Name: lipidomics and transcriptomics in neurological diseases
Uploaded: 2022-03-18T12:30:00.000+00:00
Duration: 9 min 58 s
Description: Watch this Scientific Journal Video about Lipidomics and Transcriptomics in Neurological Diseases at JoVE.com

We dedicate this article to Dr. Ermelinda Lomazzo. During the finalization of this manuscript, Dr. Ermelinda Lomazzo passed away. She is the embodiment of passion for science and selfless engagement in team work to fulfill a meaningful research purpose. She always dreamed of contributing meaningfully to the greater well-being of humans. Her goodhearted nature was never compromised by the strenuous roads of science and life. She will remain invaluable, and forever, in our hearts.
Julia M. Post was funded by Focus Program for Translational Neuroscience (FTN) at University Medical Center of the Johannes Gutenberg University Mainz and is currently funded by the SPP-2225 EXIT project to LB. Raissa Lerner was partially funded by DZHK project 81X2600250 to LB and Lipidomics Core Facility. Partial funding for these studies was provided by the Lipidomics Core Facility, Institute of Physiological Chemistry, and Intramural funds (to LB) from the University Medical Center of the Johannes Gutenberg University Mainz.

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The authors declare no conflict of interest.

The neurolipidomic and transcriptomic methodology described here is a viable mean to investigate any disease or healthy development at high and low spatial resolution in the brain and peripheral organs. Due to the optimized plasma sampling and handling procedures, plasma lipidomic analysis can also be carried out from the same animals sacrificed for tissue lipidomics and transcriptomics, thus improving the reliability of tissue blood molecular correlates and biomarker discovery. The provision of a broad set of data by ap...

Recent advances in the function of lipids and their role in the onset and progression of neurological diseases open new research and development venues of new therapeutic targets and disease mechanism elucidation1. Documented differences in lipid composition in different brain regions, emphasized by modern molecular imaging techniques such as mass spectrometry imaging and advanced mass spectrometry profiling, shifts the paradigm of lipid investigation from whole brain toward functionally distinct and discrete brain regions. The fact that lipid composition varies in different brain regions prompts new conceptualization of both membrane lipid sensitivity and downstream lipid signaling in response to a brain insult or stimuli across the functionally distinct brain regions. Hence, lipid protocols require new developments to address the challenge of low tissue amounts for higher spatial resolution detection and quantification, and concurrently, analysis of multiple lipid components of cell membranes and signaling pathways. Also, determination of enzymes, lipid ligands, and receptors involved in the regulation of their levels and function is paramount to elucidate the signaling pathways affected in a neurological disease and guide new mechanistic investigations in a pathophysiological context.
In addition to the increased brain spatial resolution, there are two major difficulties challenging the development of new neurolipidomic approaches. First, the lipid signaling molecules are typically of very low abundance compared to membrane constitutive lipids. Second, the lipidome exhibits a high structural heterogeneity, difficult to dissect using a single analytical approach. Hence, extraction and analytical methods are tailored to different lipid categories and commonly performed in distinct tissue samples2. Shotgun lipidomic methods3 are excellent tools to rapidly reveal a broad profile of membrane lipids, while increased sensitivity and selectivity afforded by the targeted discovery and quantification mass spectrometric methods are capitalized upon for investigation of low abundant signaling lipids including: i) inflammatory lipids and ii) lipids involved in the modulation of neuronal activity, such as endocannabinoids (eCBs), amino acid-linked lipids, etc.4,5. To encompass lipid changes at both the cell membrane and&#160;signaling level occurring in brain regions of neurological disease models, typically the lipid extraction and analysis are carried out in distinct tissue samples, obtained from distinct animal batches or from different hemispheres, or by dissecting a larger tissue region into multiple pieces. When mRNA levels of enzyme receptors are also of interest, their investigation typically requires the procurement of a distinct tissue sample. For example, the investigation of membrane lipids, endogenous cannabinoids, and mRNA would require three different tissue samples, (e.g., two samples for the two lipid extraction methods-membrane lipids and signaling lipids- and subsequent two lipid analysis methods- and one sample for mRNA analysis). Investigation of inflammatory lipids and endogenous cannabinoids require two distinct tissue samples, extraction methods, and analysis methods, respectively. Another example is the investigation of mRNA and of any lipid category in a brain punch or laser microdissection sample which consequently requires two distinct animals to procure two samples per brain (sub)region. A substantial extent of variability and/or poor reproducibility of the results frequently occur in such cases, originating from biological variability and/or tissue heterogeneity. Guided by these practical limitations of multimolecular analysis, occurring particularly at high spatial resolution in the brain, a three-module neurolipidomics protocol was designed encompassing: 1) coextraction and co-analysis by LC/MRM of inflammatory lipids (e.g., eicosanoids (eiCs)) and lipids involved in modulation of neuronal activity, such as eCBs2; 2) co-extraction of phospholipids (PLs) and eCBs with subsequent multiscan LC/MRM and precursor/neutral loss scan analysis2; and 3) dual extraction of membrane (phospho)lipids and eCBs as well as mRNA, with subsequent LC/MRM and qPCR or RNA sequencing analysis6. Depending on the biological question to be addressed in a neurological disease and the brain region of interest, a combination of the first and the second protocol, or the first and the third protocol, can be applied on the same tissue specimen for tissues weighing around 4 mg. The first and third protocols can be independently applied for tissues around 2 mg. The second protocol can be applied for tissues weighing as little as 0.5 mg. Irrespective of the neurolipidomic protocol module selected, the tissue sampling and pre-analytical processing, the brain isolation and region dissection, as well as the procedure for sacrificing the animal model are standardized and identical for all three modules of the protocol. In our investigation of neurological diseases, peripheral organs that are relevant for the pathological consequences of the disease are always also collected and analyzed using these modular protocols. Additionally, blood is regularly sampled for plasma lipidomics to serve as a readout tool of neurological diseases with a view on prospective translational applications. The here presented modular lipidomics protocol is very versatile: scaleable to larger tissue amounts and readily applicable for virtually any tissue type and disease. For the application of the modular protocol (Figure 1) in neurological diseases, any standardized rodent model of onset and progression of neurological disorders, such as traumatic brain injury, Parkinson&#39;s disease, Alzheimer&#39;s disease, or epilepsy are amenable.
These protocols have been extensively applied to study changes in the tissue lipidome and/or transcriptome at the acute phase of epilepsy in the kainic acid (KA)-induced mouse model of epilepsy2,7, a model widely used in preclinical studies due to the resemblance to human temporal lobe epilepsy (TLE)8,9,10,11. Using these protocols, the therapeutic potential of drugs such as Palmitoylethanolamide (PEA)12,13 was assessed in the same mouse model of epilepsy. The study identified lipid and mRNA changes at high and low spatial resolution in the brain and periphery, at the time point of maximal acute seizure intensities (at 60 min postseizure induction), and upon subchronic and acute treatment with PEA at four different timepoints (20, 60, 120, and 180 min) post KA-seizure induction, a time-window covering the acute phase of epilepsy. Plasma, brains, and peripheral organs of untreated KA-injected mice, acute and subchronically PEA-treated mice, as well as vehicle and PEA-vehicle control mice, were collected at each time point12,13, and investigated with this molecular analysis. The molecular data were correlated with behavioral phenotypes obtained by seizure scoring, as well as with immunohistochemistry-derived data on neurodegenerative processes, in order to unravel the progression of the acute epilepsy phase and PEA&#39;s potential to alleviate it.

<table><tbody><tr><td>12(S)-HETE</td><td>Biomol</td><td>Cay10007248-25</td><td>Lipid Std</td></tr><tr><td>12(S)-HETE-d8</td><td>Biomol</td><td>Cay334570-25</td><td>Lipid Std</td></tr><tr><td>1200 series LC System</td><td>Agilent</td><td></td><td>Instrumentation/LCMS</td></tr><tr><td>2100 Bioanalyzer</td><td>Agilent</td><td></td><td>Instrumentation/qPCR</td></tr><tr><td>5(S)-HETE-d8</td><td>Biomol</td><td>Cay 334230</td><td>Lipid Std</td></tr><tr><td>ABI 7300 Real-Time PCR cycler</td><td>Applied Biosystems</td><td></td><td>Instrumentation/qPCR</td></tr><tr><td>Acetonitrile LC-MS Chroma Solv</td><td>Honeywell</td><td>9814920</td><td>Solvent/LCMS</td></tr><tr><td>amber eppendorf tubes</td><td>Eppendorf</td><td></td><td>Sample Prep.</td></tr><tr><td>Analyst 1.6.2 Software</td><td>AB SCIEX, Darmstadt</td><td></td><td>Software</td></tr><tr><td>Analytical balance</td><td>Mettler Toledo</td><td></td><td>Instrumentation/Sample prep.</td></tr><tr><td>Arachidonic Acid-d8 MS Standard</td><td>Biomol</td><td>Cay-10007277</td><td>Lipid Std</td></tr><tr><td>Bessmann Tissue Pulverizer</td><td>Spectrum Laboratories, Inc. (Breda, Netherlands)</td><td></td><td>Instrumentation/Sample prep.</td></tr><tr><td>Bino</td><td>Zeiss</td><td></td><td>Microscopy</td></tr><tr><td>cleaved Caspase 3 antibody</td><td>Cellsignaling</td><td>9661S</td><td>Microscopy</td></tr><tr><td>Cryostat, Leica CM3050 S</td><td>Leica Biosystems</td><td></td><td>Instrumentation/Sample prep.</td></tr><tr><td>CTC HTC PAL autosampler</td><td>CTC Analytics AG</td><td></td><td>Instrumentation/LCMS</td></tr><tr><td>Dumont Curved Forceps Dumoxel #7</td><td>FST</td><td>11271-30</td><td>Surgical Tools</td></tr><tr><td>Dumont Forceps Super fine tip #5SF (x2)</td><td>FST</td><td>11252-00</td><td>Surgical Tools</td></tr><tr><td>EDTA 1000 A R&amp;ouml;hrchen</td><td>Kabe Labortechnik</td><td>078001</td><td>Sample Prep.</td></tr><tr><td>EP-1 EconoPump</td><td>BioRAD</td><td>700BR07757</td><td>Instrumentation/Sample prep.</td></tr><tr><td>Fine Forceps Mirror Finish</td><td>FST</td><td>11412-11</td><td>Surgical Tools</td></tr><tr><td>Fine Iris Scissors straight sharp</td><td>FST</td><td>14094-11</td><td>Surgical Tools</td></tr><tr><td>Fine Scissor Tungsten Carbide straight</td><td>FST</td><td>14568-09</td><td>Surgical Tools</td></tr><tr><td>Iris Spatulae</td><td>FST</td><td>10094-13</td><td>Surgical Tools</td></tr><tr><td>Kainic acid</td><td>Abcam</td><td>ab120100</td><td>Epileptic drug</td></tr><tr><td>Lipid View software</td><td>AB SCIEX, Darmstadt</td><td></td><td>Software</td></tr><tr><td>LPC 17:0</td><td>Avanis Polaris</td><td>855676P</td><td>Lipid Std</td></tr><tr><td>LPC 18:0</td><td>Avanis Polaris</td><td>855775P</td><td>Lipid Std</td></tr><tr><td>Luna 2,5&amp;micro;m C18(2)- HAST 100A LC column</td><td>Phenomenex</td><td>00D-4446-B0</td><td>Instrumentation/LCMS</td></tr><tr><td>Magnifying lamp</td><td>Maul GmbH</td><td></td><td>Instrumentation/Sample prep.</td></tr><tr><td>Methanol LC-MS Chroma Solv 99.9%</td><td>Honeywell</td><td>9814920</td><td>Solvent/LCMS</td></tr><tr><td>Motic Camara</td><td>Motic</td><td></td><td>Microscopy</td></tr><tr><td>MTBE</td><td>Honeywell</td><td>34875-1L</td><td>Solvent/LCMS</td></tr><tr><td>MultiQuant 3.0 quantitation software package</td><td>AB SCIEX, Darmstadt</td><td></td><td>Software</td></tr><tr><td>NanoDrop 2000c Spectrophotometer</td><td>Thermo Scientific</td><td></td><td>Instrumentation/qPCR</td></tr><tr><td>PA 16:0-18:1</td><td>Avanis Polaris</td><td>840857P</td><td>Lipid Std</td></tr><tr><td>PA 17:0-14:1</td><td>Avanis Polaris</td><td>LM-1404</td><td>Lipid Std</td></tr><tr><td>Palmitoyl Ethanolamide</td><td>Biomol</td><td>Cay90350-100</td><td>Lipid Std</td></tr><tr><td>Palmitoyl Ethanolamide-d5</td><td>Biomol</td><td>Cay9000573-5</td><td>Lipid Std</td></tr><tr><td>PC 16:0-18:1</td><td>Avanis Polaris</td><td>850457P</td><td>Lipid Std</td></tr><tr><td>PC 16:0-18:1</td><td>Avanis Polaris</td><td>850457P</td><td>Lipid Std</td></tr><tr><td>PC 17:0-14:1</td><td>Avanis Polaris</td><td>LM-1004</td><td>Lipid Std</td></tr><tr><td>PE 16:0-18:1</td><td>Avanis Polaris</td><td>850757P</td><td>Lipid Std</td></tr><tr><td>PE 17:0-14:1</td><td>Avanis Polaris</td><td>LM-1104</td><td>Lipid Std</td></tr><tr><td>PG 16:0-18:1</td><td>Avanis Polaris</td><td>840457P</td><td>Lipid Std</td></tr><tr><td>PG 17:0-14:1</td><td>Avanis Polaris</td><td>LM-1204</td><td>Lipid Std</td></tr><tr><td>PI 17:0-14:1</td><td>Avanis Polaris</td><td>LM-1504</td><td>Lipid Std</td></tr><tr><td>Precelleys 24</td><td>Peqlab</td><td></td><td>Instrumentation/Sample prep.</td></tr><tr><td>Precellys Keramik-K&amp;uuml;gelchen</td><td>Peqlab</td><td>91-pcs-ck14p</td><td>Sample Prep.</td></tr><tr><td>Precellys Stahlkugeln 2,8mm</td><td>Peqlab</td><td>91-PCS-MK28P</td><td>Sample Prep.</td></tr><tr><td>Precellys-keramik-kit 1,4 mm</td><td>VWR</td><td>91-PCS-CK14</td><td>Sample Prep.</td></tr><tr><td>Prostaglandin D2</td><td>Biomol</td><td>Cay 12010</td><td>Lipid Std</td></tr><tr><td>Prostaglandin D2-d4</td><td>Biomol</td><td>Cay 312010</td><td>Lipid Std</td></tr><tr><td>Prostaglandin E2</td><td>Biomol</td><td>Cay10007211-1</td><td>Lipid Std</td></tr><tr><td>Prostaglandin E2-d9</td><td>Biomol</td><td>Cay10581-50</td><td>Lipid Std</td></tr><tr><td>PS 17:0-14:1</td><td>Avanis Polaris</td><td>LM-1304</td><td>Lipid Std</td></tr><tr><td>Q Trap 5500 triple-quadrupole linear ion trap MS</td><td>AB SCIEX</td><td>AU111609004</td><td>Instrumentation/LCMS</td></tr><tr><td>Real Time PCR System</td><td>Appliert Biosystem</td><td></td><td>Instrumentation/qPCR</td></tr><tr><td>Resolvin D1</td><td>Biomol</td><td>Cay10012554-11</td><td>Lipid Std</td></tr><tr><td>Rneasy Mini Kit - RNAase-Free DNase Set (50)</td><td>Qiagen</td><td>79254</td><td>Sample Prep.</td></tr><tr><td>Security Guard precolumn</td><td>Phenomenex</td><td></td><td>Instrumentation/LCMS</td></tr><tr><td>Shandon coverplates</td><td>Thermo Fisher</td><td>72110017</td><td>Microscopy</td></tr><tr><td>Shandon slide rack and lid</td><td>Thermo Fisher</td><td>73310017</td><td>Microscopy</td></tr><tr><td>SM 18:0</td><td>Avanis Polaris</td><td>860586P</td><td>Lipid Std</td></tr><tr><td>SM d18:1/12:0</td><td>Avanis Polaris</td><td>LM-2312</td><td>Lipid Std</td></tr><tr><td>Standard Forceps straight Smooth</td><td>FST</td><td>11016-17</td><td>Surgical Tools</td></tr><tr><td>Surgical Scissor ToughCut Standard Pattern</td><td>FST</td><td>14130-17</td><td>Surgical Tools</td></tr><tr><td>T3000 Thermocycler</td><td>Biometra</td><td></td><td>Instrumentation/qPCR</td></tr><tr><td>Thromboxane B2</td><td>Biomol</td><td>Cay19030-5</td><td>Lipid Std</td></tr><tr><td>Thromboxane B2-d4</td><td>Biomol</td><td>Cay319030-25</td><td>Lipid Std</td></tr><tr><td>Tissue Lyser II</td><td>Qiagen/ Retsch</td><td>12120240804</td><td>Instrumentation/Sample prep.</td></tr><tr><td>Tissue Tek</td><td>Sakura Finetek</td><td>4583</td><td>Microscopy</td></tr><tr><td>Toluidinblau</td><td>Roth</td><td>0300.2</td><td>Microscopy</td></tr><tr><td>Vapotherm</td><td>Barkey</td><td>4004734</td><td>Instrumentation/Sample prep.</td></tr><tr><td>Wasser LC-MS Chroma Solv</td><td>VWR</td><td>9814920</td><td>Solvent/LCMS</td></tr></tbody></table>

lipidomics and transcriptomics in neurological diseases

Lipids serve as the primary interface to brain insults or stimuli conducive to neurological diseases and are a reservoir for the synthesis of lipids with various signaling or ligand function that can underscore the onset and progression of diseases. Often changing at the presymptomatic level, lipids are an emerging source of drug targets and biomarkers. Many neurological diseases exhibit neuroinflammation, neurodegeneration, and neuronal excitability as common hallmarks, partly modulated by specific lipid signaling systems. The interdependence and interrelation of synthesis of various lipids prompts a multilipid, multienzyme, and multireceptor analysis in order to derive the commonalities and specificities of neurological contexts and to expedite the unravelling of mechanistic aspects of disease onset and progression. Ascribing lipid roles to distinct brain regions advances the determination of lipid molecular phenotype and morphology associated with a neurological disease.
Presented here is a modular protocol suitable for the analysis of membrane lipids and downstream lipid signals along with mRNA of enzymes and mediators underlying their functionality, extracted from discrete brain regions that are relevant for a particular neurological disease and/or condition. To ensure accurate comparative lipidomic profiling, the workflows and operating criteria were optimized and standardized for: i) brain sampling and dissection of regions of interest, ii) co-extraction of multiple lipid signals and membrane lipids, iii) dual lipid/mRNA extraction, iv) quantification by liquid chromatography multiple reaction monitoring (LC/MRM), and v) standard mRNA profiling. This workflow is amenable for the low tissue amounts obtained by sampling of the functionally discrete brain subregions (i.e. by brain punching), thus preventing bias in multimolecular analysis due to tissue heterogeneity and/or animal variability. To reveal peripheral consequences of neurological diseases and establish translational molecular readouts of neurological disease states, peripheral organ sampling, processing, and their subsequent lipidomic analysis, as well as plasma lipidomics, are also pursued and described. The protocol is demonstrated on an acute epilepsy mouse model.

The set of described protocols may be combined on different levels in an aim-specific fashion, such as choice of animal model, route of sampling, method of extraction and profiling (Figure 1).
In order to determine lipid level changes in the brain and periphery over a time course of an acute epileptic seizure state and to unravel the potential antiepileptic effect13 of PEA an...

Watch this Scientific Journal Video about Lipidomics and Transcriptomics in Neurological Diseases at JoVE.com

Lipidomics and Transcriptomics in Neurological Diseases

This article presents a modular protocol for tissue lipidomics and transcriptomics, and plasma lipidomics in neurological disease mouse models targeting lipids underlying inflammation and neuronal activity, membrane lipids, downstream messengers, and mRNA-encoding enzymes/receptors underlying lipid function. Sampling, sample processing, extraction, and quantification procedures are outlined.

Lipids serve as the primary interface to brain insults or stimuli conducive to neurological diseases and are a reservoir for the synthesis of lipids with ...

lipidomics-and-transcriptomics-in-neurological-diseases

Research

JoVE Journal

Neuroscience

3.4K Views.  University Medical Center of the Johannes Gutenberg University Mainz. This article presents a modular protocol for tissue lipidomics and transcriptomics, and plasma lipidomics in neurological disease mouse models targeting lipids underlying inflammation and neuronal activity, membrane lipids, downstream messengers, and mRNA-encoding enzymes/receptors underlying lipid function. Sampling, sample processing, extraction, and quantification procedures are outlined.

Video: Lipidomics and Transcriptomics in Neurological Diseases

Detection of MicroRNAs in Microglia by Real-time PCR in Normal CNS and During Neuroinflammation

Microglia are cells of the myeloid lineage that reside in the central nervous system (CNS)1. These cells play an important role in pathologies of many diseases associated with neuroinflammation such as multiple sclerosis (MS)2. Microglia in a normal CNS express macrophage marker CD11b and exhibit a resting phenotype by expressing low levels of activation markers such as CD45. During pathological events in the CNS, microglia become activated as determined by upregulation of CD45 and other markers3. The factors that affect microglia phenotype and functions in the CNS are not well studied. MicroRNAs (miRNAs) are a growing family of conserved molecules (~22 nucleotides long) that are involved in many normal physiological processes such as cell growth and differentiation4 and pathologies such as inflammation5. MiRNAs downregulate the expression of certain target genes by binding complementary sequences of their mRNAs and play an important role in the activation of innate immune cells including macrophages6 and microglia7. In order to investigate miRNA-mediated pathways that define the microglial phenotype, biological function, and to distinguish microglia from other types of macrophages, it is important to quantitatively assess the expression of particular microRNAs in distinct subsets of CNS-resident microglia. Common methods for measuring the expression of miRNAs in the CNS include quantitative PCR from whole neuronal tissue and in situ hybridization. However, quantitative PCR from whole tissue homogenate does not allow the assessment of the expression of miRNA in microglia, which represent only 5-15% of the cells of neuronal tissue. Hybridization in situ allows the assessment of the expression of microRNA in specific cell types in the tissue sections, but this method is not entirely quantitative. In this report we describe a quantitative and sensitive method for the detection of miRNA by real-time PCR in microglia isolated from normal CNS or during neuroinflammation using experimental autoimmune encephalomyelitis (EAE), a mouse model for MS. The described method will be useful to measure the level of expression of microRNAs in microglia in normal CNS or during neuroinflammation associated with various pathologies including MS, stroke, traumatic injury, Alzheimer's disease and brain tumors.

Microglia are resident macrophages that provide the first line of defense and immune surveillance of the central nervous system. MicroRNAs are regulatory molecules that play an important role in many physiological processes including activation and differentiation of macrophages. In this article, we describe the method for measurement of microRNAs in microglia.

Microglia are cells of the myeloid lineage that reside in the central nervous system (CNS)1. These cells play an important role in pathologies of many ...

Immunology and Infection

Production of RNA for Transcriptomic Analysis from Mouse Spinal Cord Motor Neuron Cell Bodies by Laser Capture Microdissection

Preparation of high-quality RNA from cells of interest is critical to precise and meaningful analysis of transcriptional differences among cell types or between the same cell type in health and disease or following pharmacologic treatments.&#160;In the spinal cord, such preparation from motor neurons, the target of interest in many neurologic and neurodegenerative diseases, is complicated by the fact that motor neurons represent &#60;10% of the total cell population.&#160;Laser capture microdissection (LMD) has been developed to address this problem.&#160;Here, we describe a protocol to quickly recover, freeze, and section mouse spinal cord to avoid RNA damage by endogenous and exogenous RNases, followed by staining with Azure B in 70% ethanol to identify the motor neurons while keeping endogenous RNase inhibited.&#160;LMD is then used to capture the stained neurons directly into guanidine thiocyanate lysis buffer, maintaining RNA integrity.&#160;Standard techniques are used to recover the total RNA and measure its integrity.&#160;This material can then be used for downstream analysis of the transcripts by RNA-seq and qRT-PCR.

High-quality total RNA has been prepared from cell bodies of mouse spinal cord motor neurons by laser capture microdissection after staining spinal cord sections with Azure B in 70% ethanol.&#160;Sufficient RNA (~40-60 ng) is recovered from 3,000-4,000 motor neurons to allow downstream RNA analysis by RNA-seq and qRT-PCR.

Preparation of high-quality RNA from cells of interest is critical to precise and meaningful analysis of transcriptional differences among cell types or ...

In vivo Interrogation of Central Nervous System Translatome by Polyribosome Fractionation

Multiple processes are involved in gene expression including transcription, translation and stability of mRNAs and proteins. Each of these steps are tightly regulated, affecting the final dynamics of protein abundance. Various regulatory mechanisms exist at the translation step, rendering mRNA levels alone an unreliable indicator of gene expression. In addition, local regulation of mRNA translation has been particularly implicated in neuronal functions, shifting &#39;translatomics&#39; to the focus of attention in neurobiology. The presented method can be used to bridge transcriptomics and proteomics.
Here we describe essential modifications to the technique of polyribosome fractionation, which interrogates the translatome based on the association of actively translated mRNAs to multiple ribosomes and their differential sedimentation in sucrose gradients. Traditionally, working with in vivo samples, particularly of the central nervous system (CNS), has proven challenging due to the restricted amounts of material and the presence of fatty tissue components. In order to address this, the described protocol is specifically optimized for use with minimal amount of CNS material, as demonstrated by the use of single mouse spinal cord and brain. Briefly, CNS tissues are extracted and translating ribosomes are immobilized on mRNAs with cycloheximide. Myelin flotation is then performed to remove lipid rich components. Fractionation is performed on a sucrose gradient where mRNAs are separated according to their ribosomal loading. Isolated fractions are suitable for a range of downstream assays, including new genome wide assay technologies.

In vivo Interrogation of Central Nervous System Translatome by Polyribosome Fractionation

This protocol illustrates essential modifications of polyribosome fractionation in order to study the translatome of in vivo CNS samples. It allows global assessment of translation and transcription regulation through the isolation and comparison of total RNA to ribosome bound RNA fractions.

Multiple processes are involved in gene expression including transcription, translation and stability of mRNAs and proteins. Each of these steps are ...

A High Throughput, Multiplexed and Targeted Proteomic CSF Assay to Quantify Neurodegenerative Biomarkers and Apolipoprotein E Isoforms Status

Many neurodegenerative diseases are&#160;still lacking effective treatments. Reliable biomarkers for identifying and classifying these diseases will be important in the development of future novel therapies. Often&#160;potential new biomarkers do not make it&#160;into the clinic&#160;due to limitations&#160;in their development and&#160;high costs.&#160;However,&#160;targeted proteomics using Multiple Reaction Monitoring Liquid Chromatography-tandem/Mass Spectrometry (MRM LC-MS/MS), specifically using triple quadrupole mass spectrometers, is one method that can be used to rapidly evaluate and validate biomarkers&#160;for clinical translation into diagnostic laboratories. Traditionally, this platform has been used extensively for measurement of small molecules&#160;in clinical laboratories, but it is the potential to analyze proteins, that makes it an attractive alternative to ELISA (Enzyme-Linked Immunosorbent Assay)-based methods. We describe here how targeted proteomics can be used to measure multiplexed markers of dementia, including the detection and quantitation of the known risk factor apolipoprotein E isoform 4 (ApoE4).
In order to make the assay suitable for translation, it is designed to be rapid, simple, highly specific and cost effective. To achieve this, every step in the development of the assay must be optimized for the individual proteins and tissues they are analyzed in. This method describes a typical workflow including various tips and tricks to developing a targeted proteomics MRM LC-MS/MS for translation.
The method development is optimized using custom synthesized versions of tryptic quantotypic peptides, which calibrate&#160;the MS for detection and then spiked into CSF to determine correct identification of the endogenous peptide in the chromatographic separation prior to analysis in the MS. To achieve absolute quantitation, stable isotope-labeled internal standard versions of the peptides with short amino acid sequence tags and containing a trypsin cleavage site, are included in the assay.

We describe a high-throughput, multiplex, and targeted proteomic cerebrospinal fluid (CSF) assay developed with potential for clinical translation. The test can quantitate potential markers and risk factors for neurodegeneration, such as the apolipoprotein E variants (E2, E3 and E4), and measure their allelic expression.

Many neurodegenerative diseases are&#160;still lacking effective treatments. Reliable biomarkers for identifying and classifying these diseases will be ...

Medicine

Purification of Transcripts and Metabolites from Drosophila Heads

For the last decade, we have tried to understand the molecular and cellular mechanisms of neuronal degeneration using Drosophila as a model organism. Although fruit flies provide obvious experimental advantages, research on neurodegenerative diseases has mostly relied on traditional techniques, including genetic interaction, histology, immunofluorescence, and protein biochemistry. These techniques are effective for mechanistic, hypothesis-driven studies, which lead to a detailed understanding of the role of single genes in well-defined biological problems. However, neurodegenerative diseases are highly complex and affect multiple cellular organelles and processes over time. The advent of new technologies and the omics age provides a unique opportunity to understand the global cellular perturbations underlying complex diseases. Flexible model organisms such as Drosophila are ideal for adapting these new technologies because of their strong annotation and high tractability. One challenge with these small animals, though, is the purification of enough informational molecules (DNA, mRNA, protein, metabolites) from highly relevant tissues such as fly brains. Other challenges consist of collecting large numbers of flies for experimental replicates (critical for statistical robustness) and developing consistent procedures for the purification of high-quality biological material. Here, we describe the procedures for collecting thousands of fly heads and the extraction of transcripts and metabolites to understand how global changes in gene expression and metabolism contribute to neurodegenerative diseases. These procedures are easily scalable and can be applied to the study of proteomic and epigenomic contributions to disease.

Purification of Transcripts and Metabolites from Drosophila Heads

We describe here the procedures for the extraction and purification of mRNA and metabolites from Drosophila heads. We are applying these techniques to better understand the cellular perturbations underlying neuronal degeneration. These methodologies can be easily scaled and adapted for other "omic" projects.

For the last decade, we have tried to understand the molecular and cellular mechanisms of neuronal degeneration using Drosophila as a model organism. ...

Biology

Comprehensive Autopsy Program for Individuals with Multiple Sclerosis

We describe a rapid tissue donation program for individuals with multiple sclerosis (MS) that requires scientists and technicians to be on-call 24/7, 365 days a year. Participants consent to donate their brain and spinal cord. Most patients were followed by neurologists at the Cleveland Clinic Mellen Center for MS Treatment and Research. Their clinical courses and neurological disabilities are well-characterized. Soon after death, the body is transported to the MS Imaging Center, where the brain is scanned in situ by 3 T magnetic resonance imaging (MRI). The body is then transferred to the autopsy room, where the brain and spinal cord are removed. The brain is divided into two hemispheres. One hemisphere is immediately placed in a slicing box and alternate 1 cm-thick slices are either fixed in 4% paraformaldehyde for two days or rapidly frozen in dry ice and 2-methylbutane. The short-fixed brain slices are stored in a cryopreservation solution and used for histological analyses and immunocytochemical detection of sensitive antigens. Frozen slices are stored at -80 &#176;C and used for molecular, immunocytochemical, and in situ hybridization/RNA scope studies. The other hemisphere is placed in 4% paraformaldehyde for several months, placed in the slicing box, re-scanned in the 3 T magnetic resonance (MR) scanner and sliced into centimeter-thick slices. Postmortem in situ MR images (MRIs) are co-registered with 1 cm-thick brain slices to facilitate MRI-pathology correlations. All brain slices are photographed and brain white-matter lesions are identified. The spinal cord is cut into 2 cm segments. Alternate segments are fixed in 4% paraformaldehyde or rapidly frozen. The rapid procurement of postmortem MS tissues allows pathological and molecular analyses of MS brains and spinal cords and pathological correlations of brain MRI abnormalities. The quality of these rapidly-processed postmortem tissues (usually within 6 h of death) is of great value to MS research and has resulted in many high-impact discoveries.

Multiple sclerosis is an inflammatory demyelinating disease with no cure. Analysis of brain tissue provides important clues to understanding the pathogenesis of the disease. Here we discuss the methodology and downstream analysis of MS brain tissue collected through a unique rapid autopsy program in operation at the Cleveland Clinic.

We describe a rapid tissue donation program for individuals with multiple sclerosis (MS) that requires scientists and technicians to be on-call 24/7, 365 ...

Sex-Specific Glial Biomarkers in Alzheimer&#39;s Disease: A Transcriptome Analysis

Biomarker Identification for Gender Specificity of Alzheimer's Disease Based on the Glial Transcriptome Profiles

Many sex-specific biomarkers have been recently revealed in Alzheimer&#39;s disease (AD); however, cerebral glial cells were rarely reported. This study analyzed 220,095 single-nuclei transcriptomes from the frontal cortex of thirty-three AD individuals in the GEO database. Sex-specific Differentially Expressed Genes (DEGs) were identified in glial cells, including 243 in astrocytes, 1,154 in microglia, and 572 in oligodendrocytes. Gene Ontology (GO) functional annotation analyses and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses revealed functional concentration in synaptic, neural, and hormone-related pathways. Protein-protein interaction network (PPI) identified MT3, CALM2, DLG2, KCND2, PAKACB, CAMK2D, and NLGN4Y in astrocytes, TREM2, FOS, APOE, APP, and NLGN4Y in microglia, and GRIN2A, ITPR2, GNAS, and NLGN4Y in oligodendrocytes as key genes. NLGN4Y was the only gene shared by the three glia and was identified as the biomarker for the gender specificity of AD. Gene-transcription factor (TF)-miRNA coregulatory network identified key regulators for NLGN4Y and its target TCMs. Ecklonia kurome Okam (Kunbu) and Herba Ephedrae (Mahuang) were identified, and the effects of the active ingredients on AD were displayed. Finally, enrichment analysis of Kunbu and Mahuang suggested that they might act as therapeutic candidates for gender specificity of AD.

Author Spotlight: Exploring Sex-Specific Glial Signatures and Therapeutic Leads for Alzheimer&#39;s Disease

This study analyzed single-nuclei transcriptomes of thirty-three individuals with Alzheimer's disease (AD), revealing sex-specific DEGs in glial cells. Functional enrichment analysis highlighted synaptic, neural, and hormone-related pathways. Key genes, namely NLGN4Y and its regulators, were identified, and potential therapeutic candidates for gender-specific AD were proposed.

Many sex-specific biomarkers have been recently revealed in Alzheimer&#39;s disease (AD); however, cerebral glial cells were rarely reported. This study ...

Quantitative Proteomics Workflow using Multiple Reaction Monitoring Based Detection of Proteins from Human Brain Tissue

The proteomic analysis of the human brain tissue over the last decade has greatly enhanced our understanding of the brain. However, brain related disorders continue to be a major contributor of deaths around the world, necessitating the need for even greater understanding of their pathobiology. Traditional antibody-based techniques like western blotting or immunohistochemistry suffer from being low-throughput besides being labor-intensive and qualitative or semi-quantitative. Even conventional mass spectrometry-based shotgun approaches fail to provide conclusive evidence to support a certain hypothesis. Targeted proteomics approaches are largely hypothesis driven and differ from the conventional shotgun proteomics approaches that have been long in use. Multiple reaction monitoring is one such targeted approach that requires the use of a special mass spectrometer called the tandem quadrupole mass spectrometer or triple quadrupole mass spectrometer. In the current study, we have systematically highlighted the major steps involved in performing a successful tandem quadrupole mass spectrometry-based proteomics workflow using human brain tissue with an aim to introduce this workflow to a broader research community.

The protocol aims to introduce the use of a triple quadrupole mass spectrometer for Multiple Reaction Monitoring (MRM) of proteins from clinical samples. We have provided a systematic workflow starting from sample preparation to data analysis for clinical samples with all the necessary precautions to be taken.

The proteomic analysis of the human brain tissue over the last decade has greatly enhanced our understanding of the brain. However, brain related ...

Sample Preparation for Rapid Lipid Analysis in Drosophila Brain Using Matrix-Assisted Laser Desorption/Ionization Mass Spectrometry Imaging

Lipid profiling, or lipidomics, is a well-established technique used to study the entire lipid content of a cell or tissue. Information acquired from&#160;lipidomics is valuable in studying the pathways involved in development, disease, and cellular metabolism. Many tools and instrumentations have aided lipidomics projects, most notably various combinations of mass spectrometry and liquid chromatography techniques. Matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI MSI) has recently emerged as a powerful imaging technique that complements conventional approaches. This novel technique provides unique information on the spatial distribution of lipids within tissue compartments, which was previously unattainable without the use of excessive modifications. The sample preparation of the MALDI MSI approach is critical and, therefore, is the focus of this paper. This paper presents a rapid lipid analysis of a large number of Drosophila brains embedded in optimal cutting temperature compound (OCT) to provide a detailed protocol for the preparation of small tissues for lipid analysis or metabolite and small molecule analysis through MALDI MSI.

Sample Preparation for Rapid Lipid Analysis in Drosophila Brain Using Matrix-Assisted Laser Desorption/Ionization Mass Spectrometry Imaging

The aim of this protocol is to provide detailed guidance on the proper sample preparation for lipid and metabolite analysis in small tissues, such as the Drosophila brain, using matrix-assisted laser desorption/ionization (MALDI) mass spectrometry imaging.

Lipid profiling, or lipidomics, is a well-established technique used to study the entire lipid content of a cell or tissue. Information acquired ...

Isolation of the Brain Secretome from Ex Vivo Brain Slice Cultures

The brain secretome consists of proteins either actively secreted or shed from the cell surface by proteolytic cleavage in the extracellular matrix of the nervous system. These proteins include growth factor receptors and transmembrane proteins, among others, covering a broad spectrum of roles in the development and normal functioning of the central nervous system. The current procedure to extract the secretome from cerebrospinal fluid is complicated and time-consuming, and it is difficult to isolate these proteins from experimental animal brains. In this study, we present a novel protocol for isolating the brain secretome from mouse brain slice cultures. First, the brains were isolated, sliced, and cultured ex vivo. The culture medium was then filtered and concentrated for isolating proteins by centrifugation after a few days. Finally, the isolated proteins were resolved using sodium dodecyl-sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and subsequently probed for purity characterization by western blot. This isolation procedure of the brain secretome from ex vivo brain slice cultures can be used to investigate the effects of the secretome on a variety of neurodevelopmental diseases, such as autism spectrum disorders.

Isolation of the Brain Secretome from Ex Vivo Brain Slice Cultures

The brain secretome plays a pivotal role in the development and normal functioning of the central nervous system. In this article, we provide a detailed protocol for isolation of the brain secretome from ex vivo brain slice cultures.

The brain secretome consists of proteins either actively secreted or shed from the cell surface by proteolytic cleavage in the extracellular matrix of the ...

Lipidomics and Transcriptomics in Neurological Diseases

Summary

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Chapters in this video

Detection of MicroRNAs in Microglia by Real-time PCR in Normal CNS and During Neuroinflammation

Production of RNA for Transcriptomic Analysis from Mouse Spinal Cord Motor Neuron Cell Bodies by Laser Capture Microdissection

In vivo Interrogation of Central Nervous System Translatome by Polyribosome Fractionation

A High Throughput, Multiplexed and Targeted Proteomic CSF Assay to Quantify Neurodegenerative Biomarkers and Apolipoprotein E Isoforms Status

Purification of Transcripts and Metabolites from Drosophila Heads

Comprehensive Autopsy Program for Individuals with Multiple Sclerosis

Biomarker Identification for Gender Specificity of Alzheimer's Disease Based on the Glial Transcriptome Profiles

Quantitative Proteomics Workflow using Multiple Reaction Monitoring Based Detection of Proteins from Human Brain Tissue

Sample Preparation for Rapid Lipid Analysis in Drosophila Brain Using Matrix-Assisted Laser Desorption/Ionization Mass Spectrometry Imaging

Isolation of the Brain Secretome from Ex Vivo Brain Slice Cultures