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Postnatal development of the molecular complex underlying astrocyte polarization.

Lunde LK, Camassa LM, Hoddevik EH, Khan FH, Ottersen OP, Boldt HB, Amiry-Moghaddam M - Brain Struct Funct (2014)

Bottom Line: The endfoot membrane domains facing microvessels and pia are enriched with aquaporin-4 water channels (AQP4) and other members of the dystrophin associated protein complex (DAPC).Through a combination of methodological approaches, including light microscopic and high resolution immunogold cytochemistry, quantitative RT-PCR, and Western blotting, we demonstrate that the different members of this complex exhibit distinct ontogenic profiles—with the extracellular matrix (ECM) proteins laminin and agrin appearing earlier than the other members of the complex.Specifically, while laminin and agrin expression peak at P7, quantitative immunoblot analyses indicate that AQP4, α-syntrophin, and the inwardly rectifying K(+) channel Kir4.1 expression increases towards adulthood.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Molecular Neuroscience, Institute of Basic Medical Sciences, University of Oslo, Oslo, Norway.

ABSTRACT
Astrocytes are highly polarised cells with processes that ensheath microvessels, cover the brain surface, and abut synapses. The endfoot membrane domains facing microvessels and pia are enriched with aquaporin-4 water channels (AQP4) and other members of the dystrophin associated protein complex (DAPC). Several lines of evidence show that loss of astrocyte polarization, defined by the loss of proteins that are normally enriched in astrocyte endfeet, is a common denominator of several neurological diseases such as mesial temporal lobe epilepsy, Alzheimer's disease, and stroke. Little is known about the mechanisms responsible for inducing astrocyte polarization in vivo. Here we introduce the term endfoot-basal lamina junctional complex (EBJC) to denote the proteins that consolidate and characterize the gliovascular interface. The present study was initiated in order to resolve the developmental profile of the EBJC in mouse brain. We show that the EBJC is established after the first week postnatally. Through a combination of methodological approaches, including light microscopic and high resolution immunogold cytochemistry, quantitative RT-PCR, and Western blotting, we demonstrate that the different members of this complex exhibit distinct ontogenic profiles—with the extracellular matrix (ECM) proteins laminin and agrin appearing earlier than the other members of the complex. Specifically, while laminin and agrin expression peak at P7, quantitative immunoblot analyses indicate that AQP4, α-syntrophin, and the inwardly rectifying K(+) channel Kir4.1 expression increases towards adulthood. Our findings are consistent with ECM having an instructive role in establishing astrocyte polarization in postnatal development and emphasize the need to explore the involvement of ECM in neurological disease.

No MeSH data available.


Related in: MedlinePlus

Different members of the EBJC complex have different mRNA signatures during development. a–h Quantitative real time PCR analysis of mouse brains at different stages of development. Graphs illustrate the copy number of different mRNA species, compared to the total amount of RNA (ng). The different EBJC members segregate in several groups, in regard to the developmental profile of their respective mRNAs. mRNAs encoding AQP4 and α-syntrophin are weakly expressed at birth and peak before adulthood, while mRNAs encoding agrin and Lama1 are abundant at birth with decreasing levels towards the adult stage. The remaining mRNA species show a rather stable expression throughout postnatal development (dystrophin, Dag1, Lama2) or a sharp increase in expression towards adulthood (Kir4.1). Dag1 encodes both α- and β-dystroglycan. **Significantly different from P0 and ‘x’ significantly different from previous value. Error bars indicate ±2 SE, p = 0.05. i Representative DNA agarose gel electrophoresis showing the EBJC expression profile during postnatal development of mouse brain. PCR products were generated using representative cDNA samples from developmental stage P0, P4, P7, P13, P21 and adult (A) as indicated above each lane on the gel. A DNA marker (M) was included in the first lane. The EBJC sample set includes from top to bottom: AQP4, Kir4.1, α-syntrophin, Dp71, Dag1, agrin, Lama1, Lama2 and TBP. The latter was included to verify equivalent amounts of cDNA template across samples in the 30-cycle endpoint PCRs using GoTaq Green polymerase (Promega) and specific primers (Table 2). PCR product sizes in base pairs are shown to the right of each gel insert. Two bands are visible in the gel insert for agrin, which is explained by co-expression of two mRNA species resulting from alternative splicing
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Fig6: Different members of the EBJC complex have different mRNA signatures during development. a–h Quantitative real time PCR analysis of mouse brains at different stages of development. Graphs illustrate the copy number of different mRNA species, compared to the total amount of RNA (ng). The different EBJC members segregate in several groups, in regard to the developmental profile of their respective mRNAs. mRNAs encoding AQP4 and α-syntrophin are weakly expressed at birth and peak before adulthood, while mRNAs encoding agrin and Lama1 are abundant at birth with decreasing levels towards the adult stage. The remaining mRNA species show a rather stable expression throughout postnatal development (dystrophin, Dag1, Lama2) or a sharp increase in expression towards adulthood (Kir4.1). Dag1 encodes both α- and β-dystroglycan. **Significantly different from P0 and ‘x’ significantly different from previous value. Error bars indicate ±2 SE, p = 0.05. i Representative DNA agarose gel electrophoresis showing the EBJC expression profile during postnatal development of mouse brain. PCR products were generated using representative cDNA samples from developmental stage P0, P4, P7, P13, P21 and adult (A) as indicated above each lane on the gel. A DNA marker (M) was included in the first lane. The EBJC sample set includes from top to bottom: AQP4, Kir4.1, α-syntrophin, Dp71, Dag1, agrin, Lama1, Lama2 and TBP. The latter was included to verify equivalent amounts of cDNA template across samples in the 30-cycle endpoint PCRs using GoTaq Green polymerase (Promega) and specific primers (Table 2). PCR product sizes in base pairs are shown to the right of each gel insert. Two bands are visible in the gel insert for agrin, which is explained by co-expression of two mRNA species resulting from alternative splicing

Mentions: The molecules under investigation formed two distinct groups in regard to the developmental profile of their respective messengers (Fig. 6). mRNAs encoding AQP4 and other members of the dystrophin complex (α-syntrophin, β-dystroglycan, and the dystrophin isoform DP71) were scarce at P0 and increased in abundance towards a distinct peak at P13 (AQP4 and α-syntrophin) or a broader peak at P7–P13/21 (β-dystroglycan and DP71). DP71 and Kir4.1 (both being members of the DAPC) stood out as the only molecules whose messengers continue to increase until adulthood.Fig. 6


Postnatal development of the molecular complex underlying astrocyte polarization.

Lunde LK, Camassa LM, Hoddevik EH, Khan FH, Ottersen OP, Boldt HB, Amiry-Moghaddam M - Brain Struct Funct (2014)

Different members of the EBJC complex have different mRNA signatures during development. a–h Quantitative real time PCR analysis of mouse brains at different stages of development. Graphs illustrate the copy number of different mRNA species, compared to the total amount of RNA (ng). The different EBJC members segregate in several groups, in regard to the developmental profile of their respective mRNAs. mRNAs encoding AQP4 and α-syntrophin are weakly expressed at birth and peak before adulthood, while mRNAs encoding agrin and Lama1 are abundant at birth with decreasing levels towards the adult stage. The remaining mRNA species show a rather stable expression throughout postnatal development (dystrophin, Dag1, Lama2) or a sharp increase in expression towards adulthood (Kir4.1). Dag1 encodes both α- and β-dystroglycan. **Significantly different from P0 and ‘x’ significantly different from previous value. Error bars indicate ±2 SE, p = 0.05. i Representative DNA agarose gel electrophoresis showing the EBJC expression profile during postnatal development of mouse brain. PCR products were generated using representative cDNA samples from developmental stage P0, P4, P7, P13, P21 and adult (A) as indicated above each lane on the gel. A DNA marker (M) was included in the first lane. The EBJC sample set includes from top to bottom: AQP4, Kir4.1, α-syntrophin, Dp71, Dag1, agrin, Lama1, Lama2 and TBP. The latter was included to verify equivalent amounts of cDNA template across samples in the 30-cycle endpoint PCRs using GoTaq Green polymerase (Promega) and specific primers (Table 2). PCR product sizes in base pairs are shown to the right of each gel insert. Two bands are visible in the gel insert for agrin, which is explained by co-expression of two mRNA species resulting from alternative splicing
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Related In: Results  -  Collection

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Fig6: Different members of the EBJC complex have different mRNA signatures during development. a–h Quantitative real time PCR analysis of mouse brains at different stages of development. Graphs illustrate the copy number of different mRNA species, compared to the total amount of RNA (ng). The different EBJC members segregate in several groups, in regard to the developmental profile of their respective mRNAs. mRNAs encoding AQP4 and α-syntrophin are weakly expressed at birth and peak before adulthood, while mRNAs encoding agrin and Lama1 are abundant at birth with decreasing levels towards the adult stage. The remaining mRNA species show a rather stable expression throughout postnatal development (dystrophin, Dag1, Lama2) or a sharp increase in expression towards adulthood (Kir4.1). Dag1 encodes both α- and β-dystroglycan. **Significantly different from P0 and ‘x’ significantly different from previous value. Error bars indicate ±2 SE, p = 0.05. i Representative DNA agarose gel electrophoresis showing the EBJC expression profile during postnatal development of mouse brain. PCR products were generated using representative cDNA samples from developmental stage P0, P4, P7, P13, P21 and adult (A) as indicated above each lane on the gel. A DNA marker (M) was included in the first lane. The EBJC sample set includes from top to bottom: AQP4, Kir4.1, α-syntrophin, Dp71, Dag1, agrin, Lama1, Lama2 and TBP. The latter was included to verify equivalent amounts of cDNA template across samples in the 30-cycle endpoint PCRs using GoTaq Green polymerase (Promega) and specific primers (Table 2). PCR product sizes in base pairs are shown to the right of each gel insert. Two bands are visible in the gel insert for agrin, which is explained by co-expression of two mRNA species resulting from alternative splicing
Mentions: The molecules under investigation formed two distinct groups in regard to the developmental profile of their respective messengers (Fig. 6). mRNAs encoding AQP4 and other members of the dystrophin complex (α-syntrophin, β-dystroglycan, and the dystrophin isoform DP71) were scarce at P0 and increased in abundance towards a distinct peak at P13 (AQP4 and α-syntrophin) or a broader peak at P7–P13/21 (β-dystroglycan and DP71). DP71 and Kir4.1 (both being members of the DAPC) stood out as the only molecules whose messengers continue to increase until adulthood.Fig. 6

Bottom Line: The endfoot membrane domains facing microvessels and pia are enriched with aquaporin-4 water channels (AQP4) and other members of the dystrophin associated protein complex (DAPC).Through a combination of methodological approaches, including light microscopic and high resolution immunogold cytochemistry, quantitative RT-PCR, and Western blotting, we demonstrate that the different members of this complex exhibit distinct ontogenic profiles—with the extracellular matrix (ECM) proteins laminin and agrin appearing earlier than the other members of the complex.Specifically, while laminin and agrin expression peak at P7, quantitative immunoblot analyses indicate that AQP4, α-syntrophin, and the inwardly rectifying K(+) channel Kir4.1 expression increases towards adulthood.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Molecular Neuroscience, Institute of Basic Medical Sciences, University of Oslo, Oslo, Norway.

ABSTRACT
Astrocytes are highly polarised cells with processes that ensheath microvessels, cover the brain surface, and abut synapses. The endfoot membrane domains facing microvessels and pia are enriched with aquaporin-4 water channels (AQP4) and other members of the dystrophin associated protein complex (DAPC). Several lines of evidence show that loss of astrocyte polarization, defined by the loss of proteins that are normally enriched in astrocyte endfeet, is a common denominator of several neurological diseases such as mesial temporal lobe epilepsy, Alzheimer's disease, and stroke. Little is known about the mechanisms responsible for inducing astrocyte polarization in vivo. Here we introduce the term endfoot-basal lamina junctional complex (EBJC) to denote the proteins that consolidate and characterize the gliovascular interface. The present study was initiated in order to resolve the developmental profile of the EBJC in mouse brain. We show that the EBJC is established after the first week postnatally. Through a combination of methodological approaches, including light microscopic and high resolution immunogold cytochemistry, quantitative RT-PCR, and Western blotting, we demonstrate that the different members of this complex exhibit distinct ontogenic profiles—with the extracellular matrix (ECM) proteins laminin and agrin appearing earlier than the other members of the complex. Specifically, while laminin and agrin expression peak at P7, quantitative immunoblot analyses indicate that AQP4, α-syntrophin, and the inwardly rectifying K(+) channel Kir4.1 expression increases towards adulthood. Our findings are consistent with ECM having an instructive role in establishing astrocyte polarization in postnatal development and emphasize the need to explore the involvement of ECM in neurological disease.

No MeSH data available.


Related in: MedlinePlus