RESUMEN
The accumulation of extracellular amyloid beta (Abeta42) both in brain and in cerebral vessels characterizes Alzheimer's disease (AD) pathogenesis. Recently, the possibility to functionalize nanoparticles (NPs) surface with Abeta42 binding molecules, making them suitable tools for reducing Abeta42 burden has been shown effective in models of AD. Aim of this work consisted in proving that NPs might be effective in sequestering Abeta42 in biological fluids, such as CSF and plasma. This demonstration is extremely important considering that these Abeta42 pools are in continuum with the brain parenchyma with drainage of Abeta from interstitial brain tissue to blood vessel and plasma. In this work, liposomes (LIP) were functionalized as previously shown in order to promote high-affinity Abeta binding, i.e., either with, phosphatidic acid (PA), or a modified Apolipoprotein E-derived peptide (mApo), or with a curcumin derivative (TREG); Abeta42 levels were determined by ELISA in CSF and plasma samples. mApo-PA-LIP (25 and 250 µM) mildly albeit significantly sequestered Abeta42 proteins in CSF samples obtained from healthy subjects (p < 0.01). Analogously a significant binding (â¼20%) of Abeta42 (p < 0.001) was demonstrated following exposure to all functionalized liposomes in plasma samples obtained from selected AD or Down's syndrome patients expressing high levels of Abeta42. The same results were obtained by quantifying Abeta42 content after removal of liposome-bound Abeta by using gel filtration chromatography or ultracentrifugation on a discontinuous sucrose density gradient. In conclusion, we demonstrate that functionalized liposomes significantly sequester Abeta42 in human biological fluids. These data may be critical for future in vivo administration tests using NPs for promoting sink effect.
Asunto(s)
Enfermedad de Alzheimer/sangre , Enfermedad de Alzheimer/líquido cefalorraquídeo , Péptidos beta-Amiloides/sangre , Péptidos beta-Amiloides/líquido cefalorraquídeo , Liposomas/metabolismo , Fragmentos de Péptidos/sangre , Fragmentos de Péptidos/líquido cefalorraquídeo , Anciano , Anciano de 80 o más Años , Biomarcadores/sangre , Biomarcadores/líquido cefalorraquídeo , Femenino , Humanos , MasculinoRESUMEN
The CRISPR/Cas9 system is a rapid and customizable tool for gene editing in mammalian cells. In particular, this approach has widely opened new opportunities for genetic studies in neurological disease. Human neurons can be differentiated in vitro from hPSC (human Pluripotent Stem Cells), hNPCs (human Neural Precursor Cells) or even directly reprogrammed from fibroblasts. Here, we described a new platform which enables, rapid and efficient CRISPR/Cas9-mediated genome targeting simultaneously with three different paradigms for in vitro generation of neurons. This system was employed to inactivate two genes associated with neurological disorder (TSC2 and KCNQ2) and achieved up to 85% efficiency of gene targeting in the differentiated cells. In particular, we devised a protocol that, combining the expression of the CRISPR components with neurogenic factors, generated functional human neurons highly enriched for the desired genome modification in only 5 weeks. This new approach is easy, fast and that does not require the generation of stable isogenic clones, practice that is time consuming and for some genes not feasible.
Asunto(s)
Diferenciación Celular/genética , Reprogramación Celular/genética , Células Madre Pluripotentes Inducidas/citología , Células-Madre Neurales/clasificación , Sistemas CRISPR-Cas/genética , Fibroblastos/citología , Fibroblastos/metabolismo , Silenciador del Gen , Vectores Genéticos , Humanos , Células Madre Pluripotentes Inducidas/metabolismo , Células-Madre Neurales/citología , Células-Madre Neurales/metabolismo , Neuronas/citología , Neuronas/metabolismoRESUMEN
OBJECTIVE: Donepezil (DNPZ) is a drug commonly used for Alzheimer's disease (AD) that may favour a T helper 2 phenotype leading to increased naturally occurring auto-antibodies (NAb) against beta-amyloid (Aß). We hypothesized the involvement of the cholinergic receptors [α7-nicotnic acetylcholine receptor (α7nAChR)] expressed on peripheral blood mononuclear cells (PBMC). METHODS: Fifty patients with mild-to-moderate AD, DNPZ treated (DNPZ+, n = 25) or not (DNPZ-, n = 25), and 25 matched controls were enrolled and PBMC extracted for both in vitro cultures, and real-time polymerase chain reaction and chromatin immunoprecipitation assay. Plasma samples were also obtained for Aß and NAb determination. RESULTS: Donepezil increased in vitro the expression of the transcription factor GATA binding protein 3 (GATA3) through α7nAChR, because prevented by the specific antagonist methyllycaconitine. Ex vivo PBMC α7nAChR mRNA expression was increased in both AD groups, while GATA3 expression was not. A significant increase in the GATA3/interleukin 5 promoter association was found in DNPZ+ patients. Finally, DNPZ+ patients showed both significantly higher plasma levels of anti-Aß NAb with respect to DNPZ- patients and Aß 1-42 with respect to normal controls. CONCLUSIONS: Donepezil might modulate a T helper 2 bias via α7nAChR leading to increased expression of NAb. Further studies on the role of the modulation of the immune response against Aß may pave the way to innovative therapeutic strategies for AD. Copyright © 2016 John Wiley & Sons, Ltd.