MyD88 deficiency ameliorates ß-amyloidosis in an animal model of Alzheimer's disease.

The accumulation of ß-amyloid protein (Aß) in the brain is thought to be a primary etiologic event in Alzheimer's disease (AD). Fibrillar Aß plaques, a hallmark of AD abnormality, are closely associated with activated microglia. Activated microglia have contradictory roles in the pathogenesis of AD, being either neuroprotective (by clearing harmful Aß and repairing damaged tissues) or neurotoxic (by producing proinflammatory cytokines and reactive oxygen species). Aß aggregates can activate microglia by interacting with multiple toll-like receptors (TLRs), the pattern-recognition receptors of the innate immune system. Because the adapter protein MyD88 is essential for the downstream signaling of all TLRs, except TLR3, we investigated the effects of MyD88 deficiency (MyD88(-/-)) on Aß accumulation and microglial activation in an AD mouse model. MyD88 deficiency decreased Aß load and microglial activation in the brain. The decrease in Aß load in an MyD88(-/-) AD mouse model was associated with increased and decreased protein expression of apolipoprotein E (apoE) and CX3CR1, respectively, compared with that in an MyD88 wild-type AD mouse model. These results suggest that MyD88 deficiency may reduce Aß load by enhancing the phagocytic capability of microglia through fractalkine (the ligand of CX3CR1) signaling and by promoting apoE-mediated clearance of Aß from the brain. These findings also suggest that chronic inflammatory responses induced by Aß accumulation via the MyD88-dependent signaling pathway exacerbate ß-amyloidosis in AD.

TLR4 mutation reduces microglial activation, increases Aß deposits and exacerbates cognitive deficits in a mouse model of Alzheimer's disease.

BACKGROUND: 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 Aß 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 Aß deposits and buffer-soluble Aß 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 Aß 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 Aß deposits start. METHODS: Microglial activation and amyloid deposition in the brain were determined by immunohistochemistry in the AD models. Levels of cerebral soluble Aß were determined by ELISA. mRNA levels of cytokines and chemokines in the brain and Aß-stimulated monocytes were quantified by real-time PCR. Cognitive functions were assessed by the Morris water maze. RESULTS: While no difference was found in cerebral Aß 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 Aß deposition and soluble Aß42 in the brain, which were associated with decrements in cognitive functions and expression levels of IL-1ß, CCL3, and CCL4 in the hippocampus compared to TLR4W Tg mice. TLR4 mutation diminished Aß-induced IL-1ß, CCL3, and CCL4 expression in monocytes. CONCLUSION: This is the first demonstration of TLR4-dependent activation of microglia at the early stage of ß-amyloidosis. Our results indicate that TLR4 is not involved in the initiation of Aß deposition and that, as Aß deposits start, microglia are activated via TLR4 signaling to reduce Aß deposits and preserve cognitive functions from Aß-mediated neurotoxicity.

A Multifunctional Therapy Approach for Cancer: Targeting Raf1- Mediated Inhibition of Cell Motility, Growth, and Interaction with the Microenvironment.

Prostate cancer cells move from their primary site of origin, interact with a distant microenvironment, grow, and thereby cause death. It had heretofore not been possible to selectively inhibit cancer cell motility. Our group has recently shown that inhibition of intracellular activation of Raf1 with the small-molecule therapeutic KBU2046 permits, for the first time, selective inhibition of cell motility. We hypothesized that simultaneous disruption of multiple distinct functions that drive progression of prostate cancer to induce death would result in advanced disease control. Using a murine orthotopic implantation model of human prostate cancer metastasis, we demonstrate that combined treatment with KBU2046 and docetaxel retains docetaxel's antitumor action, but provides improved inhibition of metastasis, compared with monotherapy. KBU2046 does not interfere with hormone therapy, inclusive of enzalutamide-mediated inhibition of androgen receptor (AR) function and cell growth inhibition, and inclusive of the ability of castration to inhibit LNCaP-AR cell outgrowth in mice. Cell movement is necessary for osteoclast-mediated bone degradation. KBU2046 inhibits Raf1 and its downstream activation of MEK1/2 and ERK1/2 in osteoclasts, inhibiting cytoskeleton rearrangement, resorptive cavity formation, and bone destruction in vitro, with improved effects observed when the bone microenvironment is chemically modified by pretreatment with zoledronic acid. Using a murine cardiac injection model of human prostate cancer bone destruction quantified by CT, KBU2046 plus zoledronic exhibit improved inhibitory efficacy, compared with monotherapy. The combined disruption of pathways that drive cell movement, interaction with bone, and growth constitutes a multifunctional targeting strategy that provides advanced disease control.

Genistein treatment duration effects biomarkers of cell motility in human prostate.

BACKGROUND: Long term dietary consumption of genistein by Chinese men is associated with decreased PCa metastasis and mortality. Short term treatment of US men with prostate cancer (PCa) with genistein decreases MMP-2 in prostate tissue. MEK4 regulates MMP-2 expression, drives PCa metastasis, and genistein inhibits MEK4, decreases MMP-2 expression and dietary dosing inhibits human PCa metastasis in mice. This study examines short- versus long-term treatment effects of genistein in humans and in vitro. METHODS AND FINDINGS: US men with localized PCa were treated on a phase II trial with genistein (N = 14) versus not (N = 14) for one month prior to radical prostatectomy. Prostate epithelial cells were removed from fresh frozen tissue by laser capture microdissection, and the expression of 12,000 genes profiled. Genistein significantly altered the expression of four genes, three had established links to cancer cell motility and metastasis. Of these three, one was a non-coding transcript, and the other two were BASP1 and HCF2. Genistein increased BASP1 expression in humans, and its engineered over expression and knockdown demonstrated that it suppressed cell invasion in all six human prostate cell lines examined. Genistein decreased HCF2 expression in humans, and it was shown to increase cell invasion in all cell lines examined. The expression of MMP-2, MEK4 and BASP1 was then measured in formalin fixed prostate tissue from N = 38 Chinese men living in China and N = 41 US men living in the US, both cohorts with localized PCa. MMP-2 was 52% higher in Chinese compared to US tissue (P < 0.0001), MEK4 was 48% lower (P < 0.0001), and BASP1 was unaltered. Treatment of PC3 human PCa cells in vitro for up to 8 weeks demonstrated that short term genistein treatment decreased MMP-2, while long term treatment increased it, both changes being significant (P<0.05) compared to untreated control cells. Long term genistein-treated cells retained their responsiveness to genistein's anti-motility effect. CONCLUSIONS: Genistein inhibits pathways in human prostate that drive transformation to a lethal high motility phenotype. Long term treatment induces compensatory changes in biomarkers of efficacy. The current strategy of using such biomarkers after short term intervention as go/no-go determinants in early phase chemoprevention trials should be carefully examined.

Precision therapeutic targeting of human cancer cell motility.

Increased cancer cell motility constitutes a root cause of end organ destruction and mortality, but its complex regulation represents a barrier to precision targeting. We use the unique characteristics of small molecules to probe and selectively modulate cell motility. By coupling efficient chemical synthesis routes to multiple upfront in parallel phenotypic screens, we identify that KBU2046 inhibits cell motility and cell invasion in vitro. Across three different murine models of human prostate and breast cancer, KBU2046 inhibits metastasis, decreases bone destruction, and prolongs survival at nanomolar blood concentrations after oral administration. Comprehensive molecular, cellular and systemic-level assays all support a high level of selectivity. KBU2046 binds chaperone heterocomplexes, selectively alters binding of client proteins that regulate motility, and lacks all the hallmarks of classical chaperone inhibitors, including toxicity. We identify a unique cell motility regulatory mechanism and synthesize a targeted therapeutic, providing a platform to pursue studies in humans.

Removal of arsenic from drinking water by chemical precipitation--a modeling and simulation study of the physical-chemical processes.

A dynamic mathematical model was developed for removal of arsenic from drinking water by chemical coagulation-precipitation and was validated experimentally in a bench-scale set-up. While examining arsenic removal efficiency of the scheme under different operating conditions, coagulant dose, pH and degree of oxidation were found to have pronounced impact. Removal efficiency of 91-92% was achieved for synthetic feed water spiked with 1 mg/L arsenic and pre-oxidized by potassium permanganate at optimum pH and coagulant dose. Model predictions corroborated well with the experimental findings (the overall correlation coefficient being 0.9895) indicating the capability of the model in predicting performance of such a treatment plant under different operating conditions. Menu-driven, user-friendly Visual Basic software developed in the study will be very handy in quick performance analysis. The simulation is expected to be very useful in full-scale design and operation of the treatment plants for removal of arsenic from drinking water.

Catalytic immunoglobulin gene delivery in a mouse model of Alzheimer's disease: prophylactic and therapeutic applications.

Accumulation of amyloid beta-peptide (Aß) in the brain is hypothesized to be a causal event leading to dementia in Alzheimer's disease (AD). Aß vaccination removes Aß deposits from the brain. Aß immunotherapy, however, may cause T cell- and/or Fc-receptor-mediated brain inflammation and relocate parenchymal Aß deposits to blood vessels leading to cerebral hemorrhages. Because catalytic antibodies do not form stable immune complexes and Aß fragments produced by catalytic antibodies are less likely to form aggregates, Aß-specific catalytic antibodies may have safer therapeutic profiles than reversibly-binding anti-Aß antibodies. Additionally, catalytic antibodies may remove Aß more efficiently than binding antibodies because a single catalytic antibody can hydrolyze thousands of Aß molecules. We previously isolated Aß-specific catalytic antibody, IgVL5D3, with strong Aß-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 Aß load in the ipsilateral hippocampus of AD model mice. No evidence of hemorrhages, increased vascular amyloid deposits, increased proinflammatory cytokines, or infiltrating T-cells in the brains was found in the experimental animals. AAV9-mediated anti-Aß catalytic antibody brain delivery can be prophylactic and therapeutic options for AD.

CD95 and CD95L promote and protect cancer stem cells.

CD95 (APO-1/Fas) is a death receptor used by immune cells to kill cancer cells through induction of apoptosis. However, the elimination of CD95 or its ligand, CD95L, from cancer cells results in death induced by CD95R/L elimination (DICE), a type of cell death that resembles a necrotic form of mitotic catastrophe suggesting that CD95 protects cancer cells from cell death. We now report that stimulation of CD95 on cancer cells or reducing miR-200c levels increases the number of cancer stem cells (CSCs), which are more sensitive to induction of DICE than non-CSC, while becoming less sensitive to CD95-mediated apoptosis. In contrast, induction of DICE or overexpression of miR-200c reduces the number of CSCs. We demonstrate that CSCs and non-CSCs have differential sensitivities to CD95-mediated apoptosis and DICE, and that killing of cancer cells can be maximized by concomitant induction of both cell death mechanisms.

Death induced by CD95 or CD95 ligand elimination.

CD95 (Fas/APO-1), when bound by its cognate ligand CD95L, induces cells to die by apoptosis. We now show that elimination of CD95 or CD95L results in a form of cell death that is independent of caspase-8, RIPK1/MLKL, and p53, is not inhibited by Bcl-xL expression, and preferentially affects cancer cells. All tumors that formed in mouse models of low-grade serous ovarian cancer or chemically induced liver cancer with tissue-specific deletion of CD95 still expressed CD95, suggesting that cancer cannot form in the absence of CD95. Death induced by CD95R/L elimination (DICE) is characterized by an increase in cell size, production of mitochondrial ROS, and DNA damage. It resembles a necrotic form of mitotic catastrophe. No single drug was found to completely block this form of cell death, and it could also not be blocked by the knockdown of a single gene, making it a promising way to kill cancer cells.

Muscle-directed anti-Aß single-chain antibody delivery via AAV1 reduces cerebral Aß load in an Alzheimer's disease mouse model.

We previously reported that anti-amyloid-beta (Aß) single-chain antibody (scFv59) brain delivery via recombinant adeno-associated virus (rAAV) was effective in reducing cerebral Aß load in an Alzheimer's disease (AD) mouse model without inducing inflammation. Here, we investigated the prophylactic effects and mechanism of a muscle-directed gene therapy modality in an AD mouse model. We injected rAAV serotype 1 encoding scFv59 into the right thigh muscles of 3-month-old mice. Nine months later, high levels of scFv59 expression were confirmed in the thigh muscles by both immunoblotting and immunohistochemistry. As controls, model mice were similarly injected with rAAV1 encoding antihuman immunodeficiency virus Gag antibody (scFvGag). AAV1-mediated scFv59 gene delivery was effective in decreasing Aß deposits in the brain. Compared with the scFvGag group, levels of Aß in cerebrospinal fluid (CSF) decreased significantly while Aß in serum tended to increase in the scFv59 group. AAV1-mediated scFv59 gene delivery may alter the equilibrium of Aß between the blood and brain, resulting in an increased efflux of Aß from the brain owing to antibody-mediated sequestration/clearance of peripheral Aß. Our results suggest that muscle-directed scFv59 delivery via rAAV1 may be a prophylactic option for AD and that levels of CSF Aß may be used to evaluate the efficacy of anti-Aß immunotherapy.

Combined treatment of Aß immunization with statin in a mouse model of Alzheimer's disease.

We evaluated the therapeutic efficacy of combined treatment of Aß-immunization with simvastatin in an Alzheimer mouse model at age 22 months. DNA prime-adenovirus boost immunization induced modest anti-Aß titers and simvastatin increased the seropositive rate. Aß-KLH was additionally administered to boost the titers. Irrespective of simvastatin, the immunization did not decrease cerebral Aß deposits but increased soluble Aß and tended to exacerbate amyloid angiopathy in the hippocampus. The immunization increased cerebral invasion of leukocytes and simvastatin counteracted the increase. Thus, modest anti-Aß titers can increase soluble Aß and simvastatin may reduce inflammation associated with vaccination in aged Alzheimer mouse models.

The effects of MyD88 deficiency on exploratory activity, anxiety, motor coordination, and spatial learning in C57BL/6 and APPswe/PS1dE9 mice.

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 Aß deposits and soluble Aß in the brain as compared with MyD88 sufficient AD mouse models. Because accumulation of Aß 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.

Anti-Amyloid-ß Single-Chain Antibody Brain Delivery Via AAV Reduces Amyloid Load But May Increase Cerebral Hemorrhages in an Alzheimer's Disease Mouse Model.

Accumulation of amyloid-ß protein (Aß) in the brain is thought to be a causal event in Alzheimer's disease (AD). Immunotherapy targeting Aß holds great promise for reducing Aß in the brain. Here, we evaluated the efficacy and safety of anti-Aß single-chain antibody (scFv59) delivery via recombinant adeno-associated virus (rAAV) on reducing Aß deposits in an AD mouse model (TgAßPPswe/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 TgAßPPswe/PS1dE9 mice. rAAV5 was similarly injected into the ventricle of 10-month-old TgAßPPswe/PS1dE9 mice and 5 months later its efficacy and safety were evaluated. Immunoreactive Aß deposits reduced in the hippocampus. Aß42 levels in cerebrospinal fluid (CSF) tended to increase and the Aß40 : 42 ratio decreased in CSF, suggesting that Aß42 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 Aß, caution for cerebrovascular effects should be exercised when rAAV-mediated anti-Aß immunotherapy is applied.