Author(s): Thiruma V Arumugam [1, 8]; Sic L Chan [1, 8]; Dong-Gyu Jo [1, 8]; Gokhan Yilmaz ; Sung-Chun Tang [1, 3]; Aiwu Cheng ; Marc Gleichmann ; Eitan Okun ; Vishwa D Dixit ; Srinivasulu Chigurupati ; Mohamed R Mughal ; Xin Ouyang ; Lucio Miele ; Tim Magnus ; Suresh Poosala ; D Neil Granger ; Mark P Mattson (corresponding author) [1, 7]
Mice transgenic for antisense Notch and normal mice treated with inhibitors of the Notch-activating enzyme [gamma]-secretase showed reduced damage to brain cells and improved functional outcome in a model of focal ischemic stroke. Notch endangers neurons by modulating pathways that increase their vulnerability to apoptosis, and by activating microglial cells and stimulating the infiltration of proinflammatory leukocytes. These findings suggest that Notch signaling may be a therapeutic target for treatment of stroke and related neurodegenerative conditions.
Notch-1 (Notch) is a cell-surface receptor that regulates cell-fate decisions in the developing nervous system and may also have roles in synaptic plasticity in the adult brain [1, 2, 3]. Binding of ligands such as Delta and Jagged results in proteolytic cleavages of Notch: first in an extracellular domain and then in a transmembrane domain. The latter cleavage is accomplished by presenilin-1 and the [gamma]-secretase enzyme complex, resulting in the release of a Notch intracellular domain (NICD) that translocates to the nucleus, where it regulates transcription . Ischemic stroke results from damage and death of neurons in the perfusion territory of the affected blood vessel; the neurodegenerative mechanism involves metabolic and oxidative stress, excitotoxicity and apoptosis , and inflammatory processes including infiltration of activated leukocytes .
We measured activity of [gamma]-secretase and levels of NICD in brain tissue samples from mice transgenic for antisense Notch (NAS)  and nontransgenic control mice that had been subjected to ischemia-reperfusion induced by occlusion of the middle cerebral artery (Supplementary Methods online). Activity of [gamma]-secretase was significantly elevated 2 h after ischemia-reperfusion and returned to basal levels 6 h after ischemia-reperfusion (Fig. 1a). NICD levels were significantly elevated within 1 h of ischemia and remained so through 6 h (Fig. 1b,c). As expected, the NICD band was present in neuroblastoma cells overexpressing NICD, was absent from presenilin-deficient cells and was present in wild-type cells, where its levels increased in response to Jagged peptide (Fig. 1d).
We investigated the efficacy of [gamma]-secretase inhibitors in suppressing activation of Notch in the brain in vivo by measuring levels of NICD in cortical tissue samples from mice treated with the [gamma]-secretase inhibitors DAPT (N -[N -(3,5-difluorophenacetyl-L-alanyl)]-S-phenylglycine t -butyl ester)7 and DBZ ((S,S)-2-[2-(3,5-difluorophenyl)acetylamino]- N -(5-methyl-6-oxo-6,7-dihydro-5H-dibenzo[b,d]azepin-7-yl)propionamide) . The ischemia-reperfusion-induced increase in NICD was significantly less in NAS mice and in nontransgenic mice treated with either DAPT or DBZ, compared to vehicle-treated nontransgenic mice (Fig. 1e).
The amount of brain damage and neurological deficits resulting from ischemia-reperfusion, measured 3 d after ischemia-reperfusion, were significantly less in the NAS mice compared to nontransgenic mice (Fig. 1f,g). Treatment with DAPT or DBZ by intravenous infusion either 30 min before or...