Increasing Cu bioavailability inhibits Aβ oligomers and tau phosphorylation

Cognitive decline in Alzheimer's disease (AD) involves pathological accumulation of synaptotoxic amyloid-β (Aβ) oligomers and hyperphosphorylated tau. Because recent evidence indicates that glycogen synthase kinase 3β (GSK3β) activity regulates these neurotoxic pathways, we developed an AD therapeutic strategy to target GSK3β. The strategy involves the use of copper-bis(thiosemicarbazonoto) complexes to increase intracellular copper bioavailability and inhibit GSK3β through activation of an Akt signaling pathway. Our lead compound Cu^II(gtsm) significantly inhibited GSK3β in the brains of APP/PS1 transgenic AD model mice. Cu^II(gtsm) also decreased the abundance of Aβ trimers and phosphorylated tau, and restored performance of AD mice in the Y-maze test to levels expected for cognitively normal animals. Improvement in the Y-maze correlated directly with decreased Aβ trimer levels. This study demonstrates that increasing intracellular copper bioavailability can restore cognitive function by inhibiting the accumulation of neurotoxic Aβ trimers and phosphorylated tau.     

Surveillance and control of African Swine Fever in free‐ranging pigs in Sardinia

African swine fever (ASF) is a notifiable infectious disease, caused by the ASF virus (ASFV), which is a DNA virus belonging to the family Asfarviridae, genus Asfivirus. This disease has gained importance in the last decade after its spread in several countries in Eastern and Central Europe, and more recently, in China. Despite the efforts made to eradicate it, ASF is still present on the Mediterranean island of Sardinia (Italy) and has been since 1978. ASF risk factors on the island have been analysed in previous studies; the role of free‐ranging pigs in virus persistence has been suggested, but has not been fully elucidated. The most recent eradication plan provides more stringent measures to combat free‐ranging pigs and any kind of illegality in the pig sector. From December 2017 to June 2018, a total of 29 depopulation actions were performed in 13 municipalities in central Sardinia, during which 2,281 free‐ranging pigs were culled and more than 50% of them were tested for ASFV and antibody presence (1,218 and 1,416, respectively). A total of 651 pigs were seropositive, with a mean seroprevalence of 53.4% (CI 95% = 50.6–56.3), and 38 were ASFV positive (virus prevalence = 2.6%; CI 95% = 2.1–3.0). To the best of our knowledge, the present study is the first to provide a complete evaluation of this millennial system of pig farming and ASFV prevalence in free‐ranging pigs. Furthermore, it has emphasised the necessity of combining the maintenance of an epidemiological surveillance program with continuous education of farmers and other people involved in pig husbandry, based on cultural and economic aspects.     

Neurotoxicity from glutathione depletion is dependent on extracellular trace copper

Glutathione (GSH) is an important antioxidant, and its depletion in neurons has been implicated in several neurodegenerative disorders. Aberrant copper metabolism is also implicated in neurodegeneration and may result in the generation of toxic free radicals. However, little is known about the relationship between GSH depletion and copper homeostasis. In the present study, we examined the role of extracellular trace biometals in neuronal cell death induced by GSH depletion. Treatment of primary cortical neurons with buthionine sulfoximine (BSO), an inhibitor of GSH synthesis, induced a rapid loss of intracellular GSH, leading to decreased neuronal cell viability. Neuronal cell death induced by GSH depletion was dependent on trace levels of extracellular copper in the culture medium (1.6 microM). Neurons were protected against GSH depletion-mediated toxicity when cultured in Chelex 100-treated medium containing tenfold less copper (0.16 microM) than normal medium. The addition of copper, but not iron or zinc, to Chelex 100-treated medium restored the neurotoxicity induced by GSH depletion. Moreover, BSO toxicity in normal medium was inhibited by copper chelators. The neurotoxic effects of copper in GSH-depleted neurons involved generation of copper(I) and subsequent free radical-mediated oxidative stress. These studies demonstrate a critical role for extracellular trace copper in neuronal cell death caused by GSH depletion and may have important implications for the understanding of toxic processes in neurodegenerative diseases.     

Immunization with DNA vaccines encoding different mycobacterial antigens elicits a Th1 type immune response in lambs and protects against Mycobacterium avium subspecies paratuberculosis infection

Paratuberculosis, or Johne's disease, is a disease of domestic and wild ruminants that culminate with a chronic enteritis caused by Mycobacterium avium subsp. paratuberculosis. The aim of this work was to evaluate the type of immune response, Th1 or Th2, induced by DNA vaccinations in lambs of Sarda breed. Twenty-five lambs, serum negative for M. paratuberculosis, were selected at birth from equally serum negative mothers. The lambs were inoculated at 5 months of age with three different mycobacterial antigens cloned into a mammalian expression vector as fusion protein with the enhanced green fluorescent protein (pEGFP-N1). The animals were divided in five groups containing each five lambs. Each group was vaccinated as following (A: physiological solution; B: Gudair; C: p-85A-Mav; D: p-85A-BCG; E: p-Hsp65). Immune response was evaluated by measuring the expression of INF-gamma (Th1 type response) and IL-10 (Th2 type response) by real-time PCR. Gene expression was estimated by comparing the results with that of beta-actin. INF-gamma expression level was increased in lambs vaccinated with plasmids codifying mycobacterial antigens, in particular with p-Hsp65, in comparison with the controls suggesting stimulation of a Th1 immune response similar to that supported by natural infection of M. paratuberculosis. Moreover, animals were infected orally with live M. paratuberculosis. Three months after vaccination and again INF-gamma and IL-10 expression was evaluated in order to verify in vivo the protection level of the vaccines. Plasmids p-85A-BCG and p-Hsp65 seem to elicit a stronger protective immune response against M. paratuberculosis by evaluating the expression level of INF-gamma and evaluating the presence of M. paratuberculosis and animal cell organ damage post-mortem.     

It all sticks together--the APP-related family of proteins and Alzheimer's disease

In the present review, we shall discuss the pros and cons of a possible functional relationship and contribution of the APP family members (APP, APLP1 and APLP2) to the development of Alzheimer's disease: (1) APP, APLP1 and APLP2 are highly homologous proteins with similar protein domain organization. (2) All APP family proteins have been found to be aggregated in typical Alzheimer's disease lesions. (3) Several other proteins have been implied to provide a functional link among the APP-related proteins. In normal adult brain APP, APLP1 and APLP2 are involved in synaptic processes important for memory function. We hypothesize that the functional loss of members of the APP family contributes to the gradual cognitive decline in Alzheimer's disease patients.     

Generation of a recombinant Fab antibody reactive with the Alzheimer's disease-related Abeta peptide

A recombinant Fab antibody, designated 1E8-4b, which reacts with the Alzheimer's disease (AD)-related Abeta peptides, Abeta[1-40], Abeta[1-42] and Abeta[1-43] has been developed. The 1E8-4b Fab was constructed by cloning the V(H)C(H1) and V(L)C(L) domains from the parent hybridoma 1E8 antibody, reported previously to recognize these Abeta peptides. Briefly, a C-terminal Flag tag sequence was incorporated into this construct, which was ligated into the vector pHFA2 and expressed in Escherichia coli. Following purification on an M2 anti-Flag affinity column, the 1E8-4b recombinant Fab antibody was shown to bind plaques within sections of brain tissue from CERAD-defined AD patients by immunohistochemistry. ELISA, epitope mapping and immunoblotting confirmed the recognition of the Abeta1-40/42/43] peptides by the 1E8-4b Fab. The 1E8-4b Fab did not recognize APP695 or APP770 which contain the Abeta sequence. The Abeta specificity of the recombinant 1E8-4b Fab antibody was identical to the parent 1E8 monoclonal antibody.     

Identification of a new allele, HLA-DRB1*1366*

High-resolution polymerase chain reaction sequence-specific primer typing of the human leucocyte antigen (HLA)-DRB1 gene of an Italian patient candidate for bone marrow transplantation revealed a new allelic variant of HLA-DRB1*13. The sequence was named DRB1*1366, and comparison with previously described DRB1 alleles demonstrated the two closely related sequences were HLA-DRB1*1330 and HLA-DRB1*130302.     

An update on the toxicity of Aβ in Alzheimer’s disease

Alzheimer’s disease is characterized histopathologically by deposition of insoluble forms of the peptide Aβ and the protein tau in brain. Aβ is the principal component of amyloid plaques and tau of neurofibrillary tangles. Familial cases of AD are associated with causal mutations in the gene encoding the amyloid precursor protein, APP, from which the amyloidogenic Aβ peptide is derived, and this supports a role for Aβ in disease. Aβ can promote tau pathology and at the same time its toxicity is also tau-dependent. Aβ can adopt different conformations including soluble oligomers and insoluble fibrillar species present in plaques. We discuss which of these conformations exert toxicity, highlight molecular pathways involved and discuss what has been learned by applying functional genomics.