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1.  Catalytic Immunoglobulin Gene Delivery in a Mouse Model of Alzheimer's Disease: Prophylactic and Therapeutic Applications 
Molecular neurobiology  2014;51(1):43-56.
Accumulation of amyloid beta-peptide (Aβ) in the brain is hypothesized to be a causal event leading to dementia in Alzheimer's disease (AD). Aβ vaccination removes Aβ deposits from the brain. Aβ immunotherapy, however, may cause T cell- and/or Fc-receptor-mediated brain inflammation and relocate parenchymal Aβ deposits to blood vessels leading to cerebral hemorrhages. Because catalytic antibodies do not form stable immune complexes and Aβ fragments produced by catalytic antibodies are less likely to form aggregates, Aβ-specific catalytic antibodies may have safer therapeutic profiles than reversibly-binding anti-Aβ antibodies. Additionally, catalytic antibodies may remove Aβ more efficiently than binding antibodies because a single catalytic antibody can hydrolyze thousands of Aβ molecules. We previously isolated Aβ-specific catalytic antibody, IgVL5D3, with strong Aβ-hydrolyzing activity. Here, we evaluated the prophylactic and therapeutic efficacy of brain-targeted IgVL5D3 gene delivery via recombinant adeno-associated virus serotype 9 (rAAV9) in an AD mouse model. One single injection of rAAV9-IgVL5D3 into the right ventricle of AD model mice yielded widespread, high expression of IgVL5D3 in the unilateral hemisphere. IgVL5D3 expression was readily detectable in the contralateral hemisphere but to a much lesser extent. IgVL5D3 expression was also confirmed in the cerebrospinal fluid. Prophylactic and therapeutic injection of rAAV9-IgVL5D3 reduced Aβ load in the ipsilateral hippocampus of AD model mice. No evidence of hemorrhages, increased vascular amyloid deposits, increased pro-inflammatory cytokines or infiltrating T cells in the brains was found in the experimental animals. AAV9-mediated anti-Aβ catalytic antibody brain delivery can be prophylactic and therapeutic options for AD.
PMCID: PMC4198531  PMID: 24733587
Alzheimer's disease; amyloid; inflammation; antibody; adeno-associated virus; immunotherapy
2.  Combined treatment of Aβ immunization with statin in a mouse model of Alzheimer’s disease 
Journal of Neuroimmunology  2012;244(1-2):70-83.
We evaluated the therapeutic efficacy of combined treatment of Aβ-immunization with simvastatin in an Alzheimer mouse model at age 22 months. DNA prime-adenovirus boost immunization induced modest anti-Aβ titers and simvastatin increased the seropositive rate. Aβ-KLH was additionally administered to boost the titers. Irrespective of simvastatin, the immunization did not decrease cerebral Aβ deposits but increased soluble Aβ and tended to exacerbate amyloid angiopathy in the hippocampus. The immunization increased cerebral invasion of leukocytes and simvastatin counteracted the increase. Thus, modest anti-Aβ titers can increase soluble Aβ and simvastatin may reduce inflammation associated with vaccination in aged Alzheimer mouse models.
PMCID: PMC3298635  PMID: 22326143
Alzheimer’s disease; statin; adenovirus; vaccine; amyloid; inflammation
3.  The effects of MyD88 deficiency on exploratory activity, anxiety, motor coordination and spatial learning in C57BL/6 and APPswe/PS1dE9 mice 
Behavioural brain research  2011;227(1):36-42.
Toll-like receptors (TLRs) are a family of pattern-recognition receptors in innate immunity and provide a first line defense against pathogens and tissue injuries. In addition to important roles in infection, inflammation, and immune diseases, recent studies show that TLR signaling is involved in modulation of learning, memory, mood, and neurogenesis. Because MyD88 is essential for the downstream signaling of all TLRs, except TLR3, we investigated the effects of MyD88 deficiency (MyD88-/-) on behavioral functions in mice. Additionally, we recently demonstrated that a mouse model of Alzheimer’s disease (AD) deficient for MyD88 had decreases in Aβ deposits and soluble Aβ in the brain as compared with MyD88 sufficient AD mouse models. Because accumulation of Aβ in the brain is postulated to be a causal event leading to cognitive deficits in AD, we investigated the effects of MyD88 deficiency on behavioral functions in the AD mouse model at 10 months of age. MyD88 deficient mice showed more anxiety in the elevated plus-maze. In the motor coordination tests, MyD88 deficient mice remained on a beam and a bar for a longer time, but with slower initial movement on the bar. In the Morris water maze test, MyD88 deficiency appeared to improve spatial learning irrespective of the transgene. Our findings suggest that the MyD88-dependent pathway contributes to behavioral functions in an AD mouse model and its control group.
PMCID: PMC3242934  PMID: 22051943
MyD88; toll-like receptors; Alzheimer’s disease; memory; anxiety; cognitive function
4.  Anti-Aβ single-chain antibody brain delivery via AAV reduces amyloid load but may increase cerebral hemorrhages in an Alzheimer mouse model 
Accumulation of amyloid β-protein (Aβ) in the brain is thought to be a causal event in Alzheimer’s disease (AD). Immunotherapy targeting Aβ holds great promise for reducing Aβ in the brain. Here, we evaluated the efficacy and safety of anti-Aβ single-chain antibody (scFv59) delivery via recombinant adeno-associated virus (rAAV) on reducing Aβ deposits in an AD mouse model (TgAPPswe/PS1dE9). First, delivery of scFv59 to the brain was optimized by injecting rAAV serotypes 1, 2, and 5 into the right lateral ventricle. Symmetrical high expression of scFv59 was found throughout the hippocampus and partly in the neocortex in both hemispheres via rAAV1 or rAAV5 while scFv59 expression via rAAV2 was mostly limited to one hemisphere. rAAV1, however, induced apoptosis and microglial activation but rAAV5 did not. Therefore, rAAV5 was selected for therapeutic scFv59 delivery in TgAPPswe/PS1dE9 mice. rAAV5 was similarly injected into the ventricle of 10-month-old TgAPPswe/PS1dE9 mice and 5 months later its efficacy and safety were evaluated. Immunoreactive Aβ deposits reduced in the hippocampus. Aβ42 levels in cerebrospinal fluid (CSF) tended to increase and the Aβ40:42 ratio decreased in CSF, suggesting that Aβ42 was relocated from the parenchyma to CSF. Hemorrhages associated with a focal increase in blood vessel amyloid were found in the brain. While immunotherapy has great potential for clearing cerebral Aβ, caution for cerebrovascular effects should be exercised when rAAV-mediated anti-Aβ immunotherapy is applied.
PMCID: PMC3560395  PMID: 21709371
Alzheimer disease; immunotherapy; single-chain antibodies; amyloid; adeno-associated virus; cerebral hemorrhage
5.  TLR4 mutation reduces microglial activation, increases Aβ deposits and exacerbates cognitive deficits in a mouse model of Alzheimer's disease 
Amyloid plaques, a pathological hallmark of Alzheimer's disease (AD), are accompanied by activated microglia. The role of activated microglia in the pathogenesis of AD remains controversial: either clearing Aβ deposits by phagocytosis or releasing proinflammatory cytokines and cytotoxic substances. Microglia can be activated via toll-like receptors (TLRs), a class of pattern-recognition receptors in the innate immune system. We previously demonstrated that an AD mouse model homozygous for a loss-of-function mutation of TLR4 had increases in Aβ deposits and buffer-soluble Aβ in the brain as compared with a TLR4 wild-type AD mouse model at 14-16 months of age. However, it is unknown if TLR4 signaling is involved in initiation of Aβ deposition as well as activation and recruitment of microglia at the early stage of AD. Here, we investigated the role of TLR4 signaling and microglial activation in early stages using 5-month-old AD mouse models when Aβ deposits start.
Microglial activation and amyloid deposition in the brain were determined by immunohistochemistry in the AD models. Levels of cerebral soluble Aβ were determined by ELISA. mRNA levels of cytokines and chemokines in the brain and Aβ-stimulated monocytes were quantified by real-time PCR. Cognitive functions were assessed by the Morris water maze.
While no difference was found in cerebral Aβ load between AD mouse models at 5 months with and without TLR4 mutation, microglial activation in a TLR4 mutant AD model (TLR4M Tg) was less than that in a TLR4 wild-type AD model (TLR4W Tg). At 9 months, TLR4M Tg mice had increased Aβ deposition and soluble Aβ42 in the brain, which were associated with decrements in cognitive functions and expression levels of IL-1β, CCL3, and CCL4 in the hippocampus compared to TLR4W Tg mice. TLR4 mutation diminished Aβ-induced IL-1β, CCL3, and CCL4 expression in monocytes.
This is the first demonstration of TLR4-dependent activation of microglia at the early stage of β-amyloidosis. Our results indicate that TLR4 is not involved in the initiation of Aβ deposition and that, as Aβ deposits start, microglia are activated via TLR4 signaling to reduce Aβ deposits and preserve cognitive functions from Aβ-mediated neurotoxicity.
PMCID: PMC3169468  PMID: 21827663
6.  Polymorphisms affecting gene transcription and mRNA processing in pharmacogenetic candidate genes: detection through allelic expression imbalance in human target tissues 
Pharmacogenetics and genomics  2008;18(9):781-791.
Genetic variation in mRNA expression plays a critical role in human phenotypic diversity, but it has proven difficult to detect regulatory polymorphisms - mostly single nucleotide polymorphisms (rSNPs). Additionally, variants in the transcribed region, termed here ‘structural RNA SNPs’ (srSNPs), can affect mRNA processing and turnover. Both rSNPs and srSNPs cause allelic mRNA expression imbalance (AEI) in heterozygous individuals. We have applied a rapid and accurate AEI methodology for testing 42 genes implicated in human diseases and drug response, specifically cardiovascular and CNS diseases, and affecting drug metabolism and transport. Each gene was analyzed in physiologically relevant human autopsy tissues, including brain, heart, liver, intestines, and lymphocytes. Substantial AEI was observed in ∼55% of the surveyed genes. Focusing on cardiovascular candidate genes in human hearts, AEI analysis revealed frequent cis-acting regulatory factors in SOD2 and ACE mRNA expression, having potential clinical significance. SNP scanning to locate regulatory polymorphisms in a number of genes failed to support several previously proposed promoter SNPs discovered with use of reporter gene assays in heterologous tissues, while srSNPs appear more frequent than expected. Computational analysis of mRNA folding indicates that ∼90% of srSNPs affects mRNA folding, and hence potentially function. Our results indicate that both rSNPs and srSNPs represent a still largely untapped reservoir of variants that contribute to human phenotypic diversity.
PMCID: PMC2779843  PMID: 18698231

Results 1-6 (6)