Lipidomics and Transcriptomics in Neurological Diseases

Summary

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.

Abstract

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.

Introduction

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 sen....

Protocol

All experimental procedures described here are in accordance with The European Community's Council Directive of 22 September 2010 (2010/63EU) and were approved by the local animal committee of the state Rhineland-Palatinate, Germany (file reference: 23 177-07/G16-1-075).

1. Animal model of acute and prophylactically treated KA-induced epilepsy

1. Perform seizure induction, treatment, and behavioral scoring.
   1. Separate mice (minimum n = 6 mice per group) in single cages.<.......

Discussion

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.......

Materials

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