Interaction of ARC and Daxx: a novel endogenous target to preserve motor function and cell loss after focal brain ischemia in mice

The aim of this study was to explore the signaling and neuroprotective effect of transactivator of transcription (TAT) protein transduction of the apoptosis repressor with CARD (ARC) in in vitro and in vivo models of cerebral ischemia in mice. In mice, transient focal cerebral ischemia reduced endogenousARCprotein in neurons in the ischemic striatum at early reperfusion time points, and in primary neuronal cultures, RNA interference resulted in greater neuronal susceptibility to oxygen glucose deprivation (OGD).TAT.ARC protein delivery led to a dose-dependent better survival after OGD. Infarct sizes 72 h after 60 min middle cerebral artery occlusion (MCAo) were on average 30±8% (mean±SD; p=0.005; T2-weighted MRI) smaller in TAT.ARC-treated mice (1ug intraventricularly during MCAo) compared with controls. TAT.ARC-treated mice showed better performance in the pole test compared with TAT.β-Gal-treated controls. Importantly, post-stroke treatment (3 h after MCAo) was still effective in affording reduced lesion volume by 20±7% (mean±SD; p˃0.05) and better functional outcome compared with controls. Delayed treatment in mice subjected to 30 min MCAo led to sustained neuroprotection and functional behavior benefits for at least 28 d. Functionally, TAT.ARC treatment inhibited DAXX–ASK1–JNK signaling in the ischemic brain. ARC interacts with DAXX in a CARD-dependent manner to block DAXX trafficking and ASK1–JNK activation. Our work identifies for the first time ARC–DAXX binding to block ASK1–JNK activation as an ARC-specific endogenous mechanism that interferes with neuronal cell death and ischemic brain injury. Delayed delivery of TAT.ARC may present a promising target for stroke therapy

Dose response curve shows better survival of TAT.ARC protein transduced primary cortical neurons in comparison to TAT beta galactosidase transduced neurons after oxygen glucose deprivation

Primary mice cortical neuronal cultures on DIV10. OGD for 150 min. Analysis of cell survival after 24h. Exogenous ARC protein transduction leads to a dose-dependent increase in neuronal survival following OGD compared to TAT.β-Gal-treated neurons. 4 independent experiments

Delayed administration of TAT.ARC protein attenuates focal ischemic brain injury and fosters recovery in the long-term: behavioral data of neuroscores and rota rod tests, daily temperature and body weight measures

<p>Mice were subjected to 30 min MCAo or sham surgery and TAT.b-Gal or TAT.ARC were administered in the contralateral ventricle after 3h after the onset of ischemia and mice were observed over 28 days. DeSimoni Neuroscore was performed at indicated time points (Fig. 8) as described (De Simoni et al., 2003; Orsini et al., 2012) with some modifications. In brief, general health (Table 2) and specific focal assessments (Table 3) were separately scored, analyzed and finally added to form a summation score. Summative scores added up to  a maximum of 43 points with more points meaning more deficits. </p> <p>Rota rod was assessed as described recently (Hoffmann et al., 2015) and the best run out of three replicates at a given time point was used for statistical analysis. Body temperature was measured daily as survival was documented. </p> Body temperature was non-invasively assessed at the same time of the day prior to body weight measurements using subcutaneous transponders (IPTT-300, Bio Medic Data Systems), for unambiguous identification of mice in their home cages as described (Kort et al., 1998; Langer and Fietz, 2014)

Striatal volume and ventricle area was assessed in NeuN DAB sections at 28 days after 30 min MCAo in sham and MCAo mice treated with TAT proteins 3 hours after the onset of ischemia.

Striatal volume and ventricle area was assessed in NeuN DAB sections at 28 days after 30 min tMCAo in sham and MCAo mice treated with TAT proteins 3 hours after the onset of ischemia

Multiple image alignment of neuronal staining using anti-NeuN-DAB staining of sham mice and 28 days after 30 min MCAo and delayed treatment with 5 µg TAT.ARC or control protein.

Multiple image alignment of neuronal stainings using anti-NeuN-DAB of sham mice and 28 days after 30 min MCAo. Mouse brains were cut in coronal sections using a mouse brain atlas (Franklin and Paxinos, 2007) to analyze direct and indirect infarct volumes, striatal area in a3 (area 3) and neuronal densities within the striatum in a3: a1: Bregma = 2.80; a2: Bregma = 1.54; a3: Bregma = 0.14; a4: Bregma = -1.94; a5: Bregma = -3.88. Image collection of NeuN-DAB-stained brain slices from mice 28 days post MCAo were collected as transmission images using a Leica DMI8 microscope equipped with LED light source, a Fluotar 10x/030 dry objective  and a DFC300G camera and stitched within the Leica LAS X2.0 software. The code of mice ID can be found in the excel spread sheet attached. a1-a5 and mouse ID is used in the file name

Delayed administration of TAT.ARC protein attenuates focal ischemic brain injury: Mice were subjected to 60 min MCAo and 5 µg TAT.ARC or control protein was injected 3 h after the onset of ischemia and indirect lesion volume was assessed after 3 days.

Mice were treated 3h after the onset 60 min middle cerebral artery occlusion (MCAo) with TAT.b-Gal or TAT.ARC [5µg] in the contralateral ventricle of the brain and were perfused after 72 h. Infarkt volume assessment using haematoxylin staining and the following brain coordinates to select coronal slices. Mouse brains were cut in coronal sections using a mouse brain atlas (Franklin and Paxinos, 2007) to analyze direct and indirect infarct volumes, striatal area in a3 (area 3) and neuronal densities within the striatum in a3: a1 =  interaural 6.6 mm/ Bregma = 2.80; a2 =  interaural 5.34 mm/ Bregma = 1.54; a3 =  interaural 3.94 mm/ Bregma = 0.14; a4 =  interaural 1.86 mm/ Bregma = -1.94; a5 = interaural -0.08 mm/ Bregma = -3.88

TAT.ARC protein transduction or RNA interference with ARC impact on survival of primary cortical neurons subjected to 150 min oxygen-glucose deprivation (OGD): Lactate dehydrogenase activity was measured as a marker of cell membrane integrity loss and neuronal cell death 24 h afterwards.

Primary neuronal cultures were transduced with lentiviral particles for knockdown of apoptosis repressor with CARD domain (ARC) or non-targeting control or treated with TAT proteins for ARC or beta Galaktosidase before 150 min of oxygen glucose deprivation (OGD). Cell death was monitored by the release of lactate dehydrogenase (LDH) in the supernatant medium