Calcium-Sensing Receptors of Human Astrocyte-Neuron Teams: Amyloid-ß-Driven Mediators and Therapeutic Targets of Alzheimer's Disease.

It is generally assumed that the neuropathology of sporadic (late-onset or nonfamilial) Alzheimer's disease (AD) is driven by the overproduction and spreading of first Amyloid-ßx-42 (Aß42) and later hyperphosphorylated (hp)-Tau oligomeric "infectious seeds". Hitherto, only neurons were held to make and spread both oligomer types; astrocytes would just remove debris. However, we have recently shown that exogenous fibrillar or soluble Aß peptides specifically bind and activate the Ca(2+)-sensing receptors (CaSRs) of untransformed human cortical adult astrocytes and postnatal neurons cultured in vitro driving them to produce, accrue, and secrete surplus endogenous Aß42. While the Aß-exposed neurons start dying, astrocytes survive and keep oversecreting Aß42, nitric oxide (NO), and vascular endothelial growth factor (VEGF)-A. Thus astrocytes help neurons' demise. Moreover, we have found that a highly selective allosteric CaSR agonist ("calcimimetic"), NPS R-568, mimics the just mentioned neurotoxic actions triggered by Aß●CaSR signaling. Contrariwise, and most important, NPS 2143, a highly selective allosteric CaSR antagonist ("calcilytic"), fully suppresses all the Aß●CaSR signaling-driven noxious actions. Altogether our findings suggest that the progression of AD neuropathology is promoted by unceasingly repeating cycles of accruing exogenous Aß42 oligomers interacting with the CaSRs of swelling numbers of astrocyte-neuron teams thereby recruiting them to overrelease additional Aß42 oligomers, VEGF-A, and NO. Calcilytics would beneficially break such Aß/CaSR-driven vicious cycles and hence halt or at least slow the otherwise unstoppable spreading of AD neuropathology.

Opposite effects of cell differentiation and apoptosis on Ap3A/Ap4A ratio in human cell cultures.

The biological role of diadenosine oligophosphates (DAOP) remains obscure in spite of numerous attempts to solve this enigma. It is known that Ap3A contrary to Ap4A accumulates in human cultured cells treated with interferons (IFNs) alpha or gamma. Since IFNs are considered as antiproliferative regulators, we assumed that different cell status may be associated with varying intracellular levels of DAOP. Promyelocytic human cell line HL60 induced by phorbol ester (TPA) to differentiate to macrophage-like cells in culture exhibits a profound loss of proliferative potential. Here we have shown a 4-5-fold increase in Ap3A concentration in HL60 cells induced by TPA, similar to the effect of IFN, while the Ap4A concentration remained unchanged. On the contrary, in cells undergoing apoptosis induced by VP16, a topoisomerase II inhibitor, the Ap3A concentration considerably decreased, while the Ap4A concentration increased. These findings combined with earlier results suggest an involvement of the Ap3A/Ap4A ratio in signal transduction pathways controlling the cell status.

Investigations into mechanisms modulating proliferation, differentiation, and apoptosis in cultured liver, adrenal, skin, and bone cells.

The intricate modulatory roles played by manifold hormones, growth factors, cytokines, extracellular calcium concentrations, intracellular second messengers, protein kinases, and nuclear poly(ADP-ribose) polymerase in proliferative, differentiative, and apoptotic processes have been the subject of investigations that were carried out by means of in vitro either primary or secondary/tertiary cultures of differentiated epithelial (hepatocytes, keratinocytes, and adrenocytes) and connective tissue cells (osteoblasts and fibroblasts) obtained from man and/or other mammalians. In most cases, an ad hoc model system, in which cells were floated on the top of the growth medium and, hence, could enjoy nearly normal respiratory exchanges, was used. Such a system increased cell viability and the ability of parenchymal epithelial cells to respond to extremely low concentrations of growth factors, hormones, and pharmaco-toxicological agents in a way conceivably very close to their behaviour in vivo.

Increased activity of the protein kinase C-delta holoenzyme in the cytoplasmic particulate fraction precedes the activation of caspases in polyomavirus-transformed pyF111 rat fibroblasts exposed to calphostin C or topoisomerase-II inhibitors.

A caspase-mediated release of the 40-kDa catalytic fragment of the delta isoform (CF-delta) of protein kinase C (PKC-delta) is involved in apoptosis, but its actual role in apoptosis development is still unknown. In an effort to understand this role, we have used polyomavirus-transformed pyF111 rat fibroblasts, which are hypersusceptible to apoptosis as they constitutively hyperexpress PKC-delta, but cannot make the antiapoptotic Bcl-2 and Bcl-X(L) proteins, while making the proapoptotic Bax protein. Calphostin C is reportedly both a specific inhibitor of PKC-delta activity (C. Keenan, N. Goode, and C. Pears, 1997, FEBS Lett. 415, 101-108) and an effective apoptogen (M. Murata et al., 1997, Cell. Mol. Life Sci. 53, 737-743). Exposure of pyF111 cells to calphostin C (75 nM) stimulated the translocation of the PKC-delta holoenzyme (holo-PKC-delta) onto the cytoplasmic particulate (CP) fraction between 15 and 45 min, which was after the release of mitochondrial cytochrome c but before the activation of cytoplasmic DEVD-specific caspases. The CF-delta fragment started accumulating only between 2 and 4 h, while apoptosis occurred mostly within 6 h. Incubating pyF111 cells with the much slower acting, apoptogenic topoisomerase-II inhibitors etoposide (VP-16) and teniposide (VM-26) also caused within 6 h a doubling of the CP-bound holo-PKC-delta-related activity but with no significant translocation of the holoenzyme to the CP fraction. Again this occurred after the release of cytochrome c but before the activation of DEVDases and the accumulation of the CF-delta. However, while calphostin C did not affect the delta-related activity in the nuclear membrane (NM) and nucleoplasmic (NP) fractions, VP-16 and VM-26 caused a prompt, large, and irreversible drop in the delta activity at the NM and a transient surge followed by a fall in the NP-associated activity. Hence, a surge of CP-anchored holo-PKC-delta activity is a common part of the signals given by various apoptogenic drugs to pyF111 cells. On the other hand, inhibition of delta-related activity, first at the NM and then in the NP fraction, is a specific feature only of the signals given by apoptogenic DNA-damaging agents.

beta-amyloid activates the O-2 forming NADPH oxidase in microglia, monocytes, and neutrophils. A possible inflammatory mechanism of neuronal damage in Alzheimer's disease.

The deposition of beta-amyloid in the brain is the key pathogenetic event in Alzheimer's disease. Among the various mechanisms proposed to explain the neurotoxicity of beta-amyloid deposits, a new one, recently identified in our and other laboratories, suggests that beta-amyloid is indirectly neurotoxic by activating microglia to produce toxic inflammatory mediators such as cytokines, nitric oxide, and oxygen free radicals. Three findings presented here support this mechanism, showing that beta-amyloid peptides (25-35), (1-39), and (1-42) activated the classical NADPH oxidase in rat primary culture of microglial cells and human phagocytes: 1) The exposure of the cells to beta-amyloid peptides stimulates the production of reactive oxygen intermediates; 2) the stimulation is associated with the assembly of the cytosolic components of NADPH oxidase on the plasma membrane, the process that corresponds to the activation of the enzyme; 3) neutrophils and monocytes of chronic granulomatous disease patients do not respond to beta-amyloid peptides with the stimulation of reactive oxygen intermediate production. Data are also presented that the activation of NADPH oxidase requires that beta-amyloid peptides be in fibrillary state, is inhibited by inhibitors of tyrosine kinases or phosphatidylinositol 3-kinase and by dibutyryl cyclic AMP, and is potentiated by interferon-gamma or tumor necrosis factor-alpha.

Changes in nuclear protein kinase C-delta holoenzyme, its catalytic fragments, and its activity in polyomavirus-transformed pyF111 rat fibroblasts while proliferating and following exposure to apoptogenic topoisomerase-II inhibitors.

Protein kinase C-delta (PKC-delta) appears to be variously involved in proliferation and apoptosis. To compare the changes of this enzyme in these two processes, we have determined the levels and activities of the 79-kDa PKC-delta holoenzyme and its catalytically active 47- and 40-kDa C-terminal fragments in the nuclei of proliferating untreated polyomavirus-transformed pyF111 rat fibroblasts and pyF111 cells treated with the apoptogenic topoisomerase-II inhibitors VP-16 (etoposide), VM-26 (teniposide), and doxorubicin. PyF111 cells were chosen because they hyperexpress PKC-delta and they are hypersusceptible to apoptosis because they do not express the antiapoptotic proteins Bcl-2 and Bcl-XL. The highest PKC-delta activity in cells before they started proliferating or were exposed to one of the inhibitors was in the NM (nuclear envelope-containing) fraction, which contained the holoenzyme and both C-terminal fragments, while only the two fragments were in the nucleoplasmic (NP) fraction where they were tightly associated with chromatin. When the cells began proliferating the amounts of the PKC-delta holoenzyme and the two fragments increased in the NM and the NP fractions and the already high PKC-delta activity either increased or stayed the same in these fractions until the end of the 72-h incubation. And there was no leakage of cytochrome c from the mitochondria into the cytoplasm. VP-16 exposure caused a prompt release of cytochrome c from the mitochondria into the cytosol and at the same time triggered a sharp drop (35% by 3 h and 60% by 6 h) in the PKC-delta activity in the NM fraction without changing the actual amounts of the holoenzyme or its fragments. This prompt inactivation of PKC-delta and its fragments during the first 6 h of exposure to the drug was not due to their dephosphorylation and could not be reversed by phosphatidylserine and/or 12-O-tetradecanoylphorbol 13-acetate (TPA). Between 6 and 24 h the PKC-delta activity in the NM fraction dropped a further 20%, the kinase's activity transiently surged in the NP fraction, and cytoplasmic CPP-32-like (DEVD-specific caspase) activity increased without an increase in the proteolysis of nuclear PKC-delta or PARP. Between 24 and 72 h nuclear CPP-32-like activity increased along with a massive proteolysis of PKC-delta, an accumulation of various PKC-delta fragments, and the cleavage of PARP. But despite this proteolysis, the cells were still able to maintain or even increase the amounts of holoenzyme and 40- and 47-kDa fragments in the NM and NP fractions before dying. VM-26 and doxorubicin caused the same prompt release of cytochrome c from the mitochondria and dramatic drop of NM PKC-delta activity as did VP-16. Thus, high levels of activity of nuclear PKC-delta, particularly PKC-delta in the nuclear membrane, might have a role driving the cell cycle of pyF111 cells. On the other hand, the prompt and sustained large drop in the activity of PKC-delta at this site that precedes the onset of the caspase-mediated proteolysis of the isoform may be involved in starting and driving apoptogenesis in pyF111 fibroblasts exposed to topoisomerase-II inhibitors.

Expression of protein kinase C isoforms and interleukin-1beta in myofibrillar myopathy.

BACKGROUND: The term myofibrillar myopathy refers to a rare and clinically heterogeneous group of muscle disorders. The pathogenesis of this myopathy is not well understood. The morphologic hallmark is myofibrillar destruction with abnormal expression of numerous proteins, most consistently of desmin. METHODS: The authors investigated eight patients with myofibrillar myopathy belonging to four families. They studied the role of different protein kinase C isoforms and of interleukin-1beta, a cytokine that might activate protein kinase C and, in addition, mediate myofibrillar proteolysis. RESULTS: Immunohistochemical analysis showed the expression of alpha, eta, and zeta isoforms of protein kinase C and of interleukin-1beta in abnormal muscle fibers. Immunoblots confirmed the immunohistochemical data and revealed the absence of protein kinase C delta and epsilon in muscle fibers from patients and controls. CONCLUSIONS: These data suggest that protein kinase C and interleukin-1beta may play a role in the pathogenesis of myofibrillar myopathy.

Neurotrophin p75 receptor is involved in neuronal damage by prion peptide-(106-126).

In this work we have investigated the molecular basis of the neuronal damage induced by the prion peptide by searching for a surface receptor whose activation could be the first step of a cascade of events responsible for cell death. By using a human neuroblastoma cell line lacking all the neurotrophin receptors and derived clones expressing the full-length or truncated forms of the low affinity neurotrophin receptor (p75(NTR)), we have been able to demonstrate that the neuronal death induced by the prion protein fragment PrP-(106-126) is an active process mediated by a) the binding of the peptide to the extracellular region of p75(NTR), b) the signaling function of the intracytoplasmic region of the receptor, and c) the activation of caspase-8 and the production of oxidant species.