Fluorometric analysis of the assembly of the calcium-permeable cation channel TRPC6

In Analogie zu anderen 6-Transmembrandomänen-Kanälen lagern sich TRPC- Untereinheiten zu homo- und heterotetrameren Komplexen zusammen, was bisher mittels biochemischer Methoden sowie durch funktionelle Untersuchungen belegt werden konnte. Diese Protein-Interaktionsnachweise waren häufig mit der Zerstörung der untersuchten Zellen verbunden oder blieben auf die Untersuchung plasmamembranständiger Kanalkomplexe beschränkt, womit dynamische Untersuchungen an lebenden Zellen oder Aussagen über die Kanalassemblierung in subzellulären Kompartimenten nicht möglich waren. In letzter Zeit wird der Fluoreszenz-Resonanz-Energietransfer (FRET) vermehrt zur Darstellung von Protein-Protein-Interaktion in lebenden Zellen genutzt. Im Rahmen der vorliegenden Arbeit wurden weitere Einsatzmöglichkeiten des FRET zur Untersuchung von Proteininteraktionen dargestellt und auf ihre Validität überprüft. Die räumlich aufgelöste FRET-Darstellung zu verschiedenen Zeitpunkten der Proteinexpression belegt die frühe Zusammenlagerung der TRPC6-Untereinheiten im endoplasmatischen Retikulum und im Golgi-Apparat. Die konfokale FRET-Darstellung bestätigt FRET-Signale über den Endomembranen bei hoher räumlicher Auflösung. Aufgrund der durch fluorescence recovery after photobleaching (FRAP) belegten Mobilität der TRPC6-Kanäle innerhalb der Plasmamembran erfolgte die konfokale FRET-Darstellung zur Minimierung von Bewegungsartefakten auf Basis eines spektralen Dekonvolutionsverfahrens. Die Quantifizierung der FRET-Effizienz erfolgte unter Berücksichtigung der photophysikalischen Eigenschaften der verwendeten Fluoreszenproteine mittels Akzeptor-Bleichen im Rahmen der digitalen Videomikroskopie. Der hier erbrachte, nicht-destruktive Assemblierungsnachweis ermöglicht die Untersuchung der Dynamik der Kanalassemblierung (z.B. nach Agonisten-Zugabe) auch in subzellulären Kompartimenten. Die schwache Abhängigkeit der FRET- Effizienz vom Expressionsniveau der fluoreszierenden TRPC6-Untereinheiten belegt die Spezifität dieses Protein-Protein-Interaktionsnachweises. Die Wahrscheinlichkeit eines FRET-Signals als Folge lokaler Proteinanhäufungen war angesichts der konfokalmikroskopisch belegten homogenen Membranverteilung und der oben genannten Mobilität der fluoreszierenden TRPC6-Untereinheiten innerhalb der Plasmamembran als sehr gering einzustufen. Der hier vorgestellte kompetitive FRET-Nachweis belegt die Entkopplung von FRET zwischen fluoreszierenden TRPC6-Untereinheiten durch koexprimierte TRPC6-Wildtyp- Untereinheiten nicht jedoch durch TRPC4 beta. Er ist somit nicht nur ein weiterer Spezifitätsnachweis für den FRET, sondern ermöglicht den Interaktionsnachweis zwischen mehr als zwei verschiedenen Proteinen in nur einem Messschritt unter zusätzlicher Berücksichtigung ihrer Affinität zueinander. Dieses Verfahren kann auch die Untersuchung der Dynamik von Affinitäten zwischen unterschiedlichen Untereinheiten eines Proteinkomplexes z.B. nach Agonistenzugabe ermöglichen. Analyse der Stöchiometrie des TRPC6 Die hier vorgestellte Methode der Stöchiometrie-Analyse durch Korrelation von FRET-Effizienz und dem quantitativen Verhältnis Donor- zu Akzeptorfluorochrom- markierter TRPC6-Untereinheiten deutet auf eine tetramere Struktur des TRPC6 und deckt sich mit Ergebnissen aus biochemischen und FRETbasierten Multimerisierungsuntersuchungen der TRP-Familie. Im Gegensatz zu den meisten bisher publizierten Multimerisierungsnachweisen gestattet das hier vorgestellte Verfahren Beurteilungen von Proteinstöchiometrien sowohl in der Plasmamembran als auch in subzellulären Kompartimenten lebender Zellen und schließt die Möglichkeit von Untersuchungen einer Dynamik von Proteinstöchiometrien ein.By analogy to other cation channel subunits with six transmembrane-spanning domains, the TRPC channels are believed to assemble into homo- or heterotetrameric complexes. These complexes have been verified by biochemical or functional methods related to cell destruction or restricted to surface proteins. Thus dynamic analyses in living cells or analyses of the assembly of channel subunits in subcellular compartiments were not possible. More recently, fluorescence resonance energy transfer (FRET) - a measure for the close proximity of fluorescent molecules - has become instrumental in following protein assembly in living cells. This work demonstrates further possibilities and verification procedures of the FRET-technology to test the assembly of ion channel subunits. Temporally and spatially resolved FRET imaging demonstrates an early assembly of TRPC subunits in the endoplasmic reticulum and the Golgi apparatus. Due to the mobilitly of TRPC6-channels within the plasma membrane proven via fluorescence recovery after photobleaching (FRAP), TRPC-subunit interaction in subcellular compartiments was analysed via confocal FRET imaging based on a spectral deconvolution method. It verifies FRET signals over the plasma membrane at high spatial resolution.The non-destructive proof of subunit assembly presented in this work allows to study the dynamic of protein-interaction even in subcellular compartiments. Quantitative analysis of digital video imaging demonstrates that FRET between TRPC subunits is only weakly concentration-dependent. Thus FRET can be considered as specific proof of interaction between TRPC subunits. Moreover, the introduction of a competition-FRET assay allows to test the ability of wild-type TRPC subunits to recruit fluorescent TRPC subunits into separate channel complexes and offers the possibility to test the interaction between more than two different proteins as well as the affinity between different fluorescent proteins. Finally, correlation between the efficiency of energy transfer and the molar ratio of the FRET donor to the acceptor was exploited to verify the tetrameric stoichiometry of TRPC complexes

European Cooperative Acute Stroke Study-4: Extending the time for thrombolysis in emergency neurological deficits ECASS-4: ExTEND

Abstract Rationale and hypothesis: Thrombolytic therapy with recombinant tissue plasminogen activator (rt-PA) is an effective and approved therapy for acute ischemic stroke within 4.5 h of onset except for USA, Canada, Croatia, and Moldovia with a current 3 h label. We hypothesized that ischemic stroke patients selected with significant penumbral mismatch on magnetic resonance imaging (MRI) at 4.5–9 h after onset of stroke will have improved clinical outcomes when given intravenous rt-PA (alteplase) compared to placebo. Study design: ECASS-4: ExTEND is an investigator driven, phase 3, randomized, multi-center, double-blind, placebocontrolled study. Ischemic stroke patients presenting within 4.5 and 9 h of stroke onset, who fulfil clinical requirements (National Institutes of Health Stroke Score (NIHSS) 4–26 and pre-stroke modified Rankin Scale (mRS) 0–1) will undergo MRI. Patients who meet imaging criteria (infarct core volume <100 ml, perfusion lesion: infarct core mismatch ratio >1.2 and perfusion lesion minimum volume of 20 ml) additionally will be randomized to either rt-PA or placebo. Study outcome: The primary outcome measure will be the categorical shift in the mRS at day 90. Clinical secondary outcomes will be disability at day 90 dichotomized as favorable outcome mRS 0–1 at day 90. Tertiary endpoints include reduction in the NIHSS by 11 or more points or reaching 0–1 at day 90, reperfusion and recanalization at 24 h post stroke as well as depression, life quality, and cognitive impairment at day 90. Safety endpoints will include symptomatic intracranial hemorrhage (ICH) and death

Intravenous thrombolysis and platelet count.

To study the effect of platelet count (PC) on bleeding risk and outcome in stroke patients treated with IV thrombolysis (IVT) and to explore whether withholding IVT in PC < 100 × 10 <sup>9</sup> /L is supported. In this prospective multicenter, IVT register-based study, we compared PC with symptomatic intracranial hemorrhage (sICH; Second European-Australasian Acute Stroke Study [ECASS II] criteria), poor outcome (modified Rankin Scale score 3-6), and mortality at 3 months. PC was used as a continuous and categorical variable distinguishing thrombocytopenia (<150 × 10 <sup>9</sup> /L), thrombocytosis (>450 × 10 <sup>9</sup> /L), and normal PC (150-450 × 10 <sup>9</sup> /L [reference group]). Moreover, PC < 100 × 10 <sup>9</sup> /L was compared to PC ≥ 100 × 10 <sup>9</sup> /L. Unadjusted and adjusted odds ratios (ORs) with 95% confidence intervals (CIs) from the logistic regression models were calculated. Among 7,533 IVT-treated stroke patients, 6,830 (90.7%) had normal PC, 595 (7.9%) had thrombocytopenia, and 108 (1.4%) had thrombocytosis. Decreasing PC (every 10 × 10 <sup>9</sup> /L) was associated with increasing risk of sICH (OR <sub>adjusted</sub> 1.03, 95% CI 1.02-1.05) but decreasing risk of poor outcome (OR <sub>adjusted</sub> 0.99, 95% CI 0.98-0.99) and mortality (OR <sub>adjusted</sub> 0.98, 95% CI 0.98-0.99). The risk of sICH was higher in patients with thrombocytopenic than in patients with normal PC (OR <sub>adjusted</sub> 1.73, 95% CI 1.24-2.43). However, the risk of poor outcome (OR <sub>adjusted</sub> 0.89, 95% CI 0.39-1.97) and mortality (OR <sub>adjusted</sub> 1.09, 95% CI 0.83-1.44) did not differ significantly. Thrombocytosis was associated with mortality (OR <sub>adjusted</sub> 2.02, 95% CI 1.21-3.37). Forty-four (0.3%) patients had PC < 100 × 10 <sup>9</sup> /L. Their risks of sICH (OR <sub>unadjusted</sub> 1.56, 95% CI 0.48-5.07), poor outcome (OR <sub>adjusted</sub> 1.63, 95% CI 0.82-3.24), and mortality (OR <sub>adjusted</sub> 1.38, 95% CI 0.64-2.98) did not differ significantly from those of patients with PC ≥ 100 × 10 <sup>9</sup> /L. Lower PC was associated with increased risk of sICH, while higher PC indicated increased mortality. Our data suggest that PC modifies outcome and complications in individual patients, while withholding IVT in all patients with PC < 100 × 10 <sup>9</sup> /L is challenged

Quantitative Signal Intensity in Fluid-Attenuated Inversion Recovery and Treatment Effect in the WAKE-UP Trial

International audienceBackground and Purpose— Relative signal intensity of acute ischemic stroke lesions in fluid-attenuated inversion recovery (fluid-attenuated inversion recovery relative signal intensity [FLAIR-rSI]) magnetic resonance imaging is associated with time elapsed since stroke onset with higher intensities signifying longer time intervals. In the randomized controlled WAKE-UP trial (Efficacy and Safety of MRI-Based Thrombolysis in Wake-Up Stroke Trial), intravenous alteplase was effective in patients with unknown onset stroke selected by visual assessment of diffusion weighted imaging fluid-attenuated inversion recovery mismatch, that is, in those with no marked fluid-attenuated inversion recovery hyperintensity in the region of the acute diffusion weighted imaging lesion. In this post hoc analysis, we investigated whether quantitatively measured FLAIR-rSI modifies treatment effect of intravenous alteplase. Methods— FLAIR-rSI of stroke lesions was measured relative to signal intensity in a mirrored region in the contralesional hemisphere. The relationship between FLAIR-rSI and treatment effect on functional outcome assessed by the modified Rankin Scale (mRS) after 90 days was analyzed by binary logistic regression using different end points, that is, favorable outcome defined as mRS score of 0 to 1, independent outcome defined as mRS score of 0 to 2, ordinal analysis of mRS scores (shift analysis). All models were adjusted for National Institutes of Health Stroke Scale at symptom onset and stroke lesion volume. Results— FLAIR-rSI was successfully quantified in stroke lesions in 433 patients (86% of 503 patients included in WAKE-UP). Mean FLAIR-rSI was 1.06 (SD, 0.09). Interaction of FLAIR-rSI and treatment effect was not significant for mRS score of 0 to 1 ( P =0.169) and shift analysis ( P =0.086) but reached significance for mRS score of 0 to 2 ( P =0.004). We observed a smooth continuing trend of decreasing treatment effects in relation to clinical end points with increasing FLAIR-rSI. Conclusions— In patients in whom no marked parenchymal fluid-attenuated inversion recovery hyperintensity was detected by visual judgement in the WAKE-UP trial, higher FLAIR-rSI of diffusion weighted imaging lesions was associated with decreased treatment effects of intravenous thrombolysis. This parallels the known association of treatment effect and elapsing time of stroke onset

Functional Outcome of Intravenous Thrombolysis in Patients With Lacunar Infarcts in the WAKE-UP Trial

Importance: The rationale for intravenous thrombolysis in patients with lacunar infarcts is debated, since it is hypothesized that the microvascular occlusion underlying lacunar infarcts might not be susceptible to pharmacological reperfusion treatment. Objective: To study the efficacy and safety of intravenous thrombolysis among patients with lacunar infarcts. Design, Setting, and Participants: This exploratory secondary post hoc analysis of the WAKE-UP trial included patients who were screened and enrolled between September 2012 and June 2017 (with final follow-up in September 2017). The WAKE-UP trial was a multicenter, double-blind, placebo-controlled randomized clinical trial to study the efficacy and safety of intravenous thrombolysis with alteplase in patients with an acute stroke of unknown onset time, guided by magnetic resonance imaging. All 503 patients randomized in the WAKE-UP trial were reviewed for lacunar infarcts. Diagnosis of lacunar infarcts was based on magnetic resonance imaging and made by consensus of 2 independent investigators blinded to clinical information. Main Outcomes and Measures: The primary efficacy variable was favorable outcome defined by a score of 0 to 1 on the modified Rankin Scale at 90 days after stroke, adjusted for age and severity of symptoms. Results: Of the 503 patients randomized in the WAKE-UP trial, 108 patients (including 74 men [68.5%]) had imaging-defined lacunar infarcts, whereas 395 patients (including 251 men [63.5%]) had nonlacunar infarcts. Patients with lacunar infarcts were younger than patients with nonlacunar infarcts (mean age [SD], 63 [12] years vs 66 [12] years; P = .003). Of patients with lacunar infarcts, 55 (50.9%) were assigned to treatment with alteplase and 53 (49.1%) to receive placebo. Treatment with alteplase was associated with higher odds of favorable outcome, with no heterogeneity of treatment outcome between lacunar and nonlacunar stroke subtypes. In patients with lacunar strokes, a favorable outcome was observed in 31 of 53 patients (59%) in the alteplase group compared with 24 of 52 patients (46%) in the placebo group (adjusted odds ratio [aOR], 1.67 [95% CI, 0.77-3.64]). There was 1 death and 1 symptomatic intracranial hemorrhage according to Safe Implementation of Thrombolysis in Stroke-Monitoring Study criteria in the alteplase group, while no death and no symptomatic intracranial hemorrhage occurred in the placebo group. The distribution of the modified Rankin Scale scores 90 days after stroke also showed a nonsignificant shift toward better outcomes in patients with lacunar infarcts treated with alteplase, with an adjusted common odds ratio of 1.94 (95% CI, 0.95-3.93). Conclusions and Relevance: While the WAKE-UP trial was not powered to demonstrate the efficacy of treatment in subgroups of patients, the results indicate that the association of intravenous alteplase with functional outcome does not differ in patients with imaging-defined lacunar infarcts compared with those experiencing other stroke subtypes.status: publishe

Intravenous alteplase for stroke with unknown time of onset guided by advanced imaging: systematic review and meta-analysis of individual patient data

Background: Patients who have had a stroke with unknown time of onset have been previously excluded from thrombolysis. We aimed to establish whether intravenous alteplase is safe and effective in such patients when salvageable tissue has been identified with imaging biomarkers. Methods: We did a systematic review and meta-analysis of individual patient data for trials published before Sept 21, 2020. Randomised trials of intravenous alteplase versus standard of care or placebo in adults with stroke with unknown time of onset with perfusion-diffusion MRI, perfusion CT, or MRI with diffusion weighted imaging-fluid attenuated inversion recovery (DWI-FLAIR) mismatch were eligible. The primary outcome was favourable functional outcome (score of 0–1 on the modified Rankin Scale [mRS]) at 90 days indicating no disability using an unconditional mixed-effect logistic-regression model fitted to estimate the treatment effect. Secondary outcomes were mRS shift towards a better functional outcome and independent outcome (mRS 0–2) at 90 days. Safety outcomes included death, severe disability or death (mRS score 4–6), and symptomatic intracranial haemorrhage. This study is registered with PROSPERO, CRD42020166903. Findings: Of 249 identified abstracts, four trials met our eligibility criteria for inclusion: WAKE-UP, EXTEND, THAWS, and ECASS-4. The four trials provided individual patient data for 843 individuals, of whom 429 (51%) were assigned to alteplase and 414 (49%) to placebo or standard care. A favourable outcome occurred in 199 (47%) of 420 patients with alteplase and in 160 (39%) of 409 patients among controls (adjusted odds ratio [OR] 1·49 [95% CI 1·10–2·03]; p=0·011), with low heterogeneity across studies (I 2=27%). Alteplase was associated with a significant shift towards better functional outcome (adjusted common OR 1·38 [95% CI 1·05–1·80]; p=0·019), and a higher odds of independent outcome (adjusted OR 1·50 [1·06–2·12]; p=0·022). In the alteplase group, 90 (21%) patients were severely disabled or died (mRS score 4–6), compared with 102 (25%) patients in the control group (adjusted OR 0·76 [0·52–1·11]; p=0·15). 27 (6%) patients died in the alteplase group and 14 (3%) patients died among controls (adjusted OR 2·06 [1·03–4·09]; p=0·040). The prevalence of symptomatic intracranial haemorrhage was higher in the alteplase group than among controls (11 [3%] vs two [<1%], adjusted OR 5·58 [1·22–25·50]; p=0·024). Interpretation: In patients who have had a stroke with unknown time of onset with a DWI-FLAIR or perfusion mismatch, intravenous alteplase resulted in better functional outcome at 90 days than placebo or standard care. A net benefit was observed for all functional outcomes despite an increased risk of symptomatic intracranial haemorrhage. Although there were more deaths with alteplase than placebo, there were fewer cases of severe disability or death. Funding: None