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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

AQP4, Kir4.1, and α-syntrophin increase towards adulthood. Western blots of whole brain homogenates from the postnatal day 0 to 21 (P0–P21) and adult (A) mice. Representative immunoblots for AQP4, Kir4.1, and α-syntrophin (left panels) and corresponding quantitation (densitometric values; right panels). a The AQP4 antibody labelled two bands at about 30 kDa corresponding to the M1 and M23 isoforms of AQP4. A third band around 35 kDa was not included in the quantitative analysis. β-Actin was used as loading control. The densitometric analysis revealed an increasing immunosignal for AQP4 protein in the postnatal period. b Immunoblot of Kir4.1 revealed a major band at ≈200 kDa which corresponds to the tetrameric form of Kir4.1 (Connors and Kofuji 2006; Olsen et al. 2006). Ponceau red staining was used as loading control (not shown). The developmental pattern mimics that of AQP4 (a). c Immunoblot of α-syntrophin revealed a major band at 59 kDa and a weaker band at slightly higher molecular weight. The major band––absent from α-syntrophin knockout brains––was used for quantitative analysis. β-Actin was used as loading control. The expression pattern for α-syntrophin was similar to those of AQP4 and Kir4.1. **Significantly different from P0 and ‘x’ significantly different from previous value. Error bars indicate ±2 SE, p = 0.05
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Fig7: AQP4, Kir4.1, and α-syntrophin increase towards adulthood. Western blots of whole brain homogenates from the postnatal day 0 to 21 (P0–P21) and adult (A) mice. Representative immunoblots for AQP4, Kir4.1, and α-syntrophin (left panels) and corresponding quantitation (densitometric values; right panels). a The AQP4 antibody labelled two bands at about 30 kDa corresponding to the M1 and M23 isoforms of AQP4. A third band around 35 kDa was not included in the quantitative analysis. β-Actin was used as loading control. The densitometric analysis revealed an increasing immunosignal for AQP4 protein in the postnatal period. b Immunoblot of Kir4.1 revealed a major band at ≈200 kDa which corresponds to the tetrameric form of Kir4.1 (Connors and Kofuji 2006; Olsen et al. 2006). Ponceau red staining was used as loading control (not shown). The developmental pattern mimics that of AQP4 (a). c Immunoblot of α-syntrophin revealed a major band at 59 kDa and a weaker band at slightly higher molecular weight. The major band––absent from α-syntrophin knockout brains––was used for quantitative analysis. β-Actin was used as loading control. The expression pattern for α-syntrophin was similar to those of AQP4 and Kir4.1. **Significantly different from P0 and ‘x’ significantly different from previous value. Error bars indicate ±2 SE, p = 0.05

Mentions: The molecules under study segregate in three groups in regard to their expression at the protein level (Figs. 7 and 8). AQP4 mirrored α-syntrophin and Kir4.1 in showing a continuous increase from being close to undetectable at P0 to being strongly expressed at adult stages. DP71 and β-dystroglycan, on the other hand, are rather stable throughout postnatal development. Laminin and agrin formed a third group that peaked at P7 and thereafter displayed a sharp decline towards adulthood.Fig. 7


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)

AQP4, Kir4.1, and α-syntrophin increase towards adulthood. Western blots of whole brain homogenates from the postnatal day 0 to 21 (P0–P21) and adult (A) mice. Representative immunoblots for AQP4, Kir4.1, and α-syntrophin (left panels) and corresponding quantitation (densitometric values; right panels). a The AQP4 antibody labelled two bands at about 30 kDa corresponding to the M1 and M23 isoforms of AQP4. A third band around 35 kDa was not included in the quantitative analysis. β-Actin was used as loading control. The densitometric analysis revealed an increasing immunosignal for AQP4 protein in the postnatal period. b Immunoblot of Kir4.1 revealed a major band at ≈200 kDa which corresponds to the tetrameric form of Kir4.1 (Connors and Kofuji 2006; Olsen et al. 2006). Ponceau red staining was used as loading control (not shown). The developmental pattern mimics that of AQP4 (a). c Immunoblot of α-syntrophin revealed a major band at 59 kDa and a weaker band at slightly higher molecular weight. The major band––absent from α-syntrophin knockout brains––was used for quantitative analysis. β-Actin was used as loading control. The expression pattern for α-syntrophin was similar to those of AQP4 and Kir4.1. **Significantly different from P0 and ‘x’ significantly different from previous value. Error bars indicate ±2 SE, p = 0.05
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Fig7: AQP4, Kir4.1, and α-syntrophin increase towards adulthood. Western blots of whole brain homogenates from the postnatal day 0 to 21 (P0–P21) and adult (A) mice. Representative immunoblots for AQP4, Kir4.1, and α-syntrophin (left panels) and corresponding quantitation (densitometric values; right panels). a The AQP4 antibody labelled two bands at about 30 kDa corresponding to the M1 and M23 isoforms of AQP4. A third band around 35 kDa was not included in the quantitative analysis. β-Actin was used as loading control. The densitometric analysis revealed an increasing immunosignal for AQP4 protein in the postnatal period. b Immunoblot of Kir4.1 revealed a major band at ≈200 kDa which corresponds to the tetrameric form of Kir4.1 (Connors and Kofuji 2006; Olsen et al. 2006). Ponceau red staining was used as loading control (not shown). The developmental pattern mimics that of AQP4 (a). c Immunoblot of α-syntrophin revealed a major band at 59 kDa and a weaker band at slightly higher molecular weight. The major band––absent from α-syntrophin knockout brains––was used for quantitative analysis. β-Actin was used as loading control. The expression pattern for α-syntrophin was similar to those of AQP4 and Kir4.1. **Significantly different from P0 and ‘x’ significantly different from previous value. Error bars indicate ±2 SE, p = 0.05
Mentions: The molecules under study segregate in three groups in regard to their expression at the protein level (Figs. 7 and 8). AQP4 mirrored α-syntrophin and Kir4.1 in showing a continuous increase from being close to undetectable at P0 to being strongly expressed at adult stages. DP71 and β-dystroglycan, on the other hand, are rather stable throughout postnatal development. Laminin and agrin formed a third group that peaked at P7 and thereafter displayed a sharp decline towards adulthood.Fig. 7

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