Recombinant adeno-associated viruses (rAAV) have been widely used in gene therapy
applications for central nervous system diseases. Though rAAV can efficiently target
neurons and astrocytes in mouse brains, microglia, the immune cells of the brain, are
refractile to rAAV. To identify AAV capsids with microglia-specific transduction
properties, we initially screened the most commonly used serotypes, AAV1–9 and
rh10, on primary mouse microglia cultures. While these capsids were not permissive, we
then tested the microglial targeting properties of a newly characterized set of modified
rAAV6 capsid variants with high tropism for monocytes. Indeed, these newly characterized
rAAV6 capsid variants, specially a triply mutated Y731F/Y705F/T492V form, carrying a
self-complementary genome and microglia-specific promoters (F4/80 or CD68) could
efficiently and selectively transduce microglia in vitro. Delivery of these
constructs in mice brains resulted in microglia-specific expression of green fluorescent
protein, albeit at modest levels. We further show that CD68 promoter–driven
expression of the inflammatory cytokine, interleukin-6, using this capsid variant leads to
increased astrogliosis in the brains of wild-type mice. Our study describes the first
instance of AAV-targeted microglial gene expression leading to functional modulation of
the innate immune system in mice brains. This provides the rationale for utilizing these
unique capsid/promoter combinations for microglia-specific gene targeting for modeling or
functional studies.
In Alzheimer’s disease (AD) models, AD risk variants in the microglial-expressed TREM2 gene decrease Aβ plaque–associated microgliosis and increase neuritic dystrophy as well as plaque-associated seeding and spreading of tau aggregates. Whether this Aβ-enhanced tau seeding/spreading is due to loss of microglial function or a toxic gain of function in TREM2-deficient microglia is unclear. Depletion of microglia in mice with established brain amyloid has no effect on amyloid but results in less spine and neuronal loss. Microglial repopulation in aged mice improved cognitive and neuronal deficits. In the context of AD pathology, we asked whether microglial removal and repopulation decreased Aβ-driven tau seeding and spreading. We show that both TREM2KO and microglial ablation dramatically enhance tau seeding and spreading around plaques. Interestingly, although repopulated microglia clustered around plaques, they had a reduction in disease-associated microglia (DAM) gene expression and elevated tau seeding/spreading. Together, these data suggest that TREM2-dependent activation of the DAM phenotype is essential in delaying Aβ-induced pathological tau propagation.
There is considerable interest in harnessing innate immunity to treat Alzheimer's disease (AD). Here, we explore whether a decoy receptor strategy using the ectodomain of select TLRs has therapeutic potential in AD. AAV-mediated expression of human TLR5 ectodomain (sTLR5) alone or fused to human IgG4 Fc (sTLR5Fc) results in robust attenuation of amyloid β (Aβ) accumulation in a mouse model of Alzheimer-type Aβ pathology. sTLR5Fc binds to oligomeric and fibrillar Aβ with high affinity, forms complexes with Aβ, and blocks Aβ toxicity. Oligomeric and fibrillar Aβ modulates flagellin-mediated activation of human TLR5 but does not, by itself, activate TLR5 signaling. Genetic analysis shows that rare protein coding variants in human TLR5 may be associated with a reduced risk of AD. Further, transcriptome analysis shows altered TLR gene expression in human AD. Collectively, our data suggest that TLR5 decoy receptor-based biologics represent a novel and safe Aβ-selective class of biotherapy in AD.
Introduction
Triggering receptor expressed on myeloid cells‐2 (TREM2) is an immune receptor expressed on microglia that also can become soluble (sTREM2). How TREM2 engages different ligands remains poorly understood.
Methods
We used comprehensive biolayer interferometry (BLI) analysis to investigate TREM2 and sTREM2 interactions with apolipoprotein E (apoE) and monomeric amyloid beta (Aβ) (mAβ42).
Results
TREM2 engagement of apoE was protein mediated with little effect of lipidation, showing slight affinity differences between isoforms (E4 > E3 > E2). Another family member, TREML2, did not bind apoE. Disease‐linked TREM2 variants within a “basic patch” minimally impact apoE binding. Instead, TREM2 uses a unique hydrophobic surface to bind apoE, which requires the apoE hinge region. TREM2 and sTREM2 directly bind mAβ42 and potently inhibit Aβ42 polymerization, suggesting a potential role for soluble sTREM2 in preventing AD pathogenesis.
Discussion
These findings demonstrate that TREM2 has at least two ligand‐binding surfaces that might be therapeutic targets and uncovers a potential function for sTREM2 in directly inhibiting Aβ polymerization.
INTRODUCTION:TREM2 is an innate immune receptor expressed on myeloid cells including microglia in the brain. How TREM2 engages different ligands remains poorly understood. METHODS: We used comprehensive BLI analysis to investigate the TREM2 interactions with ApoE and monomeric amyloid beta (mAβ42). RESULTS: TREM2 binding did not depend on ApoE lipidation, and there were only slight differences in affinity observed between ApoE isoforms (E4 > E3 > E2). Surprisingly, diseaselinked TREM2 variants within a "basic patch" minimally impact ApoE binding. Instead, TREM2 has a unique hydrophobic surface that can bind to ApoE. This direct engagement requires the hinge region of ApoE. TREM2 directly binds mAβ42 and can potently inhibit Aβ42 polymerization, suggesting a potential mechanism for soluble TREM2 (sTREM2) in preventing AD pathogenesis. DISCUSSION: These findings demonstrate that TREM2 has at least two separate surfaces to engage ligands and uncovers a potential function for sTREM2 in directly inhibiting Aβ polymerization.
Brain expression of AAV-Ifn-γ leads to reactive gliosis, nigrostriatal degeneration and midbrain calcification in wild type mice. This mouse model phenocopies idiopathic basal ganglia calcification which is associated with Parkinsonian symptoms. To understand how the nigro-striatal pathway is selectively vulnerable to Ifn-γ, we determined if the phenotype is driven by canonical signaling intermediates, Ifngr1 and Stat1. Using focused bioinformatic analysis and rotarod testing, we show that neuroinflammation and motor abnormalities precede the appearance of midbrain neuropathologies in the brains of Ifn-γ mouse model. To test whether canonical Ifn-γ signaling is a key driver of progressive nigrostriatal degeneration, we overexpressed Ifn-γ in the brains of Ifngr1 and Stat1 mice. Expression of Ifn-γ in Ifngr1 mice did not result in any neuroinflammation, midbrain calcinosis or nigrostriatal degenerative pathology. Interestingly, in Stat1 mice, Ifn-γ expression resulted in gliosis without recapitulating the neurodegenerative phenotype. Overall, our data shows that canonical Ifn-γ signaling triggers midbrain calcinosis and nigrostriatal neurodegeneration, providing mechanistic insights into cytokine-driven selective neuronal vulnerability. Our study establishes the broader relevance of inflammatory signaling in neurodegenerative diseases and can potentially identify novel immunological targets for Parkinsonian syndromes.
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