Combination therapy with transductive anti‐death FNK protein and FK506 ameliorates brain damage with focal transient ischemia in rat

Abstract

Many practical therapies have been explored as clinical applications for ischemic cerebral infarction; however, most are still insufficient to treat acute stroke. We show here a potential combination therapy in a rat focal ischemic model to improve neurological symptoms as well as to reduce infarct volumes at the maximum level. We applied protein transduction technology using artificial anti-death Bcl-X L derivative with three amino acidsubstitutions (Y22F, Q26N and R165K) (FNK) protein fused with a protein-transduction-domain peptide (PTD-FNK). When PTD-FNK was administrated 1 h after initiating ischemia followed by the administration of an immunosuppressant FK506 with a 30-min time lag, infarct volumes of the total brain and cortex were markedly reduced to 27% and 14%, respectively. This procedure not only reduced the infarct volume and edema, but also markedly improved neurological symptoms. The therapeutic effect continued for at least 1 week after ischemia. FK506 inhibited the transduction of PTD-FNK in vitro, which explains the requirement of a time lag for the administration of FK506. An additional in vitro experiment showed that PTD-FNK, when administered 30 min before FK506, gave the maximal protective effect by reducing the intracellular calcium concentration. We propose that this combination therapy would provide a synergistic protective effect by both drugs, reducing adverse the effects of FK506. has not yet been investigated. We proceeded with experiments using a focal ischemic model in rats to prevent brain injury after transient focal ischemia by the post-administration of PTD-FNK. FK506 (Tacrolimus) is an immunosuppressant to prevent allograft rejection after organ transplantation. In addition, FK506 has a protective effect against ischemia-reperfusion injury in organs, including the heart (Nishinaka et al. 1993), liver (Garcia-Criado et al. 1997;Matsuda et al. 1998), and kidney (Sakr et al. 1992). After Sharkey and Butcher (1994) first showed that post ischemic administration of FK506 mitigates brain injury following permanent ligation of the rat middle cerebral artery (MCA), many studies have confirmed the neuroprotective effect of FK506 in various experimental models, including a focal ischemia model using a filament (Kuroda and Siesjö 1996;Arii et al. 2001) and a transient global ischemia model (Drake et al. 1996;Ide et al. 1996;Yagita et al. 1996;Katsura et al. 2003). In addition, FK506 is shown to display a neurotrophic effect (Gold et al. 1994;Lyons et al. 1994).It is estimated that there are 730 000 and 1 500 000 initial strokes each year in the United States and Japan, respectively, and that stroke is the third leading cause of death in many countries (Warlow 1998). Moreover, about 40% of bedridden aged people who are hospitalized in Japan are bedridden because of stroke (Ministry of Health, Labour and Welfare, Japan 2002), which makes stroke the leading cause of increasing medical expenses.Recently, with progress in new therapeutic approaches, a worldwide campaign was initiated ...

Combination therapy with bone marrow stromal cells and FK506 enhanced amelioration of ischemic brain damage in rats

“…FK506 has also been found to confer neuroprotective effects against various types of ischemic injury, including a focal ischemia model (Sharkey and Butcher, 1994;Kuroda et al, 1999;Arii et al, Life Sciences 89 (2011) (Yagita et al, 1996;Katsura et al, 2003), and a chronic cerebral ischemia model (Wakita et al, 1998). Our recent studies have also shown that hypothermia or transductive anti-death FNK protein in combination with FK506 enhances neuroprotection against brain damage with focal transient ischemia in rats, the two treatments acting additively or synergistically against ischemic damage (Nito et al, 2004;Katsura et al, 2008). Although the precise mechanism producing the neuroprotective effects of FK506 is still unclear, previous studies have shown that FK506 exerts anti-apoptotic and anti-inflammatory effects on cerebral ischemic injury (Furuichi et al, 2004;Noto et al, 2007).…”

“…Recently, numerous new drugs have been put through clinical trials, despite little success, probably because cerebral tissue damage may be the result of a complex pathophysiological cascade; however, combination therapies bring about additive or synergistic effects in animal studies (Lyden et al, 2000;Okubo et al, 2007;Katsura et al, 2008).…”

Transduction of Human Recombinant Proteins into Mitochondria as a Protein Therapeutic Approach for Mitochondrial Disorders

“…Ohta's group (143) ligated the TAT domain to the super-antiapoptotic factor FNK to produce the PTD-FNK fusion protein. PTD-FNK fusion protein was transduced efficiently into cells, localized into mitochondria, and protected both neuronal and cardiac cells from apoptosis induced by ischemia in culture as well as in animal models (71,143,160). Similarly, TAT-Bcl-2 fusion protein protected neurons from apoptotic cell death in culture (161).…”

“…In other cases of "primary" mitochondrial disorders, therapeutics should be targeted to the IMS or inner membrane, whereas the respiratory complexes are embedded (69). In cases of cancer or ischemia (cardiac or brain), where apoptosis can be manipulated, PTs should target mitochondria in order to influence the mitochondrial outer membrane permeabilization and thus the release of anti-apoptotic proteins (cytochrome c, apoptosis-inducing factor, endonuclease G) from the mitochondrial IMS to the cytoplasm (51,70,71).…”

N-acetylaspartate Decrease in Acute Stage of Ischemic Stroke:A Perspective from Experimental and Clinical Studies

“…72,73 One important difference between these 2 models of ischemia is the complete absence of blood flow in the global model and the maintenance of residual blood flow to the ischemic core of the focal model. 74 A massive amount of aspartate is also released into the ECS during ischemia. 75 Excretion of the aspartate from the ECS into the blood can occur through alanine-serine-cysteine transporter 2 (ASCT2) channels located on the basal membranes of the vascular epithelium, which are activated by ischemic acidosis.…”