Analysis of the hippocampal proteome in ME7 prion disease reveals a predominant astrocytic signature and highlights the brain-restricted production of clusterin in chronic neurodegeneration

Prion diseases are characterized by accumulation of misfolded protein, gliosis, synaptic dysfunction, and ultimately neuronal loss. This sequence, mirroring key features of Alzheimer disease, is modeled well in ME7 prion disease. We used iTRAQ(TM)/mass spectrometry to compare the hippocampal proteome in control and late-stage ME7 animals. The observed changes associated with reactive glia highlighted some specific proteins that dominate the proteome in late-stage disease. Four of the up-regulated proteins (GFAP, high affinity glutamate transporter (EAAT-2), apo-J (Clusterin), and peroxiredoxin-6) are selectively expressed in astrocytes, but astrocyte proliferation does not contribute to their up-regulation. The known functional role of these proteins suggests this response acts against protein misfolding, excitotoxicity, and neurotoxic reactive oxygen species. A recent convergence of genome-wide association studies and the peripheral measurement of circulating levels of acute phase proteins have focused attention on Clusterin as a modifier of late-stage Alzheimer disease and a biomarker for advanced neurodegeneration. Since ME7 animals allow independent measurement of acute phase proteins in the brain and circulation, we extended our investigation to address whether changes in the brain proteome are detectable in blood. We found no difference in the circulating levels of Clusterin in late-stage prion disease when animals will show behavioral decline, accumulation of misfolded protein, and dramatic synaptic and neuronal loss. This does not preclude an important role of Clusterin in late-stage disease, but it cautions against the assumption that brain levels provide a surrogate peripheral measure for the progression of brain degeneration

Osmotic behaviour of human mesenchymal stem cells: implications for cryopreservation

Aimed at providing a contribution to the optimization of cryopreservation processes, the present work focuses on the osmotic behavior of human mesenchymal stem cells (hMSCs). Once isolated from the umbilical cord blood (UCB) of three different donors, hMSCs were characterized in terms of size distribution and their osmotic properties suitably evaluated through the exposure to hypertonic and isotonic aqueous solutions at three different temperatures. More specifically, inactive cell volume and cell permeability to water and di-methyl sulfoxide (DMSO) were measured, being cell size determined using impedance measurements under both equilibrium and dynamic conditions. Experimental findings indicate that positive cell volume excursions are limited by the apparent increase of inactive volume, which occurs during both the shrink-swell process following DMSO addition and the subsequent restoration of isotonic conditions in the presence of hypertonic solutions of impermeant or permeant solutes. Based on this evidence, hMSCs must be regarded as imperfect osmometers, and their osmotic behavior described within a scenario no longer compatible with the simple two-parameter model usually utilized in the literature. In this respect, the activation of mechano-sensitive ion-channels seemingly represents a reasonable hypothesis for rationalizing the observed osmotic behavior of hMSCs from UCB

Ecological correlates of Golden eagle (Aquila chrysaetos) breeding occurrence in Sardinia (Italy).

Capsule Golden Eagles Aquila chrysaetos in Sardinia are clustered across the main mountain ranges of the island, with a preference for undisturbed and homogeneous inland habitats. Aims To analyse habitat preferences of the Golden Eagle in Sardinia, Italy, at the landscape and home range spatial scales. Methods Landscape scale habitat preferences were analysed using the 10 × 10 km Universal Transverse Mercator grid and the home range scale was based on the spatial distribution of breeding territories. Generalized linear models were fitted with three different sets of environmental predictors (topographic, bioclimatic and land use variables) to analyse the spatial distribution of Golden Eagles with a case-control design. Results Eagles showed a preference for rugged and elevated areas, characterized by a certain degree of humidity and surrounded by areas of forest. The distribution of Golden Eagles on this Mediterranean island was negatively affected by the occurrence of arable farmland and coastal areas, as well as by the effects of habitat fragmentation. Conclusions The results of this study could contribute to future management strategies and conservation projects aimed to protect this species, and may be used to identify the most suitable conservation areas for this and other competing species, such as the Bonelli's Eagle Aquila fasciata, which is currently the subject of a reintroduction project in Sardinia

Immune complex formation impairs the elimination of solutes from the brain: implications for immunotherapy in Alzheimer's disease

Background: Basement membranes in the walls of cerebral capillaries and arteries form a major lymphatic drainage pathway for fluid and solutes from the brain. Amyloid-β (Aβ) draining from the brain is deposited in such perivascular pathways as cerebral amyloid angiopathy (CAA) in Alzheimer's disease (AD). CAA increases in severity when Aβ is removed from the brain parenchyma by immunotherapy for AD. In this study we investigated the consequences of immune complexes in artery walls upon drainage of solutes similar to soluble Aβ. We tested the hypothesis that, following active immunization with ovalbumin, immune complexes form within the walls of cerebral arteries and impair the perivascular drainage of solutes from the brain. Mice were immunized against ovalbumin and then challenged by intracerebral microinjection of ovalbumin. Perivascular drainage of solutes was quantified following intracerebral microinjection of soluble fluorescent 3kDa dextran into the brain at different time intervals after intracerebral challenge with ovalbumin. Results: Ovalbumin, IgG and complement C3 co-localized in basement membranes of artery walls 24 hrs after challenge with antigen; this was associated with significantly reduced drainage of dextran in immunized mice. Conclusions: Perivascular drainage along artery walls returned to normal by 7 days. These results indicate that immune complexes form in association with basement membranes of cerebral arteries and interfere transiently with perivascular drainage of solutes from the brain. Immune complexes formed during immunotherapy for AD may similarly impair perivascular drainage of soluble Aβ and increase severity of CAA

Glycogen Synthase Kinase 3 (GSK3) Inhibitor, SB-216763, Promotes Pluripotency in Mouse Embryonic Stem Cells

Canonical Wnt/β-catenin signaling has been suggested to promote self-renewal of pluripotent mouse and human embryonic stem cells. Here, we show that SB-216763, a glycogen synthase kinase-3 (GSK3) inhibitor, can maintain mouse embryonic stem cells (mESCs) in a pluripotent state in the absence of exogenous leukemia inhibitory factor (LIF) when cultured on mouse embryonic fibroblasts (MEFs). MESCs maintained with SB-216763 for one month were morphologically indistinguishable from LIF-treated mESCs and expressed pluripotent-specific genes Oct4, Sox2, and Nanog. Furthermore, Nanog immunostaining was more homogenous in SB-216763-treated colonies compared to LIF. Embryoid bodies (EBs) prepared from these mESCs expressed early-stage markers for all three germ layers, and could efficiently differentiate into cardiac-like cells and MAP2-immunoreactive neurons. To our knowledge, SB-216763 is the first GSK3 inhibitor that can promote self-renewal of mESC co-cultured with MEFs for more than two months

Soluble forms of tau are toxic in Alzheimer's disease

Accumulation of neurofibrillary tangles (NFT), intracellular inclusions of fibrillar forms of tau, is a hallmark of Alzheimer Disease. NFT have been considered causative of neuronal death, however, recent evidence challenges this idea. Other species of tau, such as soluble misfolded, hyperphosphorylated, and mislocalized forms, are now being implicated as toxic. Here we review the data supporting soluble tau as toxic to neurons and synapses in the brain and the implications of these data for development of therapeutic strategies for Alzheimer’s disease and other tauopathies

Change in tau phosphorylation associated with neurodegeneration in the ME7 model of prion disease

Hyperphosphorylation of the microtubule-associated protein tau is a significant determinant in AD (Alzheimer's disease), where it is associated with disrupted axonal transport and probably causes synaptic dysfunction. Although less well studied, hyperphosphorylation has been observed in prion disease. We have investigated the expression of hyperphosphorylated tau in the hippocampus of mice infected with the ME7 prion agent. In ME7-infected animals, there is a selective loss of CA1 synapse, first discernable at 13 weeks of disease. There is a potential that dysfunctional axonal transport contributes to this synaptopathy. Thus investigating hyperphosphorylated tau that is dysfunctional in AD could illuminate whether and how they are significant in prion disease. We observed no differences in the levels of phosphorylated tau (using MC1, PHF-1 and CP13 antibodies) in detergent-soluble and detergent-insoluble fractions extracted from ME7- and NBH- (normal brain homogenate) treated animals across disease. In contrast, we observed an increase in phospho-tau staining for several epitopes using immunohistochemistry in ME7-infected hippocampal sections. Although the changes were not of the magnitude seen in AD tissue, clear differences for several phospho-tau species were seen in the CA1 and CA3 of ME7-treated animals (pSer(199-202)>pSer(214)>PHF-1 antibody). Temporally, these changes were restricted to animals at 20 weeks and none of the disease-related staining was associated with the axons or dendrites that hold CA1 synapses. These findings suggest that phosphorylation of tau at the epitopes examined does not underpin the early synaptic dysfunction. These data suggest that the changes in tau phosphorylation recorded here and observed by others relate to end-stage prion pathology when early dysfunctions have progressed to overt neuronal loss