Connexin 30 Expression and Frequency of Connexin Heterogeneity in Astrocyte Gap Junction Plaques Increase with Age in the Rat Retina

Abstract

We investigated age-associated changes in retinal astrocyte connexins (Cx) by assaying Cx numbers, plaque sizes, protein expression levels and heterogeneity of gap junctions utilizing six-marker immunohistochemistry (IHC). We compared Wistar rat retinal wholemounts in animals aged 3 (young adult), 9 (middle-aged) and 22 months (aged). We determined that retinal astrocytes have gap junctions composed of Cx26, -30, -43 and -45. Cx30 was consistently elevated at 22 months compared to younger ages both when associated with parenchymal astrocytes and vascular-associated astrocytes. Not only was the absolute number of Cx30 plaques significantly higher (P<0.05) but the size of the plaques was significantly larger at 22 months compared to younger ages (p<0.05). With age, Cx26 increased significantly initially, but returned to basal levels; whereas Cx43 expression remained low and stable with age. Evidence that astrocytes alter connexin compositions of gap junctions was demonstrated by the significant increase in the number of Cx26/Cx45 gap junctions with age. We also found gap junctions comprised of 1, 2, 3 or 4 Cx proteins suggesting that retinal astrocytes use various connexin protein combinations in their gap junctions during development and aging. These data provides new insight into the dynamic and extensive Cx network utilized by retinal astrocytes for communication within both the parenchyma and vasculature for the maintenance of normal retinal physiology with age. This characterisation of the changes in astrocytic gap junctional communication with age in the CNS is crucial to the understanding of physiological aging and age-related neurodegenerative diseases.

Cx30 exhibits unique characteristics including a long half-life when assembled into gap junctions

“…Evidence for this can be found in skin where Cx30 is expressed in terminally differentiated keratinocytes both in human (Di et al, 2001;Richard, 2003;Churko and Laird, 2013) and murine (Coutinho et al, 2003;Kretz et al, 2003) epidermis, in non-proliferating astrocytes within the brain (Kunzelmann et al, 1999) and retina (Mansour et al, 2013), in cochlear supporting cells (Lowenheim et al, 1999;Chen et al, 2003) and mature lateral wall fibrocytes that have become quiescent. In addition, Cx30 is linked to differentiation of mammary gland cells during lactation (Talhouk et al, 2005) and to calcium-induced differentiation of corneal cells (Shurman et al, 2005).…”

“…Connexin 30 (Cx30; also known as GJB6) is specifically expressed in skin keratinocytes (Di et al, 2001), brain astrocytes (Dahl et al, 1996), retinal astrocytes (Mansour et al, 2013) and various inner ear cell types (Forge et al, 2003a). Mutations in the Cx30-encoding gene GJB6 cause skin disease or hearing loss, or a combination of both (Xu and Nicholson, 2013), suggesting an essential role for Cx30 in these systems.…”

P2X7 Receptors Mediate Innate Phagocytosis by Human Neural Precursor Cells and Neuroblasts

“…For detection of cytoplasmic antigens, cells were permeabilized with 0.5% Triton X‐100 (30 minutes) before blocking with 1% BSA (45 minutes) as per our previous studies . Antibodies used included βIII‐tubulin (βIIIT) (chicken IgG; dilution 1:1,500; Chemicon #AB9354, Billerica, MA, http://www.millipore.com); nestin (rabbit IgG; 1:200; Chemicon #AB5922); vimentin (vim; Cy3 conjugate; 1:800); s100β (mouse IgG 1 ; 1:1,000); glial fibrillary acidic protein (GFAP) (Cy3 conjugate, mouse IgG; 1:800); Sox2 and doublecortin (DCX) (rabbit IgG; dilution 1:500; Cell Signalling Technology, Danvers, MA, http://www.cellsignal.com).…”

Heterogeneous astrocytes: Active players in CNS

“…For example, the expression of Cx30 in rat brain doesn't increase until postnatal and then reaches the peak at about 4 weeks (Kunzelmann et al 1999). Compared with the youth, the gap junction between astrocytes in aged mice sharply reduces, and the connexin protein combinations also change during the aging process (Mansour et al 2013;Peters et al 2009), suggestive of the defect of astrocytes in maintaining homeostasis and supporting neuronal function in old age.…”