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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

Agrin and laminin decrease with postnatal age. Western blots of whole mouse brain homogenate. Representative immunoblots of DP71, β-dystroglycan, agrin and laminin at different postnatal ages and adult (A) mice (left panels). The immunoblots were subjected to densitometric analysis (right panels). a Immunoblot of DP71 revealed a band at ≈71 kDa. There were also bands at ≈65 kDa and ≈55 kDa which could represent other dystrophin isoforms or degradation products. β-Actin was used as loading control. The immunosignal for DP71 was rather stable in the postnatal period. b Immunoblot of β-dystroglycan revealed a major band at ≈42 kDa. Ponceau red staining was used as loading control (not shown). The protein level is stable in the postnatal period. c Immunoblot of agrin revealed a major band at ≈300 kDa as shown previously (Stephan et al. 2008). Ponceau red staining was used as loading control (not shown). The immunosignal for agrin reached a peak at P7 (significantly higher than P0) with a sharp decline towards the adult level. d Immunoblot of laminin with whole brain homogenate revealed a major band at ≈200 kDa and two weaker bands at ≈400 kDa and ≈600–700 kDa. The 200 kDa band corresponds to the β- and γ-chain of laminin while the 400 kDa band corresponds to the α1-chain (Zhang et al. 2007). Ponceau red staining was used as loading control (not shown). Quantitative analysis of the 200 kDa band shows a pattern similar to that of agrin. Quantitation of the other two bands revealed no significant differences between the different postnatal stages. **Significantly different from P0 and ‘x’ significantly different from previous value. Error bars indicate ±2 SE, p = 0.05
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Fig8: Agrin and laminin decrease with postnatal age. Western blots of whole mouse brain homogenate. Representative immunoblots of DP71, β-dystroglycan, agrin and laminin at different postnatal ages and adult (A) mice (left panels). The immunoblots were subjected to densitometric analysis (right panels). a Immunoblot of DP71 revealed a band at ≈71 kDa. There were also bands at ≈65 kDa and ≈55 kDa which could represent other dystrophin isoforms or degradation products. β-Actin was used as loading control. The immunosignal for DP71 was rather stable in the postnatal period. b Immunoblot of β-dystroglycan revealed a major band at ≈42 kDa. Ponceau red staining was used as loading control (not shown). The protein level is stable in the postnatal period. c Immunoblot of agrin revealed a major band at ≈300 kDa as shown previously (Stephan et al. 2008). Ponceau red staining was used as loading control (not shown). The immunosignal for agrin reached a peak at P7 (significantly higher than P0) with a sharp decline towards the adult level. d Immunoblot of laminin with whole brain homogenate revealed a major band at ≈200 kDa and two weaker bands at ≈400 kDa and ≈600–700 kDa. The 200 kDa band corresponds to the β- and γ-chain of laminin while the 400 kDa band corresponds to the α1-chain (Zhang et al. 2007). Ponceau red staining was used as loading control (not shown). Quantitative analysis of the 200 kDa band shows a pattern similar to that of agrin. Quantitation of the other two bands revealed no significant differences between the different postnatal stages. **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)

Agrin and laminin decrease with postnatal age. Western blots of whole mouse brain homogenate. Representative immunoblots of DP71, β-dystroglycan, agrin and laminin at different postnatal ages and adult (A) mice (left panels). The immunoblots were subjected to densitometric analysis (right panels). a Immunoblot of DP71 revealed a band at ≈71 kDa. There were also bands at ≈65 kDa and ≈55 kDa which could represent other dystrophin isoforms or degradation products. β-Actin was used as loading control. The immunosignal for DP71 was rather stable in the postnatal period. b Immunoblot of β-dystroglycan revealed a major band at ≈42 kDa. Ponceau red staining was used as loading control (not shown). The protein level is stable in the postnatal period. c Immunoblot of agrin revealed a major band at ≈300 kDa as shown previously (Stephan et al. 2008). Ponceau red staining was used as loading control (not shown). The immunosignal for agrin reached a peak at P7 (significantly higher than P0) with a sharp decline towards the adult level. d Immunoblot of laminin with whole brain homogenate revealed a major band at ≈200 kDa and two weaker bands at ≈400 kDa and ≈600–700 kDa. The 200 kDa band corresponds to the β- and γ-chain of laminin while the 400 kDa band corresponds to the α1-chain (Zhang et al. 2007). Ponceau red staining was used as loading control (not shown). Quantitative analysis of the 200 kDa band shows a pattern similar to that of agrin. Quantitation of the other two bands revealed no significant differences between the different postnatal stages. **Significantly different from P0 and ‘x’ significantly different from previous value. Error bars indicate ±2 SE, p = 0.05
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Fig8: Agrin and laminin decrease with postnatal age. Western blots of whole mouse brain homogenate. Representative immunoblots of DP71, β-dystroglycan, agrin and laminin at different postnatal ages and adult (A) mice (left panels). The immunoblots were subjected to densitometric analysis (right panels). a Immunoblot of DP71 revealed a band at ≈71 kDa. There were also bands at ≈65 kDa and ≈55 kDa which could represent other dystrophin isoforms or degradation products. β-Actin was used as loading control. The immunosignal for DP71 was rather stable in the postnatal period. b Immunoblot of β-dystroglycan revealed a major band at ≈42 kDa. Ponceau red staining was used as loading control (not shown). The protein level is stable in the postnatal period. c Immunoblot of agrin revealed a major band at ≈300 kDa as shown previously (Stephan et al. 2008). Ponceau red staining was used as loading control (not shown). The immunosignal for agrin reached a peak at P7 (significantly higher than P0) with a sharp decline towards the adult level. d Immunoblot of laminin with whole brain homogenate revealed a major band at ≈200 kDa and two weaker bands at ≈400 kDa and ≈600–700 kDa. The 200 kDa band corresponds to the β- and γ-chain of laminin while the 400 kDa band corresponds to the α1-chain (Zhang et al. 2007). Ponceau red staining was used as loading control (not shown). Quantitative analysis of the 200 kDa band shows a pattern similar to that of agrin. Quantitation of the other two bands revealed no significant differences between the different postnatal stages. **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