydrogel improves border zone contractility after 2 days, which is maintained for 28 days. RNA sequencing shows that hydrogel treatment decreases the expression of erythroid differentiation regulator 1, a factor associated with apoptosis. Hydrogel treatment reduces cardiomyocyte apoptosis and oxidative stress at 2 days with greater myocardial salvage seen at 28 days. The hydrogel located at the epicardial surface is modified by MG, and less MG-modified proteins are observed in the underlying myocardium of hydrogel-treated mice. Biomaterials that can be a target for MG glycation may act as a sponge to remove MG from the myocardium post-MI. This leads to less oxidative stress, greater survival and contractility of cardiomyocytes, which altogether suggests a novel mechanism by which biomaterials improve function of the infarcted heart.

Nucleic Acid Pharmaceutical Agents

Haschek and Rousseaux's Handbook of Toxicologic Pathology, Volume 2 : Safety Assessment Environmental Toxicologic Pathology

2023 Feb 27

Kohnken, R;Harbison, C;Klein, S;Engelhardt, J;
| DOI: 10.1016/B978-0-12-821047-5.00017-8

Nucleic acid pharmaceutical (NAP) agents are a relatively recent class of therapeutics that are uniquely capable of inhibiting protein translation through direct interaction with RNA. These classes of pharmaceuticals have demonstrated clinical benefit for diseases previously considered untreatable by small molecules and biologics by their theoretical ability to target any cellular RNA associated with disease. This chapter provides an overview of the major types of NAPs and the types of indications for which they are being developed, with examples of therapeutics on the market and in clinical development. The reader is also provided with a review of the most common delivery systems and chemical modifications that enable the biodistribution and efficacy of these drugs. A brief discussion of the nonclinical safety package is provided, and finally the most common toxicities that have been observed in preclinical species are discussed.

Reagents for Mass Cytometry

Chemical reviews

2023 Jan 25

Arnett, LP;Rana, R;Chung, WW;Li, X;Abtahi, M;Majonis, D;Bassan, J;Nitz, M;Winnik, MA;
PMID: 36696538 | DOI: 10.1021/acs.chemrev.2c00350

Mass cytometry (cytometry by time-of-flight detection [CyTOF]) is a bioanalytical technique that enables the identification and quantification of diverse features of cellular systems with single-cell resolution. In suspension mass cytometry, cells are stained with stable heavy-atom isotope-tagged reagents, and then the cells are nebulized into an inductively coupled plasma time-of-flight mass spectrometry (ICP-TOF-MS) instrument. In imaging mass cytometry, a pulsed laser is used to ablate ca. 1 μm2 spots of a tissue section. The plume is then transferred to the CyTOF, generating an image of biomarker expression. Similar measurements are possible with multiplexed ion bean imaging (MIBI). The unit mass resolution of the ICP-TOF-MS detector allows for multiparametric analysis of (in principle) up to 130 different parameters. Currently available reagents, however, allow simultaneous measurement of up to 50 biomarkers. As new reagents are developed, the scope of information that can be obtained by mass cytometry continues to increase, particularly due to the development of new small molecule reagents which enable monitoring of active biochemistry at the cellular level. This review summarizes the history and current state of mass cytometry reagent development and elaborates on areas where there is a need for new reagents. Additionally, this review provides guidelines on how new reagents should be tested and how the data should be presented to make them most meaningful to the mass cytometry user community.

Targeting Peripheral μ-opioid Receptors or μ-opioid Receptor-Expressing Neurons Does not Prevent Morphine-induced Mechanical Allodynia and Anti-allodynic Tolerance

Neuroscience bulletin

2023 Jan 09

Du, F;Yin, G;Han, L;Liu, X;Dong, D;Duan, K;Huo, J;Sun, Y;Cheng, L;
PMID: 36622575 | DOI: 10.1007/s12264-022-01009-2

The chronic use of morphine and other opioids is associated with opioid-induced hypersensitivity (OIH) and analgesic tolerance. Among the different forms of OIH and tolerance, the opioid receptors and cell types mediating opioid-induced mechanical allodynia and anti-allodynic tolerance remain unresolved. Here we demonstrated that the loss of peripheral μ-opioid receptors (MORs) or MOR-expressing neurons attenuated thermal tolerance, but did not affect the expression and maintenance of morphine-induced mechanical allodynia and anti-allodynic tolerance. To confirm this result, we made dorsal root ganglia-dorsal roots-sagittal spinal cord slice preparations and recorded low-threshold Aβ-fiber stimulation-evoked inputs and outputs in superficial dorsal horn neurons. Consistent with the behavioral results, peripheral MOR loss did not prevent the opening of Aβ mechanical allodynia pathways in the spinal dorsal horn. Therefore, the peripheral MOR signaling pathway may not be an optimal target for preventing mechanical OIH and analgesic tolerance. Future studies should focus more on central mechanisms.

Leveraging circuits to understand addiction

Neurocircuitry of Addiction

2023 Jan 19

Salling, M;
| DOI: 10.1016/B978-0-12-823453-2.00002-3

Advancements in neuroscientific methods often drive new waves of insight into our understanding of addiction. While addiction research questions persist, technical improvements can augment our observational sensitivity, allowing us to update and extend existing addiction models through method development, creative application, and scientific discovery. As a result of this iterative process, we have reached the point where neuroscientists can now readily identify, monitor, and control specific neural circuits during behavior, thereby opening new windows of inquiry into the neurobiology of addiction. The objective of this chapter is to familiarize the reader with standard and emerging techniques used to observe and interrogate neural circuitry that are prevalent in contemporary clinical and preclinical addiction neuroscience labs and that are presented throughout the book. This chapter will further discuss the historical context, benefits, and limitations of these techniques with a look forward into how they can be applied to questions of addiction neurocircuitry.

Polony gels enable amplifiable DNA stamping and spatial transcriptomics of chronic pain

Cell

2022 Nov 23

Fu, X;Sun, L;Dong, R;Chen, JY;Silakit, R;Condon, LF;Lin, Y;Lin, S;Palmiter, RD;Gu, L;
PMID: 36368323 | DOI: 10.1016/j.cell.2022.10.021

Methods for acquiring spatially resolved omics data from complex tissues use barcoded DNA arrays of low- to sub-micrometer features to achieve single-cell resolution. However, fabricating such arrays (randomly assembled beads, DNA nanoballs, or clusters) requires sequencing barcodes in each array, limiting cost-effectiveness and throughput. Here, we describe a vastly scalable stamping method to fabricate polony gels, arrays of ∼1-micrometer clonal DNA clusters bearing unique barcodes. By enabling repeatable enzymatic replication of barcode-patterned gels, this method, compared with the sequencing-dependent array fabrication, reduced cost by at least 35-fold and time to approximately 7 h. The gel stamping was implemented with a simple robotic arm and off-the-shelf reagents. We leveraged the resolution and RNA capture efficiency of polony gels to develop Pixel-seq, a single-cell spatial transcriptomic assay, and applied it to map the mouse parabrachial nucleus and analyze changes in neuropathic pain-regulated transcriptomes and cell-cell communication after nerve ligation.

Inadequate Analgesia in African Americans with Cancer Pain

The Journal of Pain

2022 May 01

Singh, N;Vallerand, A;
| DOI: 10.1016/j.jpain.2022.03.141

African Americans have been found to receive less medication and experience more pain than Caucasians. Unfortunately, many studies simply highlight the disparities that exist between African Americans and Caucasian in pain management, but there is a lack of understanding at the etiology of these disparities. The purpose of this study was to determine the adequacy of analgesia that was prescribed for African Americans with cancer pain and elucidate any potential characteristics that contributed to receiving appropriate analgesia. This was a secondary analysis of baseline data of an intervention study of African Americans with cancer pain. In order to determine the adequacy of analgesics received, the Pain Management Index (PMI) was calculated for 302 African Americans with cancer pain. Structure equation modeling was utilized to determine which patient factors (age, gender, presence of caregiver, employment status, educational level, perceived control over pain, pain-related distress, pain intensity, functional status) led to adequate analgesia. Forty percent of African Americans with cancer pain did not receive adequate analgesia based on the drug class alone. African Americans with cancer pain who reported higher levels of pain received higher levels of analgesics. Cancer metastasis (p = .03) was the only significant predictor of an increased likelihood of receiving adequate analgesia. Despite national attention to racial disparities, African Americans with cancer pain continue to receive inadequate analgesia. Forty percent of African Americans did not get the appropriate drug based on their reported pain score. The only significant predictor of receiving appropriate analgesia was cancer metastasis. Ensuing studies testing whether these results would be the same for other races will clarify if this disparity was due to race alone. Interventions should then be created and utilized to decrease these disparities.

Fluorescence Microscopy in Adeno-Associated Virus Research

Viruses

2023 May 16

Golm, SK;Hübner, W;Müller, KM;
PMID: 37243260 | DOI: 10.3390/v15051174

Research on adeno-associated virus (AAV) and its recombinant vectors as well as on fluorescence microscopy imaging is rapidly progressing driven by clinical applications and new technologies, respectively. The topics converge, since high and super-resolution microscopes facilitate the study of spatial and temporal aspects of cellular virus biology. Labeling methods also evolve and diversify. We review these interdisciplinary developments and provide information on the technologies used and the biological knowledge gained. The emphasis lies on the visualization of AAV proteins by chemical fluorophores, protein fusions and antibodies as well as on methods for the detection of adeno-associated viral DNA. We add a short overview of fluorescent microscope techniques and their advantages and challenges in detecting AAV.

Reporter Selection and Postmortem Methods to Verify Transgene Expression

Vectorology for Optogenetics and Chemogenetics

2023 Feb 07

Heffernan, K;Smith, Y;Galvan, A;
| DOI: 10.1007/978-1-0716-2918-5_15

The accurate localization of transgene expression after viral vector delivery is essential to the interpretation of experiments based on genetic-based approaches, such as chemo- or optogenetics. Postmortem histological analysis can be used to examine the injection target, the extent of the virus transduction, the types of cells expressing the transgene, and the subcellular localization of the protein. In this chapter, we will provide a general description of methods to identify transgene expression, immunocytochemistry protocols, and examples of specific protocols. We close the chapter with an example of an application of electron microscopy to identify the localization of transgene expression.

Towards a definition of microglia heterogeneity

Communications biology

2022 Oct 20

Healy, LM;Zia, S;Plemel, JR;
PMID: 36266565 | DOI: 10.1038/s42003-022-04081-6

High dimensional single-cell analysis such as single cell and single nucleus RNA sequencing (sc/snRNAseq) are currently being widely applied to explore microglia diversity. The use of sc/snRNAseq provides a powerful and unbiased approach to deconvolve heterogeneous cellular populations. However, sc/snRNAseq and analyses pipelines are designed to find heterogeneity. Indeed, cellular heterogeneity is often the most frequently reported finding. In this Perspective, we consider the ubiquitous concept of heterogeneity focusing on its application to microglia research and its influence on the field of neuroimmunology. We suggest that a clear understanding of the semantic and biological implications of microglia heterogeneity is essential for mitigating confusion among researchers.

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	Description
sense Example: Hs-LAG3-sense	Standard probes for RNA detection are in antisense. Sense probe is reverse complent to the corresponding antisense probe.
Intron# Example: Mm-Htt-intron2	Probe targets the indicated intron in the target gene, commonly used for pre-mRNA detection
Pool/Pan Example: Hs-CD3-pool (Hs-CD3D, Hs-CD3E, Hs-CD3G)	A mixture of multiple probe sets targeting multiple genes or transcripts
No-XSp Example: Hs-PDGFB-No-XMm	Does not cross detect with the species (Sp)
XSp Example: Rn-Pde9a-XMm	designed to cross detect with the species (Sp)
O# Example: Mm-Islr-O1	Alternative design targeting different regions of the same transcript or isoforms
CDS Example: Hs-SLC31A-CDS	Probe targets the protein-coding sequence only
EnEm	Probe targets exons n and m
En-Em	Probe targets region from exon n to exon m
Retired Nomenclature
tvn Example: Hs-LEPR-tv1	Designed to target transcript variant n
ORF Example: Hs-ACVRL1-ORF	Probe targets open reading frame
UTR Example: Hs-HTT-UTR-C3	Probe targets the untranslated region (non-protein-coding region) only
5UTR Example: Hs-GNRHR-5UTR	Probe targets the 5' untranslated region only
3UTR Example: Rn-Npy1r-3UTR	Probe targets the 3' untranslated region only
Pan Example: Pool	A mixture of multiple probe sets targeting multiple genes or transcripts

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