Transcription Protein Transduction Domain Research Articles

In Situ Tissue Labeling of Cerebral Amyloid Using HIV-Related Tat Peptide.

Delivering peptide-based drugs to the brain is a major challenge because of the existence of the blood-brain barrier (BBB). To overcome this problem, cell-penetrating peptides derived from proteins that are able to cross biological membranes have been used as cell-permeable and brain-penetrant compounds. An example is the transactivator of transcription protein transduction domain (Tat) of the human immunodeficiency virus. The basic domain of Tat is formed of arginine and lysine amino acid residues. Tat has been used as brain-penetrant carrier also in therapies for Alzheimer disease (AD), the most common form of dementia characterized by extracellular cerebral deposits of amyloid made up of Aβ peptide. The aim of our study was to assess whether Tat bind to amyloid deposits of AD and other amyloidoses. An in situ labeling using biotinylated Tat 48-57 peptide was employed in the brain tissue with amyloid deposits made up of Aβ (patients with AD and transgenic AD mice), of prion protein (patients with Gerstmann-Straussler-Scheinker disease), and other amyloidosis, processed by different fixations and pretreatments of histological sections. Our results showed that Tat peptide binds amyloid deposits made up of Aβ, PrP, and immunoglobulin lambda chains in the brain and other tissues processed by alcoholic fixatives but not in formalin-fixed tissue. The fact that biotinylated Tat peptide stains amyloid of different biochemical composition and the specific charge characteristics of the molecules suggests that Tat may bind to heparan sulfate glicosaminoglicans, that are present in amyloid deposits. Inhibition of the binding by Tat pre-incubation with protamine reinforces this hypothesis. Binding of Tat to amyloid deposits should be kept in mind in interpreting the results of studies employing this molecule as brain-penetrating compound for the treatment of cerebral amyloidoses. Our results also suggest that Tat may be helpful for the analysis of the mechanisms of amyloidogenesis, and in particular, the interactions between specific amyloid peptides and glicosaminoglicans.

Intranasal administration of TAT-haFGF14–154 attenuates disease progression in a mouse model of Alzheimer’s disease

Human acidic fibroblast growth factor (haFGF), a neurotrophin-like growth factor in the brain, plays important roles in the development, differentiation and regeneration of brain neurons, which makes it potential to treat Alzheimer’s disease (AD). In this study, haFGF14–154 and TAT-haFGF14–154 (haFGF14–154 fused with the cell-penetrating peptide transactivator of transcription protein transduction domain (TAT-PTD)) were intranasally administrated for 5weeks to investigate the effects on senescence-accelerated mouse prone-8 (SAMP8) mice (a mouse model of AD). Results showed that TAT-PTD could increase the concentration of haFGF in the brain significantly, and TAT-haFGF14–154 was more effective than haFGF14–154 in the same dosage (300μg/kg). Importantly, TAT-haFGF14–154 improved the learning and memory abilities of SAMP8 mice in the behavioral test, and promoted the function of cholinergic system by measuring the relevant biomarkers (acetylcholine (ACh) level, acetylcholinesterase (AChE) and choline acetyltransferase (ChAT) activities). TAT-haFGF14–154 also significantly reduced β-amyloid protein1–42 (Aβ1–42) deposits as well as the levels of Aβ soluble forms in the mice brains and prevented the neurons from apoptosis. Besides, the oxidative stress impairment in the brain and serum was also ameliorated. The results suggest that TAT-haFGF14–154 could attenuate the disease progression of SAMP8 AD mice, and the mechanism is related to the regulation of neurons microenvironment including neurotransmitters, Aβ pathology and oxidative stress.

TAT-mediated delivery of neuroglobin protects against focal cerebral ischemia in mice

Neuroglobin (Ngb), a newly identified globin in vertebrate brain, has been suggested to be able to protect against brain hypoxic-ischemic injury. However, owing to its large size, the impermeability of the blood-brain barrier (BBB) to Ngb limits its application in brain injury. Recently, the 11-amino-acid human immunodeficiency virus trans-activator of transcription (TAT) protein transduction domain was shown to successfully deliver macromolecules into the brain. This study explored whether the TAT-Ngb fusion protein can cross the BBB and protect the brain from cerebral ischemia. The TAT-Ngb fusion protein generated from Escherichia coli BL21 (DE3) was efficiently delivered into mice brain tissues by intravenous injection as demonstrated by immunohistochemistry and Western blotting. Two groups of mice were treated with filamentous middle cerebral artery occlusion (MCAO) for 30min or 2h followed by reperfusion. Each group was then divided into sub-groups and was injected intravenously with TAT-Ngb, Ngb, or saline respectively before MCAO or immediately after reperfusion. Compared with the Ngb- and saline-treated group, the group with TAT-Ngb treated 2h before MCAO showed significantly less brain infarct volume and had better neurologic outcomes (p<0.05). Furthermore, a TAT-Ngb injection following a 30-min MCAO treatment significantly increased neuronal survival in the striatum (p<0.05). Our results demonstrated that the exogenous Ngb fusion protein containing the TAT protein transduction domain could be efficiently transduced into neurons in the mouse and protect the brain from mild or moderate ischemic injury.

TAT-mediated delivery of neuroglobin protects against focal cerebral ischemia in mice

Neuroglobin (Ngb), a newly identified globin in vertebrate brain, has been suggested to be able to protect against brain hypoxic–ischemic injury. However, owing to its large size, the impermeability of the blood–brain barrier (BBB) to Ngb limits its application in brain injury. Recently, the 11-amino-acid human immunodeficiency virus trans-activator of transcription (TAT) protein transduction domain was shown to successfully deliver macromolecules into the brain. This study explored whether the TAT–Ngb fusion protein can cross the BBB and protect the brain from cerebral ischemia. The TAT–Ngb fusion protein generated from Escherichia coli BL21 (DE3) was efficiently delivered into mice brain tissues by intravenous injection as demonstrated by immunohistochemistry and Western blotting. Two groups of mice were treated with filamentous middle cerebral artery occlusion (MCAO) for 30min or 2h followed by reperfusion. Each group was then divided into sub-groups and was injected intravenously with TAT–Ngb, Ngb, or saline respectively before MCAO or immediately after reperfusion. Compared with the Ngb- and saline-treated group, the group with TAT–Ngb treated 2h before MCAO showed significantly less brain infarct volume and had better neurologic outcomes (p<0.05). Furthermore, a TAT–Ngb injection following a 30-min MCAO treatment significantly increased neuronal survival in the striatum (p<0.05). Our results demonstrated that the exogenous Ngb fusion protein containing the TAT protein transduction domain could be efficiently transduced into neurons in the mouse and protect the brain from mild or moderate ischemic injury.

Ciliary neurotrophic factor infused intracerebroventricularly shows reduced catabolic effects when linked to the TAT protein transduction domain

Ciliary neurotrophic factor (CNTF) regulates the differentiation and survival of a wide spectrum of developing and adult neurons, including motor neuron loss after injury. We recently described a cell-penetrant recombinant human CNTF (rhCNTF) molecule, formed by fusion with the human immunodeficiency virus-1 transactivator of transcription (TAT) protein transduction domain (TAT-CNTF) that, upon subcutaneous administration, retains full neurotrophic activity without cytokine-like side-effects. Although the CNTF receptor is present in hypothalamic nuclei, which are involved in the control of energy, rhCNTF but not TAT-CNTF stimulates signal transducers and activators of transcription 3 phosphorylation in the rat hypothalamus after subcutaneous administration. This could be due limited TAT-CNTF distribution in the hypothalamus and/or altered intracellular signaling by the fusion protein. To explore these possibilities, we examined the effect of intracerebroventricular administration of TAT-CNTF in male adult rats. TAT-CNTF-induced weight loss, although the effect was smaller than that seen with either rhCNTF or leptin (which exerts CNTF-like effects via its receptor). In contrast to rhCNTF and leptin, TAT-CNTF neither induced morphological changes in adipose tissues nor increased uncoupling protein 1 expression in brown adipose tissue, a characteristic feature of rhCNTF and leptin. Acute intracerebroventricular administration of TAT-CNTF induced a less robust phosphorylation of signal transducers and activators of transcription 3 in the hypothalamus, compared with rhCNTF. The data show that fusion of a protein transduction domain may change rhCNTF CNS distribution, while further strengthening the utility of cell-penetrating peptide technology to neurotrophic factor biology beyond the neuroscience field.

TAT Opens the Door

TAT Opens the Door

TAT-PAX6 protein transduction in neural progenitor cells: A novel approach for generation of dopaminergic neurones in vitro

Neural stem cells (NSCs) have the potential to be used for the treatment of Parkinson's disease (PD), as they can be expanded, manipulated and differentiated in vitro to generate dopaminergic neurones which are suitable for transplantation. Since NSCs have a tendency to follow an astrocytic lineage after differentiation in vitro, researchers are investigating ways to induce a neuronal phenotype in these cells. In this study, the human immunodeficiency virus 1 (HIV-1) transactivator of transcription (TAT) protein transduction domain (PTD) system was used in an attempt to promote neuronal differentiation in rodent NSCs. A fusion protein that incorporated both the TAT PTD and the Pax6 protein (a determinant of neurogenesis) was created and added to the differentiation phase of embryonic day (E) 12 rat ventral mesencephalic (VM) neurosphere cultures. Subsequently, application of dopaminergic growth factors (GFs) was used in an attempt to induce the newly-generated neuronal progenitors to adopt a dopaminergic phenotype. In addition, a technique involving the differentiation of intact neurospheres (instead of the differentiation of neurosphere-derived dissociated cells) was investigated for its ability to promote dopaminergic neurogenesis. Immunocytochemical analysis of the differentiated neurosphere cultures indicated that both of these techniques had a significant effect on the emergence of dopaminergic neurones. Moreover, upon combination of these techniques, a further increase in dopaminergic neuronal generation was observed. Based on the findings of the present study, it is clear that NSCs are greatly influenced by their environment and that optimised in vitro conditions can support the potential of these cells to differentiate into dopaminergic neurones.

Transactivator of Transcription Fusion Protein Transduction Causes Membrane Inversion

The transactivator of transcription (TAT) protein transduction domain is an 11-amino acid positively charged peptide that has been shown to pull diverse molecules across cell membranes in vitro and in vivo. Fusion proteins constructed with TAT rapidly enter and exit cells and have been shown to cross intracellular membranes as well. Electrostatic interactions between TAT and the cell membrane have been implicated as a part of the mechanism of transduction. Here, we report that TAT transduction causes membrane phospholipid rearrangement as evidenced by detection of phosphatidylserine on the outer surface of the cell membrane. Furthermore, these rearrangements can be blocked by positively charged polylysine, further implicating electrostatic interactions as a part of the mechanism. Neither apoptosis nor necrosis is induced in these cells after exposure to TAT. We conclude that the process of TAT.GFP transduction causes phosphatidylserine to translocate from the inner to the outer leaflet of the plasma membrane. These results provide insight into the mechanism of TAT protein transduction domain transduction.