Ribozyme-mediated inhibition of caspase-3 protects cerebellar granule cells from apoptosis induced by serum-potassium deprivation.

Apoptosis is an important mechanism of physiological and pathological cell death. It is regulated by several gene products, including caspases and the bcl-2-like proteins, whose roles have been demonstrated in numerous systems. One of these is a model of cerebellar granule cells (CGCs) in which apoptosis is induced by acute removal of serum and depolarizing concentrations of potassium. Previous work by several authors showed that benzyloxycarbonyl-DEVD-fluoromethylketone, a somewhat selective caspase inhibitor, significantly protected CGCs from apoptosis; however, because this molecule targets multiple caspases, it is not known whether a single caspase is primarily responsible for effecting cell death in this model. We attempted to answer this question by cotransfecting CGCs with green fluorescent protein reporter and a hammerhead ribozyme directed against caspase-3 mRNA. Maximal protection by this ribozyme was observed after 24 hr of deprivation, at which time apoptosis was 18 +/- 0.7% compared with 32 +/- 2% in control cells. Significant protection was also observed with human inhibitor of apoptosis (IAP)-like protein-X-linked IAP, a specific inhibitor of caspase-3, -7, and -9, and with p35, a general caspase inhibitor. Overexpression of bcl-2 produced almost complete protection from apoptosis after 24 hr of serum-K(+) deprivation (5 +/- 2 vs 44 +/- 2% in control cells). These results confirm that caspases play an important role in CGC apoptosis and indicate that caspase-3 itself is a significant mediator of this process.

Differential effects of transforming growth factor-beta(s) and glial cell line-derived neurotrophic factor on gene expression of presenilin-1 in human post-mitotic neurons and astrocytes.

Mutations in the presenilin-1 gene are linked to the majority of early-onset familial Alzheimer's disease cases. We have previously shown that the expression of transforming growth factor-beta is altered in Alzheimer's patients, compared to controls. Here we examine presenilin- expression in human post-mitotic neurons (hNT cells), normal human astrocytes, and human brain tumor cell lines following treatment with three isoforms of transforming growth factor-beta, or glial cell line-derived neurotrophic factor, a member of the transforming growth factor-beta superfamily. As the NT2/D1 teratocarcinoma cell line is treated with retinoic acid to induce differentiation to hNT cells, presenilin-1 messenger RNA expression is dramatically increased. Furthermore, there is a 2-3-fold increase in presenilin-1 messenger RNA expression following treatment of hNT cells with growth factors and similar results are found by Western blotting and with immunohistochemical staining for presenilin-1 protein. However, treatment of normal human astrocytes with cytokines results in minimal changes in presenilin-1 messenger RNA and protein. Interestingly, the expression of presenilin-1 in human U87 MG astrocytoma and human SK-N-SH neuroblastoma cells is only increased when cells are treated with glial cell line-derived neurotrophic factor or transforming growth factor-beta3. These findings suggest that endogenous presenilin-1 gene expression in human neurons can be induced by growth factors present in normal and diseased brain tissue. Cytokines may play a major role in regulating expression of presenilin-1 which may affect its biological actions in physiological and pathological conditions.

A novel synthetic oleanane triterpenoid, 2-cyano-3,12-dioxoolean-1,9-dien-28-oic acid, with potent differentiating, antiproliferative, and anti-inflammatory activity.

The new synthetic oleanane triterpenoid 2-cyano-3,12-dioxoolean-1,9-dien-28-oic acid (CDDO) is a potent, multifunctional molecule. It induces monocytic differentiation of human myeloid leukemia cells and adipogenic differentiation of mouse 3T3-L1 fibroblasts and enhances the neuronal differentiation of rat PC12 pheochromocytoma cells caused by nerve growth factor. CDDO inhibits proliferation of many human tumor cell lines, including those derived from estrogen receptor-positive and -negative breast carcinomas, myeloid leukemias, and several carcinomas bearing a Smad4 mutation. Furthermore, it suppresses the abilities of various inflammatory cytokines, such as IFN-gamma, interleukin-1, and tumor necrosis factor-alpha, to induce de novo formation of the enzymes inducible nitric oxide synthase (iNos) and inducible cyclooxygenase (COX-2) in mouse peritoneal macrophages, rat brain microglia, and human colon fibroblasts. CDDO will also protect rat brain hippocampal neurons from cell death induced by beta-amyloid. The above activities have been found at concentrations ranging from 10(-6) to 10(-9) M in cell culture, and these results suggest that CDDO needs further study in vivo, for either chemoprevention or chemotherapy of malignancy as well as for neuroprotection.

Transforming growth factor-beta inhibits apoptosis induced by beta-amyloid peptide fragment 25-35 in cultured neuronal cells.

Previously, we demonstrated that transforming growth factor-beta (TGF-beta) pretreatment protects neuroblastoma cell lines, human hNT neurons, and primary rat embryo hippocampal neurons (REHIPs) from degeneration caused by incubation with beta-amyloid peptide (Abeta). Here we present evidence suggesting that TGF-beta interferes with an apoptotic pathway induced by Abeta. TGF-beta preteatment decreases the amount of DNA laddering seen following Abeta treatment in neuroblastoma cells, while in REHIPs, TGF-beta decreases the number of positive cells detected in situ by Klenow labelling following Abeta treatment. RT-PCR shows that in REHIPs, Abeta decreases mRNA expression of Bcl-2, as well as the ratio of Bcl-xL/Bcl-xS, with little effect on Bax expression. These changes are expected to promote apoptosis. When REHIPs are incubated with TGF-beta before addition of Abeta, the Bcl-xL/Bcl-xS ratio and Bcl-2 levels are increased compared to cells treated with Abeta alone. Again there is little effect on Bax expression. Western blotting and immunohistochemistry experiments also show that TGF-beta maintains increased levels of Bcl-2 and Bcl-xL protein in REHIPs even in the presence of Abeta. This pattern of gene expression should function to decrease apoptosis. Similarly, RT-PCR analysis of mRNA prepared from hNT cells shows that TGF-beta pretreatment before addition of Abeta maintains a higher level of Bcl-2 expression and an increased Bcl-xL/Bcl-xS ratio as compared to cells treated with Abeta alone. In neuronal cell types treated with Abeta, TGF-beta appears to regulate expression of genes in the Bcl-2 family to favor an anti-apoptotic pathway.

Transforming growth factor-betas in neurodegenerative disease.

Transforming growth factors-betas (TGF-betas), a family of multifunctional peptide growth factors, affect cells of the central nervous system (CNS). The three mammalian TGF-beta isoforms, TGF-betas 1, 2 and 3, are expressed in adult human brain. Since neuronal degeneration is a defining feature of CNS degenerative diseases, TGF-beta may be important because it can influence neuronal survival. In vitro TGF-beta promotes survival of rat spinal cord motoneurons and dopaminergic neurons. In addition to direct effects on neuronal survival, TGF-beta treatment of cultured astrocytes induces a reactive phenotype. Thus, TGF-beta may also normalize the extracellular matrix environment in degenerative diseases. The expression of TGF-betas change in response to neuronal injury. TGF-beta 1 expression increases in astrocytes and microglia in animal models of cerebral ischemia, while TGF-beta 2 expression increases in activated astroglial cells in human neurodegenerative diseases. TGF-betas protect neurons from a variety of insults. TGF-beta maintains survival of chick telencephalic neurons made hypoxic by treatment with cyanide and decreases the area of infarction when administered in animal models of cerebral ischemia. In vitro TGF-beta protects neurons from damage induced by treatment with beta-amyloid peptide, FeSO4 (induces production of reactive oxygen species), Ca2+ ionophores, glutamate, glutamate receptor agonists and MPTP (toxic for dopaminergic neurons). TGF-beta maintains mitochondrial potential and Ca2+ homeostasis and inhibits apoptosis in neurons. TGF-beta does not prevent neuronal degeneration in a rat model of Parkinson's disease and has yet to be tested in newly developed transgenic mouse models of Alzheimer's disease. TGF-beta is a potent neuroprotective agent which may affect the pathogenesis of neurodegenerative diseases of the CNS.

Transforming growth factor-beta protects human hNT cells from degeneration induced by beta-amyloid peptide: involvement of the TGF-beta type II receptor.

Post-mitotic, human neurons (hNT cells) which have a phenotype similar to that of terminally differentiated neurons of the central nervous system were generated by treating the NT2/D1 human teratocarcinoma cell line with retinoic acid. Treatment of both hNT and NT2/D1 cells with 10(-5) M beta-amyloid peptide fragment 25-35 (A beta P) for 24 h resulted in a decrease in cell viability as determined by MTT incorporation and Trypan blue exclusion, and also induced an apoptotic morphology in hNT cells. Pre-treatment of cells for 24 h with 10 ng/ml TGF-beta 1 or 2 before addition of A beta P reduced the apoptotic morphology of hNT cells and increased cell viability in hNT cells, but not in NT2/D1 cells. Results of RT-PCR, immunohistochemistry and analysis of receptor cross-linking of [125I]TGF-beta 1 to the cell membrane, all showed that the TGF-beta type II receptor is expressed by hNT cells, but not NT2/D1 cells. These results suggest that TGF-beta can protect human, terminally differentiated, TGF-beta type II receptor-positive neurons from A beta P toxicity. We propose that the increased expression of TGF-beta in brains of patients with Alzheimer's disease may offer some degree of neuroprotection if neurons also express a functional TGF-beta type II receptor.

Transforming growth factors-beta protect primary rat hippocampal neuronal cultures from degeneration induced by beta-amyloid peptide.

Treatment of primary rat embryo hippocampal neuronal cultures with 10(-5) M beta-amyloid peptide fragment 25-35 (A beta P) for 24 h resulted in a 60% decrease in cell viability as determined by MTT incorporation. When these cells were treated with 0.1-10 ng/ml of either transforming growth factor-beta (TGF-beta) 1, 2 or 3 for 24 h before exposure to A beta P, there was a 2.9-, 1.9-, and 3.2-fold increase in cell survival, respectively, compared to cells treated with A beta P alone. The viability of cells treated with A beta P and 0.1-10 ng/ml TGF-beta was comparable to that of cells not treated with A beta P. The protective effects were less pronounced at lower TGF-beta concentrations. The protective effects of pretreatment with TGF-beta were less striking in mouse CCL-N-2a and human SK-N-SH neuroblastoma cell lines. When all cells were treated with TGF-beta for 24 h following a 24 h exposure to A beta P, there was a trend toward increased cell viability which was less significant than pretreatment with TGFs-beta. An isoform-specific TGF-beta SELISA showed that primary hippocampal neuronal cultures and the neuroblastoma cell lines secrete all 3 TGF-beta isoforms. Based on our results, we propose that the increased expression of TGF-beta observed in brains of patients with Alzheimer's disease may offer some degree of neuroprotection.