Aims

Genetics strongly determines the risk of developing Alzheimer’s disease (AD). Interestingly, AD risk genes mainly operate in a limited number of pathways, mostly microglia-related. Microglia and inflammation have a central role in AD pathogenesis, but the molecular understanding is missing how genetic risk translates into functional consequences in microglia responses to AD pathology. We developed a humanized microglial chimeric model which allows to study genetic risk and AD pathogenesis in vivo in a unique way. Here, we propose to investigate the role of lipid-associated risk genes, which show also rare loss-of-function mutations in AD patients, by using our chimeric AD model.

Methods

We are employing inducible genetic editing of human microglia cells by CRISPR-Cas9 knock-outs to assess for functional consequences of lipid genes deletions in the context of amyloid pathology.

Results

We have previously generated an inducible iCas9 stem cell line that allows timed Cas9 activation upon doxycycline treatment. We have confirmed that human PSCs-derived microglial progenitors display inducible activation of Cas9 and can be efficiently transduced with a lentiviral library of gRNAs in vitro. We confirmed that the in vitro pooled single-cell screening shows combinatorial distribution of gRNAs, and tested the editing efficiency at the genomic level. We also analyzed the impact of mutations at phenotypic profile by single-cell RNAseq.

Conclusions

We provided proof-of-concept that Human PSCs-derived microglial progenitors are suitable for genetic screens such as single-cell pooled CRISPR-Cas9 knock-out approaches. We believe this constitutes a powerful tool to study genetic risk of AD in human microglia in vitro and in vivo.

Aims

Microglia are tissue-resident macrophages of the central nervous system and are essential for homeostasis, immune responses, neurogenesis, and plasticity. Genetic studies have strongly implicated microglial dysfunction in multiple neurodegenerative diseases. However, investigations of genetically-driven changes in gene expression in microglia have been limited by lack of access to these cells in sufficient numbers.

Methods

We performed RNA-seq and genotyping on 236 primary human CD11b+ microglia samples isolated at autopsy from multiple brain regions of 111 human subjects. We investigated the effects of brain region, age, sex, and disease on gene expression, and integrated genetic data to map expression and splicing QTLs.

Results

We observed widespread transcriptomic variation between microglia from different brain regions and across chronological age, with many affected genes found in AD or PD GWAS loci. We then identified 3,611 eQTLs, of which 50% show region-specific effects. 300 QTLs colocalized with a GWAS locus for a neurodegenerative or neuropsychiatric disease, nearly half of which are not found in prefrontal cortex or in peripheral monocytes. We prioritized 7 and 13 putative causal genes for AD and PD, respectively, many of which are novel (ITGAX, USP6NL, TSPOAP1, P2RY12, FCGR2C, and FGF20). Fine-mapping of these colocalized loci with epigenomic data nominated genetic variants within microglia-specific enhancers. These are likely to modify disease susceptibility by regulating gene expression and/or splicing in microglia.

Conclusions

In summary, we have built the most comprehensive catalog to date of genetic effects on the microglial transcriptome and propose molecular mechanisms of action for several disease-associated genes.

Aims

Microglia are strongly implicated in the development and progression of Alzheimer’s disease (AD). To further understand how microglia impact AD pathology, we crossed 5xfAD mice with ‘FIRE’ mice, a novel model that genetically lacks microglia. Compared to other genetic or pharmacological methods, this new approach allows one to understand how a consistent absence of microglia impacts disease progression. The resulting empty microglia niche also enables the robust repopulation of microglia from genetically modified donors.

Methods

‘FIRE’ mice harboring a deletion in the fms-intronic regulatory element of CSF1R and lack microglia were backcrossed with 5xfAD mice. Immunohistochemical and biochemical analysis was used to determine the impact of microglial absence on disease pathology. In addition, transplantation of wild-type donor microglia was performed to determine the impact of microglial replacement on these endpoints.

Results

Pharmacological depletion of microglial has not been reported to effect AD mouse mortality. In contrast, we found that FIREKO-5xFAD mice that genetically lack microglia, exhibit a significantly shortened lifespan, with few mice reaching 6 months of age. Interestingly, this increased mortality is accompanied by a significant reduction in parenchymal beta-amyloid plaques, but a dramatic increase in cerebral amyloid angiopathy (CAA) and other signs of blood brain barrier (BBB) pathology and disruption. Remarkably, transplantation of Wild Type donor microglia rescues many, but not all, of these changes.

Conclusions

The FIREKO-5xFAD model provides a novel platform to determine how microglia influence the relationship between plaque formation and blood vessel pathology and the impact of varying microglial donor cells on these outcomes.

Aims

Objectives: We analyzed the long-term liraglutide (LRGT) as an alternative treatment to retard or slow down metabolic, amyloid-β (Aβ) and inflammatory pathologies in a mixed model of AD and T2D (APP/PS1xdb/db mouse).

Methods

Methods: AD-T2D mice were produced by cross-breeding APPswe/PS1dE9 with db/db mice. We administered LRGT to these mice (500μg/kg/day, sc) for 20 weeks. Body weight, glucose and insulin levels were evaluated before treatment and every 4 weeks until sacrifice. Amyloid pathology was studied and senile plaques (SP) were analyzed by immunohistochemistry with 4G8 antibody and thioflavin-S staining. Soluble and insoluble Aβ40 and Aβ42 levels, as well as Aβ oligomers were determined by ELISA. The inflammatory pathology was analyzed by immunostaining for microglia with Iba-1 antibody, in the proximity and far from SP.

Results

Results: LRGT treatment maintains insulin levels and decreases glucose levels in the long term. Interestingly, LRGT reduces Aβ pathology and SP burden is reduced in APP/PS1 and SP size is reduced in APP/PS1 and APP/PS1xdb/db mice in the cortex. Moreover, it is also disclosed that LRGT treatment lessens oligomeric Aβ in the cortex from APP/PS1xdb/db mice. Likewise, LRGT treatment reduces cortical microglia burden in the proximity of SP from AD mice, whereas microglia burden is reduced in SP free areas from diabetic mice.

Conclusions

Conclusions: LRGT treatment limits metabolic compromise in diabetic mice. LRGT reduces oligomeric Aβ levels and the treatment limits microglia activation in APP/PS1xdb/db mice, supporting that LRGT might be as an alternative to slow down amyloid and inflammatory pathologies associated with AD and T2D.

Aims

The TAM (Tyro3, Axl, MerTK) receptor tyrosine kinases are known for their roles in phagocytosis and suppression of inflammation. Axl is upregulated on plaque-associated microglia and is regularly reported as a disease-associated microglial marker. Furthermore, Gas6, the primary CNS TAM ligand, has demonstrated efficacy in reducing neuroinflammation and alleviating pathology in multiple CNS diseases. We sought to determine the effects of overexpression of Gas6 on Alzheimer’s disease pathology with the following aims:

1. Determine whether overexpression of Gas6 alters behavior and plaque pathology in the APP/PS1 model of Alzheimer’s disease.

2. Determine effect of Gas6 on microglial activation using immunohistochemistry and bulk RNAseq of FACS-sorted microglia for transcriptional analyses following Gas6 overexpression.

Methods

APP/PS1 mice and nontransgenic littermates were treated with intrahippocampal injections of AAV containing Gas6 or an attenuated Gas6 protein as control. One month later, mice performed behavioral tasks including open field, novel object recognition, and fear conditioning. Immunohistochemical analyses for plaque pathology and microglial markers were completed. RNAseq was performed on FACS-sorted microglia from Gas6 and control-treated mice.

Results

Gas6 overexpression worsens performance in the contextual fear conditioning task and reduces plaque number in male APP/PS1 mice. Gas6 induces a proinflammatory microglial transcriptional signature.

Conclusions

Overexpression of Gas6 worsens behavior and reduces plaque number in male APP/PS1 mice. Contrary to literature describing an anti-inflammatory effect of Gas6, we found that overexpression of Gas6 in aged APP/PS1 mice induces a proinflammatory microglial signature, which may suggest a novel role for Gas6.

Aims

Adiponectin (APN) is a circulatory adipokine, which possesses anti-inflammatory effects. APN does not expressed in the brain, but low-molecular-weight APN includes trimeric and hexameric forms crosses the blood-brain barrier. We have reported that APN level was reduced in human AD brains and AD mice. Microglia expresses adiponectin receptors and is activated under chronic APN deficiency. However, the molecular implication of APN in microglia-mediated neuroinflammation has not been elucidated.

Methods

APN-deficient 5xFAD mice were generated by crossing APN^-/- and 5xFAD mice. Microglia was isolated and RNA was extracted for RNA sequencing. AAV2/8 serotype carrying APN gene with C39S mutation under hAAT/APOE promoter, which allowed liver specific expression of APN^Tri. 5xFAD mice were given with liver-specific AAV-APN^Tri (1 x 10¹¹ viral particles) by intravenous injection. Brains were collected for immunofluorescent staining, ELISA and western blot analysis to investigate the role of APN in microglia.

Results

APN modulates neuroinflammatory pathways as shown by RNA sequencing analysis. We have found that APN deficiency increases NLRP3 activation in 5xFAD mice. Microglia activation and IL1β levels are exacerbated under APN-deficiency. In addition, liver-specific expression of APN^Tri by AAV delivery increases the circulatory APN levels and improves memory functions in AD mice. AAV-APN^Tri treatment reduces microglia activation and IL1b levels in the brain of 5xFAD in which NLRP3, ASC and caspase-1 levels are also reduced in microglia.

Conclusions

Taken altogether, our study demonstrates the circulatory APN inhibits IL1β secretion from microglia through NLRP3 inhibition, suggesting that increasing periphery trimeric APN can be a potential AD therapy.

Aims

Aim 1. Investigating the roles of CD33 isoforms in controlling microglia cell function

Aim 2. Elucidating the roles of CD33 in regulating plaque accumulation

Methods

We are working with both cell culture models as well as mouse models. For cell culture models, we are using U937 cells in which we have deleted the CD33 gene by CRISPR/Cas9 and complemented these cells with different CD33 variants and mutants. These are being tested in phagocytosis assay and cellular signaling assays. We have also developed transgenic mice expressing either the long or short isoform of CD33 in the microglial cell lineage, which have been crossed on 5XFAD mice. Plaque accumulation, scRNAseq, and the distribution of the microglia around the plaques are being assessed.

Results

We find CD33 isoforms differentially control phagocytosis in microglia. We reveal, for the first time, a novel gain-of-function for the short isoform of CD33 that is connected with AD protection. This gain-of-function is dominant and manifests as enhanced phagocytosis and transcriptional skewing. Ongoing findinds with these transgenic mice crossed onto a 5XFAD background will be presented .

Conclusions

Elucidating whether the short isoform of CD33, encoded preferentially by an AD-protective single nucleotide polymorphism, is a loss-of-funciton or gain-of-function variant has been challenging. Our results in two different model systems reveal that there are both loss-of-funciton or gain-of-function phenotypes at play. Which of these phenotypes is responsible for AD protection will be revealed, in part, through ongoing studies with these two isoforms in mouse models of AD.

Aims

Neuroinflammation is part of the pathophysiology in numerous neurodegenerative diseases including Alzheimer’s disease (AD). AD neuroimaging studies highlight increased inflammation markers, while GWAS studies indicate that many AD-associated genes are expressed in glial cells. The hormone and neurotransmitter Glucagon-Like Peptide-1 (GLP-1) was originally developed for treatment of type 2 diabetes but have pleiotropic effects. GLP-1 receptor agonists (GLP-1RAs) also reduce cardiovascular disease, highlighting reduced inflammation as a benefit of GLP-1RAs. Small clinical studies have indicated that GLP-1RAs may have clinical relevance in AD and PD. Here, we investigated the effects of the GLP-1RA semaglutide on lipopolysaccharide (LPS) induced hippocampal neuroinflammation in mice as a model of early AD pathophysiology.

Methods

Mice were treated with semaglutide or vehicle for 28 days, and LPS or vehicle was administered on days 15-17. Quantitative assessment of the microglial-specific marker Iba1 (ionized calcium binding adaptor molecule 1) was performed to measure the area of microglia in the hippocampus at Day 19 and Day 28.

Results

LPS significantly increased hippocampal Iba1 area on Day 19 in mice dosed with LPS/vehicle compared to vehicle/vehicle controls (p<0.001). On Day 28, LPS/vehicle-treated mice continued to have a significantly higher area of hippocampal Iba1 (p<0.05 vs vehicle/vehicle controls), whereas semaglutide treatment significantly reduced Iba1 area in LPS/semaglutide-treated mice (p<0.01 vs LPS + vehicle treated mice); Figure 1.

Conclusions

In an LPS-induced neuroinflammation model, semaglutide reduced hippocampal neuroinflammation as measured by microglial area (Iba1). This could be a novel mechanism through which semaglutide may affect neuronal integrity and function relevant for AD pathophysiology.