Tiffany L. Sudduth

Neuroinflammatory phenotype in early Alzheimer’s disease

Alzheimers disease (AD) involves progressive neurodegeneration in the presence of misfolded proteins and poorly-understood inflammatory changes. However, research has shown that AD is genetically, clinically, and pathologically heterogeneous. In frozen brain samples of frontal cortex (diseased) and cerebellum (nondiseased) from the University of Kentucky Alzheimers Disease Center autopsy cohort, we performed gene expression analysis for genes categorizing inflammatory states (termed M1 and M2) from early and late stage AD, and age-matched nondemented controls. We performed analysis of the serum samples for a profile of inflammatory proteins and examined the neuropathologic data on these samples. Striking heterogeneity was found in early AD. Specifically, early-stage AD brain samples indicated apparent polarization toward either the M1 or M2 brain inflammatory states when compared with age-matched nondisease control tissue. This polarization was observed in the frontal cortex and not in cerebellar tissue. We were able to detect differences in AD neuropathology, and changes in serum proteins that distinguished the individuals with apparent M1 versus M2 brain inflammatory polarization.

Induction of hyperhomocysteinemia models vascular dementia by induction of cerebral microhemorrhages and neuroinflammation.

Vascular dementia (VaD) is the second leading cause of dementia behind Alzheimers disease (AD) and is a frequent comorbidity with AD, estimated to occur in as many as 40% of AD patients. The causes of VaD are varied and include chronic cerebral hypoperfusion, microhemorrhages, hemorrhagic infarcts, or ischemic infarcts. We have developed a model of VaD by inducing hyperhomocysteinemia (HHcy) in wild-type mice. By placing wild-type mice on a diet deficient in folate, B6, and B12 and supplemented with excess methionine, we induced a moderate HHcy (plasma level homocysteine 82.93 ± 3.561 μmol). After 11 weeks on the diet, the hyperhomocysteinemic mice showed a spatial memory deficit as assessed by the 2-day radial-arm water maze. Also, magnetic resonance imaging and subsequent histology revealed significant microhemorrhage occurrence. We found neuroinflammation induced in the hyperhomocysteinemic mice as determined by elevated interleukin (IL)-1β, tumor necrosis factor (TNF)α, and IL-6 in brain tissue. Finally, we found increased expression and increased activity of the matrix metalloproteinase 2 (MMP2) and MMP9 systems that are heavily implicated in the pathogenesis of cerebral hemorrhage. Overall, we have developed a dietary model of VaD that will be valuable for studying the pathophysiology of VaD and also for studying the comorbidity of VaD with other dementias and other neurodegenerative disorders.

Intracranial Injection of Gammagard, a Human IVIg, Modulates the Inflammatory Response of the Brain and Lowers Aβ in APP/PS1 Mice Along a Different Time Course than Anti-Aβ Antibodies

Gammagard IVIg is a therapeutic approach to treat Alzheimers 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.

Determining the role of IL-4 induced neuroinflammation in microglial activity and amyloid-β using BV2 microglial cells and APP/PS1 transgenic mice.

BackgroundMicroglia 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.MethodsIn 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.ResultsBoth 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.ConclusionsIn summary, this study offers insight into the therapeutic potential of microglial immune response in AD.

Lithium Treatment of APPSwDI/NOS2−/− Mice Leads to Reduced Hyperphosphorylated Tau, Increased Amyloid Deposition and Altered Inflammatory Phenotype

Lithium is an anti-psychotic that has been shown to prevent the hyperphosphorylation of tau protein through the inhibition of glycogen-synthase kinase 3-beta (GSK3β). We recently developed a mouse model that progresses from amyloid pathology to tau pathology and neurodegeneration due to the genetic deletion of NOS2 in an APP transgenic mouse; the APPSwDI/NOS2−/− mouse. Because this mouse develops tau pathology, amyloid pathology and neuronal loss we were interested in the effect anti-tau therapy would have on amyloid pathology, learning and memory. We administered lithium in the diets of APPSwDI/NOS2−/− mice for a period of eight months, followed by water maze testing at 12 months of age, immediately prior to sacrifice. We found that lithium significantly lowered hyperphosphorylated tau levels as measured by Western blot and immunocytochemistry. However, we found no apparent neuroprotection, no effect on spatial memory deficits and an increase in histological amyloid deposition. Aβ levels measured biochemically were unaltered. We also found that lithium significantly altered the neuroinflammatory phenotype of the brain, resulting in enhanced alternative inflammatory response while concurrently lowering the classical inflammatory response. Our data suggest that lithium may be beneficial for the treatment of tauopathies but may not be beneficial for the treatment of Alzheimers disease.

β-amyloid deposition is shifted to the vasculature and memory impairment is exacerbated when hyperhomocysteinemia is induced in APP/PS1 transgenic mice

IntroductionVascular dementia is the second most common cause of dementia after Alzheimer’s 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.MethodsWe 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.ResultsWhile 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 6xa0months 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.ConclusionsThese 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.

Transition from an M1 to a mixed neuroinflammatory phenotype increases amyloid deposition in APP/PS1 transgenic mice

BackgroundThe polarization to different neuroinflammatory phenotypes has been described in early Alzheimer’s 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.MethodsWild-type and APP/PS1 transgenic mice aged 3 to 4xa0months 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 6xa0months 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.ResultsAAV expressing IFNγ induced an M1 neuroinflammatory phenotype at 4xa0months and a mixed phenotype along with an increase in Aβ at 6xa0months. Microglial staining was increased at 6xa0months and astrocyte staining was decreased at 4 and 6xa0months in mice receiving AAV expressing IFNγ.ConclusionsExpression of IFNγ through AAV successfully induced an M1 phenotype at 4xa0months that transitioned to a mixed phenotype by 6xa0months. 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.

Down syndrome individuals with Alzheimer's disease have a distinct neuroinflammatory phenotype compared to sporadic Alzheimer's disease.

Down syndrome (DS) is the most common genetic cause of intellectual disability and is primarily caused by the triplication of chromosome 21. The overexpression of amyloid precursor protein gene may be sufficient to drive Alzheimers disease (AD) neuropathology that is observed in virtually all individuals with DS by the age of 40 years. There is relatively little information about inflammation in the DS brain and how the genetics of DS may alter inflammatory responses and modify the course of AD pathogenesis in this disorder. Using the macrophage classification system of M1, M2a, M2b, and M2c inflammatory phenotypes, we have shown that the early stages of AD are associated with a bias toward an M1 or M2a phenotype. In later stages of AD, markers of M1, M2a and M2c are elevated. We now report the inflammatory phenotype in a DS autopsy series to compare this with the progression in sporadic AD. Tissue from young DS cases (under 40 years of age, pre-AD) show a bias toward M1 and M2b states with little M2a or M2c observed. Older DS cases (over 40 with AD pathology) show a distinct bias toward an M2b phenotype. Importantly, this is distinct from sporadic AD where the M2b phenotype has been rarely, if ever observed in postmortem studies. Stimulated by immune complex activation of microglial cells and toll-like receptor activation, the M2b phenotype represents a unique neuroinflammatory state in diseased brain and may have significant implications for therapeutic intervention for persons with DS.

Reduced Efficacy of Anti-Aβ 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 Alzheimers 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. SIGNIFICANCE STATEMENT Despite significant mouse model data demonstrating both pathological and cognitive efficacy of anti-Aβ immunotherapy for the treatment of Alzheimers disease, clinical trial outcomes have been underwhelming, failing to meet any primary endpoints. We show here that vascular cognitive impairment and dementia (VCID) comorbidity eliminates cognitive efficacy of anti-Aβ immunotherapy, despite amyloid clearance. Further, cerebrovascular adverse events of the anti-Aβ immunotherapy are significantly exacerbated by the VCID comorbidity. These data suggest that VCID comorbidity with Alzheimers disease may mute the response to anti-Aβ immunotherapy.

Time-course of glial changes 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 Alzheimers 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 hyperhomocysteinemia (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.