Margherita Ruggerone

Chronic wasting disease and atypical forms of bovine spongiform encephalopathy and scrapie are not transmissible to mice expressing wild-type levels of human prion protein

The association between bovine spongiform encephalopathy (BSE) and variant Creutzfeldt-Jakob disease (vCJD) has demonstrated that cattle transmissible spongiform encephalopathies (TSEs) can pose a risk to human health and raises the possibility that other ruminant TSEs may be transmissible to humans. In recent years, several novel TSEs in sheep, cattle and deer have been described and the risk posed to humans by these agents is currently unknown. In this study, we inoculated two forms of atypical BSE (BASE and H-type BSE), a chronic wasting disease (CWD) isolate and seven isolates of atypical scrapie into gene-targeted transgenic (Tg) mice expressing the human prion protein (PrP). Upon challenge with these ruminant TSEs, gene-targeted Tg mice expressing human PrP did not show any signs of disease pathology. These data strongly suggest the presence of a substantial transmission barrier between these recently identified ruminant TSEs and humans.

MM2-thalamic Creutzfeldt-Jakob disease: neuropathological, biochemical and transmission studies identify a distinctive prion strain

In Creutzfeldt–Jakob disease (CJD), molecular typing based on the size of the protease resistant core of the disease‐associated prion protein (PrPSc) and the M/V polymorphism at codon 129 of the PRNP gene correlates with the clinico‐pathologic subtypes. Approximately 95% of the sporadic 129MM CJD patients are characterized by cerebral deposition of type 1 PrPSc and correspond to the classic clinical CJD phenotype. The rare 129MM CJD patients with type 2 PrPSc are further subdivided in a cortical and a thalamic form also indicated as sporadic fatal insomnia.

Brain delivery of AAV9 expressing an anti-PrP monovalent antibody delays prion disease in mice.

Prion diseases are caused by a conformational modification of the cellular prion protein (PrPC) into disease-specific forms, termed PrPSc, that have the ability to interact with PrPC promoting its conversion to PrPSc. In vitro studies demonstrated that anti-PrP antibodies inhibit this process. In particular, the single chain variable fragment D18 antibody (scFvD18) showed high efficiency in curing chronically prion-infected cells. This molecule binds the PrPC region involved in the interaction with PrPSc thus halting further prion formation. These findings prompted us to test the efficiency of scFvD18 in vivo. A recombinant Adeno-Associated Viral vector serotype 9 was used to deliver scFvD18 to the brain of mice that were subsequently infected by intraperitoneal route with the mouse-adapted scrapie strain RML. We found that the treatment was safe, prolonged the incubation time of scrapie-infected animals and decreased the burden of total proteinase-resistant PrPSc in the brain, suggesting that scFvD18 interferes with prion replication in vivo. This approach is relevant for designing new therapeutic strategies for prion diseases and other disorders characterized by protein misfolding.

Mutations in MAPT give rise to aneuploidy in animal models of tauopathy

Tau is a major microtubule-associated protein in brain neurons. Its misfolding and accumulation cause neurodegenerative diseases characterized by brain atrophy and dementia, named tauopathies. Genetic forms are caused by mutations of microtubule-associated protein tau gene (MAPT). Tau is expressed also in nonneural tissues such as lymphocytes. Tau has been recently recognized as a multifunctional protein, and in particular, some findings supported a role in genome stability. In fact, peripheral cells of patients affected by frontotemporal dementia carrying different MAPT mutations showed structural and numerical chromosome aberrations. The aim of this study was to assess chromosome stability in peripheral cell from two animal models of genetic tauopathy, JNPL3 and PS19 mouse strains expressing the human tau carrying the P301L and P301S mutations, respectively, to confirm the previous data on humans. After demonstrating the presence of mutated tau in spleen, we performed standard cytogenetic analysis of splenic lymphocytes from homozygous and hemizygous JNPL3, hemizygous PS19, and relevant controls. Losses and gains of chromosomes (aneuploidy) were evaluated. We detected a significantly higher level of aneuploidy in JNPL3 and PS19 than in control mice. Moreover, in JNPL3, the aneuploidy was higher in homozygotes than in hemizygotes, demonstrating a gene dose effect, which appeared also to be age independent. Our results show that mutated tau is associated with chromosome instability. It is conceivable to hypothesize that in genetic tauopathies the aneuploidy may be present also in central nervous system, possibly contributing to neurodegeneration.

Infectivity in Skeletal Muscle of Cattle with Atypical Bovine Spongiform Encephalopathy

The amyloidotic form of bovine spongiform encephalopathy (BSE) termed BASE is caused by a prion strain whose biological properties differ from those of typical BSE, resulting in a clinically and pathologically distinct phenotype. Whether peripheral tissues of BASE-affected cattle contain infectivity is unknown. This is a critical issue since the BASE prion is readily transmissible to a variety of hosts including primates, suggesting that humans may be susceptible. We carried out bioassays in transgenic mice overexpressing bovine PrP (Tgbov XV) and found infectivity in a variety of skeletal muscles from cattle with natural and experimental BASE. Noteworthy, all BASE muscles used for inoculation transmitted disease, although the attack rate differed between experimental and natural cases (∼70% versus ∼10%, respectively). This difference was likely related to different prion titers, possibly due to different stages of disease in the two conditions, i.e. terminal stage in experimental BASE and pre-symptomatic stage in natural BASE. The neuropathological phenotype and PrPres type were consistent in all affected mice and matched those of Tgbov XV mice infected with brain homogenate from natural BASE. The immunohistochemical analysis of skeletal muscles from cattle with natural and experimental BASE showed the presence of abnormal prion protein deposits within muscle fibers. Conversely, Tgbov XV mice challenged with lymphoid tissue and kidney from natural and experimental BASE did not develop disease. The novel information on the neuromuscular tropism of the BASE strain, efficiently overcoming species barriers, underlines the relevance of maintaining an active surveillance.

Tackling amyloidogenesis in Alzheimer's disease with A2V variants of Amyloid-β

We developed a novel therapeutic strategy for Alzheimer’s disease (AD) exploiting the properties of a natural variant of Amyloid-β (Aβ) carrying the A2V substitution, which protects heterozygous carriers from AD by its ability to interact with wild-type Aβ, hindering conformational changes and assembly thereof. As prototypic compound we designed a six-mer mutated peptide (Aβ1-6A2V), linked to the HIV-related TAT protein, which is widely used for brain delivery and cell membrane penetration of drugs. The resulting molecule [Aβ1-6A2VTAT(D)] revealed strong anti-amyloidogenic effects in vitro and protected human neuroblastoma cells from Aβ toxicity. Preclinical studies in AD mouse models showed that short-term treatment with Aβ1-6A2VTAT(D) inhibits Aβ aggregation and cerebral amyloid deposition, but a long treatment schedule unexpectedly increases amyloid burden, although preventing cognitive deterioration. Our data support the view that the AβA2V-based strategy can be successfully used for the development of treatments for AD, as suggested by the natural protection against the disease in human A2V heterozygous carriers. The undesirable outcome of the prolonged treatment with Aβ1-6A2VTAT(D) was likely due to the TAT intrinsic attitude to increase Aβ production, avidly bind amyloid and boost its seeding activity, warning against the use of the TAT carrier in the design of AD therapeutics.

Targeting ß-amyloid by the A2V Aß variant: a novel disease-modifying strategy for the treatment of Alzheimer’s disease

Abstracts of the IX Congresso SindemItalian Association for the study of Dementia linked to the Italian Neurological Society (SIN)Firenze, Palazzo dei Congressi, Villa Vittoria March 13-15, 2014Comitato Scientifico: Vincenzo Bonavita, Alessandro Padovani, Amalia Bruni, Leonardo Pantoni, Carlo Caltagirone, Lucilla Parnetti, Francesca Clerici, Daniela Perani, Monica Di Luca, Sandro Sorbi, Gianluigi Forloni, Francesco Tagliavini, Giovanni Frisoni, Marilu Gorno Tempini, Claudio Mariani, Annalena Venneri, Massimo Musicco.