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.

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.

The architecture of the spinal cord makes efficient delivery of recombinant adeno-associated virus (rAAV) vectors throughout the neuraxis challenging. We describe a paradigm in which small amounts of virus delivered intraspinally to newborn mice result in robust rAAV-mediated transgene expression in the spinal cord. We compared the efficacy of rAAV2/1, 2/5, 2/8, and 2/9 encoding EGFP delivered to the hindlimb muscle (IM), cisterna magna (ICM), or lumbar spinal cord (IS) of neonatal pups. IS injection of all four capsids resulted in robust transduction of the spinal cord with rAAV2/5, 2/8, and 2/9 vectors appearing to be transported to brain. ICM injection resulted in widespread expression of EGFP in the brain, and upper spinal cord. IM injection resulted in robust muscle expression, with only rAAV2/8 and 2/9 transducing spinal motor and sensory neurons. As proof of concept, we use the IS paradigm to express murine Interleukin (IL)-10 in the spinal cord of the SOD1-G93A transgenic mouse model of amyotrophic lateral sclerosis. We show that expression of IL-10 in the spinal axis of SOD1-G93A mice altered the immune milieu and significantly prolonged survival. These data establish an efficient paradigm for somatic transgene delivery of therapeutic biologics to the spinal cord of mice.

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.

Increasing evidence suggests that the renin-angiotensin system (RAS) plays an important role in tumorigenesis. The interaction between Angiotensin II (AngII) and angiotensin type 1 receptor (AT1R) may have a pivotal role in hepatocellular carcinoma (HCC) and therefore, AT1R blocker and angiotensin I-converting enzyme (ACE) inhibitors may have therapeutic potential in the treatment of hepatic cancer. Although the involvement of AT1R has been well explored, the role of the angiotensin II Type 2 receptor (AT2R) in HCC progression remains poorly understood. Thus, the aim of this study was to explore the effects of AT2R overexpression on HCC cells in vitro and in mouse models of human HCC. An AT2R recombinant adenoviral vector (Ad-G-AT2R-EGFP) was transduced into HCC cell lines and orthotopic tumor grafts. The results indicate that the high dose of Ad-G-AT2R-EGFP–induced overexpression of AT2R in transduced HCC cell lines produced apoptosis. AT2R overexpression in SMMC7721 cells inhibited cell proliferation with a significant reduction of S-phase cells and an enrichment of G1-phase cells through changing expression of CDK4 and cyclinD1. The data also indicate that overexpression of AT2R led to apoptosis via cell death signaling pathway that is dependent on activation of p38 MAPK, pJNK, caspase-8 and caspase-3 and inactivation of pp42/44 MAPK (Erk1/2). Finally, we demonstrated that moderately increasing AT2R expression could increase the growth of HCC tumors and the proliferation of HCC cells in vivo. Our findings suggest that AT2R overexpression regulates proliferation of hepatocellular carcinoma cells in vitro and in vivo, and the precise mechanisms of this phenomenon are yet to be fully determined.