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Background Cerebral microhemorrhages (CMH) are commonly found in the aging brain. CMH are also the neuropathological substrate of cerebral microbleeds (CMB), demonstrated on brain MRI. Recent studies demonstrate the importance of systemic inflammation in CMH development, but the relationships among inflammation, aging, and CMH development are not well-defined. In the current study, we hypothesized that the pathogenesis of inflammation-induced CMH in mice differs by age. Methods We studied young (3 months, n = 20) and old (18 months, n = 25) C57BL/6 mice injected with low-dose lipopolysaccharide (LPS; 1 mg/kg, i.p.) or saline at 0, 6, and 24 h. Seven days after the first LPS/saline injection, brains were harvested, sectioned, and stained with hematoxylin and eosin (H&E) and Prussian blue (PB) to estimate acute/fresh and sub-acute CMH development, respectively. The relationships between microglial/macrophage activation (ionized calcium-binding adapter molecule-1), astrocyte activation (glial fibrillary acidic protein), blood-brain barrier (BBB) disruption (brain immunoglobulin G), aging, and CMH development were examined using immunohistochemistry. Results Aging alone did not increase spontaneous H&E-positive CMH development but significantly increased the number, size, and total area of LPS-induced H&E-positive CMH in mice. LPS- and saline-treated aged mice had significantly larger PB-positive CMH compared with young mice, but the total area of PB-positive CMH was increased only in LPS-treated aged mice. Aged mice had significantly increased microglial/macrophage activation, which correlated with H&E- and PB-positive CMH development. Aged mice treated with LPS had significantly increased astrocyte activation and BBB disruption compared with young LPS-treated mice. Conclusions Aging makes the brain more susceptible to inflammation-induced CMH in mice, and this increase in CMH with aging is associated with microglial/macrophage activation.

Tumor necrosis factor alpha (TNF-α) driven processes are involved at multiple stages of Alzheimer's disease (AD) pathophysiology and disease progression. Biologic TNF-α inhibitors (TNFIs) are the most potent class of TNFIs but cannot be developed for AD since these macromolecules do not cross the blood-brain barrier (BBB). A BBB-penetrating TNFI was engineered by the fusion of the extracellular domain of the type II human TNF receptor (TNFR) to a chimeric monoclonal antibody (mAb) against the mouse transferrin receptor (TfR), designated as the cTfRMAb-TNFR fusion protein. The cTfRMAb domain functions as a molecular Trojan horse, binding to the mouse TfR and ferrying the biologic TNFI across the BBB via receptor-mediated transcytosis. The aim of the study was to examine the effect of this BBB-penetrating biologic TNFI in a mouse model of AD. Six-month-old APPswe, PSEN 1dE9 (APP/PS1) transgenic mice were treated with saline (n = 13), the cTfRMAb-TNFR fusion protein (n = 12), or etanercept (non-BBB-penetrating biologic TNFI; n = 11) 3 days per week intraperitoneally. After 12 weeks of treatment, recognition memory was assessed using the novel object recognition task, mice were sacrificed, and brains were assessed for amyloid beta (Aβ) load, neuroinflammation, BBB damage, and cerebral microhemorrhages. The cTfRMAb-TNFR fusion protein caused a significant reduction in brain Aβ burden (both Aβ peptide and plaque), neuroinflammatory marker ICAM-1, and a BBB disruption marker, parenchymal IgG, and improved recognition memory in the APP/PS1 mice. Fusion protein treatment resulted in low antidrug-antibody formation with no signs of either immune reaction or cerebral microhemorrhage development with chronic 12-week treatment. Chronic treatment with the cTfRMAb-TNFR fusion protein, a BBB-penetrating biologic TNFI, offers therapeutic benefits by targeting Aβ pathology, neuroinflammation, and BBB-disruption, overall improving recognition memory in a transgenic mouse model of AD.

We have previously demonstrated that the use of a commercially-available immersion-based optical clearing agent (OCA) enables, within 3-6 hours, three-dimensional visualization of subsurface exogenous fluorescent and absorbing markers of vascular architecture and neurodegenerative disease in thick (0.5-1.0mm) mouse brain sections. Nonetheless, the relative performance of immersion-based OCAs has remained unknown. Here, we show that immersion of brain sections in specific OCAs (FocusClear, RIMS, sRIMS, or ScaleSQ) affects both their transparency and volume; the optical clearing effect occurs over the entire visible spectrum and is reversible; and that ScaleSQ had the highest optical clearing potential and increase in imaging depth of the four evaluated OCAs, albeit with the largest change in sample volume and a concomitant decrease in apparent microvascular density of the sample. These results suggest a rational, quantitative framework for screening and characterization of the impact of optical clearing, to streamline experimental design and enable a cost-benefit assessment.

OBJECTIVE: Erythropoietin (EPO), a glycoprotein cytokine essential to hematopoiesis, has neuroprotective effects in rodent models of Alzheimer’s disease (AD). However, high therapeutic doses or invasive routes of administration of EPO are required to achieve effective brain concentrations due to low blood-brain barrier (BBB) penetrability, and high EPO doses result in hematopoietic side effects. These obstacles can be overcome by engineering a BBB-penetrable analog of EPO, which is rapidly cleared from the blood, by fusing EPO to a chimeric monoclonal antibody targeting the transferrin receptor (cTfRMAb), which acts as a molecular Trojan horse to ferry the EPO into the brain via the transvascular route. In the current study, we investigated the effects of the BBB-penetrable analog of EPO on AD pathology in a double transgenic mouse model of AD. METHODS: 5.5 month old male APPswe PSEN1dE9 (APP/PS1) transgenic mice were treated with saline (n=10), or the BBB-penetrable EPO (n=10) 3 days/week intraperitoneally for 8 weeks, compared to same-aged C57BL/6J wild-type mice treated with saline (n=8) with identical regiment. At 9 weeks following treatment initiation, exploration and spatial memory were assessed with the open-field and Y-maze test, mice were sacrificed, and brains were evaluated for Aβ peptide load, synaptic loss, BBB disruption, microglial activation and microhemorrhages. RESULTS: APP/PS1 mice treated with the BBB-penetrable cTfRMAb-EPO fusion protein had significantly lower cortical and hippocampal Aβ peptide number (p

Brain microbleeds are increased in chronic kidney disease (CKD) and their presence increases risk of cognitive decline and stroke. We examined the interaction between CKD and brain microhemorrhages (the neuropathological substrate of microbleeds) in mouse and cell culture models and studied progression of microbleed burden on serial brain imaging from humans. Mouse studies: Two CKD models were investigated: adenine-induced tubulointerstitial nephritis and surgical 5/6 nephrectomy. Cell culture studies: bEnd.3 mouse brain endothelial cells were grown to confluence, and monolayer integrity was measured after exposure to 5-15% human uremic serum or increasing concentrations of urea. Human studies: Progression of brain microbleeds was evaluated on serial MRI from control, pre-dialysis CKD, and dialysis patients. Microhemorrhages were increased 2-2.5-fold in mice with CKD independent of higher blood pressure in the 5/6 nephrectomy model. IgG staining was increased in CKD animals, consistent with increased blood-brain barrier permeability. Incubation of bEnd.3 cells with uremic serum or elevated urea produced a dose-dependent drop in trans-endothelial electrical resistance. Elevated urea induced actin cytoskeleton derangements and decreased claudin-5 expression. In human subjects, prevalence of microbleeds was 50% in both CKD cohorts compared with 10% in age-matched controls. More patients in the dialysis cohort had increased microbleeds on follow-up MRI after 1.5 years. CKD disrupts the blood-brain barrier and increases brain microhemorrhages in mice and microbleeds in humans. Elevated urea alters the actin cytoskeleton and tight junction proteins in cultured endothelial cells, suggesting that these mechanisms explain (at least in part) the microhemorrhages and microbleeds observed in the animal and human studies.

Cerebral microbleeds (CMB), a common MRI finding that may predict intracerebral hemorrhage, are based on a pathological substrate of cerebral microhemorrhages (CMH). Recent clinical and laboratory studies have demonstrated the importance of systemic inflammation in the development of CMB. The effects of age and sex on CMB development have not been well-defined. In the current study, we hypothesized that there are age and sex differences in the pathogenesis of inflammation-induced CMB in mice. We studied young (3 months, n=39) and old (17 months, n=25) male and female C57BL/6 mice injected with LPS (1mg/kg, i.p.) or saline at 0, 6, and 24h. 7 days after first LPS injection, cardiac perfusion was performed, mice were sacrificed, and brains were harvested. CMH grossly visible on the brain surface were examined and 40 μm brain sections were prepared and stained with hematoxylin and eosin (H&E) to estimate the acute/fresh parenchymal CMH number, size and total area in young versus old mice. No surface CMH and negligible H&E-positive parenchymal CMH were observed in young and old saline controls. LPS treatment significantly (p2 ; p

Background Cerebral microbleeds (CMB) are MRI-demonstrable cerebral microhemorrhages (CMH) which commonly coexist with ischemic stroke. This creates a challenging therapeutic milieu, and a strategy that simultaneously protects the vessel wall and provides anti-thrombotic activity is an attractive potential approach. Phosphodiesterase 3A (PDE3A) inhibition is known to provide cerebral vessel wall protection combined with anti-thrombotic effects. As an initial step in the development of a therapy that simultaneously treats CMB and ischemic stroke, we hypothesized that inhibition of the PDE3A pathway is protective against CMH development. Methods The effect of PDE3A pathway inhibition was studied in the inflammation-induced and cerebral amyloid angiopathy (CAA)-associated mouse models of CMH. The PDE3A pathway was modulated using two approaches: genetic deletion of PDE3A and pharmacological inhibition of PDE3A by cilostazol. The effects of PDE3A pathway modulation on H&E- and Prussian blue (PB)-positive CMH development, BBB function (IgG, claudin-5, and fibrinogen), and neuroinflammation (ICAM-1, Iba-1, and GFAP) were investigated. Results Robust development of CMH in the inflammation-induced and CAA-associated spontaneous mouse models was observed. Inflammation-induced CMH were associated with markers of BBB dysfunction and inflammation, and CAA-associated spontaneous CMH were associated primarily with markers of neuroinflammation. Genetic deletion of the PDE3A gene did not alter BBB function, microglial activation, or CMH development, but significantly reduced endothelial and astrocyte activation in the inflammation-induced CMH mouse model. In the CAA-associated CMH mouse model, PDE3A modulation via pharmacological inhibition by cilostazol did not alter BBB function, neuroinflammation, or CMH development. Conclusions Modulation of the PDE3A pathway, either by genetic deletion or pharmacological inhibition, does not alter CMH development in an inflammation-induced or in a CAA-associated mouse model of CMH. The role of microglial activation and BBB injury in CMH development warrants further investigation.

The deposition of amyloid-β peptides (Aβ) in the cerebral vasculature, a condition known as cerebral amyloid angiopathy, is increasingly recognized as an important component leading to intracerebral hemorrhage, neuroinflammation, and cognitive impairment in Alzheimer disease (AD) and related disorders. Recent studies demonstrated a role for the bradykinin B1 receptor (B1R) in cognitive deficits induced by Aβ in mice; however, its involvement in AD and cerebral amyloid angiopathy is poorly understood. Herein, we investigated the effect of B1R inhibition on AD-like neuroinflammation and amyloidosis using the transgenic mouse model (Tg-SwDI). B1R expression was found to be up-regulated in brains of Tg-SwDI mice, specifically in the vasculature, neurons, and astrocytes. Notably, administration of the B1R antagonist, R715, to 8-month-old Tg-SwDI mice for 8 weeks resulted in higher Aβ40 levels and increased thioflavin S–positive fibrillar Aβ deposition. Moreover, blockage of B1R inhibited neuroinflammation, as evidenced by the decreased accumulation of activated microglia and reactive astrocytes, diminished NF-κB activation, and reduced cytokine and chemokine levels. Together, our results indicate that B1R activation plays an important role in limiting the accumulation of Aβ in AD-like brain, likely through the regulation of activated glial cell accumulation and release of pro-inflammatory mediators. Therefore, the modulation of the receptor may represent a novel therapeutic approach for AD.

Cerebral amyloid angiopathy (CAA) is a neurovascular disease that is strongly associated with an increase in the number and size of spontaneous microhemorrhages. Conventional methods, such as magnetic resonance imaging (MRI), can detect microhemorrhages while positron emission tomography (PET) with Pittsburgh Compound B can detect amyloid deposits. MRI and PET can separately demonstrate the presence of microhemorrhages and CAA in affected brains in vivo; however, there is still a lack of strong evidence for the direct involvement of CAA in the presence of microhemorrhage formation. In this study, we use optical histology, a method which combines histochemical staining, chemical optical clearing, and optical imaging, in a Tg2576 mouse model of Alzheimer’s disease to enable simultaneous, co-registered three-dimensional visualization of cerebral microvasculature, microhemorrhages, and amyloid deposits. Our data strongly suggest that microhemorrhages are localized within the brain regions affected by amyloid deposits. All but two observed microhemorrhages (n=18) were closely localized with vessels affected by CAA whereas no microhemorrhages or amyloid deposits were observed in wild type mouse brain sections. Our data also suggest that the predominant type of CAA-related microhemorrhage is associated with leaky or ruptured hemorrhagic microvasculature within the hippocampus and cerebral cortex rather than occluded ischemic microvasculature. The proposed optical histology method will allow future studies about the relationship between CAA and microhemorrhages during disease development and in response to treatment strategies.

Introduction Individuals with Down syndrome (DS) exhibit Alzheimer's disease (AD) neuropathology and dementia early in life. Blood biomarkers of AD neuropathology would be valuable, as non-AD intellectual disabilities of DS and AD dementia overlap clinically. We hypothesized that elevations of amyloid β (Aβ) peptides and phosphorylated-tau in neuronal exosomes may document preclinical AD. Methods AD neuropathogenic proteins Aβ 1–42 , P-T181-tau, and P-S396-tau were quantified by enzyme-linked immunosorbent assays in extracts of neuronal exosomes purified from blood of individuals with DS and age-matched controls. Results Neuronal exosome levels of Aβ 1–42 , P-T181-tau, and P-S396-tau were significantly elevated in individuals with DS compared with age-matched controls at all ages beginning in childhood. No significant gender differences were observed. Discussion These early increases in Aβ 1–42 , P-T181-tau, and P-S396-tau in individuals with DS may provide a basis for early intervention as targeted treatments become available.

Introduction: Multiple epidemiological studies link vasculature-affecting disorders, such as hypertension, diabetes mellitus, kidney failure and stroke, with dementia and Alzheimer’s disease (AD). Hypertension is an important modifiable risk factor for late-onset sporadic AD, and often associated with increased incidents of intracerebral hemorrhages (ICH) and cerebral amyloid angiopathy (CAA). Hypothesis: To examine the association between short-term mild experimental hypertension and the progression of AD pathology later in life, we pharmacologically induced mild chronic hypertension in the APP/Tg2576 mouse model of AD. Methods: Twenty-month old APP/Tg2576 mice (n=6) received N-Nitro-l-arginine methyl ester hydrochloride (L-NAME) in drinking water, for the daily doses of 100 mg/kg body weight for each mouse. Control mice (n=4) had regular drinking water. Treatment continued for 4 weeks. Blood pressure was measured 4 days before termination of the experiment with tail-cuff system (CODA). At the end of experiment mouse brains were collected and analyzed for amyloid-related AD pathology, inflammatory markers of glial activation and cerebrovascular pathology. Differences between groups were tested by t-test. Results: There was a significant mild increase in diastolic (109±5.1 vs 123±2.4 mmHg, p Conclusions: Our results suggest that short-term mild experimental hypertension late in life in APP/Tg mouse model of amyloidosis exacerbated amyloid pathology and cerebral microhemorrhages. Managing the blood pressure in the population at risk of AD should be considered as a potential preventive therapy for this neurological disorder.

Stroke prevention efforts typically focus on either ischemic or hemorrhagic stroke. This approach is overly simplistic due to the frequent coexistence of ischemic and hemorrhagic cerebrovascular disease. This coexistence, termed “mixed cerebrovascular disease”, offers a conceptual framework that appears useful for stroke prevention strategies. Mixed cerebrovascular disease incorporates clinical and subclinical syndromes, including ischemic stroke, subclinical infarct, white matter disease of aging (leukoaraiosis), intracerebral hemorrhage, and cerebral microbleeds. Reliance on mixed cerebrovascular disease as a diagnostic entity may assist in stratifying risk of hemorrhagic stroke associated with platelet therapy and anticoagulants. Animal models of hemorrhagic cerebrovascular disease, particularly models of cerebral amyloid angiopathy and hypertension, offer novel means for identifying underlying mechanisms and developing focused therapy. Phosphodiesterase (PDE) inhibitors represent a class of agents that, by targeting both platelets and vessel wall, provide the kind of dual actions necessary for stroke prevention, given the spectrum of disorders that characterizes mixed cerebrovascular disease.

Background and Purpose— The aging brain demonstrates frequent MRI and pathological evidence of cerebral microbleeds, which are often associated with cerebral amyloid angiopathy. To develop new therapeutic strategies for this disorder, we studied cerebral microhemorrhage in a well-characterized mouse model of cerebral amyloid angiopathy. Methods— Tg2576 mice were studied at ages ranging from 2 to 21 months. Spontaneous and induced microscopic bleeding was analyzed with and without a passive anti-amyloid immunization regimen and dietary supplementation of ischemic stroke prevention medication dipyridamole. Results— Areas of microhemorrhage were easily demonstrated and were significantly more prominent in the oldest mice and in animals treated with anti-amyloid immunotherapy. Dipyridamole supplementation in the diet generated plasma levels >790 ng/mL within the range seen clinically. Dipyridamole treatment did not worsen frequency and size of cerebral microscopic bleeding. Conclusions— The Tg2576 mouse is a useful model to study progression and modification of spontaneous and immunotherapy-induced cerebral microhemorrhage. Absence of microhemorrhage worsening with dipyridamole treatment suggests a potential therapeutic role of this agent when ischemic and microhemorrhagic lesions coexist.

N-truncated/modified forms of amyloid beta (Aß) peptide are found in diffused and dense core plaques in Alzheimer's disease (AD) and Down's syndrome patients as well as animal models of AD, and represent highly desirable therapeutic targets. In the present study we have focused on N-truncated/modified Aβ peptide bearing amino-terminal pyroglutamate at position 11 (AβN11(pE)). We identified two B-cell epitopes recognized by rabbit anti-AβN11(pE) polyclonal antibodies. Interestingly, rabbit anti-AβN11(pE) polyclonal antibodies bound also to full-length Aβ1-42 and N-truncated/modified AβN3(pE), suggesting that the three peptides may share a common B-cell epitope. Importantly, rabbit anti-AβN11(pE) antibodies bound to naturally occurring Aβ aggregates present in brain samples from AD patients. These results are potentially important for developing novel immunogens for targeting N-truncated/modified Aβ aggregates as well, since the most commonly used immunogens in the majority of vaccine studies have been shown to induce antibodies that recognize the N-terminal immunodominant epitope (EFRH) of the full length Aβ, which is absent in N-amino truncated peptides.

Age-related cerebrovascular dysfunction contributes to ischemic stroke, intracerebral hemorrhages (ICHs), microbleeds, cerebral amyloid angiopathy (CAA), and cognitive decline. Importantly, there is increasing recognition that this dysfunction plays a critical secondary role in many neurodegenerative diseases, including Alzheimer's disease (AD). Atherosclerosis, hypertension, and CAA are the most common causes of blood-brain barrier (BBB) lesions. The accumulation of amyloid beta (Aβ) in the cerebrovascular system is a significant risk factor for ICH and has been linked to endothelial transport failure and blockage of perivascular drainage. Moreover, recent anti-Aβ immunotherapy clinical trials demonstrated efficient clearance of parenchymal amyloid deposits but have been plagued by CAA-associated adverse events. Although management of hypertension and atherosclerosis can reduce the incidence of ICH, there are currently no approved therapies for attenuating CAA. Thus, there is a critical need for new strategies that improve BBB function and limit the development of β-amyloidosis in the cerebral vasculature.

Previously we showed that anti-Abeta peptide immunotherapy significantly attenuated Alzheimer's-like amyloid deposition in the central nervous system of aged canines. In this report we have characterized the changes that occurred in the humoral immune response over 2.4years in canines immunized repeatedly with aggregated Abeta(1-42) (AN1792) formulated in alum adjuvant. We observed a rapid and robust induction of anti-Abeta antibody titers, which were associated with an anti-inflammatory T helper type 2 (Th2) response. The initial antibody response was against dominant linear epitope at the N-terminus region of the Abeta(1-42) peptide, which is identical to the one in humans and vervet monkeys. After multiple immunizations the antibody response drifted toward the elevation of antibodies that recognized conformational epitopes of assembled forms of Abeta and other types of amyloid. Our findings indicate that prolonged immunization results in distinctive temporal changes in antibody profiles, which may be important for other experimental and clinical settings.

An estimated 5.3 million Americans are living with a disability from a traumatic brain injury (TBI). There is emerging evidence of the detrimental effects from repeated mild TBIs (rmTBIs). rmTBI manifests its own unique set of behavioral and neuropathological changes. A subset of individuals exposed to rmTBI develop permanent behavioral and pathological consequences, defined postmortem as chronic traumatic encephalopathy. We have combined components of two classic rodent models of TBI, the controlled cortical impact model and the weight drop model, to develop a repeated mild closed head injury (rmCHI) that produces long-term deficits in several behaviors that correlate with neuropathological changes. Mice receiving rmCHI performed differently from 1-hit or sham controls on the elevated plus maze; these deficits persist up to 6 months postinjury (MPI). rmCHI mice performed worse than 1-hit and control sham mice at 2 MPI and 6 MPI on the Morris water maze. Mice receiving rmCHI exhibited significant atrophy of the corpus callosum at both 2 MPI and 6 MPI, as assessed by stereological volume analysis. Stereological analysis also revealed significant loss of cortical neurons in comparison with 1-hit and controls. Moreover, both of these pathological changes correlated with behavioral impairments. In human tau transgenic mice, rmCHI induced increases in hyperphosphorylated paired helical filament 1 tau in the hippocampus. This suggests that strategies to restore myelination or reduce neuronal loss may ameliorate the behavioral deficits observed following rmCHI and that rmCHI may model chronic traumatic encephalopathy in human tau mice.

N-truncated and N-modified forms of amyloid beta (Abeta) peptide are found in diffused and dense core plaques in Alzheimer's disease (AD) and Down's syndrome patients as well as transgenic mouse models of AD. Although the pathological significance of these shortened forms Abeta is not completely understood, previous studies have demonstrated that these peptides are significantly more resistant to degradation, aggregate more rapidly in vitro and exhibit similar or, in some cases, increased toxicity in hippocampal neuronal cultures compared to the full length peptides. In the present study we further investigated the mechanisms of toxicity of one of the most abundant N-truncated/modified Abeta peptide bearing amino-terminal pyroglutamate at position 3 (AbetaN3(pE)). We demonstrated that AbetaN3(pE) oligomers induce phosphatidyl serine externalization and membrane damage in SH-SY5Y cells. Also, we produced AbetaN3(pE)-specific polyclonal antibodies in rabbit and identified an immunodominant epitope recognized by anti-AbetaN3(pE) antibodies. Our results are important for developing new immunotherapeutic compounds specifically targeting AbetaN3(pE) aggregates since the most commonly used immunogens in the majority of vaccines for AD have been shown to induce antibodies that recognize the N-terminal immunodominant epitope (EFRH) of the full length Abeta, which is absent in N-amino truncated peptides.

Inclusion body myositis (IBM), the most common muscle disease to afflict the elderly, causes slow but progressive degeneration of skeletal muscle and ultimately paralysis. Hallmark pathological features include T-cell mediated inflammatory infiltrates and aberrant accumulations of proteins, including amyloid-β (Aβ), tau, ubiquitinated-proteins, apolipoprotein E, and α-synuclein in skeletal muscle. A large body of work indicates that aberrant Aβ accumulation contributes to the myodegeneration. Here, we investigated whether active immunization to promote clearance of Aβ from affected skeletal muscle fibers mitigates the IBM-like myopathological features as well as motor impairment in a transgenic mouse model. We report that active immunization markedly reduces intracellular Aβ deposits and attenuates the motor impairment compared with untreated mice. Results from our current study indicate that Aβ oligomers contribute to the myopathy process as they were significantly reduced in the affected skeletal muscle from immunized mice. In addition, the anti-Aβ antibodies produced in the immunized mice blocked the toxicity of the Aβ oligomersin vitro, providing a possible key mechanism for the functional recovery. These findings provide support for the hypothesis that Aβ is one of the key pathogenic components in IBM pathology and subsequent skeletal muscle degeneration.

Alzheimer disease (AD) is the most common form of dementia in the elderly and the number of individuals developing the disease is rapidly rising. Interventions focused on reducing beta-amyloid (Abeta), a component of senile plaques within the AD brain offer a promising approach to prevent or slow disease progression. In this review, we describe the immune system and cognitive and neurobiological features of a natural model of human brain aging, the beagle. The immune system of dogs shares many features of the human immune system, including developmental and aging characteristics. Further, dogs naturally accumulate human sequence Abeta as they age, which coincides with declines in learning and memory. A longitudinal study (approximately 2 years) of the response of aged beagles to vaccination with fibrillar Abeta1-42 indicated that despite significant clearance of Abeta, there were limited benefits in cognitive function. However, there was evidence for maintenance of executive function over time. These results are strikingly similar to reports of human clinical immunotherapy trials. We propose that the canine model complements existing animal models and will be helpful in developing new vaccine approaches to slowing or preventing Abeta pathology that can be translated to human clinical trials.

Active and passive immunotherapy targeted at the amyloid-beta (Abeta) peptide has been proposed as therapeutic approach against Alzheimer's disease (AD), and efforts towards the generation and application of antibody-based reagents that are capable of preventing and clearing amyloid aggregates are currently under active investigation. Previously, we selected and characterized a new anti-Abeta1-42 phage-displayed scFv antibody, designated clone b4.4, using a non-immune human scFv antibody library and demonstrated that a peptide based on the sequence of the Ig heavy chain (VH) complementarity-determining region (HCDR3) of this antibody fragment bound to Abeta1-42)and had neuroprotective potential against Abeta1-42 mediated neurotoxicity in rat hippocampal cultured neurons. In the present study, using novel computational methods and in vitro experiments we demonstrated that b4.4 binds to the central region of Abeta1-42. We also demonstrated that this scFv antibody binds to Abeta-derived diffusible ligands (ADDLs) and neutralizes the toxicity of both fibrillar and oligomeric forms of Abeta1-42 tested in vitro in SH-SY5Y cell cultures.

Novel amyloid precursor protein transgenic mice, which contain the Swedish as well as the vasculotropic Dutch and Iowa mutations (Tg-SwDI), were used to investigate the mechanisms of antibody-mediated clearance of amyloid-β (Aβ) from the brain. Export of the Aβ-DI peptide across the blood–brain barrier is severely reduced because of the vasculotropic mutations. Therefore, antibody-mediated clearance of Aβ-DI is dependent on antibodies entering the brain. In this report, we immunized Tg-SwDI mice with various peptide antigens, including Aβ40-DI, Aβ42, and an Aβ epitope vaccine. Immunization of Tg-SwDI mice with substantial cortical diffuse and vascular fibrillar deposits failed to promote clearance of parenchymal or vascular amyloid deposits. We then immunized young Tg-SwDI mice before the accumulation of Aβ and saw no evidence that anti-Aβ antibodies could diminish deposition of parenchymal or vascular amyloid deposits. However, injection of anti-Aβ antibodies, affinity-purified from immunized Tg-SwDI mice, into the hippocampus induced a rapid clearance of diffuse Aβ deposits but not vascular amyloid deposits. These results further support the “peripheral sink hypothesis” as a legitimate mechanism of antibody-mediated clearance of Aβ when the blood–brain barrier remains intact. Thus, approaches that deliver immunotherapy to the brain may be more effective at clearing Aβ than immunization strategies in which the majority of the antibodies are in the periphery.

Cerebral amyloid angiopathy (CAA) is a neurovascular disease that is strongly associated with an increase in the number and size of spontaneous microbleeds. Conventional methods of magnetic resonance imaging for detection of microbleeds, and positron emission tomography with Pittsburgh Compound B imaging for amyloid deposits, can separately demonstrate the presence of microbleeds and CAA in affected brains in vivo; however, there still is a critical need for strong evidence that shows involvement of CAA in microbleed formation. Here, we show in a Tg2576 mouse model of Alzheimer's disease, that the combination of histochemical staining and an optical clearing method called optical histology, enables simultaneous, co-registered three-dimensional visualization of cerebral microvasculature, microbleeds, and amyloid deposits. Our data suggest that microbleeds are localized within the brain regions affected by vascular amyloid deposits. All observed microhemorrhages (n = 39) were in close proximity (0 to 144 µm) with vessels affected by CAA. Our data suggest that the predominant type of CAA-related microbleed is associated with leaky or ruptured hemorrhagic microvasculature. The proposed methodological and instrumental approach will allow future study of the relationship between CAA and microbleeds during disease development and in response to treatment strategies.

Introduction: Cerebral microhemorrhages (CMH) are tiny deposits of blood degradation products in the brain and are the pathological substrate of cerebral microbleeds (MB). The existing animal models of MB are either β-amyloid-, anti-amyloid antibody-, or hypertension-induced; however, MB may develop independent of hypertension or amyloid deposition. In fact, MB are associated with normal aging, sepsis and neurodegenerative conditions. One common factor among the above pathologies is inflammation, and recent studies show a link between systemic inflammation and MB. Hence, we hypothesize that systemic inflammation (induced by lipopolysaccharide; LPS) will result in MB development, and an inflammation-induced animal model will be appropriate to study MB. In the current study we used two different dosing regimens for LPS and observed rapid and robust development of CMH and MB. Methods: Adult C57BL/6 mice were injected with LPS (1mg/kg or 3mg/kg, i.p.) or saline at 0, 6, and 24h. At 2 or 7 days after first LPS injection, brains were harvested. Hematoxylin and eosin (H&E) and Prussian blue (PB) were used to stain fresh (acute) hemorrhages and hemosiderin (subacute hemorrhages), respectively. Results: No surface and negligible H&E-positive CMH were observed in saline controls (n=4). LPS (3mg/kg; n=13) resulted in significantly higher number (p Conclusion: In summary, LPS produced rapid development of H&E-positive CMH (at 2 and 7 days) and PB-positive CMH (at 7 days) with low mortality. Thus, this appears to be an appropriate model to study the pathophysiology of CMH and MB.

Hypertension and cerebral amyloid angiopathy (CAA) are major risk factors for intracerebral hemorrhage (ICH); however the mechanisms of interplay between the two are not fully understood. We investigated the effect of hypertension in a transgenic mouse model with Alzheimer’s-like pathology (Tg2576) treating them with angiontensin II and L-NG-nitroarginine methyl ester. A similar increase in systolic blood pressure was observed in both Tg2576 and control mice; however Tg2576 mice developed signs of stroke with a markedly shorter latency. Cerebral deposition of amyloid beta promotes the hypertension-induced ICH, thus supporting the notion that hypertension is a risk factor for ICH among patients with CAA.

Increasing evidence points to soluble assemblies of aggregating proteins as a major mediator of neuronal and synaptic dysfunction. In Alzheimer disease (AD), soluble amyloid-beta (Abeta) appears to be a key factor in inducing synaptic and cognitive abnormalities. Here we report the novel finding that soluble tau also plays a role in the cognitive decline in the presence of concomitant Abeta pathology. We describe improved cognitive function following a reduction in both soluble Abeta and tau levels after active or passive immunization in advanced aged 3xTg-AD mice that contain both amyloid plaques and neurofibrillary tangles (NFTs). Notably, reducing soluble Abeta alone did not improve the cognitive phenotype in mice with plaques and NFTs. Our results show that Abeta immunotherapy reduces soluble tau and ameliorates behavioral deficit in old transgenic mice.

We describe a study testing fibrillar beta-amyloid(1-42) (Abeta42) vaccination in dogs. Three young beagles (4.6 years) were immunized twice with Abeta42 and a Th1 adjuvant (TiterMax Gold). Animals generated primarily IgG2 and IgM antibody responses, which were specific for the Abeta(11-30) region of Abeta(1-42). Next, 3 aged beagles (8.9-13.8 years) were immunized 4 times with Abeta(42) and a Th2 adjuvant (Alum). We observed an acute increase in IgG2, a slower increase in IgG1 and Abeta antibodies of broader specificity (Abeta(1-15>) Abeta(11-30>) Abeta(6-20)). A nonsignificant increase in CSF Abeta(1-40) and decrease in Abeta(1-40/1-42) in cortex was detected. Canines may be a useful system for testing an Abeta vaccine.

Immunization of amyloid precursor protein transgenic mice with fibrillar β-amyloid (Aβ) prevents Alzheimer’s disease (AD)-like neuropathology. The first immunotherapy clinical trial used fibrillar Aβ, containing the B and T cell self epitopes of Aβ, as the immunogen formulated with QS21 as the adjuvant in the vaccine. Unfortunately, the clinical trial was halted during the phase II stage when 6% of the participants developed meningoencephalitis. The cause of the meningoencephalitis in the patients that received the vaccine has not been definitively determined; however, analysis of two case reports from the AN-1792 vaccine trial suggest that the meningoencephalitis may have been caused by a T cell-mediated autoimmune response, whereas production of anti-Aβ Abs may have been therapeutic to the AD patients. Therefore, to reduce the risk of an adverse T cell-mediated immune response to Aβ immunotherapy we have designed a prototype epitope vaccine that contains the immunodominant B cell epitope of Aβ in tandem with the synthetic universal Th cell pan HLA DR epitope, pan HLA DR-binding peptide (PADRE). Importantly, the PADRE-Aβ1–15 sequence lacks the T cell epitope of Aβ. Immunization of BALB/c mice with the PADRE-Aβ1–15 epitope vaccine produced high titers of anti-Aβ Abs. Splenocytes from immunized mice showed robust T cell stimulation in response to peptides containing PADRE. However, splenocytes from immunized mice were not reactivated by the Aβ peptide. New preclinical trials in amyloid precursor protein transgenic mouse models may help to develop novel immunogen-adjuvant configurations with the potential to avoid the adverse events that occurred in the first clinical trial.

Active immunization with fibrillar beta-amyloid peptide (Abeta(42)) as well as passive transfer of anti-Abeta antibodies significantly reduces Abeta plaque deposition, neuritic dystrophy, and astrogliosis in the brain of mutant amyloid precursor protein (APP)-transgenic mice. Although the mechanism(s) of clearance of Abeta from the brain following active or passive immunization remains to be determined, it is clear that anti-Abeta antibodies are critical for clearance. DNA immunization provides an attractive alternative to direct peptide and adjuvant approaches for inducing a humoral response to Abeta. We constructed a DNA minigene with Abeta fused to mouse interleukin-4 (pAbeta(42)-IL-4) as a molecular adjuvant to generate anti-Abeta antibodies and enhance the Th2-type of immune responses. Gene gun immunizations induced primarily IgG1 and IgG2b anti-Abeta antibodies. Fine epitope analysis with overlapping peptides of the Abeta(42) sequence identified the 1-15 region as a dominant B cell epitope. The DNA minigene-induced anti-Abeta antibodies bound to Abeta plaques in brain tissue from an Alzheimer's disease patient demonstrating functional activity of the antibodies and the potential for therapeutic efficacy.

A careful analysis of the immune response to immunization of amyloid precursor protein/transgenic (APP/Tg) mice with beta-amyloid (Abeta) may provide insights into why a subset of the patients in a clinical trial receiving Abeta-immunotherapy developed encephalomyelitis. Characterization of isotypic immune responses have been reported in different APP/Tg models. In these studies the relative ratios of IgG1 to IgG2a anti-Abeta antibodies has been used as an indirect measure of T helper 1 (Th1) and Th2 types immune responses. However, it has previously been shown that certain strains of mice, C57Bl/6, C57Bl/10, SJL, and NOD, have an IgG2c rather than an IgG2a gene. Since a substantial number of Abeta-immunization studies rely on APP/Tg mice that have at least one parental C57Bl/6 strain, we have investigated whether antibodies specific for IgG2a can be used for characterization of antibody isotypes in APP/Tg2576 mice. Our results suggest that APP/Tg2576 and major histocompatibilty complex-matched parental strains are not expressing IgG2a, producing instead IgG2c anti-Abeta antibodies.

Human epithelial mucin (MUC1) is expressed by many carcinomas, including breast cancer cells. This breast cancer-associated antigen has been widely used for immunotherapy, despite the fact that cellular immune responses to MUC1 are impaired in breast cancer patients and MUC1 transgenic animals. Previously, we found that immunogenicity to MUC1 was also impaired in BALB/c mice injected with a mammary tumor cell line (410.4) expressing human MUC1. We suggested that one reason for its weak immunogenicity was the lack of expression of B7 molecules by 410.4 cells. Recognition of antigenic epitopes in conjunction with MHCI/II by the T-cell receptor without co-stimulation by B7/CD28 association resulted in T-cell anergy. Therefore, we attempted to enhance protective anti-MUC1-specific immunity in mice using B7 co-stimulatory molecules as a component of the MUC1 vaccine. We also compared cell-based with DNA-based vaccination strategies. One group of mice was vaccinated with an irradiated, 410.4 syngeneic mammary tumor cell line co-expressing human MUC1 and CD80 or CD86 co-stimulatory molecules, and a second group of mice was vaccinated with plasmids encoding MUC1 and CD80 or CD86. These mice along with appropriate controls were challenged with mammary tumor cell line 4T1, which expresses MUC1. There were significant inhibition on rates of tumor growth and survival in mice vaccinated with irradiated 410.4/MUC1 cells co-expressing either CD80 or CD86 molecules, compared to non-vaccinated animals. In addition, there were also significant delays in the appearance of measurable tumors and their growth in mice vaccinated by gene-gun immunization with plasmids encoding MUC1 and CD80 or CD86.

Cerebral microbleeds are microscopic hemorrhages with deposits of blood products in the brain, which can be visualized with MRI and are implicated in cerebrovascular diseases. Hematoxylin and eosin (H&E) and Perl’s Prussian blue are popular staining methods used to localize cerebral microbleeds in pathology. This paper compared these two staining techniques in a mouse model of cerebral microbleeds. We used lipopolysaccharide (LPS) to induce cerebral microhemorrhages. C57B6 mice were treated with LPS (5 mg/kg, i.p.) or vehicle at baseline and at 24 hr. The brains were extracted 48 hr after the first injection and adjacent coronal sections were stained with H&E and Prussian blue to compare the effectiveness of the two staining techniques. H&E-positive stains were increased with LPS treatment and were correlated with grossly visible microhemorrhages on the brain surface; Prussian blue-positive stains, by comparison, showed no significant increase with LPS treatment and did not correlate with either H&E-positive stains or surface microhemorrhages. H&E staining is thus a more reliable indicator of acute bleeding events induced by LPS in this model within a short time span.

Alzheimer's disease (AD) is a proteinopathy characterized by the accumulation of β-amyloid (Aβ) and tau. To date, clinical trials indicate that Aβ immunotherapy does not improve cognition. Consequently, it is critical to modulate other aspects of AD pathology. As such, tau represents an excellent target, as its accumulation better correlates with cognitive impairment. To determine the effectiveness of targeting pathological tau, with Aβ pathology present, we administered a single injection of AT8, or control antibody, into the hippocampus of aged 3xTg-AD mice. Extensive data indicates that phosphorylated Ser202 and Thr205 sites of tau (corresponding to the AT8 epitope) represent a pathologically relevant target for AD. We report that immunization with AT8 reduced somatodendritic tau load, p-tau immunoreactivity, and silver stained positive neurons, without affecting Aβ pathology. We also discovered that tau pathology soon reemerges post-injection, possibly due to persistent Aβ pathology. These studies provide evidence that targeting p-tau may represent an effective treatment strategy: potentially in conjunction with Aβ immunotherapy.

Introduction: Cerebral microscopic hemorrhages are the pathologic substrate of cerebral microbleeds (CMB). Little is known about mechanisms of expansion of CMB. Here we studied the dynamics of CMB after acute inflammation, using a transgenic mouse model of cerebral amyloid angiopathy with spontaneous microscopic hemorrhages and endotoxin (lipopolysaccharide, LPS) as an inflammatory stimulus. Methods: 21-month old Tg2576 mice and wildtype littermates received a single dose of LPS, 100 or 1000 ug/kg i.p., or phosphate-buffered saline (PBS) as control. Mice were sacrificed 48 hours later and brains were collected for the analysis of microscopic hemorrhages, as well as edema formation, blood-brain barrier constituents and glial activation. Differences among groups were tested by anova and contrasts. Results: Hematoxylin and eosin staining demonstrated no freshly-formed microscopic hemorrhages in LPS-treated mice. Using Prussian blue staining, we found that total area, average size, and number of CMB differed significantly between PBS-treated wildtype and transgenic mice (p=0.03, p=.04 and p=.03 respectively). Acute inflammation did not significantly affect total area, average size, or number of CMB in wildtype animals. For transgenic mice, both doses of LPS significantly (p Conclusions: In a mouse model of cerebral amyloid angiopathy, acute inflammatory stimulus induced expansion of CMB without producing fresh hemorrhage. Increased size of CMB was associated with indices of cerebral edema, alteration of tight junction constituents, and microglial activation. These findings suggest that cerebral edema may mediate expansion of cerebral microbleeds.

The main amyloid-β peptide (Aβ) variants detected in the human brain are Aβ1-40 and Aβ1-42; however, a significant proportion of Aβ in Alzheimer's disease (AD) brain also consists of N-terminal truncated/modified species. AβN3(pE), Aβ peptide bearing amino-terminal pyroglutamate at position 3, has been demonstrated to be a major N-truncated/modified constituent of intracellular, extracellular, and vascular Aβ deposits in AD and Down syndrome brain tissue. It has been previously demonstrated that rabbits fed a diet enriched in cholesterol and given water containing trace copper levels developed AD-like pathology including intraneuronal and extracellular Aβ accumulation, tau hyperphosphorylation, vascular inflammation, astrocytosis, microgliosis, reduced levels of acetylcholine, as well as learning deficits and thus, may be used as a non-transgenic animal model of sporadic AD. In the present study, we have demonstrated for the first time the presence of AβN3(pE) in blood vessels in cholesterol-enriched diet-fed rabbit brain. In addition, we detected AβN3(pE) immunoreactivity in all postmortem AD brain samples studied. We believe that our results are potentially important for evaluation of novel therapeutic molecules/strategies targeting Aβ peptides in a suitable non-transgenic animal model.

Alzheimer's disease (AD) is hallmarked by amyloid plaques, neurofibrillary tangles, and widespread cortical neuronal loss (Selkoe, 2001). The “amyloid cascade hypothesis” posits that cerebral amyloid sets neurotoxic events into motion that precipitate Alzheimer dementia (Hardy and Allsop, 1991). Yet, faithful recapitulation of all AD features in widely used transgenic (Tg) mice engineered to overproduce Aβ peptides has been elusive. We have developed a Tg rat model (line TgF344-AD) expressing mutant human amyloid precursor protein (APPsw) and presenilin 1 (PS1ΔE9) genes, each independent causes of early-onset familial AD. TgF344-AD rats manifest age-dependent cerebral amyloidosis that precedes tauopathy, gliosis, apoptotic loss of neurons in the cerebral cortex and hippocampus, and cognitive disturbance. These results demonstrate progressive neurodegeneration of the Alzheimer type in these animals. The TgF344-AD rat fills a critical need for a next-generation animal model to enable basic and translational AD research.

Cerebral microbleeds are microscopic hemorrhages with deposits of blood products in the brain that can be visualized with MRI. Microbleeds are increased with age and in diseases such as cerebral amyloid angiopathy, hypertension, and stroke. There are few studies of either a) pathological correlates of cerebral microbleeds or b) imaging microscopic hemorrhage in brain tissue. The existing animal models of cerebral microbleeds involve transgenic mice (e.g. Tg2576) that spontaneously over-express β-amyloid, mimicking cerebral amyloid angiopathy. However, these models have limited use due to the fact that a) microbleeds may develop independent of amyloid deposition and b) the transgenic animals may require two years to fully develop microbleeds. In the current study, we used lipopolysaccharide (LPS) to induce cerebral microscopic hemorrhages over the course of several days. In this model, adult C57B6 mice (3 months of age) were injected with LPS (5mg/kg ip) or vehicle (phosphate buffered saline) at baseline and at 24 hr, and animals were sacrificed two days after initial injection. Mouse brains were analyzed for fresh hemorrhage using hematoxylin and eosin (H&E) staining, iron deposition using Prussian blue staining, and blood-brain barrier (BBB) permeability by measuring sodium flouorescein (NaF) content. H&E staining revealed small fresh hemorrhages visible grossly and microscopically in all animals treated with LPS, but not in control animals. Prussian blue staining revealed markedly increased iron deposition with LPS treatment, with positive stains in cerebral cortex, midbrain, and hippocampus regions. Quantification of Prussian blue staining revealed that LPS induced more than four-fold increase in number of positive stained foci (p

Hypertension is a major risk factor for intracerebral hemorrhage (ICH), and the accumulation of amyloid-beta (Aβ) in the cerebrovascular system, cerebral amyloid angiopathy (CAA), is also a significant risk factor for intracerebral hemorrhage ICH. Currently, there are no animal studies demonstrating a direct involvement of hypertension in the accumulation of Alzheimer’s disease-like pathology. To address this issue we have developed several mouse models that combine hypertension protocols with amyloid precursor protein (APP) transgenic mice (Tg2576), which accumulate significant CAA in the large cerebral vessels and the meninges by 18 months of age. The goal of this study was to determine the effect of acute and chronic hypertension on ICH in wildtype and a transgenic mouse model overexpressing a mutant human amyloid precursor protein (Tg2576 mice) associated with early onset AD and CAA. Fifteen-month-old Tg2576 mice and non-transgenic (nTg) littermates were treated with an angiotensin II (AngII) infusion (1000 ng/kg/min) and L-NAME (100 mg/kg/day) in drinking water to produce chronic hypertension. One week later, transient acute hypertension was induced by daily AngII injections (0.5 μg/g, s.c., twice daily) to produce ICH. A similar increase in mean blood pressure was observed in Tg2576 and nTg mice when evaluated 2 weeks after initiation of treatment. However Tg2576 mice had a higher incidence of signs of stroke compared with nTg littermates (P > 0.05). These data suggest that the accumulation of Aβ in the brain has an important role in development of ICH. Moreover, there was robust glial activation and increase in CAA in the gray matter of Tg2576 mice showing that hypertension may affect gray as well as white matter in the brain. Further studies may provide insights into the hypertension-induced changes in the cerebral vascular system that initiated the increase in CAA. The accumulation of Aβ in the cerebrovascular system is a significant risk factor for intracerebral hemorrhage (ICH), and has been linked to endothelial transport failure and blockage of perivascular drainage. While management of hypertension and atherosclerosis can reduce the incidence of ICH, there are currently no approved therapies for attenuating CAA.

Author(s): Lo, Patrick; Crouzet, Christian; Vasilevko, Vitaly; Choi, Bernard | Abstract: The authors regret omission of an acknowledgment of funding provided by National Institutes of Health grant R01 NS020989. The authors would like to apologize for any inconvenience caused.

Inflammation is a key pathological hallmark of Alzheimer’s disease (AD), although its impact on disease progression and neurodegeneration remains an area of active investigation. Among numerous inflammatory cytokines associated with AD, IL-1β in particular has been implicated in playing a pathogenic role. In this study, we sought to investigate whether inhibition of IL-1β signaling provides disease-modifying benefits in an AD mouse model and, if so, by what molecular mechanisms. We report that chronic dosing of 3xTg-AD mice with an IL-1R blocking Ab significantly alters brain inflammatory responses, alleviates cognitive deficits, markedly attenuates tau pathology, and partly reduces certain fibrillar and oligomeric forms of amyloid-β. Alterations in inflammatory responses correspond to reduced NF-κB activity. Furthermore, inhibition of IL-1 signaling reduces the activity of several tau kinases in the brain, including cdk5/p25, GSK-3β, and p38–MAPK, and also reduces phosphorylated tau levels. We also detected a reduction in the astrocyte-derived cytokine, S100B, and in the extent of neuronal Wnt/β-catenin signaling in 3xTg-AD brains, and provided in vitro evidence that these changes may, in part, provide a mechanistic link between IL-1 signaling and GSK-3β activation. Taken together, our results suggest that the IL-1 signaling cascade may be involved in one of the key disease mechanisms for AD.