A view of the genetic and proteomic profile of extracellular matrix molecules in aging and stroke

IntroductionModification of the extracellular matrix (ECM) is one of the major processes in the pathology of brain damage following an ischemic stroke. However, our understanding of how age-related ECM alterations may affect stroke pathophysiology and its outcome is still very limited.MethodsWe conducted an ECM-targeted re-analysis of our previously obtained RNA-Seq dataset of aging, ischemic stroke and their interactions in young adult (3-month-old) and aged (18-month-old) mice. The permanent middle cerebral artery occlusion (pMCAo) in rodents was used as a model of ischemic stroke. Altogether 56 genes of interest were chosen for this study.ResultsWe identified an increased activation of the genes encoding proteins related to ECM degradation, such as matrix metalloproteinases (MMPs), proteases of a disintegrin and metalloproteinase with the thrombospondin motifs (ADAMTS) family and molecules that regulate their activity, tissue inhibitors of metalloproteinases (TIMPs). Moreover, significant upregulation was also detected in the mRNA of other ECM molecules, such as proteoglycans, syndecans and link proteins. Notably, we identified 8 genes where this upregulation was enhanced in aged mice in comparison with the young ones. Ischemia evoked a significant downregulation in only 6 of our genes of interest, including those encoding proteins associated with the protective function of ECM molecules (e.g., brevican, Hapln4, Sparcl1); downregulation in brevican was more prominent in aged mice. The study was expanded by proteome analysis, where we observed an ischemia-induced overexpression in three proteins, which are associated with neuroinflammation (fibronectin and vitronectin) and neurodegeneration (link protein Hapln2). In fibronectin and Hapln2, this overexpression was more pronounced in aged post-ischemic animals.ConclusionBased on these results, we can conclude that the ratio between the protecting and degrading mechanisms in the aged brain is shifted toward degradation and contributes to the aged tissues’ increased sensitivity to ischemic insults. Altogether, our data provide fresh perspectives on the processes underlying ischemic injury in the aging brain and serve as a freely accessible resource for upcoming research

In vitro investigating of anticancer activity of new 7-MEOTA-tacrine heterodimers

A combination of biochemical, biophysical and biological techniques was used to study calf thymus DNA interaction with newly synthesized 7-MEOTA-tacrine thiourea 12–17 and urea heterodimers 18–22, and to measure interference with type I and II topoisomerases. Their biological profile was also inspected in vitro on the HL-60 cell line using different flow cytometric techniques (cell cycle distribution, detection of mitochondrial membrane potential dissipation, and analysis of metabolic activity/viability). The compounds exhibited a profound inhibitory effect on topoisomerase activity (e.g. compound 22 inhibited type I topoisomerase at 1 µM concentration). The treatment of HL-60 cells with the studied compounds showed inhibition of cell growth especially with hybrids containing thiourea (14–17) and urea moieties (21 and 22). Moreover, treatment of human dermal fibroblasts with the studied compounds did not indicate significant cytotoxicity. The observed results suggest beneficial selectivity of the heterodimers as potential drugs to target cancer cells

Image_1_The absence of AQP4/TRPV4 complex substantially reduces acute cytotoxic edema following ischemic injury.jpg

IntroductionAstrocytic Aquaporin 4 (AQP4) and Transient receptor potential vanilloid 4 (TRPV4) channels form a functional complex that likely influences cell volume regulation, the development of brain edema, and the severity of the ischemic injury. However, it remains to be fully elucidated whether blocking these channels can serve as a therapeutic approach to alleviate the consequences of having a stroke.Methods and resultsIn this study, we used in vivo magnetic resonance imaging (MRI) to quantify the extent of brain lesions one day (D1) and seven days (D7) after permanent middle cerebral artery occlusion (pMCAO) in AQP4 or TRPV4 knockouts and mice with simultaneous deletion of both channels. Our results showed that deletion of AQP4 or TRPV4 channels alone leads to a significant worsening of ischemic brain injury at both time points, whereas their simultaneous deletion results in a smaller brain lesion at D1 but equal tissue damage at D7 when compared with controls. Immunohistochemical analysis 7 days after pMCAO confirmed the MRI data, as the brain lesion was significantly greater in AQP4 or TRPV4 knockouts than in controls and double knockouts. For a closer inspection of the TRPV4 and AQP4 channel complex in the development of brain edema, we applied a real-time iontophoretic method in situ to determine ECS diffusion parameters, namely volume fraction (α) and tortuosity (λ). Changes in these parameters reflect alterations in cell volume, and tissue structure during exposure of acute brain slices to models of ischemic conditions in situ, such as oxygen-glucose deprivation (OGD), hypoosmotic stress, or hyperkalemia. The decrease in α was comparable in double knockouts and controls when exposed to hypoosmotic stress or hyperkalemia. However, during OGD, there was no decrease in α in the double knockouts as observed in the controls, which suggests less swelling of the cellular components of the brain.ConclusionAlthough simultaneous deletion of AQP4 and TRPV4 did not improve the overall outcome of ischemic brain injury, our data indicate that the interplay between AQP4 and TRPV4 channels plays a critical role during neuronal and non-neuronal swelling in the acute phase of ischemic injury.</p

Table_1_The absence of AQP4/TRPV4 complex substantially reduces acute cytotoxic edema following ischemic injury.DOCX

IntroductionAstrocytic Aquaporin 4 (AQP4) and Transient receptor potential vanilloid 4 (TRPV4) channels form a functional complex that likely influences cell volume regulation, the development of brain edema, and the severity of the ischemic injury. However, it remains to be fully elucidated whether blocking these channels can serve as a therapeutic approach to alleviate the consequences of having a stroke.Methods and resultsIn this study, we used in vivo magnetic resonance imaging (MRI) to quantify the extent of brain lesions one day (D1) and seven days (D7) after permanent middle cerebral artery occlusion (pMCAO) in AQP4 or TRPV4 knockouts and mice with simultaneous deletion of both channels. Our results showed that deletion of AQP4 or TRPV4 channels alone leads to a significant worsening of ischemic brain injury at both time points, whereas their simultaneous deletion results in a smaller brain lesion at D1 but equal tissue damage at D7 when compared with controls. Immunohistochemical analysis 7 days after pMCAO confirmed the MRI data, as the brain lesion was significantly greater in AQP4 or TRPV4 knockouts than in controls and double knockouts. For a closer inspection of the TRPV4 and AQP4 channel complex in the development of brain edema, we applied a real-time iontophoretic method in situ to determine ECS diffusion parameters, namely volume fraction (α) and tortuosity (λ). Changes in these parameters reflect alterations in cell volume, and tissue structure during exposure of acute brain slices to models of ischemic conditions in situ, such as oxygen-glucose deprivation (OGD), hypoosmotic stress, or hyperkalemia. The decrease in α was comparable in double knockouts and controls when exposed to hypoosmotic stress or hyperkalemia. However, during OGD, there was no decrease in α in the double knockouts as observed in the controls, which suggests less swelling of the cellular components of the brain.ConclusionAlthough simultaneous deletion of AQP4 and TRPV4 did not improve the overall outcome of ischemic brain injury, our data indicate that the interplay between AQP4 and TRPV4 channels plays a critical role during neuronal and non-neuronal swelling in the acute phase of ischemic injury.</p

Altered Astrocytic Swelling in the Cortex of α-Syntrophin-Negative GFAP/EGFP Mice

<div><p>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 <i>in situ</i> evoked by pathological stimuli using three dimensional confocal morphometry and changes in the extracellular space volume fraction (α) <i>in situ</i> and <i>in vivo</i> 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⁺.</p></div

Characterization of double transgenic GFAP/EGFP/α-Syn^−/− mice.

<p>(<b>A</b>) PCR of tail genomic DNA isolated from α-Syn^−/− mice, GFAP/EGFP mice, or double transgenic GFAP/EGFP/α-Syn^−/− mice. Mice lacking the <i>Snta1</i> gene coding α-syntrophin also express <i>Neo</i>, the gene of neomycin resistance, inserted in the gene construct as a positive selectable marker during homologous recombination (top). Western blot analysis detecting α-syntrophin in cortical tissue isolated from α-Syn^−/− mice, GFAP/EGFP mice, or GFAP/EGFP/α-Syn^−/− mice (bottom). (<b>B</b>) Immunohistochemical staining for aquaporin 4 (AQP4) in cortical slices isolated from α-Syn^−/− mice, (<b>C</b>) GFAP/EGFP mice or GFAP/EGFP/α-Syn^−/− mice. Note that α-syntrophin deletion is accompanied by the loss of AQP4 expression on the astrocytic membranes contacting blood vessels.</p

Volume changes in the astrocytic soma and processes during hypotonic stress, increased extracellular K⁺ concentration and OGD.

<p>(<b>A–C</b>) Time-dependent changes in the volume of the astrocytic soma (<b>top</b>) and processes (<b>bottom</b>) in GFAP/EGFP (red) and GFAP/EGFP/α-Syn^−/− mice (green) during a 30-minute application of aCSF_H-100 (<b>A</b>), a 20-minute application of aCSF_K+50 (<b>B</b>) or 20-minute OGD (<b>C</b>), followed by a 60- or 40-minute washout. (<b>D–F</b>) The contribution of the astrocytic soma and processes to the total astrocyte volume changes was expressed as a ratio of the volume changes of both compartments (V_processes/V_soma) after 30 minutes of hypotonic stress and a subsequent 60-minute washout (<b>D</b>), after a 20-minute exposure to aCSF_K+50 and a subsequent 40-minute washout (<b>E</b>), and after 20 minutes of OGD and a subsequent 40-minute washout (<b>F</b>). Note that in GFAP/EGFP mice the swelling of the astrocytic processes prevails (V_processes/V_soma = ∼1.2), while in GFAP/EGFP/α-Syn^−/− the ratio declines towards 1, indicating that the astrocytic processes swell less and the contribution of the cell soma to total astrocyte volume increases. The time-points at which the ratio was calculated are indicated by arrows in A–C. Asterisks indicate significant (*, p<0.05), very significant (**, p<0.01) and extremely significant (***, p<0.001) differences between GFAP/EGFP and GFAP/EGFP/α-Syn^−/− mice.</p

The effect of hypotonic stress or elevated K⁺ on the ECS volume <i>in situ</i>.

<p>The left side: The control values of all experiments were set to 100%, and the relative changes of the values of the extracellular volume fraction α were calculated at 5 min intervals during a 30 min application and a subsequent 60 min washout of mild (<b>A</b>) or severe (<b>B</b>) hypotonic stress or 10 mM K⁺ (<b>C</b>). Each data point represents mean ± S.E.M. The right side: volume regulation during washout at 20 min intervals is expressed as changes in the values reached in the 30^th minute of application, set as 0%. Asterisks indicate significant (*, p<0.05) and very significant (**, p<0.01) differences between GFAP/EGFP and GFAP/EGFP/α-Syn^−/− mice.</p