Unknown,Transcriptomics,Genomics,Proteomics

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RNA profiling by deepSAGE sequencing in the cortex of a migraine mouse model after induction of cortical spreading depression

ABSTRACT: Familial Hemiplegic Migraine type 1 (FHM1) is a rare monogenic subtype of migraine with aura caused by mutations in CACNA1A that encodes the a1A subunit of voltage-gated CaV2.1 calcium channels. Transgenic knock-in mice that carry the human FHM1 R192Q missense mutation (âFHM1 R192Q miceâ) exhibit an increased susceptibility to cortical spreading depression (CSD), the mechanism underlying migraine aura. Here we analysed gene expression profiles from isolated cortical tissue of FHM1 R192Q mice 24 hours after experimentally induced CSD in order to identify molecular pathways affected by CSD. Gene expression profiles were generated using deep Serial Analysis of Gene Expression sequencing. Our data reveal a signature of inflammatory signalling upon CSD in the cortex of both mutant and wild-type mice. However, only in the brains of FHM1 R192Q mice specific genes are up-regulated in response to CSD that are implicated in interferon-related inflammatory signalling. Our findings show that CSD modulates inflammatory processes in both wild-type and mutant brains, but that an additional unique inflammatory signature becomes expressed after CSD in a relevant mouse model of migraine. Cortical RNA expression analysis using deepSAGE sequencing of wild-type (C57BL/6J) or transgenic R192Q FHM1 mice (n=6 per experimental group) 24 hours after sham surgery or the induction of 7 cortical spreading depression episodes

ORGANISM(S): Mus musculus

SUBMITTER: Else Eising

PROVIDER: E-GEOD-67933 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Publications

Cortical Spreading Depression Causes Unique Dysregulation of Inflammatory Pathways in a Transgenic Mouse Model of Migraine.

Eising Else E Shyti Reinald R 't Hoen Peter A C PAC Vijfhuizen Lisanne S LS Huisman Sjoerd M H SMH Broos Ludo A M LAM Mahfouz Ahmed A Reinders Marcel J T MJT Ferrari Michel D MD Tolner Else A EA de Vries Boukje B van den Maagdenberg Arn M J M AMJM

Molecular neurobiology 20160331 4

Familial hemiplegic migraine type 1 (FHM1) is a rare monogenic subtype of migraine with aura caused by mutations in CACNA1A that encodes the α<sub>1A</sub> subunit of voltage-gated Ca<sub>V</sub>2.1 calcium channels. Transgenic knock-in mice that carry the human FHM1 R192Q missense mutation ('FHM1 R192Q mice') exhibit an increased susceptibility to cortical spreading depression (CSD), the mechanism underlying migraine aura. Here, we analysed gene expression profiles from isolated cortical tissue ...[more]

PMID: 27032388

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Project description:We investigated the effects of PACAP treatment, and endogenous PACAP deficiency, on infarct volume, neurological function, and the cerebrocortical transcriptional response in a mouse model of stroke, middle cerebral artery occlusion (MCAO). PACAP-38 administered i.v. or i.c.v. one hour after MCAO significantly reduced infarct volume, and ameliorated functional motor deficits measured 24 hours later in wild-type mice. Infarct volumes and neurological deficits (walking faults) were both greater in PACAP-deficient than in wild-type mice, but treatment with PACAP reduced lesion volume and neurological deficits in PACAP-deficient mice to the same level of improvement as in wild-type mice. A 35,546-clone mouse cDNA microarray was used to investigate cortical transcriptional changes associated with cerebral ischemia in wild-type and PACAP-deficient mice, and with PACAP treatment after MCAO in wild-type mice. 229 known (named) transcripts were increased (228) or decreased (1) in abundance at least 50% following cerebral ischemia in wild-type mice. 49 transcripts were significantly up-regulated only at one hour post-MCAO (acute response transcripts), 142 were up-regulated only at 24 hours post-MCAO (delayed response transcripts) and 37 transcripts were up-regulated at both times (sustained response transcripts). More than 50% of these are transcripts not previously reported to be associated with brain ischemia responses. Many of the acutely regulated neurotrauma-associated transcripts, but a larger percentage of the delayed ones, require endogenous PACAP for up-regulation by ischemia, suggesting a more prominent role for PACAP in later response to injury than in the initial response, and consistent with a neuroprotective role for PACAP in late response to injury and neuroprotective efficacy when given post-MCAO. Putative injury effector transcripts regulated by PACAP include Ã£Ã-actin, midline 2, and metallothionein 1. Potential neuroprotective transcripts include several demonstrated to be PACAP-regulated in other contexts. Prominent among these were transcripts encoding the PACAP-regulated gene Ier3, and the neuropeptides enkephalin, substance P (tachykinin 1), and neurotensin. In the hybridization method employed, equal amounts (2-5Ã£Ãg/15.5 Ã£Ãl) of total RNA obtained from brain tissue of different experimental groups were separately labeled with two different fluorescent dyes (Cy3 or Cy5) and applied on the same NIMH Mouse 36K cDNA microarray chip containing 13217 distinct Entrez gene IDs (formerly known as LocusLink) and 15888 distinct unigene IDs, the remainders are incompletely annotated IMAGE clones. Probe preparation and hybridization were performed as follows. The total RNA (15-50 Ã£Ãg in 15.5 Ã£Ãl) was incubated with amine-modified random primer (2 Ã£Ãg/Ã£Ãl, 2 Ã£Ãl) and RNase inhibitor (5 units/Ã£Ãl, 1 Ã£Ãl) at 70 Ã¤aC for 10 min. Primer-RNA solution was then incubated at 42 Ã¤aC for 2 hr with the reverse transcriptase mix containing 5X first-strand buffer, 50X aa-dUTP/dNTPs (25 mM dATP, 25 mM dGTP, 25 mM dCTP, 15 mM dTTP and 10 mM aminoallyl dUTP), 0.1 M DDT, and Superscript II reverse transcriptase. The cDNA were labeled with NHS-ester Cy3 or Cy5 dye in the presence of 1 M sodium bicarbonate. Array slides placed in a hybridization chamber (Corning, Corning, NY) were incubated at 42 Ã¤aC for 16-24 hr, and successively washed with 0.5X SSC, 0.01% SDS, and 0.06X SSC at room temperature for 10 min each. Arrays were scanned with a GenePix 4000A scanner (Axon, Foster City, CA), and the resulting images were analyzed using IPlab (Fairfax, VA) and a FileMaker Pro 5 (Santa Clara, CA) relational database designed by Zedong Chen, NHGRI. Following background subtraction and normalization, a calculated ratio of Cy3 to Cy5 signal intensities was used to define the relative increase or decrease of a particular transcript. Ratios were calculated only from those spots with a combined ratio quality above 0.3.

Dataset Information

RNA profiling by deepSAGE sequencing in the cortex of a migraine mouse model after induction of cortical spreading depression

Publications

Cortical Spreading Depression Causes Unique Dysregulation of Inflammatory Pathways in a Transgenic Mouse Model of Migraine.

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