Neuro-Immunology: The Impact of Immune Function on Alzheimer’s Disease

The rapid elimination of dying neurons and nonfunctional synapses in the brain is carried out by microglia, the resident myeloid cells of the brain. We found that microglia clearance activity in the adult brain is regionally regulated and depends on the rate of neuronal attrition. Cerebellar, but not striatal or cortical, microglia exhibited high levels of basal clearance activity, which correlated with an elevated degree of cerebellar neuronal attrition. Exposing forebrain microglia to apoptotic cells activated gene-expression programs supporting clearance activity. We show that the polycomb repressive complex 2 (PRC2) epigenetically restricts the expression of genes that support clearance activity in striatal and cortical microglia. Loss of PRC2 leads to aberrant activation of a microglia clearance phenotype, which triggers changes in neuronal morphology and behavior. Our data highlight a key role of epigenetic mechanisms in preventing microglia-induced neuronal alterations that are frequently associated with neurodegenerative and psychiatric diseases.

Recently it has been suggested that the potential microglia phenotypic states form a multidimensional space, rather than a linear spectrum encompassing three phenotypes (resting, M1 and M2), as historically held. To explore microglia phenotype diversity in an unbiased way, we performed high-throughput single cell RNA sequencing of human microglia. By profiling 16,000 CD45+ cells isolated from the cerebral cortices of 6 aged and 8 middle-aged individuals, we have identified several unique subpopulations of microglia. While some smaller subpopulations were demarcated based on mutually exclusive marker combinations, the bulk of the microglia cells belonged to 5 large clusters that fell along gene expression gradients. We have confirmed the existence of these subpopulations in situ. We also investigated the functional significance of these microglia subpopulations by exploring their relationship to traits associated with Alzheimer’s disease and aging. Marker genes of the disease associated microglia (DAM) phenotype, which has been recently described in mouse, did not segregate clearly to one microglia subpopulation, suggesting that fundamental differences exist between mouse and human in terms microglia phenotype diversity. Importantly, additional non-microglial cell populations were also identified that, based on their marker combinations, likely belonged to the T cell, NKT cell and B cell category. These minor non-microglial clusters accounted for less than 1% of the total number of profiled cells and they originated primarily from the aged donor samples, suggesting that their presence in the parenchyma is characteristic of the aging brain.

Coauthors: Vilas Menon, PhD^1,2,3,4, Mariko Taga, PhD^1,2,3,4, Christina Yung, BSc^1,2,3,4, Rani Sarkis, MD⁵, Wassim Elyaman, PhD^1,2,3,4, Julie A. Schneider, MD⁶, David A. Bennett, MD⁶, Elizabeth M. Bradshaw, PhD^1,2,3,4 and Philip L. De Jager, MD, PhD^1,2,3,4.

1 Center for Translational and Computational Neuroimmunology, Columbia University Medical Center, New York, New York, USA

2 Taub Institute for Research on Alzheimer’s disease and the Aging Brain, Columbia University Medical Center, New York, New York, USA

3 Department of Neurology, Columbia University Medical Center, New York, New York, USA

4 Broad Institute, Cambridge, Massachusetts, USA

5 Brigham and Women’s Hospital, Boston, Massachusetts, USA

6 Rush University Medical Center, Chicago, Illinois, USA

Genome-wide association studies have identified and validated several genes, such as CD33, associated with AD susceptibility that directly implicate the innate immune system. CD33 is a sialic acid binding protein expressed on the surface of myeloid cells, and higher CD33 expression levels in the brain have been associated with more advanced cognitive decline and AD. We demonstrated that individuals with the Alzheimer’s disease associated rs3865444^CC risk genotype have increased expression of full-length CD33, the isoform containing the sialic acid binding domain, on the surface of their monocytes compared to those with the rs3865444^AA protective genotype. The risk allele is also associated with diminished internalization of amyloid-β1-42 peptide, accumulation of neuritic amyloid pathology and fibrillar amyloid on in vivo imaging, and increased numbers of human microglia with small, thick processes, and a rounded morphology.  Using a crosslinking strategy combined with mass spectrometry we identified immune cell specific sialic acid-dependent and independent CD33 binding partners. Using proximity ligation assays, we have validated these results in vitro as well as in situ. For one of the binding partners, we confirmed that the CD33 ITIM domain was also involved in the interaction. Using a known inhibitor for the binding partner, we can disrupt the interaction and correct the functional reduction in uptake seen with the risk genotype. In conclusion, we have identified and validated the functional relevance of CD33 binding partners that are specific to the sialic acid binding domain of CD33, the domain that is modulated by the Alzheimer’s disease association.

Recent studies on the pathogenesis of dementia have established a close link between Alzheimer’s disease (AD) and neuroinflammation. This is pertinent especially to the Asian population, where emerging studies have shown high incidences of co-existence between the two. It is postulated that inflammatory processes play significant roles in the pathogenesis of AD. This study investigates the expression of inflammatory cytokines and presence of white matter hyperintensities (WMH) in brain tissues and blood from healthy subjects, MCI and mild AD patients. Differential expressions of the inflammatory markers were seen in the post-mortem grey and white matter brain tissues at different AD stages. For example, mild AD patients had higher WMH scores than the control patients, and had higher expression levels of CRP and IL-6 mRNA in their blood samples, but lower levels of IL-1β and TNF-α, than the Control group. MCI patients also had higher WMH scores than the Control group, but no significant changes in the expression of many inflammatory marker mRNAs examined. We used principal component analysis and hierarchical clustering to demonstrate correlations between the groups in the 15 biomarkers expressions that we examined so far. Positive correlations were seen between WMH scores for all four brain regions scanned in all three groups, and these correlated more weakly with cytokine expression levels in the MCI and mild AD groups than the Control group. Taken together, this study reports an expression profile of inflammatory markers and presence of WMH characteristic to the diagnosis of the individual. It illustrates the potential of using both inflammatory marker expression and WMH scores in distinguishing MCI and mild AD patients from healthy controls. Such characterization of inflammatory biomarkers could aid in pinpointing specific time periods for therapeutic intervention, thereby improving the diagnosis and management of AD.

Increasing evidence supports a significant role of the complement pathway in Alzheimer’s disease (AD). While a great number of studies have implicated its effect in modulating amyloid pathology and associated synaptic dysfunction, the role of complement in tau pathology is much less understood. Here, we show that the deletion of the C3a receptor (C3aR) in the PS19 tau transgenic mice results in a significant reduction of tau pathology and rescue of synaptic and behavioral deficits. Using RNA sequencing and cell-type specific profiling, we demonstrate that C3aR critically controls glial transcription factor network and its inactivation reverses the disease-associated microglia and neurotoxic astrocyte signatures. We further identify the STAT3 signaling pathway as a potent regulator of reactive gliosis and tau pathology downstream of C3aR. Importantly, the elevated C3-C3aR signaling correlates with cognitive decline and Braak score in human AD and regulates the expression of multiple genes linked to the late-onset AD. Together, our studies demonstrate a crucial role for the C3-C3aR pathway in mediating CNS immune homeostasis and tau pathology progression. The inhibition of C3aR and its downstream signaling may be therapeutically beneficial for future AD treatment.

There are no effective disease-modifying therapies available for Alzheimer’s disease, and new therapeutic strategies are urgently needed. To address this urgent need, we explored attenuation of the dysregulated neuroinflammation response caused by proinflammatory cytokine overproduction, a key contributor to downstream synaptic dysfunction and cognitive deficits. We designed and produced novel small molecule drug candidates using the molecular fragment expansion method paired with function-based or single molecular target-based drug discovery approaches to develop CNS-penetrant therapeutic candidates that selectively attenuate injury- or disease-induced proinflammatory cytokine overproduction. The unbiased functional approach focused on early cytokine overproduction causally linked to downstream synaptic dysfunction. The single target approach focused on p38αMAPK, a druggable mediator of proinflammatory cytokine production that is also a neuronal kinase up-regulated in stress responses. Novel therapeutic candidates are now in early clinical trial stage or late stage investigational new drug-enabling preclinical drug development. Their common pharmacodynamic effect on proinflammatory cytokine overproduction via distinct signaling mechanisms attenuates neuroinflammation, synaptic dysfunction and cognitive impairments in multiple Alzheimer mouse models.

Coauthors: Saktimayee M. Roy, PhD², Ottavio Arancio, PhD³ and D. Martin Watterson, PhD²

¹University of Kentucky, Lexington, Kentucky, United States

²Northwestern University, Chicago, Illinois, United States

³Columbia University, New York, New York, United States

Metabolic syndrome (MetS) and Type 2 Diabetes (T2D) are chronic peripheral inflammatory conditions newly recognized as risk factors for Alzheimer’s (AD); yet the underlying mechanisms are not well understood. We hypothesize that soluble Tumor Necrosis Factor (solTNF) is a critical driver of mechanisms contributing to development of age-related neurodegenerative disease. Here, we investigated the extent to which psychological stress or genetic predisposition for AD (5xFAD) and high-fat high-fructose diet (HFHF) in mice synergize to impact immunophenotype and insulin and lipid metabolism. MetS-related conditions were assessed using untargeted plasma metabolomics, and structural and immune changes were evaluated in the gut-liver-brain axis via qPCR, immunoassays, and flow cytometry. The extent to which solTNF mediates these alterations was investigated by selectively blocking solTNF-dependent pathways using. XPro®1595. Psychological stress and HFHF synergistically promoted tight junction protein alterations, increased insulin resistance, plasma cholesterol, and hippocampal inflammatory factor expression. XPro®1595 reversed the elevated plasma insulin, prostaglandin 2 and cholesterol metabolites. HFHF diet and AD predisposition synergized to increase hippocampal TNF and CCL2 mRNA; XPro®1595 eliminated these effects. 5xFAD mice fed HFHF diet had increased T cell populations in the brain compared with HFHF WT mice. Collectively, these results suggest that diet,stress, and AD risk impact the central-peripheral neuroimmune cross-talk and may exacerbate MetS-related conditions through inflammatory mechanisms frequently associated with neurodegenerative diseases. A more in-depth evaluation of the mechanisms mediating the therapeutic effects of solTNF blockade in rodent models of multifactorial chronic systemic disease may provide compelling rationale to target solTNF in individuals with MetS and at risk for T2D and AD to effectively reduce their risk for development of age-related neurodegenerative disease and comorbidities.

Coauthors: Maria Elizabeth de Sousa Rodrigues, PhD¹, Lori N. Eidson, PhD¹, Kathryn P. MacPherson, PhD¹, Mary K. Herrick¹, Mandakh Bekhbat¹, Madelyn C. Houser¹, Jianjun Chang¹, Douglas I. Walker, PhD², Dean P. Jones, PhD², Meixiang Huang³, Claudia M.P. Oller do Nascimento, PhD⁴, Christopher J. Barnum, PhD⁵, Raymond J. Tesi, PhD⁵, and Thomas L. Kukar, PhD³

¹Department of Physiology, Emory University School of Medicine, Atlanta, Georgia, United States ²Division of Pulmonary, Allergy and Critical Care Medicine, Emory University School of Medicine, Atlanta, Georgia, United States, ³Department of Pharmacology, Emory University School of Medicine, Atlanta, Georgia, United States, ⁴Department of Physiology of Nutrition, Federal University of Sao Paulo, Sao Paulo, Brazil, and ⁵INmune Bio, La Jolla, California.

The pathophysiology of neurodegenerative diseases is poorly understood and there are few therapeutic options. Neurodegenerative diseases are characterized by progressive neuronal dysfunction and loss, and chronic glial activation. Whether microglial activation, which is generally viewed as a secondary process, is harmful or protective in neurodegeneration remains unclear. Late-onset neurodegenerative disease observed in patients with histiocytoses, which are clonal myeloid diseases associated with somatic mutations in the RAS-MEK-ERK pathway such as BRAF(V600E), suggests a possible role of somatic mutations in myeloid cells in neurodegeneration. Yet the expression of BRAF(V600E) in the haematopoietic stem cell lineage causes leukaemic and tumoural diseases but not neurodegenerative disease. Microglia belong to a lineage of adult tissue-resident myeloid cells that develop during organogenesis from yolk-sac erythro-myeloid progenitors (EMPs) distinct from haematopoietic stem cells. We therefore hypothesized that a somatic BRAF(V600E) mutation in the EMP lineage may cause neurodegeneration. Here we show that mosaic expression of BRAF(V600E) in mouse EMPs results in clonal expansion of tissue-resident macrophages and a severe late-onset neurodegenerative disorder. This is associated with accumulation of ERK-activated amoeboid microglia in mice, and is also observed in human patients with histiocytoses. In the mouse model, neurobehavioural signs, astrogliosis, deposition of amyloid precursor protein, synaptic loss and neuronal death were driven by ERK-activated microglia and were preventable by BRAF inhibition. These results identify the fetal precursors of tissue-resident macrophages as a potential cell-of-origin for histiocytoses and demonstrate that a somatic mutation in the EMP lineage in mice can drive late-onset neurodegeneration. Moreover, these data identify activation of the MAP kinase pathway in microglia as a cause of neurodegeneration and this offers opportunities for therapeutic intervention aimed at the prevention of neuronal death in neurodegenerative diseases. (Mass E et al. Nature. 2017 Sep 21;549(7672):389-393. doi: 10.1038/nature23672. Epub 2017 Aug 30.)

In Alzheimer’s disease (AD), the age of symptom onset is highly variable, with some patients exhibiting cognitive symptoms several decades later than predicted based on family history and genetic status. This variability cannot be explained by simple clinical or environmental factors, suggesting that additional genetic factors modify disease onset. The identification of modifier genes that confer resilience in high-risk patient populations would reveal new mechanisms and thus therapeutic strategies to delay disease onset. Disease-relevant genetic variants are difficult to identify in human populations, primarily because asymptomatic individuals rarely enter the clinic. Mouse models represent an ideal complement to human studies, as they present many advantages, such as defined genotypes, early access to brain tissue, and precise environmental control. However, the traditional mouse models of AD have failed to translate into successful treatments that improve cognition in humans. Here, we employ a novel AD mouse genetic reference panel, designed to overcome some of the barriers presented by current AD models. Analyses of whole-genome RNA expression from the hippocampus using a variety of bioinformatics approaches including differential expression, gene set enrichment, principal component, and Pearson’s correlations revealed a strong negative association between microglia transcriptional signatures and cognitive resilience to AD, when assessed using a variety of memory tasks. Overall, work here introduces a humanized mouse population as an innovative and reproducible resource for the study of AD and identifies a core set of microglial genes as a novel therapeutic targets to promote cognitive resilience to AD.

Coauthors: Sarah Heuer, BS¹*, Sarah M. Neuner, BS^1,2*, Timothy J. Hohman, PhD³, Ryan Richholt, BS⁴, David A. Bennett, MD^5,6, Julie A. Schneider, MD^5,6,7, and Philip L. De Jager, MD/PhD⁸, Matthew J. Huentelman, PhD⁴, Kristen M.S. O’Connell, PhD^2.

1. The Jackson Laboratory, Bar Harbor, Maine, United States
2. The Neuroscience Institute, University of Tennessee Health Science Center, Memphis, Tennessee, United States
3. Vanderbilt Memory and Alzheimer’s Center, Department of Neurology, Vanderbilt University Medical Center, Nashville, Tennessee, United States
4. Neurogenomics Division, Translational Genomics Research Institute, Phoenix, Arizona, United States
5. Rush Alzheimer’s Disease Center, Rush University Medical Center, Chicago, Illinois, United States
6. Department of Neurological Sciences, Rush University Medical Center, Chicago, Illinois, United States
7. Department of Pathology, Rush University Medical Center, Chicago, Illinois, United States
8. Center for Translational & Computational Neuroimmunology, Department of Neurology, Columbia University Medical Center, New York; Program in Medical and Population Genetics, Broad Institute, Cambridge, Massachusetts, United States

* contributed equally

Missense mutations in Presenilins and the amyloid precursor protein (APP) are linked to familial Alzheimer's disease (FAD). We employ gene targeting approaches in mice to investigate the normal physiological role of the Presenilin and APP families in the cerebral cortex and the dysfunction caused by the FAD mutations. We also performed similar genetic analysis in Drosophila. Our genetic studies demonstrate that complete or partial inactivation of Presenilin in excitatory neurons of the adult cerebral cortex causes age-dependent increases in apoptosis and neurodegeneration, whereas similar conditional inactivation of APP/APLP1/APLP2 does not result in age-dependent neurodegeneration. Furthermore, the essential role of Presenilin in support of neuronal survival in the aging brain is evolutionarily conserved, and conditional knockdown of the fly Presenilin homolog in adult neurons of the Drosophila brain also leads to age-dependent neurodegeneration. Interestingly, both Presenilin and APP families are required for normal synaptic plasticity in the mouse hippocampus, and the specific synaptic impairment observed in conditional knockout mice lacking either Presenilins or APP family members will be discussed. We have also developed several Presenilin-1 knockin mice carrying either an FAD mutation or an alteration of the Aspartate residue at the gamma-secretase active site, and the comparison of their phenotypes will be presented and discussed.

Increasing evidence suggests that neuroinflammation is an important contributor to Alzheimer's disease (AD) pathogenesis, as underscored by the identification of immune-related genetic risk factors for AD, including coding variants in the gene TREM2 (triggering receptor expressed on myeloid cells 2) amongst others. Understanding TREM2 (as well as other immune system genes) function promises to provide important insights into how neuroinflammation contributes to AD pathology. Notably, recent evidence suggests that development of the various AD pathologies (amyloid and tau) occurs across several decades. In the current studies we provide evidence that TREM2 plays different roles at different stages of disease progression in a transgenic mouse model of AD that develops robust amyloid pathology. By contrast, TREM2 deficiency seems to play a different role in a transgenic mouse model of tau pathology. The opposing results on amyloid and tau pathology is similar to that previously reported for other innate immune system pathways. Finally, human studies also support that TREM2 may play a unique role at different stages of disease progression. Taken together, these results suggest that TREM2 and other innate immune pathways implicated in AD, may play distinct functional roles that are both stage- and pathology-dependent. Finally, we will also provide an update on the Model Organism Development and Evaluation for Late-onset AD (MODEL-AD) Consortium, which focuses on developing, characterizing, distributing and performing preclinical testing on more accurate animal models of AD, that includes a strong focus on immune system genes and pathways.