Tetanus toxin fragments and Bcl-2 fusion proteins: cytoprotection and retrograde axonal migration.

BACKGROUND: Tetanus neurotoxin (TeNT) is taken up at nerve terminals and undergoes retrograde migration. The toxic properties of TeNT reside in the toxin light chain (L), but like complete TeNT, the TeNT heavy chain (TTH) and the C-terminal domain (TTC) alone can bind and enter into neurons. Here, we explored whether atoxic fragments of TeNT could act as drug delivery vehicles in neurons. In this study, we used Bcl-2, a protein known to have anti-apoptotic properties in vivo and in vitro, as a parcel to couple to TeNT fragments. RESULTS: We expressed Bcl-2 and the TTC fragments alone, and also attempted to express fusion proteins with the Bcl-2 coupled at the N-terminus of TTH (Bcl2-TTH) and the N- and C-terminus of TTC (TTC-Bcl2 and Bcl2-TTC) in mammalian (Cos7 cells) and Escherichia coli systems. TTC and Bcl-2 were efficiently expressed in E. coli and Cos7 cells, respectively, but Bcl-2 and the fusion proteins did not express well in E. coli. The fusion proteins were also not expressed in Cos7 cells. To improve the yield and purity of the fusion protein, we genetically deleted the N-terminal half of TTC from the Bcl2-TTC fusion to yield Bcl2-hTTC. Purified Bcl2-hTTC exhibited neuronal binding and prevented cell death of neuronal PC12 cells induced by serum and NGF deprivation, as evidenced by the inhibition of cytochrome C release from the mitochondria. For in vivo assays, Bcl2-hTTC was injected into the tongues of mice and was seen to selectively migrate to hypoglossal nuclei mouse brain stems via retrograde axonal transport. CONCLUSIONS: These results indicate that Bcl2-hTTC retains both Bcl-2 and TTC functions and therefore could be a potent therapeutic agent for various neurological conditions.

Anthocyanin suppresses the toxicity of Aß deposits through diversion of molecular forms in in vitro and in vivo models of Alzheimer's disease.

OBJECTIVES: The pathogenesis of Alzheimer's disease (AD) is strongly correlated with the aggregation and deposition of the amyloid beta (Aß1-42) peptide in fibrillar form, and many studies have shown that plant-derived polyphenols are capable of attenuating AD progression in various disease models. In this study, we set out to correlate the effects of anthocyanoside extracts (Vaccinium myrtillus anthocyanoside (VMA)) obtained from bilberry on the in vitro progression of Aß fibril formation with the in vivo effects of this compound on AD pathogenesis. METHODS: Thioflavin T fluorescence assays and atomic force microscopy were used to monitor Aß amyloid formation in in vitro assays. Effects of Aß amyloids on cellular viability were assayed using cultured Neuro2a cells. Cognitive effects were probed using mice that simultaneously expressed mutant human Aß precursor and mutant presenilin-2. RESULTS: Addition of VMA inhibited the in vitro formation of Aß peptide fibrils and also reduced the toxicity of these aggregates toward Neuro2a cells. A diet containing 1% VMA prevented the cognitive degeneration in AD mice. Curiously, this diet-derived retention of cognitive ability was not accompanied by a reduction in aggregate deposition in brains; rather, an increase in insoluble deposits was observed compared with mice raised on a control diet. DISCUSSION: The paradoxical increase in insoluble deposits caused by VMA suggests that these polyphenols divert Aß aggregation to an alternate, non-toxic form. This finding underscores the complex effects that polyphenol compounds may exert on amyloid deposition in vivo.

Impact of wound blush as an angiographic end point of endovascular therapy for patients with critical limb ischemia.

OBJECTIVES: Several reports have been published of the acceptable patency and limb salvage rates after infrapopliteal interventions for the treatment of critical limb ischemia (CLI). However, the optimal angiographic end point of endovascular therapy (EVT) remains unclear. This study assessed the relationship between the appearance of wound blush as an angiographic end point and the limb salvage rate in patients with CLI. METHODS: "Wound blush" was defined as contrast opacification of the vessels around the wound in digital subtraction angiograms obtained immediately after EVT through the catheter introduced into the popliteal artery. We analyzed the data of 77 consecutive patients (93 limbs) with ischemic ulcerations, classified as Rutherford category 5 or 6, who underwent EVT without bypass surgery. Patients were divided into two groups depending on whether wound blush was seen in the angiogram obtained immediately after the procedure. The freedom from amputation rate was compared between the two groups. RESULTS: The overall limb salvage rate was 81.7%. The limb salvage rate was significantly higher in the wound blush-positive group than in the wound blush-negative group and remained so for at least 3 years after the EVT (96.4% vs 56.8%, P < .001). CONCLUSIONS: Presence of wound blush after EVT is associated with higher skin perfusion pressure, both of which are associated with higher rates of limb salvage. Wound blush as an angiographic end point in EVT may be a novel predictor of limb salvage in patients with CLI.

SOD1-interacting proteins: Roles of aggregation cores and protein degradation systems.

Cu/Zn superoxide dismutase (SOD1) mutations are associated with amyotrophic lateral sclerosis (ALS). SOD1-positive aggregates in motor neurons, as well as proteins that interact with the aggregates are presumably involved in ALS neurotoxicity. We used a proteomics approach to compare differences in protein expression in spinal cord homogenates from non-transgenic (NTG) and ALS model mice. Using the homogenates, we identified proteins that interacted with SOD1 seeds in vitro. We assessed differences in SOD1-interacting proteins in cell cultures treated with proteasome or autophagy inhibitor. In the first experiment, intermediate filamentous and small heat shock proteins were upregulated in glial cells. We identified 26 protein types that interacted with aggregation cores in ALS model homogenates, and unexpectedly, 40 proteins in were detected in NTG mice. In cell cultures treated with proteasome and autophagy inhibitors, we identified 16 and 11 SOD1-interacting proteins, respectively, and seven proteins in untreated cells. These SOD1-interacting proteins were involved in multiple cellular functions such as protein quality control, cytoskeletal organization, and pathways involved in growth factor signaling and their downstream cascades. The complex interactions between pathways could cause further dysregulation, ultimately leading to fatal cellular dysfunction in ALS.

Enhancing the Therapeutic Efficacy of Bone Marrow-Derived Mononuclear Cells with Growth Factor-Expressing Mesenchymal Stem Cells for ALS in Mice.

Several treatments have been attempted in amyotrophic lateral sclerosis (ALS) animal models and patients. Recently, transplantation of bone marrow-derived mononuclear cells (MNCs) was investigated as a regenerative therapy for ALS, but satisfactory treatments remain to be established. To develop an effective treatment, we focused on mesenchymal stem cells (MSCs) expressing hepatocyte growth factor, glial cell line-derived neurotrophic factor, and insulin-like growth factor using human artificial chromosome vector (HAC-MSCs). Here, we demonstrated the transplantation of MNCs with HAC-MSCs in ALS mice. As per our results, the progression of motor dysfunction was significantly delayed, and their survival was prolonged dramatically. Additional analysis revealed preservation of motor neurons, suppression of gliosis, engraftment of numerous MNCs, and elevated chemotaxis-related cytokines in the spinal cord of treated mice. Therefore, growth factor-expressing MSCs enhance the therapeutic effects of bone marrow-derived MNCs for ALS and have a high potential as a novel cell therapy for patients with ALS.

Dynamics of host and graft after cell sheet transplantation: Basic study for the application of amyotrophic lateral sclerosis.

Stem cells offer great hope for the therapy of neurological disorders. Using a human artificial chromosome (HAC), we generated modified mesenchymal stem cells (MSCs), termed HAC-MSC that express 3 growth factors and 2 marker proteins including luciferase, and previously demonstrated that intrathecal administration of HAC-MSCs extended the lifespan in a mouse model of amyotrophic lateral sclerosis (ALS). However, donor cells disappeared rapidly after transplantation. To overcome this poor survival, we transplanted the HAC-MSCs as a sheet structure which retained the extracellular matrix. We investigated, here, whether cell sheet showed a longer survival than intrathecal administration. Also, the therapeutic effects on ALS model mice were examined. In vivo imaging showed that luciferase signals increased immediately after transplantation up to 7 days, and these signals were sustained for up to 14 days. In contrast, following intrathecal administration, signals were drastically decreased by day 3. Moreover, cell sheet transplantation successfully prolonged the survival of donor HAC-MSCs. Cell sheet transplantation increased the level of p-Akt at the graft area. Pathologically, none of the donor cells differentiated into neurons, astrocytes or microglial cells. When the cell sheet was transplanted into ALS model mice, there was an encouraging trend in the delayed onset of symptoms and increased lifespan. If each group was subdivided into rapid and slow progressors based on cut-off values for respective median survival, the survival of rapid progressors differed significantly between groups (treated vs. sham-operated = 145.4 ±â€¯1.4 vs. 139.2 ±â€¯1.2). The effect of HAC-MSC sheet transplantation still has a temporally narrow therapeutic window. Further improvement could be achieved by optimization of the transplantation conditions, e.g. co-transplantation of HAC-MSCs with endothelial progenitor cells.

Use of a Human Artificial Chromosome for Delivering Trophic Factors in a Rodent Model of Amyotrophic Lateral Sclerosis.

A human artificial chromosome (HAC) is maintained as an episome within a cell and avoids random integration into the host genome. It can transfer multiple and/or large transgenes along with their regulatory elements thereby resembling native chromosomes. Using this HAC system, we established mesenchymal stem cells (MSCs) that simultaneously expressed hepatocyte growth factor, glial cell line-derived neurotrophic factor, and insulin-like growth factor 1, termed HAC-MSCs. This cell line provides an opportunity for stable transplantation and thorough analyses. We then introduced the cells for the treatment of a neurodegenerative disorder, amyotrophic lateral sclerosis. The HAC-MSCs were transplanted via the fourth cerebral ventricle (CV) or intravenous (i.v.) infusion at various ages of recipient mice. Littermate- and sex-matched mice underwent a sham procedure. Compared to the controls, there was an encouraging trend of increased life span via CV transplantation and delayed onset in i.v. infusion 60 days after transplantation. Further, we confirmed a statistically significant increase in life span via CV transplantation at 100 days. This effect was not seen in mice transplanted with MSCs lacking the HAC. We successfully enhanced the trophic potential of the MSCs using the HAC. This strategy could be a promising direction for the treatment of neurodegenerative disorders.

Impact of continuous deterioration of kidney function 6 to 8 months after percutaneous coronary intervention for acute coronary syndrome.

Preprocedural chronic kidney disease and contrast-induced acute kidney injury are predictors of in-hospital death and long-term mortality. However, neither the time course of kidney function after percutaneous coronary intervention (PCI) nor the relation between the time course of kidney function and prognosis has been adequately studied. We studied 531 patients who underwent PCI for acute coronary syndrome. The continuous deterioration of kidney function (CDKF) was defined as a >25% increase in serum creatinine level or serum creatinine >0.5 mg/dl above baseline at 6 to 8 months after PCI. CDKF was observed in 87 patients (16.4%). Independent risk factors for CDKF were contrast-induced acute kidney injury, preprocedural hemoglobin level, and proteinuria. Patients with CDKF exhibited significant higher 5-year mortality rate than patients without CDKF (25% vs 9.4%, log-rank p = 0.0006). Independent risk factors for 5-year mortality were age >75 year, anemia, New York Heart Association class III or IV, low ejection fraction, and CDKF. CDKF is associated with an increased risk of all-cause mortality of 5 years in patients with acute coronary syndrome undergoing PCI.