Investigating Immune Responses to the scAAV9-HEXM Gene Therapy Treatment in Tay–Sachs Disease and Sandhoff Disease Mouse Models

GM2 gangliosidosis disorders are a group of neurodegenerative diseases that result from a functional deficiency of the enzyme β-hexosaminidase A (HexA). HexA consists of an α- and β-subunit; a deficiency in either subunit results in Tay–Sachs Disease (TSD) or Sandhoff Disease (SD), respectively. Viral vector gene transfer is viewed as a potential method of treating these diseases. A recently constructed isoenzyme to HexA, called HexM, has the ability to effectively catabolize GM2 gangliosides in vivo. Previous gene transfer studies have revealed that the scAAV9-HEXM treatment can improve survival in the murine SD model. However, it is speculated that this treatment could elicit an immune response to the carrier capsid and “non-self”-expressed transgene. This study was designed to assess the immunocompetence of TSD and SD mice, and test the immune response to the scAAV9-HEXM gene transfer. HexM vector-treated mice developed a significant anti-HexM T cell response and antibody response. This study confirms that TSD and SD mouse models are immunocompetent, and that gene transfer expression can create an immune response in these mice. These mouse models could be utilized for investigating methods of mitigating immune responses to gene transfer-expressed “non-self” proteins, and potentially improve treatment efficacy.     

Analysis of tocopherols and phytosterols in vegetable oils by HPLC with evaporative light-scattering detection

Methods were developed for the separation, detection, and quantification of tocopherols and phytosterols by high-performance liquid chromatography with an evaporative light-scattering detector. Four tocopherols— α, β, γ and δ—and four phytosterols—campesterol, β-sitosterol, brassicasterol, and stigmasterol—were analyzed in soybean, sunflower, low-erucic acid rapeseed (LEAR) and corn oils. The use of an evaporative light-scattering detector, in conjunction with modification of methods from the literature to prepare and analyze tocopherols and phytosterols by HPLC, showed consistent results between trials and levels of these minor constituents. Presented at the Annual American Oil Chemists' Society Meeting, May 3–7, 1989, Cincinnati, OH.     

Design,Synthesis, and Biological Evaluation of Enantiomeric β‐N‐Acetylhexosaminidase Inhibitors LABNAc and DABNAc as Potential Agents against Tay‐Sachs and Sandhoff Disease

Combating glycolipid storage disorders : LABNAc was prepared in an efficient 11‐step procedure from D ‐lyxonolactone. The enantiomer DABNAc was also prepared from L ‐lyxonolactone. Preliminary cellular studies indicate that these compounds may find utility as chemical chaperones for the treatment of Tay‐Sachs and Sandhoff diseases.

     

Role of Lipids in the Onset,Progression and Treatment of Periodontal Disease. A Systematic Review of Studies in Humans

The risk of different oral problems (root caries, tooth mobility, and tooth loss) can be increased by the presence of periodontal disease, which has also been associated with a growing list of systemic diseases. The presence of some bacteria is the primary etiology of this disease; a susceptible host is also necessary for disease initiation. In this respect, the progression of periodontal disease and healing of the periodontal tissues can be modulated by nutritional status. To clarify the role of lipids in the establishment, progression, and/or treatment of this pathology, a systematic review was conducted of English-written literature in PubMed until May 2016, which included research on the relationship of these dietary components with the onset and progression of periodontal disease. According to publication type, randomized-controlled trials, cohort, case-control and cross-sectional studies were included. Among all the analyzed components, those that have any effect on oxidative stress and/or inflammation seem to be the most interesting according to current evidence. On one hand, there is quite a lot of information in favor of a positive role of n-3 fatty acids, due to their antioxidant and immunomodulatory effects. On the other hand, saturated fat-rich diets increase oxidative stress as well the as intensity and duration of inflammatory processes, so they must be avoided.     

Oxidation kinetics of β-carotene in oleic acid solvent with addition of an antioxidant, α-tocopherol

Oxidation experiments with β-carotene were performed in oleic acid solvent with addition of an antioxidant, α-tocopherol. A kinetic model was proposed based on a reaction mechanism consisting of the oxidation of β-carotene, oleic acid, and α-tocopherol; the antioxidation reactions of β-carotene and oleic acid by α-tocopherol; the cross-reaction of β-carotene and oleic acid; and the radical-exchange reaction of β-carotene and α-tocopherol. The model quantitatively described the oxidation behavior of β-carotene over a wide range of temperatures, oxygen compositions, and initial antioxidant concentrations. The model simulated well the time over which β-carotene was almost totally consumed under practical storage conditions at room temperature in air.     

Inhibition of oxidation in 10% oil-in-water emulsions by β-carotene with α- and γ-tocopherols

The effects of low concentrations of β-carotene, α-, and γ-tocopherol were evaluated on autoxidation of 10% oil-in-water emulsions of rapeseed oil triacylglycerols. At concentrations of 0.45, 2, and 20 μg/g, β-carotene was a prooxidant, based on the formation of lipid hydroperoxides, hexanal, or 2-heptenal. In this emulsion, 1.5, 3, and 30 μg/g of γ-tocopherol, as well as 1.5 μg/g of α-tocopherol, acted as antioxidants and inhibited both the formation and decomposition of lipid hydroperoxides. Moreover, at a level of 1.5 μg/g, γ-tocopherol was more effective as an antioxidant than α-tocopherol. At levels of 0.5 μg/g, both α- and γ-tocopherol significantly inhibited the formation of hexanal but not the formation of lipid hydroperoxides. Oxidation was effectively retarded by combinations of 2 μg/g β-carotene and 1.5 μg/g γ- or α-tocopherol. The combination of β-carotene and α-tocopherol was significantly better in retarding oxidation than α-tocopherol alone. While γ-tocopherol was an effective antioxidant, a synergistic effect between β-carotene and γ-tocopherol could not be shown. The results indicate that there is a need to protect β-carotene from oxidative destruction by employing antioxidants, such as α- and γ-tocopherol, should β-carotene be used in fat emulsions.     

Systemic Dietary Hesperidin Modulation of Osteoclastogenesis,Bone Homeostasis and Periodontal Disease in Mice

This study aimed to evaluate the effects of hesperidin (HE) on in vitro osteoclastogenesis and dietary supplementation on mouse periodontal disease and femoral bone phenotype. RAW 264.7 cells were stimulated with RANKL in the presence or absence of HE (1, 100 or 500 µM) for 5 days, and evaluated by TRAP, TUNEL and Western Blot (WB) analyses. In vivo, C57BL/6 mice were given HE via oral gavage (125, 250 and 500 mg/kg) for 4 weeks. A sterile silk ligature was placed between the first and second right maxillary molars for 10 days and microcomputed tomography (μCT), histopathological and immunohistochemical evaluation were performed. Femoral bones subjected or not to dietary HE (500 mg/kg) for 6 and 12 weeks were evaluated using μCT. In vitro, HE 500 µM reduced formation of RANKL-stimulated TRAP-positive(+) multinucleated cells (500 µM) as well as c-Fos and NFATc1 protein expression (p < 0.05), markers of osteoclasts. In vivo, dietary HE 500 mg/kg increased the alveolar bone resorption in ligated teeth (p < 0.05) and resulted in a significant increase in TRAP+ cells (p < 0.05). Gingival inflammatory infiltrate was greater in the HE 500 mg/kg group even in the absence of ligature. In femurs, HE 500 mg/kg protected trabecular and cortical bone mass at 6 weeks of treatment. In conclusion, HE impaired in vitro osteoclastogenesis, but on the contrary, oral administration of a high concentration of dietary HE increased osteoclast numbers and promoted inflammation-induced alveolar bone loss. However, HE at 500 mg/kg can promote a bone-sparing effect on skeletal bone under physiological conditions.     

Ingested Ketone Ester Leads to a Rapid Rise of Acetyl-CoA and Competes with Glucose Metabolism in the Brain of Non-Fasted Mice

The role of ketone bodies in the cerebral energy homeostasis of neurological diseases has begun to attract recent attention particularly in acute neurological diseases. In ketogenic therapies, ketosis is achieved by either a ketogenic diet or by the administration of exogenous ketone bodies. The oral ingestion of the ketone ester (KE), (R)-3-hydroxybutyl (R)-3-hydroxybutyrate, is a new method to generate rapid and significant ketosis (i.e., above 6 mmol/L) in humans. KE is hydrolyzed into β-hydroxybutyrate (βHB) and its precursor 1,3-butanediol. Here, we investigate the effect of oral KE administration (3 mg KE/g of body weight) on brain metabolism of non-fasted mice using liquid chromatography in tandem with mass spectrometry. Ketosis (Cmax = 6.83 ± 0.19 mmol/L) was obtained at Tmax = 30 min after oral KE-gavage. We found that βHB uptake into the brain strongly correlated with the plasma βHB concentration and was preferentially distributed in the neocortex. We showed for the first time that oral KE led to an increase of acetyl-CoA and citric cycle intermediates in the brain of non-fasted mice. Furthermore, we found that the increased level of acetyl-CoA inhibited glycolysis by a feedback mechanism and thus competed with glucose under physiological conditions. The brain pharmacodynamics of this oral KE strongly suggest that this agent should be considered for acute neurological diseases.     

Dysfunction of Mitochondria in Alzheimer’s Disease: ANT and VDAC Interact with Toxic Proteins and Aid to Determine the Fate of Brain Cells

Alzheimer’s disease (AD), certainly the most widespread proteinopathy, has as classical neuropathological hallmarks, two groups of protein aggregates: senile plaques and neurofibrillary tangles. However, the research interest is rapidly gaining ground in a better understanding of other pathological features, first, of all the mitochondrial dysfunctions. Several pieces of evidence support the hypothesis that abnormal mitochondrial function may trigger aberrant processing of amyloid progenitor protein or tau and thus neurodegeneration. Here, our aim is to emphasize the role played by two ‘bioenergetic’ proteins inserted in the mitochondrial membranes, inner and outer, respectively, that is, the adenine nucleotide translocator (ANT) and the voltage-dependent anion channel (VDAC), in the progression of AD. To perform this, we will magnify the ANT and VDAC defects, which are measurable hallmarks of mitochondrial dysfunction, and collect all the existing information on their interaction with toxic Alzheimer’s proteins. The pathological convergence of tau and amyloid β-peptide (Aβ) on mitochondria may finally explain why the therapeutic strategies used against the toxic forms of Aβ or tau have not given promising results separately. Furthermore, the crucial role of ANT-1 and VDAC impairment in the onset/progression of AD opens a window for new therapeutic strategies aimed at preserving/improving mitochondrial function, which is suspected to be the driving force leading to plaque and tangle deposition in AD.     

Single antibody domains as small recognition units: design and in vitro antigen selection of camelized, human VH domains with improved protein stability

Folding stabilities of camelized human antibody VH domains werestudied through the determination of their melting points inthermodenaturation experiments. The melting point of a VH domainoriginating from a synthetic library of human VHs, which hadbeen optimized for the use as small recognition units throughthe mimicking of camelid antibody heavy chains occurring naturallywithout light chain, was 56.6C compared with 71.2C of theoriginal human VH. Its stability was improved (melting point61.6C) through three mutations to mimic camelid VHs even further:Va137 was replaced by phenylalanine and two cysteines were introducedat positions 33 and 100b. The resulting VH folded properly andformed a second intradomain disulphide between the extra cysteines.The new mutations were then built constitutively into a phage-displayVH library, from which antigen-specific VHs were selected. Twowere analysed for stability with melting points of 72.6 and75.3C. Thus secondary camelization enabled the isolation ofVHs with improved folding stabilities exceeding even that ofthe original human VH. This indicates an effect on folding stabilityfor some mutations specific in the light chain lacking camelidheavy chains.     

Activation of Endogenous Retrovirus,Brain Infections and Environmental Insults in Neurodegeneration and Alzheimer’s Disease

Chronic neurodegenerative diseases are complex, and their pathogenesis is uncertain. Alzheimer’s disease (AD) is a neurodegenerative brain alteration that is responsible for most dementia cases in the elderly. AD etiology is still uncertain; however, chronic neuroinflammation is a constant component of brain pathology. Infections have been associated with several neurological diseases and viruses of the Herpes family appear to be a probable cause of AD neurodegenerative alterations. Several different factors may contribute to the AD clinical progression. Exogeneous viruses or other microbes and environmental pollutants may directly induce neurodegeneration by activating brain inflammation. In this paper, we suggest that exogeneous brain insults may also activate retrotransposons and silent human endogenous retroviruses (HERVs). The initial inflammation of small brain areas induced by virus infections or other brain insults may activate HERV dis-regulation that contributes to neurodegenerative mechanisms. Chronic HERV activation in turn may cause progressive neurodegeneration that thereafter merges in cognitive impairment and dementia in genetically susceptible people. Specific treatment for exogenous end endogenous pathogens and decreasing pollutant exposure may show beneficial effect in early intervention protocol to prevent the progression of cognitive deterioration in the elderly.     

MicroPET Imaging Assessment of Brain Tau and Amyloid Deposition in 6 × Tg Alzheimer’s Disease Model Mice

Alzheimer’s disease (AD) is characterized by the deposition of extracellular amyloid plaques and intracellular accumulation of neurofibrillary tangles (NFT). Amyloid beta (Aβ) and tau imaging are widely used for diagnosing and monitoring AD in clinical settings. We evaluated the pathology of a recently developed 6 × Tg − AD (6 × Tg) mouse model by crossbreeding 5 × FAD mice with mice expressing mutant (P301L) tau protein using micro-positron emission tomography (PET) image analysis. PET studies were performed in these 6 × Tg mice using [¹⁸F]Flutemetamol, which is an amyloid PET radiotracer; [¹⁸F]THK5351 and [¹⁸F]MK6240, which are tau PET radiotracers; moreover, [¹⁸F]DPA714, which is a translocator protein (TSPO) radiotracer, and comparisons were made with age-matched mice of their respective parental strains. We compared group differences in standardized uptake value ratio (SUVR), kinetic parameters, biodistribution, and histopathology. [¹⁸F]Flutemetamol images showed prominent cortical uptake and matched well with 6E10 staining images from 2-month-old 6 × Tg mice. [¹⁸F]Flutemetamol images showed a significant correlation with [¹⁸F]DPA714 in the cortex and hippocampus. [¹⁸F]THK5351 images revealed prominent hippocampal uptake and matched well with AT8 immunostaining images in 4-month-old 6 × Tg mice. Moreover, [¹⁸F]THK5351 images were confirmed using [¹⁸F]MK6240, which revealed significant correlations in the cortex and hippocampus. Uptake of [¹⁸F]THK5351 or [¹⁸F]MK6240 was highly correlated with [¹⁸F]Flutemetamol in 4-month-old 6 × Tg mice. In conclusion, PET imaging revealed significant age-related uptake of Aβ, tau, and TSPO in 6 × Tg mice, which was highly correlated with age-dependent pathology.     

Intracerebral but Not Peripheral Infection of Live Porphyromonas gingivalis Exacerbates Alzheimer’s Disease Like Amyloid Pathology in APP-TgCRND8 Mice

The impact of oral microbial dysbiosis on Alzheimer’s disease (AD) remains controversial. Building off recent studies reporting that various microbes might directly seed or promote amyloid β (Aβ) deposition, we evaluated the effects of periodontal bacteria (Porphyromonas gingivalis, Treponema denticola) and supragingival commensal (Streptococcus gordonii) oral bacterial infection in the APP-transgenic CRND8 (Tg) mice model of AD. We tracked bacterial colonization and dissemination, and monitored effects on gliosis and amyloid deposition. Chronic oral infection did not accelerate Aβ deposition in Tg mice but did induce alveolar bone resorption, IgG immune response, and an intracerebral astrogliosis (GFAP: glial fibrillary acidic protein). In contrast, intracerebral inoculation of live but not heat-killed P. gingivalis increased Aβ deposition and Iba-1 (ionized calcium-binding adaptor-1) microgliosis after 8 weeks of bacterial infection but not at 4 days. These data show that there may be differential effects of infectious microbes on glial activation and amyloid deposition depending on the species and route of inoculation, and thereby provide an important framework for future studies. Indeed, these studies demonstrate marked effects on amyloid β deposition only in a fairly non-physiologic setting where live bacteria is injected directly into the brain.     

The Function of Transthyretin Complexes with Metallothionein in Alzheimer’s Disease

Alzheimer’s disease (AD) is one of the most frequently diagnosed types of dementia in the elderly. An important pathological feature in AD is the aggregation and deposition of the β-amyloid (Aβ) in extracellular plaques. Transthyretin (TTR) can cleave Aβ, resulting in the formation of short peptides with less activity of amyloid plaques formation, as well as being able to degrade Aβ peptides that have already been aggregated. In the presence of TTR, Aβ aggregation decreases and toxicity of Aβ is abolished. This may prevent amyloidosis but the malfunction of this process leads to the development of AD. In the context of Aβplaque formation in AD, we discuss metallothionein (MT) interaction with TTR, the effects of which depend on the type of MT isoform. In the brains of patients with AD, the loss of MT-3 occurs. On the contrary, MT-1/2 level has been consistently reported to be increased. Through interaction with TTR, MT-2 reduces the ability of TTR to bind to Aβ, while MT-3 causes the opposite effect. It increases TTR-Aβ binding, providing inhibition of Aβ aggregation. The protective effect, assigned to MT-3 against the deposition of Aβ, relies also on this mechanism. Additionally, both Zn₇MT-2 and Zn₇MT-3, decrease Aβ neurotoxicity in cultured cortical neurons probably because of a metal swap between Zn₇MT and Cu(II)Aβ. Understanding the molecular mechanism of metals transfer between MT and other proteins as well as cognition of the significance of TTR interaction with different MT isoforms can help in AD treatment and prevention.     

Altered Blood and Brain Expression of Inflammation and Redox Genes in Alzheimer’s Disease,Common to APPV717I × TAUP301L Mice and Patients

Despite intensive research, the pathophysiology of Alzheimer’s disease (AD) is still not fully understood, and currently there are no effective treatments. Therefore, there is an unmet need for reliable biomarkers and animal models of AD to develop innovative therapeutic strategies addressing early pathologic events such as neuroinflammation and redox disturbances. The study aims to identify inflammatory and redox dysregulations in the context of AD-specific neuronal cell death and DNA damage, using the APP^V717I× TAU^P301L (AT) mouse model of AD. The expression of 84 inflammatory and 84 redox genes in the hippocampus and peripheral blood of double transgenic AT mice was evaluated against age-matched controls. A distinctive gene expression profile in the hippocampus and the blood of AT mice was identified, addressing DNA damage, apoptosis and thrombosis, complemented by inflammatory factors and receptors, along with ROS producers and antioxidants. Gene expression dysregulations that are common to AT mice and AD patients guided the final selection of candidate biomarkers. The identified inflammation and redox genes, common to AD patients and AT mice, might be valuable candidate biomarkers for preclinical drug development that could be readily translated to clinical trials.     

Amyloid β, Lipid Metabolism,Basal Cholinergic System,and Therapeutics in Alzheimer’s Disease

The presence of insoluble aggregates of amyloid β (Aβ) in the form of neuritic plaques (NPs) is one of the main features that define Alzheimer’s disease. Studies have suggested that the accumulation of these peptides in the brain significantly contributes to extensive neuronal loss. Furthermore, the content and distribution of cholesterol in the membrane have been shown to have an important effect on the production and subsequent accumulation of Aβ peptides in the plasma membrane, contributing to dysfunction and neuronal death. The monomeric forms of these membrane-bound peptides undergo several conformational changes, ranging from oligomeric forms to beta-sheet structures, each presenting different levels of toxicity. Aβ peptides can be internalized by particular receptors and trigger changes from Tau phosphorylation to alterations in cognitive function, through dysfunction of the cholinergic system. The goal of this review is to summarize the current knowledge on the role of lipids in Alzheimer’s disease and their relationship with the basal cholinergic system, as well as potential disease-modifying therapies.     

Intersection of AHR and Wnt Signaling in Development,Health, and Disease

The AHR (aryl hydrocarbon receptor) and Wnt (wingless-related MMTV integration site) signaling pathways have been conserved throughout evolution. Appropriately regulated signaling through each pathway is necessary for normal development and health, while dysregulation can lead to developmental defects and disease. Though both pathways have been vigorously studied, there is relatively little research exploring the possibility of crosstalk between these pathways. In this review, we provide a brief background on (1) the roles of both AHR and Wnt signaling in development and disease, and (2) the molecular mechanisms that characterize activation of each pathway. We also discuss the need for careful and complete experimental evaluation of each pathway and describe existing research that explores the intersection of AHR and Wnt signaling. Lastly, to illustrate in detail the intersection of AHR and Wnt signaling, we summarize our recent findings which show that 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD)-induced disruption of Wnt signaling impairs fetal prostate development.