Alessandra Paladini

Targeting Mannose-Binding Lectin Confers Long-Lasting Protection With a Surprisingly Wide Therapeutic Window in Cerebral Ischemia

Background— The involvement of the complement system in brain injury has been scarcely investigated. Here, we document the pivotal role of mannose-binding lectin (MBL), one of the recognition molecules of the lectin complement pathway, in brain ischemic injury. Methods and Results— Focal cerebral ischemia was induced in mice (by permanent or transient middle cerebral artery occlusion) and rats (by 3-vessel occlusion). We first observed that MBL is deposited on ischemic vessels up to 48 hours after injury and that functional MBL/MBL-associated serine protease 2 complexes are increased. Next, we demonstrated that (1) MBL−/− mice are protected from both transient and permanent ischemic injury; (2) Polyman2, the newly synthesized mannosylated molecule selected for its binding to MBL, improves neurological deficits and infarct volume when given up to 24 hours after ischemia in mice; (3) anti-MBL-A antibody improves neurological deficits and infarct volume when given up to 18 hours after ischemia, as assessed after 28 days in rats. Conclusions— Our data show an important role for MBL in the pathogenesis of brain ischemic injury and provide a strong support to the concept that MBL inhibition may be a relevant therapeutic target in humans, one with a wide therapeutic window of application.

Transgenic fatal familial insomnia mice indicate prion infectivity-independent mechanisms of pathogenesis and phenotypic expression of disease.

Fatal familial insomnia (FFI) and a genetic form of Creutzfeldt-Jakob disease (CJD178) are clinically different prion disorders linked to the D178N prion protein (PrP) mutation. The disease phenotype is determined by the 129 M/V polymorphism on the mutant allele, which is thought to influence D178N PrP misfolding, leading to the formation of distinctive prion strains with specific neurotoxic properties. However, the mechanism by which misfolded variants of mutant PrP cause different diseases is not known. We generated transgenic (Tg) mice expressing the mouse PrP homolog of the FFI mutation. These mice synthesize a misfolded form of mutant PrP in their brains and develop a neurological illness with severe sleep disruption, highly reminiscent of FFI and different from that of analogously generated Tg(CJD) mice modeling CJD178. No prion infectivity was detectable in Tg(FFI) and Tg(CJD) brains by bioassay or protein misfolding cyclic amplification, indicating that mutant PrP has disease-encoding properties that do not depend on its ability to propagate its misfolded conformation. Tg(FFI) and Tg(CJD) neurons have different patterns of intracellular PrP accumulation associated with distinct morphological abnormalities of the endoplasmic reticulum and Golgi, suggesting that mutation-specific alterations of secretory transport may contribute to the disease phenotype.

Longitudinal Tracking of Human Fetal Cells Labeled with Super Paramagnetic Iron Oxide Nanoparticles in the Brain of Mice with Motor Neuron Disease

Stem Cell (SC) therapy is one of the most promising approaches for the treatment of Amyotrophic Lateral Sclerosis (ALS). Here we employed Super Paramagnetic Iron Oxide nanoparticles (SPIOn) and Hoechst 33258 to track human Amniotic Fluid Cells (hAFCs) after transplantation in the lateral ventricles of wobbler (a murine model of ALS) and healthy mice. By in vitro, in vivo and ex vivo approaches we found that: 1) the main physical parameters of SPIOn were maintained over time; 2) hAFCs efficiently internalized SPIOn into the cytoplasm while Hoechst 33258 labeled nuclei; 3) SPIOn internalization did not alter survival, cell cycle, proliferation, metabolism and phenotype of hAFCs; 4) after transplantation hAFCs rapidly spread to the whole ventricular system, but did not migrate into the brain parenchyma; 5) hAFCs survived for a long time in the ventricles of both wobbler and healthy mice; 6) the transplantation of double-labeled hAFCs did not influence mice survival.

Comparative Magnetic Resonance Imaging and Histopathological Correlates in Two SOD1 Transgenic Mouse Models of Amyotrophic Lateral Sclerosis.

Amyotrophic Lateral Sclerosis (ALS) is a progressive and fatal disease due to motoneuron degeneration. Magnetic resonance imaging (MRI) is becoming a promising non-invasive approach to monitor the disease course but a direct correlation with neuropathology is not feasible in human. Therefore in this study we aimed to examine MRI changes in relation to histopathology in two mouse models of ALS (C57BL6/J and 129S2/SvHsd SOD1G93A mice) with different disease onset and progression. A longitudinal in vivo analysis of T2 maps, compared to ex vivo histological changes, was performed on cranial motor nuclei. An increased T2 value was associated with a significant tissue vacuolization that occurred prior to motoneuron loss in the cranial nuclei of C57 SOD1G93A mice. Conversely, in 129Sv SOD1G93A mice, which exhibit a more severe phenotype, MRI detected a milder increase of T2 value, associated with a milder vacuolization. This suggests that alteration within brainstem nuclei is not predictive of a more severe phenotype in the SOD1G93A mouse model. Using an ex vivo paradigm, Diffusion Tensor Imaging was also applied to study white matter spinal cord degeneration. In contrast to degeneration of cranial nuclei, alterations in white matter and axons loss reflected the different disease phenotype of SOD1G93A mice. The correspondence between MRI and histology further highlights the potential of MRI to monitor progressive motoneuron and axonal degeneration non-invasively in vivo. The identification of prognostic markers of the disease nevertheless requires validation in multiple models of ALS to ensure that these are not merely model-specific. Eventually this approach has the potential to lead to the development of robust and validated non-invasive imaging biomarkers in ALS patients, which may help to monitor the efficacy of therapies.

The Continuing Failure of Bexarotene in Alzheimer's Disease Mice.

Alzheimers disease (AD) is the most common form of dementia characterized by synaptic dysfunction, memory loss, neuroinflammation, and neuronal cell death. Amyloid-β (Aβ), recognized as the main culprit of AD, aggregates and accumulates in the extracellular compartment as neuritic plaques, after deregulation of its production or clearance. Apolipoprotein E (ApoE) plays a major role in Aβ clearance and its expression is transcriptionally regulated by the liver X receptor and retinoid X receptors (RXRs) system. Bexarotene (BEXA), an RXR agonist that increases ApoE expression and microglia phagocytosis has been proposed as a promising therapy for AD, resolving both the amyloid pathology and memory loss. Despite the first compelling report, however, multiple failures have been documented, raising concern about whether BEXA could in fact become a novel disease-modifying strategy for AD. To help clarify this, we investigated the effect of BEXA in vivo at multiple levels in TASTPM transgenic mice. Seven-day oral administration of BEXA to these mice did not achieve any significant memory improvement, plaque reduction, or enhancement of microglial cell activation. No differences were found when specifically investigating the microglial phagocytic state in vivo. In addition, a brain structural analysis with magnetic resonance did not detect any BEXA-mediated change in the volume reduction of the main affected brain areas in our mice. These results suggest that BEXA has no beneficial effects on the multi-factorial pathologic phenotype of AD mice.

Striatum and entorhinal cortex atrophy in AD mouse models: MRI comprehensive analysis

Alzheimers disease is experimentally modeled in transgenic (Tg) mice overexpressing mutated forms of the human amyloid precursor protein either alone or combined with mutated presenilins and tau. In the present study, we developed a systematic approach to compare double (TASTPM) and triple (APP/PS2/Tau) Tg mice by serial magnetic resonance imaging and spectroscopy analysis from 4 to 26 months of age to define homologous biomarkers between mice and humans. Hippocampal atrophy was found in Tg mice compared with WT. In APP/PS2/Tau the effect was age-dependent, whereas in TASTPM it was detectable from the first investigated time point. Importantly, both mice displayed an age-related entorhinal cortex thinning and robust striatal atrophy, the latter associated with a significant loss of synaptophysin. Hippocampal magnetic resonance spectroscopy revealed lower glutamate levels in both Tg mice and a selective myo-inositol increase in TASTPM. This noninvasive magnetic resonance imaging analysis, revealed common biomarkers between humans and mice, and could, thus, be promoted as a fully translational tool to be adopted in the preclinical investigation of therapeutic approaches.

Six-Month ischemic mice show sensorimotor and cognitive deficits associated with brain atrophy and axonal disorganization

To identify long‐term sensorimotor and cognitive deficits and to evaluate structural alterations in brain ischemic mice.

MAGNETIC RESONANCE IMAGING AND ALZHEIMER'S DISEASE: A LONGITUDINAL STUDY OF STRUCTURAL CHANGES IN DIFFERENT TRANSGENIC MODELS

Alessandra Paladini, Edoardo Micotti, Angelisa Frasca, Daniele Tolomeo, Moira Marizzoni, Anna Caroli, Claudia Balducci, Sophie Dix, Michael O’Neill, Ozmen Laurence, Christian Czech, Jill Richardson, Giovanni B. Frisoni, Gianluigi Forloni, Istituto di Ricerche Farmacologiche Mario Negri, Milano, Italy; IRCCS Istituto di Ricerche Farmacologiche Mario Negri, Milano, Italy; 3 IRCCS Istituto di Ricerche Farmacologiche Mario Negri, Milan, Italy; 4 IRCCS San Giovanni di Dio Fatebenefratelli, Brescia, Italy; Eli Lilly, Lilly Research Centre, Surrey, United Kingdom; Eli Lilly, Lilly Research Centre, Windlesham, United Kingdom; 7 CNS Research, Hoffmann-La Roche AG, Basel, Switzerland; 8 GlaxoSmithKline, Stevenage, United Kingdom; 9 IRCCS San Giovanni di Dio Fatebenefratelli, Brescia, Italy. Contact e-mail: forloni@ marionegri.it

1H magnetic resonance spectroscopy longitudinal study in different transgenic mouse models of Alzheimer's disease

P1-132 1H MAGNETIC RESONANCE SPECTROSCOPY LONGITUDINAL STUDY IN DIFFERENT TRANSGENIC MOUSE MODELS OF ALZHEIMER’S DISEASE Alessandra Paladini, Marta Filibian, Edoardo Micotti, Claudia Balducci, Sophie Dix, Michael O’Neill, Ozmen Laurence, Christian Czech, Jill Richardson, Gianluigi Forloni, Istituto di Ricerche Farmacologiche Mario Negri, Milano, Italy; Istituto di Ricerche Farmacologiche Mario Negri, Department of Physics ‘A. Volta,’ University of Pavia, Milano/Pavia, Italy; 3 Istituto di Ricerche Farmacologiche Mario Negri, Milano, Italy; Istituto di Ricerche Farmacologiche Mario Negri, Milan, Italy; Eli Lilly, Lilly Research Centre, Surrey, United Kingdom; 6 CNS Research, Hoffmann-La Roche AG, Basel, Switzerland; 7 GlaxoSmithKline, Stevenage, United Kingdom.

Longitudinal magnetic resonance spectroscopy analysis of APP/PS1 transgenic mice

Background:Due to increasing numbers of patients suffering fromAlzheimer’s Disease (AD) and no effective therapies so far, there is an exigent need for a reliableand inexpensive diagnosis at anearly stageof the disease.Aggregates of amyloid ß and tau protein are the physiological markers of the disease. Therefore inhibition and reversal of this protein-aggregation are subjects of clinical studies. The established PET tracer PIB is so far limited to exclude the AD diagnosis. The outcome of clinical studies can be monitored by the combination of cognitive ability assays and the sampling of biomarkers from cerebrospinal fluid, which is supported by PIB PET imaging. A distinct AD diagnosis is possible postmortem only via the histological examination of brain tissue. Neither of these approaches is suitable for a large cohort survey. Moreover, community physicians fail to diagnose up to 33% ofmild dementia cases. Novel markers are required to establish a reliable, early diagnosis of a pathological accumulation of tau andAß in the living patient. Established radio diagnostic probes allowmonitoring individual disease progress but display restricted ability to differentiate AD patients from healthy controls. Hence an important criterion for the development of novel imaging techniques and probes is their ability to differentiate clearly tau-protein depositions from Aßaggregates. ThereforewedevelopbrainMethods:Thereforewedeveloped brain penetrating, selective fluorescent probes for the in vivo detection of tauPHFandAß-aggregates inADpatients and animalmodels.Results: Iterative cycles of ligand design, synthesis and histology of human AD tissue resulted in the identification of bright-fluorescent binders for amyloid plaques and tauPHF. Conclusions: A biomarker-supported diagnosis is of essential importance for future therapies of ADs both for clinical studies and for awidespread screening of high-risk groups with respect to an early beginning of therapy.