Intracranial Injection of Gammagard, a Human IVIg, Modulates the Inflammatory Response of the Brain and Lowers A\u3cem\u3eβ\u3c/em\u3e in APP/PS1 Mice Along a Different Time Course than Anti-A\u3cem\u3eβ\u3c/em\u3e Antibodies

Gammagard IVIg is a therapeutic approach to treat Alzheimer\u27s disease currently in phase 3 clinical trials. Despite the reported efficacy of the approach the mechanism of action is poorly understood. We have previously shown that intracranial injection of anti-Aβ antibodies into the frontal cortex and hippocampus reveals important information regarding the time course of events once the agent is in the brain. In the current study we compared IVIg, mouse-pooled IgG, and the anti-Aβ antibody 6E10 injected intracranially into the frontal cortex and hippocampus of 7-month-old APP/PS1 mice. We established a time course of events ranging from 1 to 21 d postinjection. IVIg and pooled mouse IgG both significantly reduced Aβ deposition to the same degree as the 6E10 anti-Aβ antibody; however, the clearance was much slower to occur, happening between the 3 and 7 d time points. In contrast, as we have previously shown, Aβ reductions were apparent with the 6E10 anti-Aβ group at the 1 d time point. Also, neuroinflammatory profiles were significantly altered by the antibody treatments. APP/PS1 transgenic mice at 7 months of age typically exhibit an M2a inflammatory phenotype. All antibody treatments stimulated an M2b response, yet anti-Aβ antibody was a more rapid change. Because the neuroinflammatory switch occurs before the detectable reductions in amyloid deposition, we hypothesize that the IVIg and pooled mouse IgG act as immune modulators and this immune modulation is responsible for the reductions in amyloid pathology

Hyperhomocysteinemia-Induced Gene Expression Changes in the Cell Types of the Brain

High plasma levels of homocysteine, termed hyperhomocysteinemia, are a risk factor for vascular cognitive impairment and dementia, which is the second leading cause of dementia. While hyperhomocysteinemia induces microhemorrhages and cognitive decline in mice, the specific effect of hyperhomocysteinemia on each cell type remains unknown. We took separate cultures of astrocytes, microglia, endothelial cells, and neuronal cells and treated each with moderate levels of homocysteine for 24, 48, 72, and 96 hr. We then determined the gene expression changes for cell-specific markers and neuroinflammatory markers including the matrix metalloproteinase 9 system. Astrocytes had decreased levels of several astrocytic end feet genes, such as aquaporin 4 and an adenosine triphosphate (ATP)-sensitive inward rectifier potassium channel at 72 hr, as well as an increase in matrix metalloproteinase 9 at 48 hr. Gene changes in microglia indicated a peak in proinflammatory markers at 48 hr followed by a peak in the anti-inflammatory marker, interleukin 1 receptor antagonist, at 72 hr. Endothelial cells had reduced occludin expression at 72 hr, while kinases and phosphatases known to alter tau phosphorylation states were increased in neuronal cells. This suggests that hyperhomocysteinemia induces early proinflammatory changes in microglia and astrocytic changes relevant to their interaction with the vasculature. Overall, the data show how hyperhomocysteinemia could impact Alzheimer’s disease and vascular cognitive impairment and dementia

β-Amyloid Deposition is Shifted to the Vasculature and Memory Impairment is Exacerbated When Hyperhomocysteinemia is Induced in APP/PS1 Transgenic Mice

INTRODUCTION: Vascular dementia is the second most common cause of dementia after Alzheimer\u27s disease (AD). In addition, it is estimated that almost half of all AD patients have significant cerebrovascular disease comorbid with their AD pathology. We hypothesized that cerebrovascular disease significantly impacts AD pathological progression. METHODS: We used a dietary model of cerebrovascular disease that relies on the induction of hyperhomocysteinemia (HHcy). HHcy is a significant clinical risk factor for stroke, cardiovascular disease and type 2 diabetes. In the present study, we induced HHcy in APP/PS1 transgenic mice. RESULTS: While total β-amyloid (Aβ) load is unchanged across groups, Congophilic amyloid deposition was decreased in the parenchyma and significantly increased in the vasculature as cerebral amyloid angiopathy (CAA; vascular amyloid deposition) in HHcy APP/PS1 mice. We also found that HHcy induced more microhemorrhages in the APP/PS1 mice than in the wild-type mice and that it switched the neuroinflammatory phenotype from an M2a biased state to an M1 biased state. Associated with these changes was an induction of the matrix metalloproteinase protein 2 (MMP2) and MMP9 systems. Interestingly, after 6 months of HHcy, the APP/PS1 mice were cognitively worse than wild-type HHcy mice or APP/PS1 mice, indicative of an additive effect of the cerebrovascular pathology and amyloid deposition. CONCLUSIONS: These data show that cerebrovascular disease can significantly impact Aβ distribution in the brain, favoring vascular deposition. We predict that the presence of cerebrovascular disease with AD will have a significant impact on AD progression and the efficacy of therapeutics

Neurovascular Astrocyte Degeneration in the Hyperhomocysteinemia Model of Vascular Cognitive Impairment and Dementia (VCID)

Vascular cognitive impairment and dementia (VCID) is the second leading cause of dementia behind Alzheimer’s disease (AD) and is a frequent co-morbidity with AD. Despite its prevalence, little is known about the molecular mechanisms underlying the cognitive dysfunction resulting from cerebrovascular disease. Astrocytic end-feet almost completely surround intraparenchymal blood vessels in the brain and express a variety of channels and markers indicative of their specialized functions in the maintenance of ionic and osmotic homeostasis and gliovascular signaling. These functions are mediated by end-foot enrichment of the aquaporin 4 water channel (AQP4), the inward rectifying potassium channel Kir4.1 and the calcium-dependent potassium channel MaxiK. Using our HHcy model of VCID we examined the time-course of astrocytic end-foot changes along with cognitive and neuroinflammatory outcomes. We found that there were significant astrocytic end-foot disruptions in the HHcy model. AQP4 becomes dislocalized from the end-feet, there is a loss of Kir4.1 and MaxiK protein expression, as well as a loss of the Dp71 protein known to anchor the Kir4.1, MaxiK and AQP4 channels to the end-foot membrane. Neuroinflammation occurs prior to the astrocytic changes, while cognitive impairment continues to decline with the exacerbation of the astrocytic changes. We have previously reported similar astrocytic changes in models of cerebral amyloid angiopathy (CAA) and therefore, we believe astrocytic end-foot disruption could represent a common cellular mechanism of VCID and may be a target for therapeutic development

Therapeutic Trem2 Activation Ameliorates Amyloid-Beta Deposition and Improves Cognition in the 5XFAD Model of Amyloid Deposition

BACKGROUND: Triggering receptor expressed on myeloid cell-2 (TREM2) is a lipid and lipoprotein binding receptor expressed by cells of myeloid origin. Homozygous TREM2 mutations cause early onset progressive presenile dementia while heterozygous, point mutations triple the risk of Alzheimer\u27s disease (AD). Although human genetic findings support the notion that loss of TREM2 function exacerbates neurodegeneration, it is not clear whether activation of TREM2 in a disease state would result in therapeutic benefits. To determine the viability of TREM2 activation as a therapeutic strategy, we sought to characterize an agonistic Trem2 antibody (AL002a) and test its efficacy and mechanism of action in an aggressive mouse model of amyloid deposition. METHODS: To determine whether agonism of Trem2 results in therapeutic benefits, we designed both intracranial and systemic administration studies. 5XFAD mice in the intracranial administration study were assigned to one of two injection groups: AL002a, a Trem2-agonizing antibody, or MOPC, an isotype-matched control antibody. Mice were then subject to a single bilateral intracranial injection into the frontal cortex and hippocampus and euthanized 72 h later. The tissue from the left hemisphere was histologically examined for amyloid-beta and microglia activation, whereas the tissue from the right hemisphere was used for biochemical analyses. Similarly, mice in the systemic administration study were randomized to one of the aforementioned injection groups and the assigned antibody was administered intraperitoneally once a week for 14 weeks. Mice underwent behavioral assessment between the 12- and 14-week timepoints and were euthanized 24 h after their final injection. The tissue from the left hemisphere was used for histological analyses whereas the tissue from the right hemisphere was used for biochemical analyses. RESULTS: Here, we show that chronic activation of Trem2, in the 5XFAD mouse model of amyloid deposition, leads to reversal of the amyloid-associated gene expression signature, recruitment of microglia to plaques, decreased amyloid deposition, and improvement in spatial learning and novel object recognition memory. CONCLUSIONS: These findings indicate that Trem2 activators may be effective for the treatment of AD and possibly other neurodegenerative disorders

Reduced Efficacy of Anti-A\u3cem\u3eβ\u3c/em\u3e Immunotherapy in a Mouse Model of Amyloid Deposition and Vascular Cognitive Impairment Comorbidity

Vascular cognitive impairment and dementia (VCID) is the second most common form of dementia behind Alzheimer\u27s disease (AD). It is estimated that 40% of AD patients also have some form of VCID. One promising therapeutic for AD is anti-Aβ immunotherapy, which uses antibodies against Aβ to clear it from the brain. While successful in clearing Aβ and improving cognition in mice, anti-Aβ immunotherapy failed to reach primary cognitive outcomes in several different clinical trials. We hypothesized that one potential reason the anti-Aβ immunotherapy clinical trials were unsuccessful was due to this high percentage of VCID comorbidity in the AD population. We used our unique model of VCID-amyloid comorbidity to test this hypothesis. We placed 9-month-old wild-type and APP/PS1 mice on either a control diet or a diet that induces hyperhomocysteinemia (HHcy). After being placed on the diet for 3 months, the mice then received intraperotineal injections of either IgG2a control or 3D6 for another 3 months. While we found that treatment of our comorbidity model with 3D6 resulted in decreased total Aβ levels, there was no cognitive benefit of the anti-Aβ immunotherapy in our AD/VCID mice. Further, microhemorrhages were increased by 3D6 in the APP/PS1/control but further increased in an additive fashion when 3D6 was administered to the APP/PS1/HHcy mice. This suggests that the use of anti-Aβ immunotherapy in patients with both AD and VCID would be ineffective on cognitive outcomes

Water Exchange Rate across the Blood-Brain Barrier Is Associated with CSF Amyloid-β 42 in Healthy Older Adults

INTRODUCTION: We tested if water exchange across the blood-brain barrier (BBB), estimated with a noninvasive magnetic resonance imaging (MRI) technique, is associated with cerebrospinal fluid (CSF) biomarkers of Alzheimer\u27s disease (AD) and neuropsychological function. METHODS: Forty cognitively normal older adults (67–86 years old) were scanned with diffusion‐prepared, arterial spin labeling (DP‐ASL), which estimates water exchange rate across the BBB (kw). Participants also underwent CSF draw and neuropsychological testing. Multiple linear regression models were run with kw as a predictor of CSF concentrations and neuropsychological scores. RESULTS: In multiple brain regions, BBB kw was positively associated with CSF amyloid beta (Aβ)42 concentration levels. BBB kw was only moderately associated with neuropsychological performance. DISCUSSION: Our results suggest that low water exchange rate across the BBB is associated with low CSF Aβ42 concentration. These findings suggest that kw may be a promising noninvasive indicator of BBB Aβ clearance functions, a possibility which should be further tested in future research

Transition from an M1 to a Mixed Neuroinflammatory Phenotype Increases Amyloid Deposition in APP/PS1 Transgenic Mice

BACKGROUND: The polarization to different neuroinflammatory phenotypes has been described in early Alzheimer\u27s disease, yet the impact of these phenotypes on amyloid-beta (Aβ) pathology remains unknown. Short-term studies show that induction of an M1 neuroinflammatory phenotype reduces Aβ, but long-term studies have not been performed that track the neuroinflammatory phenotype. METHODS: Wild-type and APP/PS1 transgenic mice aged 3 to 4 months received a bilateral intracranial injection of adeno-associated viral (AAV) vectors expressing IFNγ or green fluorescent protein in the frontal cortex and hippocampus. Mice were sacrificed 4 or 6 months post-injection. ELISA measurements were used for IFNγ protein levels and biochemical levels of Aβ. The neuroinflammatory phenotype was determined through quantitative PCR. Microglia, astrocytes, and Aβ levels were assessed with immunohistochemistry. RESULTS: AAV expressing IFNγ induced an M1 neuroinflammatory phenotype at 4 months and a mixed phenotype along with an increase in Aβ at 6 months. Microglial staining was increased at 6 months and astrocyte staining was decreased at 4 and 6 months in mice receiving AAV expressing IFNγ. CONCLUSIONS: Expression of IFNγ through AAV successfully induced an M1 phenotype at 4 months that transitioned to a mixed phenotype by 6 months. This transition also appeared with an increase in amyloid burden suggesting that a mixed phenotype, or enhanced expression of M2a and M2c markers, could contribute to increasing amyloid burden and disease progression

Determining the Role of IL-4 Induced Neuroinflammation in Microglial Activity and Amyloid-ß Using BV2 Microglial Cells and APP/PS1 Transgenic Mice

Background Microglia are considered the resident immune cells of the central nervous system (CNS). In response to harmful stimuli, an inflammatory reaction ensues in which microglia are activated in a sequenced spectrum of pro- and antiinflammatory phenotypes that are akin to the well-characterized polarization states of peripheral macrophages. A “classically” activated M1 phenotype is known to eradicate toxicity. The transition to an “alternatively” activated M2 phenotype encompasses neuroprotection and repair. In recent years, inflammation has been considered an accompanying pathology in response to the accumulation of extracellular amyloid-β (Aβ) in Alzheimer’s disease (AD). This study aimed to drive an M2a-biased immune phenotype with IL-4 in vitro and in vivo and to determine the subsequent effects on microglial activation and Aβ pathology. Methods In vitro, exogenous IL-4 was applied to BV2 microglial cell cultures to evaluate the temporal progression of microglial responses. In vivo, intracranial injections of an adeno-associate-virus (AAV) viral vector were performed to assess long-term expression of IL-4 in the frontal cortex and hippocampus of Aβ-depositing, APP/PS1 transgenic mice. Quantitative real-time PCR was used to assess the fold change in expression of biomarkers representing each of the microglial phenotypes in both the animal tissue and the BV2 cells. ELISAs quantified IL-4 expression and Aβ levels. Histological staining permitted quantification of microglial and astrocytic activity. Results Both in vitro and in vivo models showed an enhanced M2a phenotype, and the in vivo model revealed a trend toward a decreased trend in Aβ deposition. Conclusions In summary, this study offers insight into the therapeutic potential of microglial immune response in AD

Hierarchical Clustering Analyses of Plasma Proteins in Subjects With Cardiovascular Risk Factors Identify Informative Subsets Based on Differential Levels of Angiogenic and Inflammatory Biomarkers

Agglomerative hierarchical clustering analysis (HCA) is a commonly used unsupervised machine learning approach for identifying informative natural clusters of observations. HCA is performed by calculating a pairwise dissimilarity matrix and then clustering similar observations until all observations are grouped within a cluster. Verifying the empirical clusters produced by HCA is complex and not well studied in biomedical applications. Here, we demonstrate the comparability of a novel HCA technique with one that was used in previous biomedical applications while applying both techniques to plasma angiogenic (FGF, FLT, PIGF, Tie-2, VEGF, VEGF-D) and inflammatory (MMP1, MMP3, MMP9, IL8, TNFα) protein data to identify informative subsets of individuals. Study subjects were diagnosed with mild cognitive impairment due to cerebrovascular disease (MCI-CVD). Through comparison of the two HCA techniques, we were able to identify subsets of individuals, based on differences in VEGF (p \u3c 0.001), MMP1 (p \u3c 0.001), and IL8 (p \u3c 0.001) levels. These profiles provide novel insights into angiogenic and inflammatory pathologies that may contribute to VCID