NO measurement

NO concentrations in media were determined using a Nitric Oxide Assay Kit (Abcam, England, UK) or a Griess Reagent Kit (ThermoFisher Scientific, Waltham, Massachusetts, USA) according to manufacturer’s protocols. Briefly, freshly collected samples and standards (in duplicate) in 96 well microplate (Greiner Bio-One, Frickenhausen, Germany) were reacted with Griess reagents 1 and 2 (of each kit). Following 15 min of colour development at RT, absorbance at 540 nm was determined using an EnSpire® Multimode Plate Reader (Perkin Elmer, Massachusetts, USA). Standard curves were constructed, and sample nitrite concentrations were calculated from standard curves. Results were expressed as percentage NO released relative to TNF-α and IFNγ-stimulated positive controls.

4.4.13 X-ray fluorescence microscopy (XFM, II)

The distribution of copper was mapped at the XFM beamline at the Australian Synchrotron.

For this purpose, cell culture samples were grown on SiN X-ray transparent grids and cryofrozen after free-drying as described previously (Grubman, James et al. 2014). An incident beam of 12.7 keV X-rays was chosen to induce K-shell ionization of copper, while providing adequate separation of the Rayleigh and Compton peaks from the elemental fluorescence of interest. The incident beam was focused to a 2 µm spot (full-width at half maximum) using a Kirkpatrick–Baez mirror pair and specimens were fly- scanned through

X-ray focus. The resulting XRF was collected in event-mode using the low-latency, 384-channel Maia XRF detector in the backscatter geometry and the full XRF spectra used to reconstruct copper maps of the specimen using a virtual pixel size of 0.8 µm, giving an effective dwell time of 0.025 seconds. Single element foils of Mn and Pt (Micromatter, Canada), were scanned in the same geometry and used as references to establish elemental quantitation. Deconvolution of the Maia data was performed using the GeoPIXE v6.6.

4.4.14 Quantitative real-time polymerase chain reaction (Rt-PCR,II)

RNA extraction from cells was performed using MagMax Total RNA isolation kit and from frozen tissue samples using TRIzol (both ThermoFisher Scientific, Waltham, Massachusetts, USA). RNA concentrations were measured with Qubit 2.0 fluorometer or NanoDrop spectrophotometer (both ThermoFisher Scientific, Waltham, Massachusetts, USA). CDNA was constructed using 200 ng-2 µg RNA, random hexamers and High Capacity cDNA kit or Maxima reverse transcriptase (both ThermoFisher Scientific, Waltham, Massachusetts, USA).

Relative expression of mRNAs were measured using StepOnePlus (ThermoFisher Scientific, Waltham, Massachusetts, USA) or LightCycler 480 (Roche, Basel, Switzerland). Target gene expression levels were normalized to GAPDH or β-tubulin, and presented as fold change using the 2^-ΔΔCt method, where Ct is the threshold-cycle value.

4.5 STATISTICAL ANALYSES AND EXCLUSION CRITERIA

Animals were randomized to treatment groups using GrapPad QuickCalcs software. All the data collected from the study was analyzed blinded to the treatment groups and the statistical analysis was run with GrapPad Prim (version 5, GraphPad Software Inc, La Jolla, CA, USA) using statistical tests indicated in the figure legends to compare means of interest assuming 2-sided distribution. Animals with visible hemorrhages in MRI-images, unsuccessful MCAO or health-related problems were excluded from studies. Statistical significance level was set at was assumed if p<0.05.

5 Results

5.1 BEXAROTENE IS PROTECTIVE IN A MOUSE MODEL COMBINING AGING, TAU-PATHOLOGY AND THROMBOEMBOLIC STROKE BY MODULATING AUTOPHAGY (I)

Study (I) was carried out to study therapeutic potential of bexarotene in thromboembolic model of stroke using wildtype and P301L-Tau transgenic mice. Outcome was assessed using MRI and automated gait analysis. Samples collected from animals were analyzed by Western blotting and immunohistochemical analyses to detect the treatment effect on pathologic cascades. We also used primary neurons and cell line cells carrying the same mutation as the animals to further specify the mechanism of action of bexarotene.

5.1.1 Bexarotene protects old P301L-Tau transgenic animals against stroke

To determine whether bexarotene affects lesion volume after stroke, the lesion size of the ischemic animals was measured by MRI 3 days after thromboembolic MCAO. Volumetric analysis of MRI images showed that P301L-Tau animals that had received bexarotene after surgery had significantly smaller lesion sizes compared to their vehicle-treated littermates (Figure 1). No treatment effect was seen in wildtype animals. Moreover, there was no indication of different lesion sizes between vehicle-treated wildtype and P301L-Tau transgenic animals. To determine whether bexarotene modulates functional outcome, the motor function of the mice was tested by CatWalk gait analysis. A slight but significant improvement was observed in the CatWalk gait performance in the bexarotene-treated P301L-Tau transgenic mice in those parameters that were affected 7 days after stroke (Figure 3). Moreover, wildtype animals did not show deficits, or a treatment effect in important limb use parameters that reflect sensory and motor cortex circuit damage. No genotype or treatment-related differences were seen in blood parameters measured at 2 h after ischemia (data not shown).

5.1.2 Neuroprotective effect of bexarotene against excitotoxicity in vitro is not genotype-specific

The potential protective effect of bexarotene on glutamate-induced excitotoxicity was studied using the MTT viability assay in primary cortical neurons. 400 µM glutamate exposure resulted in approximately 50 % reduction in cell viability. A 2-h bexarotene pre-treatment prior to the addition of glutamate was protective both in wildtype and P301L-Tau neurons, increasing the viability up to 70 % (Figure 2.).

5.1.3 Bexarotene has no effect on tau, Aβ, MMP-9, gliosis and the peripheral immune response in aged, ischemic mice.

Close examination of brains after stroke revealed that bexarotene had no effect on tau pathology. This lack of effect was shown with the peri-ischemic samples of P301L-Tau animals 12 days after stroke by Western blotting using antibodies against total and Thr205-phosphorylated tau (Figure 4). Similar results were obtained in immunohistochemical stainings of stroked animals of both genotypes with the AT100 antibody that recognizes tau

phosphorylated at Ser212 and Thr214 (Figure S1). These results thus indicated that bexarotene did not mediate its ischemia-protecting effect through modulation of tau in the P301L-Tau transgenic mice.

Bexarotene has been previously shown to increase ApoE-mediated clearance of Aβ and alleviate adjacent inflammatory reactions in the brain (Cramer, Cirrito et al. 2012). Thus, a pan-Aβ immunohistochemical staining was performed for the ischemic brains at 12 days after stroke, but no treatment effect was observed (Figure S2). An increase in ApoE levels was, however, confirmed by Western blotting in the brains of P301L-Tau transgenic animals treated with bexarotene (data not shown). Similarly, the lack of a treatment effect was evident in further immunohistochemical stainings with the reactive microglia/monocyte marker Iba1 and the astrocyte marker GFAP at the same time point (Figure S2).

It has been demonstrated that the neuroprotective effect of bexarotene in stroke is mediated by modulation of immune responses in young animals (Certo, Endo et al. 2015). To assess this effect, flow cytometry was used to measure the amount and phenotype of neutrophils in the spleens of old animals 3 days after stroke. In addition, Ym-1/neutrophil double staining was performed on the brain sections at 1 dpi. No treatment effect was observed in either of the two analyses (Figures S3 and S4).

5.1.4 LC3b levels are reduced in ischemic P301L-Tau mice and this drop in protein expression is reversed by bexarotene treatment

Next, immunohistochemistry and Western blotting were applied to inspect the effect of bexarotene on cell death and survival markers. Staining of mouse brains with cleaved caspase-3 antibody at 12 days after ischemia showed no treatment or genotype-related effects (data not shown). However, there was a striking increase in the Lc3b immunoreactive area in ischemic, bexarotene-treated P301L-Tau animal brains, and based on morphology, this immunoreactivity accumulated in peri-ischemic neurons (Figure 5A). No effect was seen in wildtype animals. This finding was confirmed by Western blotting using peri-ischemic samples, which revealed a drop in LC3b-II levels in vehicle-treated P301L-Tau transgenic animals after stroke. This reduction in LC3b-II was normalized by bexarotene treatment (Figure 5B).

5.1.5 Bexarotene-induced increases in autophagy markers in N2a cells carrying the P301L-Tau mutation is caused by inhibition of autophagy flux

Because autophagy is a dynamic process, N2a cells with and without P301L-Tau mutation were used to decipher in more detail the effect of bexarotene on LC3b. In line with in vivo findings, a robust increase in LC3b-II, but also in p62, was seen in P301L-Tau cells after bexarotene treatment. In some cases, increased expression of p62 can already be described by decreased speed of autophagy flux (Bjorkoy, Lamark et al. 2009). Bexarotene treatment had no significant effect on LC3b-II or p62 in wildtype N2a cells (Figure 6). Beclin-1 and tau remained unaltered in both cell types (data not shown). Furthermore, results of the autophagy flux assay indicate that there is no basal level difference in the speed of autophagy between the genotypes, but bexarotene significantly lowers the speed of autophagy flux in cells carrying the P301L-Tau mutation (Figure 7).

5.1.6 P301L-Tau mutation alters bioenergetic functions of neurons and these alterations are alleviated by bexarotene treatment

Autophagy has an important role in cellular respiration through quality control of mitochondria (Ding, Gao et al. 2015). Because bexarotene had a significant effect on autophagy flux, primary cortical neurons were exposed to a mitochondrial stress with simultaneous measurement of oxygen consumption rate. P301L-Tau transgenic cortical neurons had a significantly lower ATP and oxidative phosphorylation-linked oxygen consumption rates (Figure 8A). These genotype-related differences in respiratory performance were normalized with bexarotene-pretreatment (Figure 8B). Despite the beneficial effect on cellular bioenergetics in primary cortical neurons, bexarotene did not affect ROS production of N2a cells as measured by the CellRox flow cytometry assay (Figure S5).

5.2 COPPER BIS(THIOSEMICARBAZONE) COMPLEXES MODULATE EXPERIMENTAL NEUROINFLAMMATION IN VITRO AND IN VIVO BY INCREASING METALLOTHIONEIN 1 (II)

We performed a study (II) to assess the therapeutic effect of copper delivery in acute (initiated with peripheral LPS injection into mice) and chronic inflammatory conditions (mice with AD pathology). The inflammatory status of the animals was measured using VCAM-1 expression with contrast MRI in acute inflammation, and with RT-PCR for cytokines in AD mice.

Primary microglia and astrocyte cultures were used to detect treatment effects on individual cell types using PCR, cytokine measurements, NO measurement, mass spectrometry and microscopy.

5.2.1 Copper bis(thiosemicarbazone) complexes scavenge acute and chronic neuroinflammation

Peripheral LPS administration is known to induce inflammation also in brain which in turn increases VCAM-1 expression in endothelial cells reflecting inflammatory status of brain (Gamal, Moawad et al. 2015). More importantly, in a neuroinflammation model of peripheral LPS injection VCAM-1 expression correlates with behavioral deficits thus allowing the peripheral monitoring of both CNS inflammation and brain function (Gamal, Moawad et al.

2015). VCAM-1 expression was measured by conjugating a VCAM-1 antibody to microsized particles of iron oxide (MPIO), injecting this conjugate intravenously into animals, and measuring hypointense brain areas in MRI images. Peripheral LPS induced a clear increase in brain signal area, indicating elevated vascular VCAM-1 expression 24 h after induction of peripheral inflammation (Figure 1). The signal was significantly reduced by p.o.

administration of Cu^II(atsm) 2 h after LPS injection.

Another copper delivery complex, Cu^II(gtsm), was previously demonstrated to have a beneficial effect in AD mouse models (Crouch, Hung et al. 2009). The anti-inflammatory potential of this compound was tested in APdE9 mice modelling AD by using quantitative RT-PCR due to lack of MRI-detectable VCAM-pathology. Results show that transcription of anti-inflammatory proteins Arginase-1 and TGF-β was increased in Cu^II(gtsm)-treated APdE9-animals (Figure 2). Transcription of pro-inflammatory MCP-1, IL-1, NOS2 and TNFα remained unaltered (data not shown). Taken together, these results indicated that copper delivery has anti-inflammatory effects in both acute and chronic inflammation.

5.2.2 Both microglia and astrocytes gain a less pro-inflammatory phenotype in the presence of Cu^II(atsm) in vitro

Cell-specific anti-inflammatory effects were tested using primary microglia and astrocyte cultures. An inflammatory milieu was induced in these models by the addition of pro-inflammatory cytokines IFN-γ and TNFα, as well as LPS (for astrocytes). Minocycline was used as a positive control as it has anti-inflammatory effects on microglia (Tikka, Fiebich et al. 2001). Co-stimulation of microglia with IFN-γ and TNFα increased NO production, which was reduced with low-dose co-treatment with Cu^II(atsm) (Figure 3). Cu^II(atsm) co-treatment also reduced MCP-1 transcription and secretion into the media, and the compound was more potent than minocycline in this assay. In addition, TNFα expression was reduced by Cu^II(atsm) treatment.

Further studies with astrocytes revealed that Cu^II(atsm) also reduced astrocytic production of MCP-1 during IFN-γ and TNFα co-stimulation (Figure 4). This effect was similar during LPS stimulation, but in addition, Cu^II(atsm) also reduced IL-6 secretion. Cu^II(atsm) was toxic for neither microglia nor astrocytes based on MTT assays (Figures 3 and 5).

5.2.3 Cu^II(atsm) increases the copper content of cytokine-stimulated cells

Increased copper content by Cu^II(atsm) treatment in primary microglia and neonatal astrocytes during IFN-γ and TNFα co-stimulation was confirmed with ICP-MS (Figure 6), indicating that the complex delivers copper into cells. Furthermore, X-ray fluorescence was used to demonstrate an elevation in microglial copper content during combined Cu^II(atsm) and IFN-γ/TNFα exposure.

5.2.4 Anti-inflammatory effects of Cu^II(atsm) are mediated by metallothionein 1

As a metal-binding protein with anti-inflammatory and antioxidant properties, metallothionein 1 was a suspected mediator of the beneficial effects of Cu^II(atsm). Indeed, both cytokine stimulated and unstimulated primary microglia treated with Cu^II(atsm) had increased levels of MT1 mRNA (Figure 7). Moreover, the anti-inflammatory effect of Cu^II(atsm) was abolished in primary microglia treated with propargyl glycine, an inhibitor of metallotionein synthesis. In this case, both the reduction in MCP-1 levels and NO release were absent, indicating that metallothionein 1 is important in the observed anti-inflammatory effect of Cu^II(atsm).

5.3 CU^II(ATSM) IMPROVES OUTCOME IN PERMANENT AND TRANSIENT MODELS OF CEREBRAL ISCHEMIA AND MODIFIES MICROGLIAL ACTIVATION (III)

In study (III) we tested therapeutic potential of Cu^II(atsm) in transient and permanent models of ischemic stroke in mice. The extent of brain injury was measured with MRI, histochemical stainings and behavioral assessment. We also used primary cortical neurons to study the effect of Cu^II(atsm) on glutamate-induced excitotoxicity. The effect of Cu^II(atsm) on post-stroke neuroinflammation was further analyzed by various methods, including immunohistochemistry, FACS and CBA.

5.3.1 Cu^II(atsm) protects cortical neurons against excitotoxicity and N2a cells against OGD in vitro

The effect of Cu^II(atsm) on excitotoxicity was studied using primary cortical neurons. In this assay, exposure to 400 µM resulted in 40 % decrease in cell viability in 24 h (Figure 1A). Co- or 2-h post-treatment with Cu^II(atsm) protected neurons partially from glutamate-induced excitotoxicity based on MTT assay, bringing the cell viability up to 70 %. Similarly, viability of N2a cells exposed to 24h OGD was reduced by 50 %, and 0,1 µM Cu^II(atsm) pretreatment had a slight protective effect, increasing the viability to 55 % (Figure 1B).

5.3.2 Cu^II(atsm) is protective in a mouse model of transient ischemia

Cu^II(atsm) was first tested in the transient MCAO model of cerebral ischemia. We found that Cu^II(atsm) treatment (15 mg/kg) both prior to MCAO and at the start of reperfusion reduced infarct sizes 24 h later (Figure 2). MCAO-induced functional impairment (assessed with neuroscore with a 5-point scale) was also alleviated by pre-treatment with Cu^II(atsm).

5.3.3 Cu^II(atsm) reduces ischemic damage in a permanent model of stroke

The therapeutic potential of Cu^II(atsm) was also tested in a permanent ischemia model. It was found out that p.o. 2 h post-treatment with 60 mg/kg significantly reduced lesion size as assessed by MRI at 24 h after stroke (Figure 3). In addition, co-treatment with Cu^II(atsm) during ischemia improved neurological status of the animals by reducing time spent in the latency to move testing at 1 dpi. Cu^II(atsm)-mediated copper delivery to the brain was confirmed using ICP-MS - animals treated with the Cu^II(atsm) complex had a significantly higher concentration of copper in the peri-ischemic area than vehicle treated controls.

5.3.4 Cu^II(atsm) reduces CD45 expression in cells located in the ischemic core

Copper bis(thiosemicarbazone) complexes have been described to modulate inflammatory reactions in animal models of neurological diseases (Crouch, Hung et al. 2009). To study the anti-inflammatory potential of Cu^II(atsm) in ischemia we applied immunohistochemical techniques to brain sections collected from animals with permanent MCAO. Astrocytic reactivity was not affected based on GFAP staining (data not shown). Instead, Cu^II(atsm) treatment significantly reduced the amount of CD45 immunoreactivity in the ischemic core both at 1 and 3 dpi (Figure 4). CD45 is highly expressed by monocytes and a lower expression is typical for reactive microglia.

5.3.5 Iba1 expression is reduced and the morphology of Iba-1 positive cells is altered by Cu^II(atsm) treatment after ischemia

Next, a set of immunohistochemical stainings were performed to distinguish the inflammatory cell types affected by Cu^II(atsm) treatment in mice that underwent permanent MCAO. No differences between treatment groups were found based on cells expressing CD68 (marker used for phagocytotic cells) or Arginase-1 (alternatively activated cells) (data not shown). However, reduced Iba1 immunoreactivity was observed 3 dpi in the peri-ischemic area of Cu^II(atsm) treated animals (Figure 5). Because microglial morphology and function are linked, a computer algorithm was used to analyse the morphology of Iba1-positive cells in detail. Results indicate that Cu^II(atsm) did not affect cellular branches, but significantly increased the cellular area of Iba-1 positive cells.

5.3.6 The proportion of resident microglia is increased in Cu^II(atsm) treated ischemic brains

To gain further information about the alterations in proportions of different inflammatory cell populations in the ischemic brain following Cu^II(atsm) treatment, cells were isolated from the ischemic hemisphere and analysed by flow cytometry 3 days after permanent MCAO.

The proportions of multiple cell types, such as lymphocytes, neutrophils and myeloid cells, were affected by stroke. Cu^II(atsm) increased the proportion of resting microglia, characterized by CD45^low CD11b⁺ Ly6G^- indicating reduced microglial activation, increased viability or increased proliferaton (Figure 6).

5.3.7 Cu^II(atsm) has beneficial effects on further inflammatory markers after ischemia Finally, immunohistochemistry and CBA were used to detect the effects of Cu^II(atsm) on inflammatory cascades. Activated p38 MAPK, a major signalling molecule mediating inflammatory activation in microglia, was found to be reduced in Cu^II(atsm) treated animals 1 day after permanent MCAO (Figure 7). We also saw a reduction in IL-12 levels at the same time point and increased IL-10 levels 3 days after ischemia.

5.4 COLLAGEN XV DEFICIENCY AMELIORATES ISHCEMIC DAMAGE IN MICE (IV)

Study (IV) aimed to assess the role of collagen XV in thromboembolic stroke. Sham and stroke animals received either vehicle or rtPA and underwent MRI for lesion size quantification.

Western blotting, cytokine measurements and immunohistrochemistry were used to detect genotype and treatment-related differences in cytokines and proteins (e.g. collagen XV and VEGF-A).

5.4.1 Α1-Collagen XV deficiency protects against thromboembolic stroke

A mechanistically relevant model of thromboembolic stroke was used to test rtPA-treatment and genotype effect on MRI-measured outcome in mice 2 days after stroke. Thrombolysis with tPa 20 minutes after MCAO significantly reduced lesion size in wildtype animals (Figure 1). However, α1-ColXV KO mice had significantly smaller lesion sizes than wildtype animals and there was no therapeutic effect of rtPA on lesion size in these animals.

To exclude obvious genotype-mediated reasons for the observed protection, cerebral vasculature, blood parameters and cerebral edema of the mice were assessed. No differences were detected between treatment groups in either cerebral vasculature or blood parameters (such as partial pressures of blood gases, Figure 2). Also, brain swelling and aquaporin-4 immunoreactivity (water channel contributing to increased edema) following stroke were similar in all groups (Figure 3).

5.4.2 RtPA increases Collagen XV levels in the plasma of WT mice but not in α1-Collagen XV KO mice.

Next, we used ELISA to measure the protein levels of ColXV in the plasma of WT and α1-ColXV KO mice with or without rtPA treatment 3 days after stroke. Interestingly, we found

that rtPA tends to increase α1-ColXV protein levels after ischemia in the plasma of WT mice (Figure 4).

5.4.3 rtPA treatment does not reduce post-stroke cytokine production in α1-Collagen XV KO mice

Immunohistochemical examination of brains with markers for astrocyte and microglial/monocyte activation (GFAP and Iba1) at 3 days after stroke did not show any

Immunohistochemical examination of brains with markers for astrocyte and microglial/monocyte activation (GFAP and Iba1) at 3 days after stroke did not show any

In document Neurogliavascular remodeling and therapeutic intervention in ischemia and inflammation (sivua 65-0)