Cerebrospinal Fluid Aβ42 Levels: When Physiological Become Pathological State

Impaired amyloid beta (Aβ) metabolism is currently considered central to understand the pathophysiology of Alzheimer's disease (AD). Measurements of cerebrospinal fluid Aβ levels remain the most useful marker for diagnostic purposes and to individuate people at risk for AD. Despite recent advances criticized the direct role in neurodegeneration of cortical neurons, Aβ is considered responsible for synaptopathy and impairment of neurotransmission and therefore remains the major trigger of AD and future pharmacological treatment remain Aβ oriented. However, experimental and clinical findings showed that Aβ peptides could have a wider range of action responsible for cell dysfunction and for appearance of clinico‐pathological entities different from AD. Such findings may induce misunderstanding of the real role played by Aβ in AD and therefore strengthen criticism on its centrality and need for CSF measurements. Aim of this review is to discuss the role of CSF Aβ levels in light of experimental, clinical pathologic, and electrophysiological results in AD and other pathological entities to put in a correct frame the value of Aβ changes.     

A 6q14.1‐q15 microdeletion in a male patient with severe autistic disorder,lack of oral language,and dysmorphic features with concomitant presence of a maternally inherited Xp22.31 copy number gain

We report on a male patient with severe autistic disorder, lack of oral language, and dysmorphic features who carries a rare interstitial microdeletion of 4.96 Mb at chromosome 6q14.1‐q15. The patient also harbors a maternally inherited copy number gain of 1.69 Mb at chromosome Xp22.31, whose pathogenicity is under debate.     

Combinatorial biosynthesis of sapogenins and saponins in Saccharomyces cerevisiae using a C-16α hydroxylase from Bupleurum falcatum

The saikosaponins comprise oleanane- and ursane-type triterpene saponins that are abundantly present in the roots of the genus Bupleurum widely used in Asian traditional medicine. Here we identified a gene, designated CYP716Y1, encoding a cytochrome P450 monooxygenase from Bupleurum falcatum that catalyzes the C-16α hydroxylation of oleanane- and ursane-type triterpenes. Exploiting this hitherto unavailable enzymatic activity, we launched a combinatorial synthetic biology program in which we combined CYP716Y1 with oxidosqualene cyclase, P450, and glycosyltransferase genes available from other plant species and reconstituted the synthesis of monoglycosylated saponins in yeast. Additionally, we established a culturing strategy in which applying methylated β-cyclodextrin to the culture medium allows the sequestration of heterologous nonvolatile hydrophobic terpenes, such as triterpene sapogenins, from engineered yeast cells into the growth medium, thereby greatly enhancing productivity. Together, our findings provide a sound base for the development of a synthetic biology platform for the production of bioactive triterpene sapo(ge)nins.Triterpene saponins are secondary metabolites that exhibit a large structural diversity and wide range of biological activities in many plant species (1, 2). Saponins are glycosides of sapogenins, which are composed of 30 carbon atoms arranged in 4- or 5-ring structures that are “decorated” by functional groups. Saponins are synthesized by multiple glycosylations of the sapogenin building blocks that are produced by multiple cytochrome P450-dependent monooxygenase (P450) or oxidoreductase-mediated modifications of basic backbones, such as β-amyrin (oleanane type), α-amyrin (ursane type), lupeol, and dammarenediol. These backbones are generated by oxidosqualene cyclase (OSC)-mediated cyclization of 2,3-oxidosqualene, which is also an intermediate in the synthesis of sterols in eukaryotes (3, 4). Both saponins and sapogenins include biologically active compounds or serve as starter molecules for the generation of novel, potentially bioactive structures by synthetic modification (5–7).The genus Bupleurum consists of perennial herbs that are used in Asian traditional medicine, either alone or in combination with other ingredients, for the treatment of common colds, fever, and inflammatory disorders (8). Saikosaponins constitute the largest class of secondary metabolites in Bupleurum and can account for up to ∼7% of root dry weight. Their accumulation can be further stimulated by jasmonate treatment (9). More than 120 closely related oleanane- and ursane-type saikosaponins have been identified from this genus and the oxidations at various positions suggest the presence of multiple enzymes, mainly P450s, capable of catalyzing specific modifications on the amyrin backbones (8, 10). To date, no P450 or oxidoreductase involved in triterpene saponin biosynthesis has been identified from Bupleurum species.P450s that modify the β-amyrin backbone on C-11; C-12,13; C-16; C-22; C-23; C-28 or C-30 have been characterized from Glycyrrhiza uralensis, Avena strigosa, Medicago truncatula, Glycine max, Vitis vinifera, and Catharanthus roseus (11–18). Hydroxylases from Panax ginseng that oxidize the dammarenediol-II backbone on C-6, C-12, or C-28 (19–21), and a C-20 hydroxylase from Lotus japonicus (22) that modifies lupeol, have also been identified. To characterize these P450s, they have been ectopically expressed in yeast strains either producing β-amyrin or externally supplied with candidate substrates. Similarly, several OSCs have been produced and functionally analyzed in yeast. From these studies, it is clear that yeast cells cannot only be used for the characterization of novel enzymes, but possibly also as a heterologous host for the production of triterpene sapogenins (23). To date only two pilot studies have aimed at engineering of β-amyrin production in yeast (24, 25), but no efforts toward engineering of sustainable production of sapogenins or saponins in yeast have been reported.Here, we identified and characterized CYP716Y1, a P450 from Bupleurum falcatum that corresponds to a C-16α oxidase, designated according to Nelson’s nomenclature (http://drnelson.uthsc.edu/cytochromeP450.html). By designing triterpene-hyperproducing starter strains, optimizing culturing conditions for triterpene synthesis, and using the CYP716Y1 gene in a combinatorial synthetic biology program, we established a platform that allows us to produce and sequester triterpene sapogenins in culture medium and to reconstitute a full saponin synthetic pathway in yeast cells.     

Testosterone exerts antiapoptotic effects against H2O2 in C2C12 skeletal muscle cells through the apoptotic intrinsic pathway

Experimental data indicate that apoptosis is activated in the aged skeletal muscle, contributing to sarcopenia. We have previously demonstrated that testosterone protects against hydrogen peroxide (H(2)O(2))-induced apoptosis in C2C12 muscle cells. Here we identified molecular events involved in the antiapoptotic effect of testosterone. At short times of exposure to H(2)O(2) cells exhibit a defense response but at longer treatment times cells undergo apoptosis. Incubation with testosterone prior to H(2)O(2) induces BAD inactivation, inhibition of poly(ADP-ribose) polymerase cleavage, and a decrease in BAX levels, and impedes the loss of mitochondrial membrane potential, suggesting that the hormone participates in the regulation of the apoptotic intrinsic pathway. Simultaneous treatment with testosterone, H(2)O(2), and the androgen receptor (AR) antagonist, flutamide, reduces the effects of the hormone, pointing to a possible participation of the AR in the antiapoptotic effect. The data presented allow us to begin to elucidate the mechanism by which the hormone prevents apoptosis in skeletal muscle.