Changes in the brain transcriptome after DNA Aß42 trimer immunization in a 3xTg-AD mouse model.

Alzheimer's disease (AD) has been associated with accumulation of amyloid beta (Aß) peptides in brain, and immunotherapy targeting Aß provides potential for AD prevention. We have used a DNA Aß42 trimer construct for immunization of 3xTg-AD mice and found previously significant reduction of amyloid and tau pathology due to the immunotherapy. We show here that DNA Aß42 immunized 3xTg-AD mice showed better performance in nest building activities and had a higher 24 months survival rate compared to the non-treated AD controls. The analysis of differently expressed genes in brains from 24 months old mice showed significant increases transcript levels between non-immunized AD mice and wild-type controls for genes involved in microglia and astrocyte function, cytokine and inflammatory signaling, apoptosis, the innate and adaptive immune response and are consistent with an inflammatory phenotype in AD. Most of these upregulated genes were downregulated in the DNA Aß42 immunized 3xTg-AD mice due to the vaccine. Transcript numbers for the immediate early genes, Arc, Bdnf, Homer1, Egr1 and cfos, involved in neuronal and neurotransmission pathways which were much lower in the non-immunized 3xTg-AD mice, were restored to wild-type mouse brain levels in DNA Aß42 immunized 3xTg-AD mice indicating positive effects of DNA Aß42 immunotherapy on synapse stability and plasticity. The immune response after immunization is complex, but the multitude of changes after DNA Aß42 immunization shows that this response moves beyond the amyloid hypothesis and into direction of disease prevention.

Cardiovascular Risk Factors Associated with Smaller Brain Volumes in Regions Identified as Early Predictors of Cognitive Decline.

PURPOSE: To determine in a large multiethnic cohort the cardiovascular and genetic risk factors associated with smaller volume in the hippocampus, precuneus, and posterior cingulate, and their association with preclinical deficits in cognitive performance in patients younger and older than 50 years. MATERIALS AND METHODS: The institutional review board approved the study and all participants provided written informed consent. Eligible for this study were 1629 participants (700 men and 929 women; mean age, 50.0 years ± 10.2 [standard deviation]) drawn from the population-based Dallas Heart Study who underwent laboratory and clinical analysis in an initial baseline visit and approximately 7 years later underwent brain magnetic resonance imaging with automated volumetry and cognitive assessment with the Montreal Cognitive Assessment (MoCA). Regression analysis showed associations between risk factors and segmental volumes, and associations between these volumes with cognitive performance in participants younger and older than 50 years. RESULTS: Lower hippocampal volume was associated with previous alcohol consumption (standardized estimate, -0.04; P = .039) and smoking (standardized estimate, -0.04; P = .048). Several risk factors correlated with lower total brain, posterior cingulate, and precuneus volumes. Higher total (standardized estimate, 0.06; P = .050), high-density lipoprotein (standardized estimate, 0.07; P = .003), and low-density lipoprotein (standardized estimate, 0.04; P = .037) cholesterol levels were associated with larger posterior cingulate volume, and higher triglyceride levels (standardized estimate, 0.06; P = .004) were associated with larger precuneus volume. Total MoCA score was associated with posterior cingulate volume (standardized estimate, 0.13; P = .001) in younger individuals and with hippocampal (standardized estimate, 0.06; P < .05) and precuneus (standardized estimate, 0.08; P < .023) volumes in older adults. CONCLUSION: Smaller volumes in specific brain regions considered to be early markers of dementia risk were associated with specific cardiovascular disease risk factors and cognitive deficits in a predominantly midlife multiethnic population-based sample. Additionally, the risk factors most associated with these brain volumes differed in participants younger and older than 50 years, as did the association between brain volume and MoCA score.

DNA Aß42 immunization via needle-less Jet injection in mice and rabbits as potential immunotherapy for Alzheimer's disease.

Alzheimer's disease (AD) is the most common form of dementia found in the elderly and disease progression is associated with accumulation of Amyloid beta 1-42 (Aß42) in brain. An immune-mediated approach as a preventive intervention to reduce amyloid plaques without causing brain inflammation is highly desirable for future clinical use. Genetic immunization, in which the immunizing agent is DNA encoding Aß42, has great potential because the immune response to DNA delivered into the skin is generally non-inflammatory, and thus differs quantitatively and qualitatively from immune responses elicited by peptides, which are inflammatory with production of IFNγ and IL-17 cytokines by activated T cells. DNA immunization has historically been proven difficult to apply to larger mammals. A potential barrier to use DNA immunization in large mammals is the method for delivery of the DNA antigen. We tested jet injection in mice and rabbits and found good antibody production and safe immune responses (no inflammatory cytokines). We found significant reduction of amyloid plaques and Aß peptides in brains of the DNA Aß42 immunized 3xTg-AD mouse model. This study was designed to optimize DNA delivery for possible testing of the DNA Aß42 vaccine for AD prevention in a clinical trial.

Glial cell transcriptome analyses in 3xTg-AD mice: Effects of aging, disease progression, and anti-Aß immunotherapy.

Background: To investigate how changes in expression of glial genes relate to a progression of Alzheimer's disease (AD) pathology, and how anti-Aß immunotherapy impact these changes, we conducted a transcriptomic analysis for brains from cohorts of 2-, 10-, and 20 month old 3xTg-AD mice, and a cross-sectional study in groups of 20 month-old mice treated with active DNA Aß42 immunization, passive immunotherapy, untreated, and wild-type (wt) controls. Methods: Twenty-four Formalin-Fixed Paraffin-Embedded (FFPE) mouse brain sections were used for the gene expression analyses (nanostring). Adjacent sections from these and additional mouse brains were stained for microglia using antibodies detecting IbaI and Gal3. For a semi-quantitative analysis of increased tau and amyloid pathology with aging and disease progression, a comparison of ELISA results from brains of 12 and 20 months old 3xTg-AD mice were shown. Results: Based on the different comparisons of transcript numbers found the 3xTg-AD age groups with the senescent 20 months old wt control mouse brains, and the 20 months old 3xTg-AD mouse brains with the 20 months old wt control mouse brains, genes were assigned as upregulated due to aging, or due to disease progression, or due to both. The immunohistochemistry of microglia markers revealed that Gal3 might be an important marker for phagocytosing microglia around amyloid plaques. The comparison of the two anti-Aß immunotherapy approaches showed a differential downregulation of inflammatory glial genes. Conclusion: These results are relevant for future clinical trials using active anti-amyloid immunotherapy.

DNA immunization against amyloid beta 42 has high potential as safe therapy for Alzheimer's disease as it diminishes antigen-specific Th1 and Th17 cell proliferation.

The pathogenesis of Alzheimer's disease (AD) has been strongly associated with the accumulation of amyloid beta (Aß) peptides in brain, and immunotherapy targeting Aß provides potential for AD prevention. A clinical trial in which AD patients were immunized with Aß42 peptide was stopped when 6% of participants showed meningoencephalitis, apparently due to an inflammatory Th1 immune response. Previously, we and other have shown that Aß42 DNA vaccination via gene gun generates a Th2 cellular immune response, which was shown by analyses of the respective antibody isotype profiles. We also determined that in vitro T cell proliferation in response to Aß42 peptide re-stimulation was absent in DNA Aß42 trimer-immunized mice when compared to Aß42 peptide-immunized mice. To further characterize this observation prospectively and longitudinally, we analyzed the immune response in wild-type mice after vaccination with Aß42 trimer DNA and Aß42 peptide with Quil A adjuvant. Wild-type mice were immunized with short-term (1-3× vaccinations) or long-term (6× vacinations) immunization strategies. Antibody titers and isotype profiles of the Aß42 specific antibodies, as well as cytokine profiles and cell proliferation studies from this longitudinal study were determined. Sufficient antibody titers to effectively reduce Aß42, but an absent T cell proliferative response and no IFNγ or IL-17 secretion after Aß42 DNA trimer immunization minimizes the risk of inflammatory activities of the immune system towards the self antigen Aß42 in brain. Therefore, Aß42 DNA trimer immunization has a high probability to be effective and safe to treat patients with early AD.

The relationship of cardiovascular risk factors to Alzheimer disease in Choctaw Indians.

OBJECTIVES: To test the hypothesis that cardiovascular risk factors (CRFs) influence predisposition to and the clinical course of Alzheimer disease (AD), the authors compared Choctaw Indians, a group with known high CRF with white persons with AD. In addition to CRF history, the authors investigated the frequency of apolipoprotein E4 (apoE4) genotype andplasma homocysteine (HC) levels. METHOD: The authors compared 39 Choctaw Indians with AD and 39 Choctaw Indians without AD to 39 white persons with AD with all groups similar in age. CRF history included diabetes, hypertension, high cholesterol or hypolipidemic agent use, or myocardial infarction. The authors also compared plasma HC concentration and apoE4 allele frequency. RESULTS: Choctaw persons with AD differed significantly from white persons with AD in history of hypertension, diabetes, and in HC values but not from Indians without AD. There was a significantly lower apoE4 allele frequency in Choctaw Indian AD than white persons with AD, and both AD groups had an affected first degree relative significantly more often than Indian controls. There was no relationship between the number of CRF and age at onset among Indians or whites, whereas HC concentration was associated with significantly earlier age of onset for Choctaw Indians but not for whites. CONCLUSIONS: This small study suggests that in Choctaw Indians modifiable risk factors may play more of a role in disease pathogenesis than in whites and that nonmodifiable risk factors such as apoE4 may play less of a role.

The universal brain code a genetic mechanism for memory.

We do not have an understanding of the fundamental mechanism of how information is stored and retrieved by the brain. A Universal Brain Code utilized for these functions is proposed here. The basic tenent of the Code is that a memory engram is propagated and guided through the connectome by specific proteins/peptides embedded within the pre-synaptic neuronal membrane corresponding to information provided by afferent electrical currents to the pre-synaptic neuron. It is intended to provide a working approach to this central brain activity and begin the process of investigation based on these ideas which are new and unexplored.

Quantification of early learning and movement sub-structure predictive of motor performance.

Time-to-fall off an accelerating rotating rod (rotarod) is widely utilized to evaluate rodent motor performance. We reasoned that this simple outcome could be refined with additional measures explicit in the task (however inconspicuously) to examine what we call movement sub-structure. Our goal was to characterize normal variation or motor impairment more robustly than by using time-to-fall. We also hypothesized that measures (or features) early in the sub-structure could anticipate the learning expected of a mouse undergoing serial trials. Using normal untreated and baclofen-treated movement-impaired mice, we defined these features and automated their analysis using paw video-tracking in three consecutive trials, including paw location, speed, acceleration, variance and approximate entropy. Spectral arc length yielded speed and acceleration uniformity. We found that, in normal mice, paw movement smoothness inversely correlated with rotarod time-to-fall for the three trials. Greater approximate entropy in vertical movements, and opposite changes in horizontal movements, correlated with greater first-trial time-to-fall. First-trial horizontal approximate entropy in the first few seconds predicted subsequent time-to-fall. This allowed for the separation, after only one rotarod trial, of different-weight, untreated mouse groups, and for the detection of mice otherwise unimpaired after baclofen, which displayed a time-to-fall similar to control. A machine-learning support vector machine classifier corroborated these findings. In conclusion, time-to-fall off a rotarod correlated well with several measures, including some obtained during the first few seconds of a trial, and some responsive to learning over the first two trials, allowing for predictions or preemptive experimental manipulations before learning completion.

DNA beta-amyloid(1-42) trimer immunization for Alzheimer disease in a wild-type mouse model.

CONTEXT: DNA beta-amyloid(1-42) (Abeta42) trimer immunization was developed to produce specific T helper 2 cell (T(H)2)-type antibodies to provide an effective and safe therapy for Alzheimer disease (AD) by reducing elevated levels of Abeta42 peptide that occur in the brain of patients with AD. OBJECTIVE: To compare the immune response in wild-type mice after immunization with DNA Abeta42 trimer and Abeta42 peptide. DESIGN AND INTERVENTION: Wild-type mice received either 4 microg of DNA Abeta42 trimer immunization administered with gene gun (n = 8) or intraperitoneal injection of 100 microg of human Abeta42 peptide with the adjuvant Quil A (n = 8). Titers, epitope mapping, and isotypes of the Abeta42-specific antibodies were analyzed. MAIN OUTCOME MEASURES: Antibody titers, mapping of binding sites (epitopes), isotype profiles of the Abeta42-specific antibodies, and T-cell activation. RESULTS: DNA Abeta42 trimer immunization resulted in antibody titers with a mean of 15 microg per milliliter of plasma. The isotype profile of the antibodies differed markedly. A predominant IgG1 antibody response was found in the DNA-immunized mice, indicating a T(H)2 type of immune response (IgG1/IgG2a ratio of 10). The peptide-immunized mice showed a mixed T(H)1/T(H)2 immune response (IgG1/IgG2a ratio of 1) (P < .001). No increased T-cell proliferation was observed in the DNA-immunized mice (P = .03). CONCLUSION: In this preliminary study in a wild-type mouse model, DNA Abeta42 trimer immunization protocol produced a T(H)2 immune response and appeared to have low potential to cause an inflammatory T-cell response.

Genome-wide association studies in Alzheimer disease.

The genetics of Alzheimer disease (AD) to date support an age-dependent dichotomous model whereby earlier age of disease onset (< 60 years) is explained by 3 fully penetrant genes (APP [NCBI Entrez gene 351], PSEN1 [NCBI Entrez gene 5663], and PSEN2 [NCBI Entrez gene 5664]), whereas later age of disease onset (> or = 65 years) representing most cases of AD has yet to be explained by a purely genetic model. The APOE gene (NCBI Entrez gene 348) is the strongest genetic risk factor for later onset, although it is neither sufficient nor necessary to explain all occurrences of disease. Numerous putative genetic risk alleles and genetic variants have been reported. Although all have relevance to biological mechanisms that may be associated with AD pathogenesis, they await replication in large representative populations. Genome-wide association studies have emerged as an increasingly effective tool for identifying genetic contributions to complex diseases and represent the next frontier for furthering our understanding of the underlying etiologic, biological, and pathologic mechanisms associated with chronic complex disorders. There have already been success stories for diseases such as macular degeneration and diabetes mellitus. Whether this will hold true for a genetically complex and heterogeneous disease such as AD is not known, although early reports are encouraging. This review considers recent publications from studies that have successfully applied genome-wide association methods to investigations of AD by taking advantage of the currently available high-throughput arrays, bioinformatics, and software advances. The inherent strengths, limitations, and challenges associated with study design issues in the context of AD are presented herein.

Prion proteins: physiological functions and role in neurological disorders.

Stanley Prusiner was the first to promote the concept of misfolded proteins as a cause for neurological disease. It has since been shown by him and other investigators that the scrapie isoform of prion protein (PrP(Sc)) functions as an infectious agent in numerous human and non-human disorders of the central nervous system (CNS). Interestingly, other organ systems appear to be less affected, and do not appear to lead to major co-morbidities. The physiological function of the endogenous cellular form of the prion protein (PrP(C)) is much less clear. It is intriguing that PrP(c) is expressed on most tissues in mammals, suggesting not only biological functions outside the CNS, but also a role other than the propagation of its misfolded isotype. In this review, we summarize accumulating in vitro and in vivo evidence regarding the physiological functions of PrP(C) in the nervous system, as well as in lymphoid organs.

Atherosclerosis risk factors in American Indians with Alzheimer disease: preliminary findings.

Factors predisposing to and associated with atherosclerosis may impact the onset and progression of Alzheimer disease (AD). The high prevalence of atherosclerosis and associated risk factors in American Indians makes them ideal subjects to test this association. We compared frequency of history of hypertension, myocardial infarction, stroke, diabetes, and high cholesterol in 34 American Indians with AD with 34 age-matched American Indian controls, and 34 age-matched whites with probable AD. We also measured waist size, height, and weight, and acquired blood for determination of plasma homocysteine and apolipoprotein E genotype. The 3 groups did not differ significantly in age or sex. History of hypertension and diabetes was significantly more common among American Indian AD patients than Indian controls or whites with AD. The 3 groups did not differ in history of stroke or myocardial infarction. Body mass index was significantly greater in both Indian groups than the white AD group. Plasma homocysteine levels were greater, but not significantly so, in the Indian AD than the Indian control group. Thus, there is preliminary evidence of a modest association between history of hypertension and diabetes and AD in a small sample of American Indians. This suggests that changes in lifestyle factors could influence the expression of AD in American Indians.

Active full-length DNA Aß42 immunization in 3xTg-AD mice reduces not only amyloid deposition but also tau pathology.

BACKGROUND: Alzheimer's disease (AD) is the most well-known and most common type of age-related dementia. Amyloid deposition and hyperphosphorylation of tau protein are both pathological hallmarks of AD. Using a triple-transgenic mouse model (3xTg-AD) that develops plaques and tangles in the brain similar to human AD, we provide evidence that active full-length DNA amyloid-ß peptide 1-42 (Aß42) trimer immunization leads to reduction of both amyloid and tau aggregation and accumulation. METHODS: Immune responses were monitored by enzyme-linked immunosorbent assay (ELISA) (antibody production) and enzyme-linked immunospot (cellular activation, cytokine production). Brains from 20-month-old 3x Tg-AD mice that had received DNA Aß42 immunotherapy were compared with brains from age- and gender-matched transgenic Aß42 peptide-immunized and control mice by histology, Western blot analysis, and ELISA. Protein kinase activation and kinase levels were studied in Western blots from mouse hemibrain lysates. RESULTS: Quantitative ELISA showed a 40% reduction of Aß42 peptide and a 25-50% reduction of total tau and different phosphorylated tau molecules in the DNA Aß42 trimer-immunized 3xTg-AD mice compared with nonimmunized 3xTg-AD control animals. Plaque and Aß peptide reductions in the brain were due to the anti-Aß antibodies generated following the immunizations. Reductions of tau were likely due to indirect actions such as less Aß in the brain resulting in less tau kinase activation. CONCLUSIONS: The significance of these findings is that DNA Aß42 trimer immunotherapy targets two major pathologies in AD-amyloid plaques and neurofibrillary tangles-in one vaccine without inducing inflammatory T-cell responses, which carry the danger of autoimmune inflammation, as found in a clinical trial using active Aß42 peptide immunization in patients with AD (AN1792).

Intradermal active full-length DNA Aß42 immunization via electroporation leads to high anti-Aß antibody levels in wild-type mice.

Aß immunotherapies with anti-Aß antibody responses have high potential as possible prevention treatment for Alzheimer's disease. We have previously shown that active DNA Aß1-42 immunization via gene gun delivery led to a non-inflammatory immune response resulting in decreased Aß levels in brains of an immunized AD mouse model. To make DNA vaccination more applicable for clinical use, we used here intradermal electroporation. With fine tuning of the electropulse parameters, high antibody levels and low levels of inflammatory cytokines in the cellular immunoassays were observed. Full-length DNA Aß1-42 immunization delivered via electroporation has potential to be used in the clinical setting.

Abeta42 gene vaccine prevents Abeta42 deposition in brain of double transgenic mice.

Abeta42 peptide aggregation and deposition is an important component of the neuropathology of Alzheimer's disease (AD). Gene-gun mediated gene vaccination targeting Abeta42 is a potential method to prevent and treat AD. APPswe/PS1DeltaE9 transgenic (Tg) mice were immunized with an Abeta42 gene construct delivered by the gene gun. The vaccinated mice developed Th2 antibodies (IgG1) against Abeta42. The Abeta42 levels in brain were decreased by 41% and increased in plasma 43% in the vaccinated compared with control mice as assessed by ELISA analysis. Abeta42 plaque deposits in cerebral cortex and hippocampus were reduced by 51% and 52%, respectively, as shown by quantitative immunolabeling. Glial cell activation was also significantly attenuated in vaccinated compared with control mice. One rhesus monkey was vaccinated and developed anti-Abeta42 antibody. These new findings advance significantly our knowledge that gene-gun mediated Abeta42 gene immunization effectively induces a Th2 immune response and reduces the Abeta42 levels in brain in APPswe/PS1DeltaE9 mice. Abeta42 gene vaccination may be safe and efficient immunotherapy for AD.

Evaluation of a DNA Aß42 vaccine in adult rhesus monkeys (Macaca mulatta): antibody kinetics and immune profile after intradermal immunization with full-length DNA Aß42 trimer.

BACKGROUND: Aggregated amyloid-ß peptide 1-42 (Aß42), derived from the cellular amyloid precursor protein, is one of the pathological hallmarks of Alzheimer's disease (AD). Although active immunization against Aß42 peptide was successful in AD mouse models and led to removal of plaques and improved memory, a similar clinical trial in humans (Aß42 peptide immunization with QS-21 adjuvant) was stopped in phase II, when 6% of the treated patients developed encephalitis. Currently ongoing passive immunizations with the injection of preformed monoclonal antibodies against different epitopes within the Aß1-42 peptide, which do not lead to activation of the immune system, have shown some effects in slowing AD pathology. Active DNA Aß42 immunizations administered with the gene gun into the skin are noninflammatory because they activate a different T-cell population (Th2) with different cytokine responses eliciting a different humoral immune response. We present our findings in rhesus macaques that underwent the DNA Aß42 immunization via gene gun delivery into the skin. METHODS: Six rhesus monkeys received two different doses of a DNA Aß42 trimer vaccine. The humoral immune response was analyzed from blood throughout the study, and cellular immune responses were determined in peripheral blood mononuclear cells (PBMCs) after three and six immunizations. RESULTS: DNA Aß42 trimer immunization led to high titer antibody responses in the nonhuman primate (NHP) model. Antibodies generated in the rhesus monkeys following DNA Aß42 immunization detected amyloid plaques consisting of human Aß42 peptide in the brain of the triple-transgenic AD mouse model. T-cell responses showed no interferon (IFN)-γ- and interleukin (IL)-17-producing cells from PBMCs in Enzyme-Linked ImmunoSpot assays after three immunization time points. At six immunization time points, IFN-γ- and IL-17-producing cells were found in immunized animals as well as in control animals and were thus considered nonspecific and not due to the immunization regimen. IFN-γ and IL-17 secretion in response to Aß42 peptide restimulation became undetectable after a 3-month rest period. CONCLUSIONS: Intradermal DNA Aß42 immunization delivered with the gene gun produces a high antibody response in NHPs and is highly likely to be effective and safe in a clinical AD prevention trial in patients.

Evaluation of a DNA Aß42 Vaccine in Aged NZW Rabbits: Antibody Kinetics and Immune Profile after Intradermal Immunization with Full-Length DNA Aß42 Trimer.

A pathological hallmark of Alzheimer's disease (AD) are amyloid plaques in the brain consisting of aggregated amyloid-ß 42 peptide (Aß42) derived from cellular amyloid-ß protein precursor (AßPP). Based on successful experiments in mouse AD models, active immunization with Aß42 peptide and passive immunizations with anti-Aß42 antibodies were started in clinical trials. Active Aß42 peptide immunization in humans had led to an inflammatory autoimmune response, and the trial was stopped. Passive immunizations had shown some effects in slowing AD pathology. Active DNA Aß42 immunizations administered with the gene gun into the skin elicits a different immune response and is non-inflammatory. While in rodents, good responses had been found for this type of immunization, positive results in larger mammals are missing. We present here results from sixteen New Zealand White Rabbits, which underwent intradermal DNA Aß42 immunization via gene gun. The humoral immune response was analyzed from blood throughout the study, and cellular immune responses were determined from spleens at the end of the study. A good anti-Aß antibody response was found in the rabbit model. The T cell response after re-stimulation in cell culture showed no IFNγ producing cells when ELISPOT assays were analyzed from PBMC, but low numbers of IFNγ and IL-17 producing cells were found in ELISPOTS from spleens (both 5 immunizations). Brains from immunized rabbits showed no signs of encephalitis. Based on these results, DNA Aß42 immunization is highly likely to be safe and effective to test in a possible clinical AD prevention trial in patients.

Laquinimod has no effects on brain volume or cellular CNS composition in the F1 3xTg-AD/C3H mouse model of Alzheimer's disease.

BACKGROUND: Laquinimod is an anti-inflammatory agent with good central nervous system (CNS) bioavailability, and neuroprotective and myelorestorative properties. A clinical trial in patients with multiple sclerosis demonstrated that laquinimod significantly reduced loss of brain volume. The cellular substrate or molecular events underlying that treatment effect are unknown. In this study, we aimed to explore laquinimod's potential effects on brain volume, animal behavior, cellular numbers and composition of CNS-intrinsic cells and mononuclear cells within the CNS, amyloid beta (Aß) accumulation and tau phosphorylation in the F1 3xTg-AD/C3H mouse model of Alzheimer's disease. METHODS: Utilizing a dose response study design, four months old F1 3xTg-AD/C3H mice were treated for 10months between ages 4 and 14months with laquinimod (5, 10, or 25mg/kg), or PBS administered by oral gavage. Brain volumes were measured in a 7 Tesla magnetic resonance imager (MRI) at ages 4 and 14months. Behavioral testing included locomotor and rearing activity and the Morris water maze task. Cell numbers and immunophenotypes were assessed by multiparameter flow cytometry. Aß deposition and tau phosphorylation were determined by immunohistochemistry. RESULTS: In the F1 3xTg-AD/C3H animal model of AD, there was no detectable reduction of brain volume over a period of 10months of treatment, as there was not brain atrophy in any of the placebo or treatment groups. Laquinimod had no detectable effects on most neurobehavioral outcomes. The number or composition of CNS intrinsic cells and mononuclear subsets isolated from the CNS were not altered by laquinimod. CONCLUSION: This is the first demonstration that there are no age-associated brain volume changes in the F1 3xTg-AD/C3H mouse model of Alzheimer's disease. Consequently, laquinimod had no effect on that outcome of this study. Most secondary outcomes on the effects of laquinimod on behavior and the number and composition of CNS-intrinsic cells and mononuclear cells within the CNS were also negative.