NH2-truncated human tau induces deregulated mitophagy in neurons by aberrant recruitment of Parkin and UCHL-1: implications in Alzheimer's disease.

Disarrangement in functions and quality control of mitochondria at synapses are early events in Alzheimer's disease (AD) pathobiology. We reported that a 20-22 kDa NH2-tau fragment mapping between 26 and 230 amino acids of the longest human tau isoform (aka NH2htau): (i) is detectable in cellular and animal AD models, as well in synaptic mitochondria and cerebrospinal fluids (CSF) from human AD subjects; (ii) is neurotoxic in primary hippocampal neurons; (iii) compromises the mitochondrial biology both directly, by inhibiting the ANT-1-dependent ADP/ATP exchange, and indirectly, by impairing their selective autophagic clearance (mitophagy). Here, we show that the extensive Parkin-dependent turnover of mitochondria occurring in NH2htau-expressing post-mitotic neurons plays a pro-death role and that UCHL-1, the cytosolic Ubiquitin-C-terminal hydrolase L1 which directs the physiological remodeling of synapses by controlling ubiquitin homeostasis, critically contributes to mitochondrial and synaptic failure in this in vitro AD model. Pharmacological or genetic suppression of improper mitophagy, either by inhibition of mitochondrial targeting to autophagosomes or by shRNA-mediated silencing of Parkin or UCHL-1 gene expression, restores synaptic and mitochondrial content providing partial but significant protection against the NH2htau-induced neuronal death. Moreover, in mitochondria from human AD synapses, the endogenous NH2htau is stably associated with Parkin and with UCHL-1. Taken together, our studies show a causative link between the excessive mitochondrial turnover and the NH2htau-induced in vitro neuronal death, suggesting that pathogenetic tau truncation may contribute to synaptic deterioration in AD by aberrant recruitment of Parkin and UCHL-1 to mitochondria making them more prone to detrimental autophagic clearance.

Ricin A chain conjugated with monoclonal antibodies selectively killing human carcinoma cells in vitro.

Ricin A chain was coupled to murine monoclonal antibodies MBr1 and MOv2 respectively raised against human breast and ovarian carcinomas. Inhibition of protein synthesis only occurred in those cultured human tumor cells bearing the appropriate target antigens, demonstrating that both components of the conjugate were unchanged in regards to specificity and toxicity. Conjugates were 125-200 times more efficient in inhibiting [3H]proline incorporation than the uncoupled ricin A chain. They were however unable to kill the entire population of the appropriate cells even after repeated treatment. Although the two monoclonal antibodies had similar binding kinetics, the conjugates differed in their cytotoxicity kinetics. The MBr1-ricin A chain conjugate had slow kinetics, and about 20 hours were needed to obtain a protein synthesis inhibition above 50% on the appropriate line (mammary carcinoma MCF-7). In contrast, the MOv2-ricin A chain conjugate showed very fast kinetics, reaching 50% inhibition after only 30 minutes of treatment on both appropriate cell lines SW626 and HT-29 from ovarian and colon carcinomas, respectively. Growth conditions of cell lines, i.e., adherent cells versus suspended cells, and plating time were found to greatly influence the conjugates' killing efficiencies. These studies confirm the possibility of preparing ricin A chain-antibody conjugates, which retain specific cytotoxicity against tumor cells; but they also underline the need for further in vitro studies of various parameters before one considers a therapeutic use of such conjugates.

12-O-tetradecanoylphorbol-13-acetate-induced differentiation of a human rhabdomyosarcoma cell line.

The effect of 12-O-tetradecanoyl phorbol-13-acetate (TPA) on proliferation and differentiation of the human embryonal rhabdomyosarcoma cell line RD was investigated. The proliferation of RD cells is drastically and reversibly inhibited by 100 nM TPA. The effect is evident after 24 h of treatment and is maximal after 50-70 h. The reduction of proliferation in treated cells is followed by increased expression of differentiative characters such as a large increase in muscle myosin expression and in the binding of 125I-alpha-bungarotoxin. Moreover TPA induces the appearance of myotube-like structures, which contain bundles of thick and thin myofilaments along with Z bodies. The described effects are not observed if the TPA-containing medium is replaced daily, thus suggesting that these effects might be related to substances secreted by treated cells. The phosphorylation of three proteins is significantly stimulated by TPA within minutes of its administration to RD cells. Although with a different pattern, the stimulation of protein phosphorylation is still clearly detectable after 6 days of incubation with TPA. These results on human rhabdomyosarcoma cells are, to our knowledge, the first evidence for a growth-inhibiting and a differentiative effect of TPA on a solid tumor of mesodermal origin.

Biochemical analysis of human ovarian cancer-associated antigens defined by murine monoclonal antibodies.

Two monoclonal antibodies (MOv1 and MOv2) raised against a membrane preparation of a human surgical specimen from a mucinous ovarian cystoadenocarcinoma were used to biochemically define their target antigens. The heating of peritumoral mucus-soluble extracts and the sialidase treatment of crude membrane preparations did not affect the binding capacity of MOv1 and MOv2, which, on the contrary, was significantly reduced by periodate oxidation of the same materials. Pronase digestion completely solubilized MOv1-defined antigens, whereas MOv2-defined antigens were only partially solubilized. This, however, did not affect antibody binding with digested products. These data suggest that carbohydrate residues of recognized molecules constitute the antigenic determinants and that sialic acid residues are not involved. Gel filtration on Sepharose 4B of the peritumoral mucus, solubilized either by 200 mM NaCl or Pronase, revealed that most of the antigenic activity eluted in the void-volume fractions with a high carbohydrate content and in the included fractions before the elution volume of the ferritin standard protein. When CsCl gradient equilibrium ultracentrifugation of the solubilized mucus was used, MOv1-recognized antigens sedimented with a density of 1.45 g/ml, while the MOv2-defined epitope was carried by molecules with a density of 1.52 g/ml as well as by molecules with a lower density. Using thin-layer chromatography of organic solvent extracts obtained from mucus and crude membrane preparations, only MOv2-positive molecules could be resolved as a single band of glycolipid. Altogether, these data suggest that the antigens detected by MOv1 are mainly mucins whereas the determinant recognized by MOv2 is carried by both mucins and a glycolipid. To analyze the diagnostic potential of MOv1- and MOv2-recognized molecules, we tested their presence, as soluble products, in supernatants of tumor cell lines and in peritoneal effusions from cancer patients. To this aim, we developed an immunoradiometric assay using the same monoclonal antibody in insolubilized and soluble form. Whereas MOv1-immunoradiometric assay was always negative, by MOv2-immunoradiometric assay it was possible to detect the relevant antigen in 8 of the 10 effusions from patients with well-differentiated ovarian tumors and in 5 of the 11 effusions from patients with poorly differentiated ovarian tumors, whereas the 10 control effusions from patients with various diseases were negative.

Affinity purification-mass spectrometry analysis of bcl-2 interactome identified SLIRP as a novel interacting protein.

Members of the bcl-2 protein family share regions of sequence similarity, the bcl-2 homology (BH) domains. Bcl-2, the most studied member of this family, has four BH domains, BH1-4, and has a critical role in resistance to antineoplastic drugs by regulating the mitochondrial apoptotic pathway. Moreover, it is also involved in other relevant cellular processes such as tumor progression, angiogenesis and autophagy. Deciphering the network of bcl-2-interacting factors should provide a critical advance in understanding the different functions of bcl-2. Here, we characterized bcl-2 interactome by mass spectrometry in human lung adenocarcinoma cells. In silico functional analysis associated most part of the identified proteins to mitochondrial functions. Among them we identified SRA stem-loop interacting RNA-binding protein, SLIRP, a mitochondrial protein with a relevant role in regulating mitochondrial messenger RNA (mRNA) homeostasis. We validated bcl-2/SLIRP interaction by immunoprecipitation and immunofluorescence experiments in cancer cell lines from different histotypes. We showed that, although SLIRP is not involved in mediating bcl-2 ability to protect from apoptosis and oxidative damage, bcl-2 binds and stabilizes SLIRP protein and regulates mitochondrial mRNA levels. Moreover, we demonstrated that the BH4 domain of bcl-2 has a role in maintaining this binding.

Proliferating and quiescent cells exhibit different subcellular distribution of protein kinase C activity.

The activity of calcium, phospholipid-dependent protein kinase (PKc), which is thought to play an important role in cell proliferation, has been measured in the particulate and soluble fractions of cultured cells, under different proliferative conditions. Our results indicate that proliferating cells display higher PKc activity than quiescent cells. Furthermore, in both normal and transformed cells, PKc is preferentially associated with the particulate fraction when the cells are proliferating, while in mitotically quiescent cells the majority of the enzyme activity is found in the soluble fraction. These data suggest tha PKc activity and subcellular distribution undergo spontaneous changes according to the proliferative state of the cells.

Identification and cellular localisation of voltage-operated calcium channels in immature rat testis.

Sertoli cells regulate the spermatogenic process mainly through the secretion of a complex fluid into the lumen of the seminiferous tubules behind the blood-testis barrier, containing many of the essential proteins necessary for maintenance and maturation of male germ cells. Thus, the study of Sertoli cell secretory processes is strictly correlated with the understanding of the regulatory mechanisms of spermatogenesis. In this work the authors have explored the voltage-sensitive calcium channel variety in the immature rat testis, their localisation and distribution within the seminiferous epithelium and peritubular and interstitial tissues as well as the possible role in the control of Sertoli cell secretion. The results reported in this paper, obtained by in situ hybridisation, immunohistology of rat testicular sections and Western blot analysis of Sertoli cell plasma membranes, show that mammalian Sertoli cells express mRNA encoding for several voltage-operated calcium channel subunits and express such proteins on their surface. Experiments performed on Sertoli cell monolayers cultured in the presence of specific toxins indicate that both N and P/Q-type Ca(2+) channels are involved in the regulation of protein secretion.

Breast cancer stem cells rely on fermentative glycolysis and are sensitive to 2-deoxyglucose treatment.

A number of studies suggest that cancer stem cells are essential for tumour growth, and failure to target these cells can result in tumour relapse. As this population of cells has been shown to be resistant to radiation and chemotherapy, it is essential to understand their biology and identify new therapeutic approaches. Targeting cancer metabolism is a potential alternative strategy to counteract tumour growth and recurrence. Here we applied a proteomic and targeted metabolomic analysis in order to point out the main metabolic differences between breast cancer cells grown as spheres and thus enriched in cancer stem cells were compared with the same cells grown in adherent differentiating conditions. This integrated approach allowed us to identify a metabolic phenotype associated with the stem-like condition and shows that breast cancer stem cells (BCSCs) shift from mitochondrial oxidative phosphorylation towards fermentative glycolysis. Functional validation of proteomic and metabolic data provide evidences for increased activities of key enzymes of anaerobic glucose fate such as pyruvate kinase M2 isoform, lactate dehydrogenase and glucose 6-phopshate dehydrogenase in cancer stem cells as well as different redox status. Moreover, we show that treatment with 2-deoxyglucose, a well known inhibitor of glycolysis, inhibits BCSC proliferation when used alone and shows a synergic effect when used in combination with doxorubicin. In conclusion, we suggest that inhibition of glycolysis may be a potentially effective strategy to target BCSCs.

ProNGF\NGF imbalance triggers learning and memory deficits, neurodegeneration and spontaneous epileptic-like discharges in transgenic mice.

ProNGF, the precursor of mature nerve growth factor (NGF), is the most abundant form of NGF in the brain. ProNGF and mature NGF differ significantly in their receptor interaction properties and in their bioactivity. ProNGF increases markedly in the cortex of Alzheimer's disease (AD) brains and proNGF\NGF imbalance has been postulated to play a role in neurodegeneration. However, a direct proof for a causal link between increased proNGF and AD neurodegeneration is lacking. In order to evaluate the consequences of increased levels of proNGF in the postnatal brain, transgenic mice expressing a furin cleavage-resistant form of proNGF, under the control of the neuron-specific mouse Thy1.2 promoter, were derived and characterized. Different transgenic lines displayed a phenotypic gradient of neurodegenerative severity features. We focused the analysis on the two lines TgproNGF#3 and TgproNGF#72, which shared learning and memory impairments in behavioral tests, cholinergic deficit and increased Aß-peptide immunoreactivity. In addition, TgproNGF#3 mice developed Aß oligomer immunoreactivity, as well as late diffuse astrocytosis. Both TgproNGF lines also display electrophysiological alterations related to spontaneous epileptic-like events. The results provide direct evidence that alterations in the proNGF/NGF balance in the adult brain can be an upstream driver of neurodegeneration, contributing to a circular loop linking alterations of proNGF/NGF equilibrium to excitatory/inhibitory synaptic imbalance and amyloid precursor protein (APP) dysmetabolism.

Aberrant splicing of Gs alpha transcript in transformed human astroglial and glioblastoma cell lines.

Alpha subunits of G proteins, which play a vital role in signal transduction, display considerable structural and functional diversity. Point mutations in two forms of alpha subunits, Gs alpha and Gi2 alpha, impairing their GTPase activity, have been detected in endocrine disorders. We report here the presence of truncated Gs alpha transcripts in a human glioblastoma cell line, HS683, and in an SV40-transformed human astroglial cell line, SVG. These transcripts were detected by polymerase chain reaction (PCR) amplification of cDNAs from the cell lines. The truncated Gs alpha transcripts, with deletions in the central region of the molecule, seem to have originated due to aberrant splicing within exonic sequences, which did not conform to the consensus GT/AG splice signals. The presence of a smaller size protein of mol.wt. around 25,000 kd in the SVG and HS683 cell lines, detected by antibodies specific for the C-terminal region of the Gs alpha subunit, seems to be consistent with the presence of truncated Gs alpha transcripts in these cell lines. These aberrantly spliced transcripts, if translated, could synthesize potentially oncogenic Gs alpha subunits deficient in GTPase activity. Whether such molecules, with sometimes relatively large deletions, retain some aspects of their function and are biologically significant remains to be seen.

Single acetylcholine-activated channels in cultured rhabdomyoblasts.

Acetylcholine receptor (AChR) was found to be present on the cell surface of the human rhabdomyoblast (RD) cell line. Two classes of ACh-activated channels have been observed, one with a large conductance and long duration and the other with smaller conductance and short duration, similar to those of human myotubes. RD membrane exhibited a specific binding to the alpha-bungarotoxin indicating the presence of nicotinic AChRs. These results support the hypothesis that rhabdomyosarcomas derive from myogenic precursors.

Myogenic conversion of mammalian fibroblasts induced by differentiating muscle cells.

Somite-derived skeletal myoblasts are supposed to be the sole source of muscle fibre nuclei during pre- and postnatal development, but evidence is accumulating for unorthodox contributions to muscle fibre nuclei from other cell types. For example, in tissue culture, fibroblasts can fuse with dysgenic myoblasts and restore correct membrane function. We report here the results of a series of experiments investigating this phenomenon and its possible mechanism. 10T1/2 cells, infected with a replication defective retrovirus encoding the bacterial enzyme beta-galactosidase, fused to form beta-galactosidase positive, differentiated myotubes when cocultured with differentiating uninfected C2C12 or primary myogenic cells, but this did not occur when they were cocultured with other cells such as 3T3 fibroblasts or PC12 pheochromocytoma cells. Myogenic conversion ranged from 1 to 10% of the 10T1/2 cell population and required close cell interaction between the different cells types: it was not induced by conditioned medium or extracellular matrix deposited by C2C12 cells. Myogenic conversion was also observed in vivo, after injection of similarly infected 10T1/2 cells into regenerating muscle. Conversion was seen also after coculture of uninfected 10T1/2 cells with primary chick myoblasts, thus demonstrating that it was not dependent upon viral infection and that there is no species or class barrier in this phenomenon. Primary fibroblasts, isolated from different organs of transgenic mice carrying a Lac Z marker under the control of a muscle-specific promoter, restricting beta-galactosidase expression to striated muscle cells, also underwent myogenic conversion, when cocultured with C2C12 myoblasts.(ABSTRACT TRUNCATED AT 250 WORDS)

l-Threonine aldolase, serine hydroxymethyltransferase and fungal alanine racemase. A subgroup of strictly related enzymes specialized for different functions.

Serine hydroxymethyltransferase (SHMT) is a member of the fold type I family of vitamin B6-dependent enzymes, a group of evolutionarily related proteins that share the same overall fold. The reaction catalysed by SHMT, the transfer of Cbeta of serine to tetrahydropteroylglutamate (H4PteGlu), represents in the cell an important link between the breakdown of amino acids and the metabolism of folates. In the absence of H4PteGlu and when presented with appropriate substrate analogues, SHMT shows a broad range of reaction specificity, being able to catalyse at appreciable rates retroaldol cleavage, racemase, aminotransferase and decarboxylase reactions. This apparent lack of specificity is probably a consequence of the particular catalytic apparatus evolved by SHMT. An interesting question is whether other fold type I members that normally catalyse the reactions which for SHMT could be considered as 'forced errors', may be close relatives of this enzyme and have a catalytic apparatus with the same basic features. As shown in this study, l-threonine aldolase from Escherichia coli is able to catalyse the same range of reactions catalysed by SHMT, with the exception of the serine hydroxymethyltransferase reaction. This observation strongly suggests that SHMT and l-threonine aldolase are closely related enzymes specialized for different functions. An evolutionary analysis of the fold type I enzymes revealed that SHMT and l-threonine aldolase may actually belong to a subgroup of closely related proteins; fungal alanine racemase, an extremely close relative of l-threonine aldolase, also appears to be a member of the same subgroup. The construction of three-dimensional homology models of l-threonine aldolase from E. coli and alanine racemase from Cochliobolus carbonum, and their comparison with the SHMT crystal structure, indicated how the tetrahydrofolate binding site might have evolved and offered a starting point for further investigations.