Summary Anti-inflammatory strategies are proposed to have beneficial effects in Alzheimer's disease. To explore how anti-inflammatory cytokine signaling affects Aβ pathology, we investigated the effects of adeno-associated virus (AAV2/1) mediated expression of Interleukin (IL)-10 in the brains of APP transgenic mouse models. IL-10 expression resulted in increased Aβ accumulation and impaired memory in APP mice. A focused transcriptome analysis revealed changes consistent with enhanced IL-10 signaling and increased ApoE expression in IL-10 expressing APP mice. ApoE protein was selectively increased in the plaque-associated insoluble cellular fraction, likely due to direct interaction with aggregated Aβ in the IL-10 expressing APP mice. Ex vivo studies also show that IL-10 and ApoE can individually impair glial Aβ phagocytosis. Our observations that IL-10 has an unexpected negative effect on Aβ proteostasis and cognition in APP mouse models demonstrate the complex interplay between innate immunity and proteostasis in neurodegenerative diseases, an interaction we call immunoproteostasis.

Adeno-associated virus (AAV) mediated gene expression is a powerful tool for gene therapy and preclinical studies. A comprehensive analysis of CNS cell type tropism, expression levels and biodistribution of different capsid serotypes has not yet been undertaken in neonatal rodents. Our previous studies show that intracerebroventricular injection with AAV2/1 on neonatal day P0 results in widespread CNS expression but the biodistribution is limited if injected beyond neonatal day P1. To extend these observations we explored the effect of timing of injection on tropism and biodistribution of six commonly used pseudotyped AAVs delivered in the cerebral ventricles of neonatal mice. We demonstrate that AAV2/8 and 2/9 resulted in the most widespread biodistribution in the brain. Most serotypes showed varying biodistribution depending on the day of injection. Injection on neonatal day P0 resulted in mostly neuronal transduction, whereas administration in later periods of development (24–84 hours postnatal) resulted in more non-neuronal transduction. AAV2/5 showed widespread transduction of astrocytes irrespective of the time of injection. None of the serotypes tested showed any microglial transduction. This study demonstrates that both capsid serotype and timing of injection influence the regional and cell-type distribution of AAV in neonatal rodents, and emphasizes the utility of pseudotyped AAV vectors for translational gene therapy paradigms.

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.

BackgroundGenetic studies have established a causative role for α-synuclein (αS) in Parkinson’s disease (PD), and the presence of αS aggregates in the form of Lewy body (LB) and Lewy neurite (LN) protein inclusions are defining pathological features of PD. Recent data has established that extracellular αS aggregates can induce intracellular αS pathologies supporting the hypothesis that αS pathology can spread via a “prion-like” self-templating mechanism.ResultsHere we investigated the potential for conformational templating of αS intracellular aggregates by seeding using recombinant wild-type and PD-linked mutant (A53T and E46K) αS in primary mixed neuronal-glial cultures. We find that wild-type and A53T αS fibrils predominantly seed flame-like inclusions in both neurons and astrocytes of mixed primary cultures; whereas the structurally distinct E46K fibrils seed punctate, rounded inclusions. Notably, these differences in seeded inclusion formation in these cultures reflect differences in inclusion pathology seen in transgenic mice expressing the A53T or E46K αS mutants. We further show that the inclusion morphology is dictated primarily by the seed applied rather than the form of αS expressed. We also provide initial evidence that αS inclusion pathology can be passaged in primary astrocyte cultures.ConclusionThese studies establish for the first time that αS aggregation in cultured cells can occur by a morphological self-templating mechanism.