Intravenous TAT-GDNF Is Protective After Focal Cerebral Ischemia in Mice

Abstract

Background and Purpose-Delivery of therapeutic proteins into tissues and across the blood-brain barrier is severely limited by their size and biochemical properties. The 11-amino acid human immunodeficiency virus TAT protein transduction domain is able to cross cell membranes and the blood-brain barrier, even when coupled with larger peptides. The present studies were done to evaluate whether TAT-glial line-derived neurotrophic factor (GDNF) fusion protein is protective in focal cerebral ischemia. Methods-Anesthetized male C57BL/6j mice were submitted to intraluminal thread occlusion of the middle cerebral artery. Reperfusion was initiated 30 minutes later by thread retraction. Laser Doppler flow was monitored during the experiments. TAT-GDNF, TAT-GFP (0.6 nmol each), or vehicle was intravenously applied over 10 minutes immediately after reperfusion. After 3 days (30 minutes of ischemia), animals were reanesthetized and decapitated. Brain injury was evaluated by histochemical stainings. Results-Immunocytochemical experiments confirmed the presence of TAT-GDNF protein in the brains of fusion protein-treated nonischemic control animals 3 to 4 hours after TAT fusion protein delivery. TAT-GDNF significantly reduced the number of caspase-3-immunoreactive and DNA-fragmented cells and increased the number of viable neurons in the striatum, where disseminated tissue injury was observed, compared with TAT-GFP-or vehicle-treated animals. Conclusions-Our results demonstrate that TAT fusion proteins are powerful tools for the treatment of focal ischemia when delivered both before and after an ischemic insult. This approach may be of clinical interest because such fusion proteins can be intravenously applied and reach the ischemic brain regions. This approach may therefore offer new perspectives for future strategies in stroke therapy.

Pretreatment with PTD-Calbindin D 28k Alleviates Rat Brain Injury Induced by Ischemia and Reperfusion

“…Overexpression of CaBD could decrease neuronal death after transient focal ischemia (Yenari et al, 2001). In contrast to the low transfection, PTD can deliver its cargo into brain cells more efficiently across the blood-brain barrier into neurons and astrocytes in the cerebral cortex, striatum, and medulla oblongata (Asoh et al, 2005;Cao et al, 2002;Kilic et al, 2003). After injection of 50 mg/mL PTDCaBD, we detected a number of anti-histidinepositive cells in the subventricular zone, cerebral cortex, and caudate putamen.…”

The taming of the cell penetrating domain of the HIV Tat: Myths and realities

“…Further, PTD-Bcl-xL demonstrated prevention of neuronal death resulting from transient focal ischemia [69]. In another successful in vivo study, Kilic et al [71] demonstrated that intravenous inoculation of PTD-GDNF fusion protein protected neurons from focal cerebral ischemic injury. Similarly, in a non-neurological model, Hotchkiss et al [77] showed that Tat-Bcl-xL fusion protein and PTD-BH4 peptide prevented E.coli-induced human lymphocyte apoptosis ex-vivo and markedly decreased lymphocyte apoptosis in an in-vivo mouse model of sepsis.…”

“…Subsequently, several other studies have also reported effective biodistribution of therapeutically important proteins in vivo by using PTD [52,[69][70][71][72][73][74][75]. In a previous study, however, no PTD mediated transduction in brain was observed, [46] possibly because beta-gal was chemically conjugated to PTD instead of recombinant fusion protein.…”

Effect of Synthetic Cell-Penetrating Peptides on TrkA Activity in PC12 Cells

“…As the present study showed that Tat-acp-SRDIYSTDYYR suppressed NGF-induced TRPV1 expression in PC12 cells, Tat-acp-SRDIYSTDYYR likely suppresses the downstream signals of TrkA, which play a crucial role for prolonged inflammatory pain (1, 2, 5). Cell-penetrating peptides including the Tat-derived amino acid sequence administered intravenously are reportedly detected intracellularly in all brain regions (21,22). Therefore in vivo administration of Tat-acp-SRDIYSTDYYR could distribute into peripheral and central neurons and could affect TrkA activity and the downstream signaling.…”