In this study, we aimed to disclose the impact of amyloid-β toxicity and tau pathology on astrocyte swelling, their volume recovery and extracellular space (ECS) diffusion parameters, namely volume fraction (α) and tortuosity (λ), in a triple transgenic mouse model of Alzheimer's disease (3xTg-AD). Astrocyte volume changes, which reflect astrocyte ability to take up ions/neurotransmitters, were quantified during and after exposure to hypo-osmotic stress, or hyperkalemia in acute hippocampal slices, and were correlated with alterations in ECS diffusion parameters. Astrocyte volume and ECS diffusion parameters were monitored during physiological aging (controls) and during AD progression in 3-, 9-, 12- and 18-month-old mice. In the hippocampus of controls α gradually declined with age, while it remained unaffected in 3xTg-AD mice during the entire time course. Moreover, age-related increases in λ occurred much earlier in 3xTg-AD animals than in controls. In 3xTg-AD mice changes in α induced by hypo-osmotic stress or hyperkalemia were comparable to those observed in controls, however, AD progression affected α recovery following exposure to both. Compared to controls, a smaller astrocyte swelling was detected in 3xTg-AD mice only during hyperkalemia. Since we observed a large variance in astrocyte swelling/volume regulation, we divided them into high- (HRA) and low-responding astrocytes (LRA). In response to hyperkalemia, the incidence of LRA was higher in 3xTg-AD mice than in controls, which may also reflect compromised K+ and neurotransmitter uptake. Furthermore, we performed single-cell RT-qPCR to identify possible age-related alterations in astrocytic gene expression profiles. Already in 3-month-old 3xTg-AD mice, we detected a downregulation of genes affecting the ion/neurotransmitter uptake and cell volume regulation, namely genes of glutamate transporters, α2β2 subunit of Na+/K+-ATPase, connexin 30 or Kir4.1 channel. In conclusion, the aged hippocampus of 3xTg-AD mice displays an enlarged ECS volume fraction and an increased number of obstacles, which emerge earlier than in physiological aging. Both these changes may strongly affect intercellular communication and influence astrocyte ionic/neurotransmitter uptake, which becomes impaired during aging and this phenomenon is manifested earlier in 3xTg-AD mice. The increased incidence of astrocytes with limited ability to take up ions/neurotransmitters may further add to a cytotoxic environment.

• Klíčová slova
• Alzheimer’s disease, ECS diffusion, astrocyte heterogeneity, astrocytes, ion uptake, volume changes,
• Publikační typ
• časopisecké články MeSH

Brain edema accompanying ischemic or traumatic brain injuries, originates from a disruption of ionic/neurotransmitter homeostasis that leads to accumulation of K(+) and glutamate in the extracellular space. Their increased uptake, predominantly provided by astrocytes, is associated with water influx via aquaporin-4 (AQP4). As the removal of perivascular AQP4 via the deletion of α-syntrophin was shown to delay edema formation and K(+) clearance, we aimed to elucidate the impact of α-syntrophin knockout on volume changes in individual astrocytes in situ evoked by pathological stimuli using three dimensional confocal morphometry and changes in the extracellular space volume fraction (α) in situ and in vivo in the mouse cortex employing the real-time iontophoretic method. RT-qPCR profiling was used to reveal possible differences in the expression of ion channels/transporters that participate in maintaining ionic/neurotransmitter homeostasis. To visualize individual astrocytes in mice lacking α-syntrophin we crossbred GFAP/EGFP mice, in which the astrocytes are labeled by the enhanced green fluorescent protein under the human glial fibrillary acidic protein promoter, with α-syntrophin knockout mice. Three-dimensional confocal morphometry revealed that α-syntrophin deletion results in significantly smaller astrocyte swelling when induced by severe hypoosmotic stress, oxygen glucose deprivation (OGD) or 50 mM K(+). As for the mild stimuli, such as mild hypoosmotic or hyperosmotic stress or 10 mM K(+), α-syntrophin deletion had no effect on astrocyte swelling. Similarly, evaluation of relative α changes showed a significantly smaller decrease in α-syntrophin knockout mice only during severe pathological conditions, but not during mild stimuli. In summary, the deletion of α-syntrophin markedly alters astrocyte swelling during severe hypoosmotic stress, OGD or high K(+).

• MeSH
• akvaporin 4 genetika   metabolismus   MeSH
• astrocyty metabolismus   patologie   MeSH
• biologický transport MeSH
• draslík metabolismus   MeSH
• draslíkové kanály genetika   metabolismus   MeSH
• edém mozku genetika   metabolismus   patologie   MeSH
• gliový fibrilární kyselý protein MeSH
• glukosa nedostatek   MeSH
• konfokální mikroskopie MeSH
• membránové proteiny nedostatek   genetika   MeSH
• mikrotomie MeSH
• mozková kůra metabolismus   patologie   MeSH
• myši transgenní MeSH
• myši MeSH
• osmolární koncentrace MeSH
• osmotický tlak MeSH
• promotorové oblasti (genetika) MeSH
• proteiny nervové tkáně genetika   metabolismus   MeSH
• proteiny vázající vápník nedostatek   genetika   MeSH
• regulace genové exprese MeSH
• signální transdukce MeSH
• stereotaktické techniky MeSH
• svalové proteiny nedostatek   genetika   MeSH
• techniky tkáňových kultur MeSH
• zelené fluorescenční proteiny genetika   metabolismus   MeSH
• zvířata MeSH
• Check Tag
• mužské pohlaví MeSH
• myši MeSH
• ženské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• akvaporin 4 MeSH
• Aqp4 protein, mouse MeSH Prohlížeč
• draslík MeSH
• draslíkové kanály MeSH
• enhanced green fluorescent protein MeSH Prohlížeč
• glial fibrillary astrocytic protein, mouse MeSH Prohlížeč
• gliový fibrilární kyselý protein MeSH
• glukosa MeSH
• membránové proteiny MeSH
• proteiny nervové tkáně MeSH
• proteiny vázající vápník MeSH
• svalové proteiny MeSH
• syntrophin alpha1 MeSH Prohlížeč
• zelené fluorescenční proteiny MeSH

Alzheimer's disease (AD) is a progressive neurodegenerative disorder characterized by the loss of cognitive functions, reflecting pathological damage to the medial prefrontal cortex (mPFC) as well as to the hippocampus and the entorhinal cortex. Astrocytes maintain the internal homeostasis of the CNS and are fundamentally involved in neuropathological processes, including AD. Here, we analysed the astrocytic cytoskeletal changes within the mPFC of a triple transgenic mouse model of AD (3 × Tg-AD) by measuring the surface area and volume of glial fibrillary acidic protein (GFAP)-positive profiles in relation to the build-up and presence of amyloid-β (Aβ), and compared the results with those found in non-transgenic control animals at different ages. 3 × Tg-AD animals showed clear astroglial cytoskeletal atrophy, which appeared at an early age (3 months; 33% and 47% decrease in GFAP-positive surface area and volume, respectively) and remained throughout the disease progression at 9, 12 and 18 months old (29% and 36%; 37% and 35%; 43% and 37%, respectively). This atrophy was independent of Aβ accumulation, as only a few GFAP-positive cells were localized around Aβ aggregates, which suggests no direct relationship with Aβ toxicity. Thus, our results indicate that the progressive reduction in astrocytic branching and domain in the mPFC can account for the integrative dysfunction leading to the cognitive deficits and memory disturbances observed in AD.

• MeSH
• Alzheimerova nemoc patologie   MeSH
• amyloidní beta-protein metabolismus   MeSH
• astrocyty cytologie   metabolismus   patologie   MeSH
• atrofie patologie   MeSH
• cytoskelet patologie   MeSH
• imunohistochemie MeSH
• modely nemocí na zvířatech MeSH
• myši transgenní MeSH
• myši MeSH
• prefrontální mozková kůra cytologie   metabolismus   patologie   MeSH
• progrese nemoci MeSH
• zvířata MeSH
• Check Tag
• myši MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• amyloidní beta-protein MeSH

Recently, we have identified two astrocytic subpopulations in the cortex of GFAP-EGFP mice, in which the astrocytes are visualized by the enhanced green-fluorescent protein (EGFP) under the control of the human glial fibrillary acidic protein (GFAP) promotor. These astrocytic subpopulations, termed high response- (HR-) and low response- (LR-) astrocytes, differed in the extent of their swelling during oxygen-glucose deprivation (OGD). In the present study we focused on identifying the ion channels or transporters that might underlie the different capabilities of these two astrocytic subpopulations to regulate their volume during OGD. Using three-dimensional confocal morphometry, which enables quantification of the total astrocytic volume, the effects of selected inhibitors of K⁺ and Cl⁻ channels/transporters or glutamate transporters on astrocyte volume changes were determined during 20 minute-OGD in situ. The inhibition of volume regulated anion channels (VRACs) and two-pore domain potassium channels (K(2P)) highlighted their distinct contributions to volume regulation in HR-/LR-astrocytes. While the inhibition of VRACs or K(2P) channels revealed their contribution to the swelling of HR-astrocytes, in LR-astrocytes they were both involved in anion/K⁺ effluxes. Additionally, the inhibition of Na⁺-K⁺-Cl⁻ co-transporters in HR-astrocytes led to a reduction of cell swelling, but it had no effect on LR-astrocyte volume. Moreover, employing real-time single-cell quantitative polymerase chain reaction (PCR), we characterized the expression profiles of EGFP-positive astrocytes with a focus on those ion channels and transporters participating in astrocyte swelling and volume regulation. The PCR data revealed the existence of two astrocytic subpopulations markedly differing in their gene expression levels for inwardly rectifying K⁺ channels (Kir4.1), K(2P) channels (TREK-1 and TWIK-1) and Cl⁻ channels (ClC2). Thus, we propose that the diverse volume changes displayed by cortical astrocytes during OGD mainly result from their distinct expression patterns of ClC2 and K(2P) channels.

• MeSH
• astrocyty cytologie   účinky léků   metabolismus   MeSH
• biologické modely MeSH
• chloridové kanály metabolismus   MeSH
• draslíkové kanály metabolismus   MeSH
• gliový fibrilární kyselý protein metabolismus   MeSH
• glukosa nedostatek   MeSH
• kotransportéry pro draslík a chloridy MeSH
• kyslík MeSH
• lidé MeSH
• modulátory membránového transportu farmakologie   MeSH
• mozková kůra cytologie   MeSH
• myši transgenní MeSH
• myši MeSH
• pohlavní dimorfismus MeSH
• regulace genové exprese účinky léků   MeSH
• sodík-draslík-chloridové symportéry metabolismus   MeSH
• stanovení celkové genové exprese MeSH
• symportéry metabolismus   MeSH
• techniky in vitro MeSH
• velikost buňky účinky léků   MeSH
• vezikulární transportní proteiny pro glutamát metabolismus   MeSH
• zelené fluorescenční proteiny metabolismus   MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• mužské pohlaví MeSH
• myši MeSH
• ženské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• chloridové kanály MeSH
• draslíkové kanály MeSH
• enhanced green fluorescent protein MeSH Prohlížeč
• gliový fibrilární kyselý protein MeSH
• glukosa MeSH
• kyslík MeSH
• modulátory membránového transportu MeSH
• sodík-draslík-chloridové symportéry MeSH
• symportéry MeSH
• vezikulární transportní proteiny pro glutamát MeSH
• zelené fluorescenční proteiny MeSH