Immunopurified aggregates of PrP^Sc support <i>in vitro</i> misfolding of PrP^C by PMCA.

<p>Aggregates of PrP^Sc immunopurified from P3 fractions of RML-infected C57BL/6 mice (lanes 1–8), or immunoprecipitates of P3 fractions of <i>Prnp</i>^0/0 mice (lanes 9–12) were resuspended in PBS (lanes 1–4 and 9–12) or immobilized on glass coverslips by ultracentrifugation (lanes 5–8), and mixed with brain homogenates of Tg(WT) mice overexpressing 3F4-tagged PrP^C. Mixtures were either rapidly frozen (lanes 1 and 2, 5 and 6, 9 and 10) or subjected to 90 cycles of sonication/incubation (lanes 3 and 4, 7 and 8, 11 and 12). Samples were then either subjected to proteinase K (PK) (lanes 2, 4, 6, 8, 10 and 12), or left undigested (lanes 1, 3, 5, 7, 9 and 11). All samples were analyzed by Western blotting with 3F4 antibody.</p

15B3 immunoprecipitation yields pure preparations of infectious and non-infectious mouse PrP aggregates.

<p>(A) PrP aggregates were immunoprecipitated from P3 fractions of Tg(PG14) brains with antibody 15B3. PG14 PrP eluted (E lanes) by incubation in 0.1 M Tris-Glycine buffer at different pH (lanes 1–7) or by sonication in PBS (lanes 8–9), and the residual protein eluted by boiling the Dynabeads in SDS (B lanes), were detected by Western blotting with anti-PrP antibody 3F4. (B) PrP aggregates, immunopurified and eluted by sonication, from un-inoculated Tg(PG14) (lane 1) and Tg(CJD) (lane 2) mice, or RML-infected C57BL/6 (lane 3) and Tg(PG14) (lane 4) mice were visualized by silver staining (top panel) or by Western blotting (bottom panel) using anti-PrP antibody 8H4. No protein bands were detected when the purification was done using <i>Prnp</i>^0/0 mouse brains (lane 5). (C) Two samples of PrP^Sc immunopurified from RML-infected C57BL/6 mice were run on SDS-PAGE, and the gels stained to saturation with silver (lanes 1–2) or immunoblotted with antibody 8H4 (lanes 3–4).</p

Surface plasmon resonance shows that monoclonal antibody 15B3 selectively captures PG14 PrP aggregates from P3 fractions.

<p>Fractions P3 prepared from whole brains of Tg(PG14) or <i>Prnp</i>^0/0 mice were perfused for 5 min (bars) over the sensor surface of SPR chips on which antibody 15B3 (A) or 3F4 (B) had been immobilized. The sensorgrams (time course of the SPR signal in Resonance Units, RU) refer to the specific binding to the antibody (binding was negligible on sensor surfaces without antibody). Significant binding was detected when P3 fractions containing PG14 PrP (black line) were flowed over the 15B3-coated chip (A), but not on the 3F4-coated chip (B). No binding was observed when a P3 fraction from <i>Prnp</i>^0/0 mice was analyzed (gray line).</p

Brain Uptake of Tetrahydrohyperforin and Potential Metabolites after Repeated Dosing in Mice

Tetrahydrohyperforin (IDN-5706) is a semisynthetic derivative of hyperforin, one of the main active components of <i>Hypericum perforatum</i> extracts. It showed remarkable positive effects on memory and cognitive performances in wild-type mice and in a transgenic mouse model of Alzheimer’s disease, but little was known about the concentrations it can reach in the brain. The investigations reported herein show that repeated treatment of mice with tetrahydrohyperforin (20 mg/kg intraperitoneally, twice daily for 4 days and once on the fifth day) results in measurable concentrations in the brain, up to 367 ng/g brain (∼700 nM) 6 h after the last dose; these concentrations have significant effects on synaptic function in hippocampal slices. The other main finding was the identification and semiquantitative analysis of tetrahydrohyperforin metabolites. In plasma, three hydroxylated/dehydrogenated metabolites were the largest (M1–3) and were also formed in vitro on incubation of tetrahydrohyperforin with mouse liver microsomes; the fourth metabolite in abundance was a hydroxylated/deisopropylated derivative (M13), which was not predicted in vitro. These metabolites were all detected in the brain, with peak areas from 10% (M1) to ∼1.5% (M2, M3, and M13) of the parent compound. In summary, repeated treatment of mice with tetrahydrohyperforin gave brain concentrations that might well underlie its central pharmacological effects. We also provide the first metabolic profile of this compound

Tetrahydro-β-carboline-Based Spirocyclic Lactam as Type II′ β‑Turn: Application to the Synthesis and Biological Evaluation of Somatostatine Mimetics

The synthesis of novel spirocyclic lactams, embodying d-tryptophan (Trp) amino acid as the central core and acting as peptidomimetics, is presented. It relies on the strategic combination of Seebach’s self-reproduction of chirality chemistry and Pictet–Spengler condensation as key steps. Investigation of the conformational behavior by molecular modeling, X-ray crystallography, and NMR and IR spectroscopies suggests very stable and highly predictable type II′ β-turn conformations for all compounds. Relying on this feature, we also pursued their application to two potential mimetics of the hormone somatostatin, a pharmaceutically relevant natural peptide, which contains a Trp-based type II′ β-turn pharmacophore

A Mouse Model of Familial ALS Has Increased CNS Levels of Endogenous Ubiquinol_9/10 and Does Not Benefit from Exogenous Administration of Ubiquinol₁₀

<div><p>Oxidative stress and mitochondrial impairment are the main pathogenic mechanisms of Amyotrophic Lateral Sclerosis (ALS), a severe neurodegenerative disease still lacking of effective therapy. Recently, the coenzyme-Q (CoQ) complex, a key component of mitochondrial function and redox-state modulator, has raised interest for ALS treatment. However, while the oxidized form ubiquinone₁₀ was ineffective in ALS patients and modestly effective in mouse models of ALS, no evidence was reported on the effect of the reduced form ubiquinol₁₀, which has better bioavailability and antioxidant properties. In this study we compared the effects of ubiquinone₁₀ and a new stabilized formulation of ubiquinol₁₀ on the disease course of SOD1^G93A transgenic mice, an experimental model of fALS. Chronic treatments (800 mg/kg/day orally) started from the onset of disease until death, to mimic the clinical trials that only include patients with definite ALS symptoms. Although the plasma levels of CoQ₁₀ were significantly increased by both treatments (from <0.20 to 3.0–3.4 µg/mL), no effect was found on the disease progression and survival of SOD1^G93A mice. The levels of CoQ₁₀ in the brain and spinal cord of ubiquinone₁₀- or ubiquinol₁₀-treated mice were only slightly higher (≤10%) than the endogenous levels in vehicle-treated mice, indicating poor CNS availability after oral dosing and possibly explaining the lack of pharmacological effects. To further examine this issue, we measured the oxidized and reduced forms of CoQ_9/10 in the plasma, brain and spinal cord of symptomatic SOD1^G93A mice, in comparison with age-matched SOD1^WT. Levels of ubiquinol_9/10, but not ubiquinone_9/10, were significantly higher in the CNS, but not in plasma, of SOD1^G93A mice, suggesting that CoQ redox system might participate in the mechanisms trying to counteract the pathology progression. Therefore, the very low increases of CoQ₁₀ induced by oral treatments in CNS might be not sufficient to provide significant neuroprotection in SOD1^G93A mice.</p></div

6‑Methoxy-7-benzofuranoxy and 6‑Methoxy-7-indolyloxy Analogues of 2‑[2-(2,6-Dimethoxyphenoxy)ethyl]aminomethyl-1,4-benzodioxane (WB4101): Discovery of a Potent and Selective α_1D-Adrenoceptor Antagonist

Previous results have shown that replacement of one of the two <i>o</i>-methoxy groups at the phenoxy residue of the potent, but not subtype-selective, α₁-AR antagonist (<i>S</i>)-WB4101 [(<i>S</i>)-<b>1</b>] by phenyl, or by ortho,meta-fused cyclohexane, or especially by ortho,meta-fused benzene preferentially elicits α_1D-AR antagonist affinity. Such observations inspired the design of four new analogues of <b>1</b> bearing, in lieu of the 2,6-dimethoxyphenoxy residue, a 6-methoxy-substituted 7-benzofuranoxy or 7-indolyloxy group or, alternatively, their corresponding 2,3-dihydro form. Of these new compounds, which maintain, rigidified, the characteristic ortho heterodisubstituted phenoxy substructure of <b>1</b>, the <i>S</i> enantiomer of the dihydrobenzofuranoxy derivative exhibited the highest α_1D-AR antagonist affinity (p<i>A</i>₂ 9.58) with significant α_1D/α_1A and α_1D/α_1B selectivity. In addition, compared both to α_1D-AR antagonists structurally related to <b>1</b> and to the well-known α_1D-AR antagonist BMY7378, this derivative had modest 5-HT_1A affinity and neutral α₁-AR antagonist behavior

Ubiquinol₁₀ chronic treatment has no effect on disease course in SOD1^G93A mice.

<p>Effect of oral treatment with 800 mg/kg/day ubiquinone₁₀, ubiquinol₁₀ or vehicle, on motor dysfunction and disease progression of 129Sv SOD1^G93A mice (n = 15 mice per group). Treatment started at the age of 91 days (arrows) until the sacrifice (at the end-stage of the disease, when mice were unable to right themselves within 10 seconds after being placed on both sides). Treatment with ubiquinone₁₀ or ubiquinol₁₀ had no significant effects on body weight (A), on the latency of rotarod (B) and PaGE test (C) (Two-way ANOVA), or on disease onset (D) and survival length (E). Each point represents the mean; for sake of clarity standard deviations are not indicated but they were always less than 15% of the value. Table reports the mean and standard deviations of symptoms onset and life-span for each group.</p

Levels of CoQ_9/10 in the plasma of SOD1^G93A mice treated chronically with ubiquinone₁₀ or ubiquinol₁₀.

<p>Female 129Sv SOD1^G93A mice were treated orally with 800 mg/kg of ubiquinol₁₀ or ubiquinone₁₀, or vehicle (sunflower seed oil), once a day, starting from age of 91 days until the last stage of the disease (age of 125 days, on average) when they were sacrificed, two hours after the last treatment. Values are means±SEM of (n) mice.</p