Maria Teresa Ferretti

A Novel Transgenic Rat Model with a Full Alzheimer's-Like Amyloid Pathology Displays Pre-Plaque Intracellular Amyloid-β-Associated Cognitive Impairment

Alzheimers disease (AD) is a neurodegenerative pathology in which amyloid-beta (Abeta) peptide accumulates in different brain areas leading to deposition of plaques and a progressive decline of cognitive functions. After a decade in which a number of transgenic (Tg) mouse models mimicking AD-like amyloid-deposition pathology have been successfully generated, few rat models have been reported that develop intracellular and extracellular Abeta accumulation, together with impairment of cognition. The generation of a Tg rat reproducing the full AD-like amyloid pathology has been elusive. Here we describe the generation and characterization of a new transgenic rat line, coded McGill-R-Thy1-APP, developed to express the human amyloid-beta precursor protein (AbetaPP) carrying both the Swedish and Indiana mutations under the control of the murine Thy1.2 promoter. The selected mono-transgenic line displays an extended phase of intraneuronal Abeta accumulation, already apparent at 1 week after birth, which is widespread throughout different cortical areas and the hippocampus (CA1, CA2, CA3, and dentate gyrus). Homozygous Tg animals eventually produce extracellular Abeta deposits and, by 6 months of age, dense, thioflavine S-positive, amyloid plaques are detected, associated with glial activation and surrounding dystrophic neurites. The cognitive functions in transgenic McGill-R-Thy1-APP rats, as assessed using the Morris water maze task, were found already altered as early as at 3 months of age, when no CNS plaques are yet present. The spatial cognitive impairment becomes more prominent in older animals (13 months), where the behavioral performance of Tg rats positively correlates with the levels of soluble Abeta (trimers) measured in the cortex.

Intracellular Aβ-oligomers and early inflammation in a model of Alzheimer's disease

Lifelong use of nonsteroidal anti-inflammatory drugs (NSAIDs) has been shown to diminish the incidence of Alzheimers disease (AD), suggesting a key role of inflammation in early stages of the pathology. While amyloid plaque-associated inflammation has been extensively studied in human and animal models, little is known about the inflammatory process prior to plaque deposition, i.e., in preclinical stages of AD. In this study we investigated microglial and neuronal inflammatory markers in preplaque transgenic McGill-Thy1-APP mice. We found evidence that prior to plaque deposition classical markers of microglial activation such as major histocompatibility complex class II (MHC-II), inducible nitric oxide synthase (i-NOS), and CD40 are already upregulated in the hippocampus of transgenic mice. Microglial cells from transgenic mice in the preplaque stage displayed intermediately activated morphology and appeared to be recruited toward intracellular amyloid-β peptide (Aβ)-oligomer burdened neurons. The inducible, neuron-specific cyclooxygenase 2 (COX-2) enzyme was found to be upregulated and specifically expressed by neurons in close relationship with Aβ-bearing cells, at this early stage of the AD-like pathology. Our study suggests that neuroinflammation might be one of the earliest pathological responses to intracellular accumulation of Aβ-oligomers.

Minocycline corrects early, pre-plaque neuroinflammation and inhibits BACE-1 in a transgenic model of Alzheimer's disease-like amyloid pathology

BackgroundA growing body of evidence indicates that inflammation is one of the earliest neuropathological events in Alzheimers disease. Accordingly, we have recently shown the occurrence of an early, pro-inflammatory reaction in the hippocampus of young, three-month-old transgenic McGill-Thy1-APP mice in the absence of amyloid plaques but associated with intracellular accumulation of amyloid beta petide oligomers. The role of such a pro-inflammatory process in the progression of the pathology remained to be elucidated.Methods and resultsTo clarify this we administered minocycline, a tetracyclic derivative with anti-inflammatory and neuroprotective properties, to young, pre-plaque McGill-Thy1-APP mice for one month. The treatment ended at the age of three months, when the mice were still devoid of plaques. Minocycline treatment corrected the up-regulation of inducible nitric oxide synthase and cyclooxygenase-2 observed in young transgenic placebo mice. Furthermore, the down-regulation of inflammatory markers correlated with a reduction in amyloid precursor protein levels and amyloid precursor protein-related products. Beta-site amyloid precursor protein cleaving enzyme 1 activity and levels were found to be up-regulated in transgenic placebo mice, while minocycline treatment restored these levels to normality. The anti-inflammatory and beta-secretase 1 effects could be partly explained by the inhibition of the nuclear factor kappa B pathway.ConclusionsOur study suggests that the pharmacological modulation of neuroinflammation might represent a promising approach for preventing or delaying the development of Alzheimers disease neuropathology at its initial, pre-clinical stages. The results open new vistas to the interplay between inflammation and amyloid pathology.

Early-Stage Inflammation and Experimental Therapy in Transgenic Models of the Alzheimer-Like Amyloid Pathology

Background: Intracellular accumulation of β-amyloid (Aβ) is one of the early features in the neuropathology of Alzheimer’s disease (AD) and Down’s syndrome. This can be reproduced in cell and transgenic animal models of the AD-like amyloid pathology. In a transgenic rat model, our lab has previously shown that the intracellular accumulation of Aβ is sufficient to provoke cognitive impairments and biochemical alterations in the cerebral cortex and hippocampus in the absence of amyloid plaques. Objective: To investigate an early, pre-plaque inflammatory process in AD-like transgenic models and establish whether the neurotoxic effects of Aβ oligomers and proinflammatory responses can be arrested with minocycline. Methods: For these studies, we used naïve mice and transgenic animal models of the AD-like amyloid pathology and applied neurochemical, immunohistochemical and behavioral experimental approaches. Results: In the early stages of the AD-like amyloid pathology, intracellular Aβ oligomers accumulate within neurons of the cerebral cortex and hippocampus. Coincidental with this, behavioral impairments occur prior to the appearance of amyloid plaques, together with an upregulation of MHC-II, i-NOS and COX-2, well-known proinflammatory markers. Treatment with minocycline corrected behavioral impairments, lowered inflammatory markers and levels of Aβ trimers. Conclusion: A pharmacological approach targeting the early neuroinflammatory effects of Aβ might be a promising strategy to prevent or delay the onset of AD.

Evidence for the accumulation of Abeta immunoreactive material in the human brain and in transgenic animal models

In this review we highlight the evidence for an intracellular origin of Abeta (Aβ) amyloid peptides as well as the observations for a pathological accumulation of these peptides in Alzheimers disease and Down syndrome, as well as in transgenic animal models. We deliberate on the controversy as to whether the intracellular Aβ immunoreactive material is simply an accumulation of unprocessed full length amyloid precursor protein (APP) or a mix of processed APP fragments including Aβ. Finally, we discuss the possible pathological significance of these intracellular APP fragments and the expected future research directions regarding this thought-provoking problem.

Preplaque (‘Preclinical’) Aβ-Induced Inflammation and Nerve Growth Factor Deregulation in Transgenic Models of Alzheimer’s Disease-Like Amyloid Pathology

Background: Alzheimer’s disease (AD) neuropathology likely begins decades before clinical symptoms are manifested. Investigations on the early stages of the amyloid pathology are crucial for the discovery of diagnostic biomarkers or new therapeutic targets. Our transgenic (tg) animal models are most suitable to study early AD pathological events, as the pathology evolves in a well-staged manner, starting with intracellular Aβ accumulation and ending with plaque deposition. Objective: To determine the occurrence of key inflammatory markers and to look for signs of nerve growth factor (NGF) dysmetabolism at preplaque and postplaque stages in tg models of AD-like amyloid pathology and in human AD brains. Methods: We used our own tg lines (mice and rat), high-quality human brain material and applied neurochemical and immunohistochemical experimental approaches. Results: In both tg models, we observed an intracellular accumulation of oligomeric Aβ in cortical and hippocampal pyramidal neurons. This coincided with an upregulation of key inflammatory markers (iNOS, MHCII, COX-2) and a recruitment of microglia towards Aβ-burdened neurons. Using human AD brains, we showed alterations in the NGF metabolic pathway, which were mirrored in our tg rat model at early and late stages of amyloid plaque generation. Conclusion: A proinflammatory process and, consequently, the deregulation of the NGF metabolic pathway could be amongst the earliest pathological events in the progression of AD.

Extravascular CD3+ T Cells in Brains of Alzheimer Disease Patients Correlate with Tau but Not with Amyloid Pathology: An Immunohistochemical Study

Background: Strong genetic and epidemiological evidence points to a crucial role of the immune system in the development of Alzheimer disease (AD). CD3+ T lymphocytes have been described in brains of postmortem AD patients and in transgenic models of AD-like cerebral amyloidosis and tau pathology. However, the occurrence of T cells in AD brains is still controversial; furthermore, the relationship between T cells and hallmarks of AD pathology (amyloid plaques and neurofibrillary tangles) remains to be established. Objectives: We have studied the occurrence of T cells in postmortem hippocampi and mid frontal gyrus (MFG) samples of AD patients (Braak stage V-VI) and nondemented control subjects and correlated it with amyloid and tau pathology burden. Methods: Confocal microscopy and bright-field immunohistochemistry were used to identify brain-associated T cells. Extravascular CD3+ T cells were quantified and compared to nondemented controls. In addition, numbers of extravascular CD3+ T cells were correlated with amyloid (6E10 staining) and tau pathology (AT8 staining) in the same sections. Results: Several CD3+, extravascular T cells were observed in the brains of AD patients, mostly of the CD8+ subtype. AD hippocampi harbored significantly increased numbers of extravascular CD3+ T cells compared to nondemented controls. CD3+ T cells significantly correlated with tau pathology but not with amyloid plaques in AD samples. Conclusions: Our data support the notion of T-cell occurrence in AD brains and suggest that, in advanced stages of AD, T-cell extravasation is driven by tau-related neurodegenerative changes rather than by cerebral amyloidosis. T cells could be crucial for driving the amyloid-independent phase of the AD pathology.

EFFECTS OF AGGREGATED HUMAN Aβ 1-42 ON IMMUNE SURVEILLANCE: EX VIVO AND IN VITRO STUDIES

Figure 1. Inflammation’s Association with dT2A in TARCC Background:It is becoming increasingly clear that immune dysfunction is a critical component of Alzheimer’s disease (AD) neuropathology. While excessive pro-inflammatory activity and decreased phagocytosis have been extensively studied, little is known about the impact of beta-amyloidosis on brain immune surveillance operated by both resident microglial cells and other antigen presenting cells (APCs). Methods:To clarify this point, we have taken advantage of pre-clinical animal models of AD-like brain amyloidosis (APP-PS1-dE9 mice), as well as in vitro assays of antigen presentation (using OVA-specific CD4+ T cells, obtained from OT-II mice, and bone marrow derived dendritic cells, BMDC, from wild type mice). Expression of major histocompatibility class II (MHC-II), interferon gamma (IFNg), co-stimulatory factors and Ki67 were studies via FACS and fluorescent microscopy. Results: MHC-II expression was significantly reduced on the surface of both microglial cells and other APCs in brains of transgenic mice, while the overall number of MHC-II-positive cells was increased, as compared to non-transgenic wild-type littermates. Comprehensive analysis of APP-PS1-dE9 mice at 3, 6, 8, 14 and 20 month of age allowed us to define the onset of such alterations; interestingly, the earliest MHC-II reduction occurred between 6 and 8months of age, coinciding with the first deposition of amyloid plaques in this model. In in vitro antigen presentation functional assays, oligomers of human amyloid beta-peptide (Ab) 1-42 significantly inhibited OVA-induced T cell activation and proliferation, as compared to scrambled Ab. The inhibitory effect in such in vitro, acute system was however not mediated by reduced surface level of MHC-II, CD80 or CD86, or by a generic toxic effect of Ab on APCs or T cells. Conclusions:These results suggest that human Ab 1-42, in addition to its well described neurotoxic effects, exerts immunomodulatory action both ex vivo and in vitro. Reduced surface expression of MHC-II per cell, and inhibited T cell activation in antigen presentation assays, both indicate an overall inhibitory effect of Ab on antigen presentation. The molecular mechanism responsible for such effects remains to be elucidated. Reduced brain immune surveillance could be a critical, novel component of AD-related immune dysfunction.

INCREASED RESILIENCE TO ALZHEIMER’S DISEASE PATHOPHYSIOLOGY IN MEN WITH SUBJECTIVE MEMORY COMPLAINTS COMPARED TO WOMEN

Constant 2730.3157 <0.001 1859.0788 3601.5525 1.5029 <0.001 1.4559 1.5498 APOE4 -327.9283 0.005 -556.4050 -99.4516 -0.0018 0.064 -0.0036 0.0001 Amyloid -283.8190 <0.001 -381.5672 -186.0708 -0.0423 <0.001 -0.0579 -0.0268 PA 22.3413 0.805 -155.8281 200.5108 0.0450 0.002 0.0163 0.0738 APOE4 x PA -143.7908 0.549 -615.2068 327.6252 0.0027 0.162 -0.0011 0.0065 Amyloid x PA 147.2199 0.126 -41.6790 336.1188 0.0002 0.987 -0.0288 0.0293 CA 103.9141 0.139 -33.7525 241.5806 0.0097 0.358 -0.0124 0.0319 APOE4x CA -356.6207 0.024 -665.8677 -47.3737 0.0002 0.876 -0.0021 0.0024 Amyloid x CA 69.0120 0.257 -50.5644 188.5883 -0.0038 0.669 -0.0215 0.0138 Sex -481.8246 <0.001 -669.3021 -294.3472 0.0088 0.565 -0.0212 0.0388 Age -50.9411 <0.001 -62.5039 -39.3783 -0.0024 0.229 -0.0064 0.0015 Educational year 0.4370 0.973 -24.5808 25.4548 -0.0157 0.389 -0.0516 0.0202

REDUCED BASAL FOREBRAIN FUNCTIONAL CONNECTIVITY IN WOMEN WITH SUBJECTIVE MEMORY COMPLAINTS COMPARED TO MEN

Background: Cardiovascular disease risk factors (CVD-RFs) are associated with decreased gray and white matter integrity and reduced cognition in older adults. Less is known regarding the interplay between CVD-RFs, cognition, and structural connectivity integrity between gray and white matter regions. This study examined whether alterations in tractbased structural connectivity mediated the known association between CVD-RFs and cognition in a cross-sectional study of cognitively-normal older adults. Methods: Ninety-six participants (mean age1⁄467.9 years; 53.1% female; 46.9% Black) underwent CVD-RF assessment, cognitive evaluation, and 3T MRI. The Framingham 10-year stroke risk (FSRP-10) quantified CVD-RFs. PCA of select cognitive variables resulted in three rotated factor scores: memory (CVLT-II Trial 1-5 Total, Delayed Free Recall, Recognition Discriminability); executive function (FAS, Trail Making Test (TMT) B-A, Letter-Number Sequencing, Matrix Reasoning); attention/information processing (AIP; TMT-A, TMT-M, Digit Symbol). Graph theory analysis integrated T1-derived Alzheimer’s disease-related gray matter regions of interest (ROIs) from FreeSurfer parcellation and DTI-derived white matter deterministic tractography via DTI-Studio into connectivity matrices. Resulting matrices were analyzed in Brain Connectivity Toolbox for local measures of efficiency, the average path length across nodes in a given region, and centrality, the influence of a node based on its associated path quantity. Results: Following standard steps for mediation, simple linear regressions adjusting for educational quality and intracranial volume revealed significant associations between FSRP-10 and AIP (p1⁄4.02), FSRP-10 and centrality in six ROIs, and efficiency in seven ROIs (p-values .05). Mediation analyses revealed significant mediation of right hippocampal centrality on FSRP-10 and AIP (p1⁄4.04; Figure 1) and left caudal middle frontal gyrus on FSRP-10 and AIP (p1⁄4.01; Figure 2). Conclusions: Stroke risk plays deleterious roles in structural connectivity metrics, which then negatively impact cognitive measures, indicating the importance of multi-modal neuroimaging biomarkers in understanding behavioral and biological pathways in preclinical aging.