Amyloid precursor protein is a restriction factor that protects against Zika virus infection in mammalian brains.

Zika virus (ZIKV) is a neurotropic flavivirus that causes several diseases including birth defects such as microcephaly. Intrinsic immunity is known to be a frontline defense against viruses through host anti-viral restriction factors. Limited knowledge is available on intrinsic immunity against ZIKV in brains. Amyloid precursor protein (APP) is predominantly expressed in brains and implicated in the pathogenesis of Alzheimer's diseases. We have found that ZIKV interacts with APP, and viral infection increases APP expression via enhancing protein stability. Moreover, we identified the viral peptide, HGSQHSGMIVNDTGHETDENRAKVEITPNSPRAEATLGGFGSLGL, which is capable of en-hancing APP expression. We observed that aging brain tissues with APP had protective effects on ZIKV infection by reducing the availability of the viruses. Also, knockdown of APP expression or blocking ZIKV-APP interactions enhanced ZIKV replication in human neural progenitor/stem cells. Finally, intracranial infection of ZIKV in APP-null neonatal mice resulted in higher mortality and viral yields. Taken together, these findings suggest that APP is a restriction factor that protects against ZIKV by serving as a decoy receptor, and plays a protective role in ZIKV-mediated brain injuries.

Subcutaneous Administration of AMD3100 into Mice Models of Alzheimer's Disease Ameliorated Cognitive Impairment, Reduced Neuroinflammation, and Improved Pathophysiological Markers.

BACKGROUND: Alzheimer's disease (AD), the prevalent dementia in the elderly, involves many related and interdependent pathologies that manifest simultaneously, leading to cognitive impairment and death. Amyloid-ß (Aß) accumulation in the brain triggers the onset of AD, accompanied by neuroinflammatory response and pathological changes. The CXCR4/CXCL12 (SDF1) axis is one of the major signal transduction cascades involved in the inflammation process and regulation of homing of hematopoietic stem cells (HSCs) within the bone marrow niche. Inhibition of the axis with AMD3100, a reversible antagonist of CXCR4 mobilizes endogenous HSCs from the bone marrow into the periphery, facilitating the recruitment of bone marrow-derived microglia-like cells into the brain, attenuates the neuroinflammation process that involves release of excitotoxic markers such as TNFα, intracellular Ca2 +, and glutamate and upregulates monocarboxylate transporter 1, the major L-lactate transporter in the brain. OBJECTIVE: Herein, we investigate if administration of a combination of AMD3100 and L-lactate may have beneficial effects in the treatment of AD. METHODS: We tested the feasibility of the combined treatment for short- and long-term efficacy for inducing endogenous stem cells' mobilization and attenuation of neuroinflammation in two distinct amyloid-ß-induced AD mouse models. RESULTS: The combined treatment did not demonstrate any adverse effects on the mice, and resulted in a significant improvement in cognitive/memory functions, attenuated neuroinflammation, and alleviated AD pathologies compared to each treatment alone. CONCLUSION: This study showed AMD3100's beneficial effect in ameliorating AD pathogenesis, suggesting an alternative to the multistep procedures of transplantation of stem cells in the treatment of AD.

Hyperbaric oxygen therapy ameliorates pathophysiology of 3xTg-AD mouse model by attenuating neuroinflammation.

There is a real need for new interventions for Alzheimer's disease (AD). Hyperbaric oxygen therapy (HBOT), the medical administration of 100% oxygen at conditions greater than 1 atmosphere absolute, has been used successfully to treat several neurological conditions, but its effects on AD pathology have never been thoroughly examined. Therefore, we exposed old triple-transgenic (3xTg) and non-transgenic mice to HBOT followed by behavioral, histological, and biochemical analyses. HBOT attenuated neuroinflammatory processes by reducing astrogliosis, microgliosis, and the secretion of proinflammatory cytokines (IL-1ß and TNFα) and increasing expression of scavenger receptor A, arginase1, and antiinflammatory cytokines (IL-4 and IL-10). Moreover, HBOT reduced hypoxia, amyloid burden, and tau phosphorylation in 3xTg mice and ameliorated their behavioral deficits. Therefore, we suggest that HBOT has multifaceted effects that reduce AD pathologies, even in old mice. Given that HBOT is used in the clinic to treat various indications, including neurological conditions, these results suggest HBOT as a novel therapeutic intervention for AD.

Chronic administration of AMD3100 increases survival and alleviates pathology in SOD1(G93A) mice model of ALS.

BACKGROUND: Amyotrophic lateral sclerosis (ALS) is a progressive fatal neurodegenerative disease, involving both upper and lower motor neurons. The disease is induced by multifactorial pathologies, and as such, it requires a multifaceted therapeutic approach. CXCR4, a chemokine receptor widely expressed in neurons and glial cells and its ligand, CXCL12, also known as stromal-cell-derived factor (SDF1), modulate both neuronal function and apoptosis by glutamate release signaling as well as hematopoietic stem and progenitor cells (HSPCs) migration into the blood and their homing towards injured sites. Inhibition approaches towards the CXCR4/CXCL12 signaling may result in preventing neuronal apoptosis and alter the HSPCs migration and homing. Such inhibition can be achieved by means of treatment with AMD3100, an antagonist of the chemokine receptor CXCR4. METHODS: We chronically treated male and female transgenic mice model of ALS, SOD1(G93A) mice, with AMD3100. Mice body weight and motor function, evaluated by Rotarod test, were recorded once a week. The most effective treatment regimen was repeated for biochemical and histological analyses in female mice. RESULTS: We found that chronic administration of AMD3100 to SOD1(G93A) mice led to significant extension in mice lifespan and improved motor function and weight loss. In addition, the treatment significantly improved microglial pathology and decreased proinflammatory cytokines in spinal cords of treated female mice. Furthermore, AMD3100 treatment decreased blood-spinal cord barrier (BSCB) permeability by increasing tight junction proteins levels and increased the motor neurons count in the lamina X area of the spinal cord, where adult stem cells are formed. CONCLUSIONS: These data, relevant to the corresponding disease mechanism in human ALS, suggest that blocking CXCR4 by the small molecule, AMD3100, may provide a novel candidate for ALS therapy with an increased safety.

Multifunctional Effect of Human Serum Albumin Reduces Alzheimer's Disease Related Pathologies in the 3xTg Mouse Model.

Alzheimer's disease (AD), the prevalent dementia in the elderly, involves many related and interdependent pathologies that manifests simultaneously, eventually leading to cognitive impairment and death. No treatment is currently available; however, an agent addressing several key pathologies simultaneously has a better therapeutic potential. Human serum albumin (HSA) is a highly versatile protein, harboring multifunctional properties that are relevant to key pathologies underlying AD. This study provides insight into the mechanism for HSA's therapeutic effect. In vivo, a myriad of beneficial effects were observed by pumps infusing HSA intracerebroventricularly, for the first time in an AD 3xTg mice model. A significant effect on amyloid-ß (Aß) pathology was observed. Aß1-42, soluble oligomers, and total plaque area were reduced. Neuroblastoma SHSY5Y cell line confirmed that the reduction in Aß1-42 toxicity was due to direct binding rather than other properties of HSA. Total and hyperphosphorylated tau were reduced along with an increase in tubulin, suggesting increased microtubule stability. HSA treatment also reduced brain inflammation, affecting both astrocytes and microglia markers. Finally, evidence for blood-brain barrier and myelin integrity repair was observed. These multidimensional beneficial effects of intracranial administrated HSA, together or individually, contributed to an improvement in cognitive tests, suggesting a non-immune or Aß efflux dependent means for treating AD.

NPT088 reduces both amyloid-ß and tau pathologies in transgenic mice.

INTRODUCTION: Alzheimer's disease (AD) is characterized by appearance of both extracellular senile plaques and intracellular neurofibrillary tangles, comprised of aggregates of misfolded amyloid-ß (Aß) and hyper-phosphorylated tau, respectively. In a previous study, we demonstrated that g3p, a capsid protein from bacteriophage M13, binds to and remodels misfolded aggregates of proteins that assume an amyloid conformation. We engineered a fusion protein ("NPT088") consisting of the active fragment of g3p and human-IgG1-Fc. METHODS: Aged Tg2576 mice or rTg4510 mice received NPT088 weekly via IP injection. Cognitive and/or functional motor endpoints were monitored during dosing. Pathology was quantified biochemically and immunohistochemically. RESULTS: NPT088-lowered Aß plaque and improved cognitive performance of aged Tg2576 mice. Moreover, NPT088 reduced phospho-tau pathology, reduced brain atrophy, and improved cognition in rTg4510 mice. DISCUSSION: These observations establish NPT088 as a novel therapeutic approach and potential drug class that targets both Aß and tau, the hallmark pathologies of AD.

Mutant SOD1 Increases APP Expression and Phosphorylation in Cellular and Animal Models of ALS.

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease and it is the most common adult onset neurodegenerative disorder affecting motor neurons. There is currently no effective treatment for ALS and our understanding of the pathological mechanism is still far away from prevention and/or treatment of this devastating disease. Amyloid precursor protein (APP) is a transmembrane protein that undergoes processing either by ß-secretase or α-secretase, followed by γ-secretase. In the present study, we show that APP levels, and aberrant phosphorylation, which is associated with enhanced ß-secretase cleavage, are increased in SOD1G93A ALS mouse model. Fluorescence resonance energy transfer (FRET) analysis suggests a close interaction between SOD1 and APP at hippocampal synapses. Notably, SOD1G93A mutation induces APP-SOD1 conformational changes, indicating a crosstalk between these two signaling proteins. Inhibition of APP processing via monoclonal antibody called BBS that blocks APP ß-secretase cleavage site, resulted in reduction of mutant SOD1G93A levels in animal and cellular models of ALS, significantly prolonged life span of SOD1G93A mice and diminished inflammation. Beyond its effect on toxic mutant SOD1G93A, BBS treatment resulted in a reduction in the levels of APP, its processing product soluble APPß and pro-apoptotic p53. This study demonstrates that APP and its processing products contribute to ALS pathology through several different pathways; thus BBS antibody could be a promising neuroprotective strategy for treatment of this disease.

Targeting glioblastoma via intranasal administration of Ff bacteriophages.

Bacteriophages (phages) are ubiquitous viruses that control the growth and diversity of bacteria. Although they have no tropism to mammalian cells, accumulated evidence suggests that phages are not neutral to the mammalian macro-host and can promote immunomodulatory and anti-tumorigenic activities. Here we demonstrate that Ff phages that do not display any proteins or peptides could inhibit the growth of subcutaneous glioblastoma tumors in mice and that this activity is mediated in part by lipopolysaccharide molecules attached to their virion. Using the intranasal route, a non-invasive approach to deliver therapeutics directly to the CNS, we further show that phages rapidly accumulate in the brains of mice and could attenuate progression of orthotopic glioblastoma. Taken together, this study provides new insight into phages non-bacterial activities and demonstrates the feasibility of delivering Ff phages intranasally to treat brain malignancies.

A bacteriophage capsid protein provides a general amyloid interaction motif (GAIM) that binds and remodels misfolded protein assemblies.

Misfolded protein aggregates, characterized by a canonical amyloid fold, play a central role in the pathobiology of neurodegenerative diseases. Agents that bind and sequester neurotoxic intermediates of amyloid assembly, inhibit the assembly or promote the destabilization of such protein aggregates are in clinical testing. Here, we show that the gene 3 protein (g3p) of filamentous bacteriophage mediates potent generic binding to the amyloid fold. We have characterized the amyloid binding and conformational remodeling activities using an array of techniques, including X-ray fiber diffraction and NMR. The mechanism for g3p binding with amyloid appears to reflect its physiological role during infection of Escherichia coli, which is dependent on temperature-sensitive interdomain unfolding and cis-trans prolyl isomerization of g3p. In addition, a natural receptor for g3p, TolA-C, competitively interferes with Aß binding to g3p. NMR studies show that g3p binding to Aß fibers is predominantly through middle and C-terminal residues of the Aß subunit, indicating ß strand-g3p interactions. A recombinant bivalent g3p molecule, an immunoglobulin Fc (Ig) fusion of the two N-terminal g3p domains, (1) potently binds Aß fibers (fAß) (KD=9.4nM); (2); blocks fAß assembly (IC50~50nM) and (3) dissociates fAß (EC50=40-100nM). The binding of g3p to misfolded protein assemblies is generic, and amyloid-targeted activities can be demonstrated using other misfolded protein systems. Taken together, our studies show that g3p(N1N2) acts as a general amyloid interaction motif.

Inhibition of amyloid precursor protein processing leads to downregulation of apoptotic genes in Alzheimer's disease animal models.

BACKGROUND AND METHODS: We previously reported the development of site-directed monoclonal antibodies able to inhibit the initiation of the amyloid precursor protein (APP) processing site, named 'blocking ß site 1' (BBS1). The beneficial effect of intracerebroventricular administration of these antibodies in the triple transgenic mouse model of Alzheimer's disease (AD) showed improvement in cognitive functions and reduction in tau and amyloid pathology. Amyloid-ß may not be the only active component of AD neurotoxicity and may involve other proteolytic APP fragments such as the APP intracellular domain, proposed to work as a transcription factor involved in the regulation of p53 and glycogen synthase kinase 3ß (GSK3ß) as well as affecting several physiological processes contributing to AD pathology. RESULTS: We show that inhibition of the ß-secretase cleavage site via site-directed antibodies resulted in a major reduction in phosphorylated GSK3ß levels, which is the active form of GSK3ß, as well as in p53 levels. CONCLUSION: A therapy that is capable of reducing not only the direct hallmarks of AD but also the components that lead to neuronal apoptosis might have neuroprotective potential in AD treatment.

Beneficial effect of antibodies against ß- secretase cleavage site of APP on Alzheimer's-like pathology in triple-transgenic mice.

The toxicity of amyloid ß and tau, the two hallmark proteins in Alzheimer's disease (AD), has been extensively studied individually. Recently new data suggest their possible interactions and synergistic effects in the disease. In this study, we investigate the ability of antibodies against the ß secretase cleavage site on APP, named BBS1, to affect tau pathology, besides their well established effect on intracellular Aß and amyloid load. For this purpose we treated the triple transgenic mice model of AD (3x Tg-AD) with mAb BBS1 intracerebroventricularly, using mini osmotic pumps for one month. The experimental data demonstrated reduction in total and phosphorylated tau levels, explained by significant reduction in GSK3ß which phosphorylates tau on sites recognized by antibodies against PHF1 and AT-8. The treatment increased the cognitive capabilities and reduced the brain inflammation levels which accompany AD pathology. The data showing that tau pathology was significantly reduced by BBS1 antibodies suggest a close interaction between tau and Aß in the development of AD, and may serve as an efficient novel immunotherapy against both hallmarks of this disease.

Inhibition of amyloid precursor protein beta-secretase cleavage site affects survival and motor functions of amyotrophic lateral sclerosis transgenic mice.

BACKGROUND: Amyotrophic lateral sclerosis (ALS) is a progressive neurodegenerative disease defined by motor neuron loss. Recent studies have reported an increase in amyloid precursor protein (APP) levels and in its cleavage products in ALS patients indicating their possible involvement in this disease. APP is a transmembrane protein processed either by ß-secretase or α-secretase followed by γ-secretase. The APP cleavage products--soluble APP-ß (sAPPß), amyloidogenic Aß, and amino-terminal fragment N-APP--mediate a reduction in synaptic transmission, synaptic loss, neurite retraction and, ultimately, programmed cell death. OBJECTIVE: To elucidate the role of APP cleavage products in the pathology of ALS. METHODS: ALS mouse models that express mutant superoxide dismutase 1 were treated intraventricularly with a monoclonal antibody that blocks the ß-secretase cleavage site on APP. Levels of the APP cleavage product called sAPPß, motor functions and survival were assessed. RESULTS: Inhibition of APP cleavage at a presymptomatic stage resulted in a decrease in the levels of sAPPß, delay of disease onset and deterioration while at the symptomatic stage there was almost no beneficial effect. CONCLUSION: APP cleavage products might contribute to the degeneration in ALS, and early inhibition of the APP process may ameliorate disease progression.

Immunomodulation of AßPP processing alleviates amyloid-ß-related pathology in Alzheimer's disease transgenic mice.

Among the different paradigms aimed at interfering with amyloid-ß (Aß)-related pathology, the attenuation of amyloid-ß protein precursor (AßPP) processing to limit Aß levels seems to be a promising one. Along with the development of BACE1 inhibitors, and the generation of its knock-out mice, accumulating data raise concerns regarding a total inhibition of the enzyme as it shares the processing of other substrates. We described a novel approach to interfere with the specific interaction between AßPP and BACE1 using monoclonal antibodies directed to the ß-secretase cleavage site upon the substrate, AßPP. Such antibodies limit AßPP cleavage in a cellular model of Alzheimer's disease (AD) and avoid the total inhibition of BACE1. Here, we demonstrate the ability of AßPP ß-site antibodies to interfere with Aß production in vivo. Systemic antibody treatment diminished Aß plaques, membrane-associated oligomers, and intracellular Aß accumulation, all of which have been implicated in cellular death and synaptic loss, suggesting that this approach may be an applicable strategy for AD treatment.

Antigen-specific therapy of EAE via intranasal delivery of filamentous phage displaying a myelin immunodominant epitope.

The presence of anti-myelin antibodies (Abs) in patients with early multiple sclerosis (MS) and in MS animal models has led to renewed interest in the role of B cells, plasma cells and their products in the pathogenesis of the disease, and in their therapeutic potential. Here, we present a novel strategy based on filamentous phage display of the myelin oligodendrocyte glycoprotein immunodominant epitope (MOG 36-44) fused to the main coat protein. Filamentous phages are well characterized, both structurally and genetically. We found that the fibrous shape of the phage (1000 nm long and 6 nm wide) enables penetration into the central nervous system (CNS) when administered nasally. Thus, intranasal treatment of experimental autoimmune encephalomyelitis (EAE) in mice, with phage MOG, showed improved neuronal function, reduced levels of proinflammatory cytokines, particularly monocyte chemoattractant protein 1 (MCP-1), interferon gamma (IFN-gamma) and IL-6, but no change in IL-10 or IL-12 levels. Moreover, the treatment induced depletion of the autoantibodies against MOG and prevented demyelination resulting in improved clinical scores and the reduced inflammation in the CNS and periphery in EAE mice compared to untreated sick animals.

Immunotherapy for Alzheimer's disease.

The amyloid cascade hypothesis states that overproduction of amyloid-beta peptide (Abeta/AbetaP) or failure to clear this peptide, leads to Alzheimer's disease (AD) primarily through amyloid deposition, presumed to be involved in neurofibrillary tangle formation; these lesions are then associated with cell death which is reflected in memory impairment, the hallmarks of this dementia. The abundant evidence that Abeta aggregation/oligomerization is an essential early event in AD pathogenesis has prompted intensive search for therapeutics that target various conformations of Abeta. Several labs have bred AD diseased models of transgenic mice that produce human Abeta and develop plaques and neuron damage in their brains as well as immunological aspects of the disease pathogenesis. The immune system appears to participate in AD pathogenesis. There is evidence for partial tolerance against Abeta in mutant amyloid precursor protein (APP) transgenic mice as well as in AD patients. Animal models of the disease enabled the immunological concept for treatment of conformational diseases to gain more attention and immunization approaches are being pursued in order to stimulate clearance of brain amyloid plaques. In spite of the first clinical setback, this research field has clearly strengthened the hypothesis that Abeta plays a central role in AD and has stimulated a new area for development of Alzheimer's therapeutics. The renewed human phase clinical trials toward improved immunotherapeutic strategies which maintain the beneficial effects without adverse side effects are under further evaluation.

Filamentous phages reduce alpha-synuclein oligomerization in the membrane fraction of SH-SY5Y cells.

BACKGROUND: alpha-Synuclein (AS) is an abundant neuronal protein predominantly localized in presynaptic terminals in the central nervous system. AS aggregation is a molecular hallmark of several neurodegenerative diseases, including Parkinson's disease, and is thought to play a significant role in the etiology of the disease. Recent experimental evidence indicates that AS exists in two forms, a membrane-bound form and a disordered cytosolic form. Much effort is dedicated to prevent and dissolve AS aggregates, specifically AS oligomers and protofibrils, which are thought to be the more toxic form of aggregates. METHODS: The effect of filamentous phages on AS aggregation in SH-SY5Y cells overexpressing wild-type AS was quantified in ELISA designed to detect and quantify AS oligomers. RESULTS: We found reduced levels of AS oligomers in the membrane fraction in cells treated with filamentous phages compared to nontreated cells. CONCLUSION: The reduction in AS oligomers from the plasma membrane in treated cells may suggest further therapeutic application.

TAR1, a human anti-p53 single-chain antibody, restores tumor suppressor function to mutant p53 variants.

The tumor suppressor gene p53 is mutated in more than half of human tumors. One important characteristic of p53 mutants is their accumulation in the nucleus of cancer cells. Thus, reactivation of mutant p53 proteins may trigger massive apoptosis in tumor cells. Pharmacologic methods are currently under development to induce mutant p53 proteins to resume their wild-type function. We have identified a human single-chain Fv fragment, designated as transcriptional transactivation and apoptosis restoring (TAR1), which specifically and with high affinity binds to mutant p53 and restores its wild-type active conformation. Binding of TAR1 to mutant p53 induced transcriptional transactivation of p53 target genes and down-regulation of mutant p53 transcriptional target genes. TAR1 treatment induced apoptosis in a variety of cell lines endogenously expressing p53 carrying different point mutations DNA contact or structural p53 mutants. Moreover, in an animal model of mice carrying human xenografts, TAR1 induced tumor regression with no apparent deleterious side effects. Thus, it may be considered as a potential candidate for anticancer treatment, targeting tumors with mutant p53.

Immunotherapeutic strategies for Alzheimer's disease treatment.

Naturally occurring antibodies against amyloid-beta peptides have been found in human cerebrospinal fluid and in the plasma of healthy individuals, but were significantly lower in Alzheimer's disease (AD) patients, suggesting that AD may be an immunodeficient disorder. The performance of anti-amyloid-beta antibodies in transgenic mice models of AD showed that they are delivered to the central nervous system, preventing and dissolving amyloid-beta plaques. Moreover, these antibodies protected the mice from learning and age-related memory deficits. Active and/or passive immunization against the amyloid-beta peptide has been proposed as a method for preventing and/or treating AD. Immunotherapy represents fascinating ways to test the amyloid hypothesis and offers genuine opportunities for AD treatment, but requires careful antigen and antibody selection to maximize efficacy and minimize adverse events.