Pathological Nuclear Hallmarks in Dentate Granule Cells of Alzheimer's Patients: A Biphasic Regulation of Neurogenesis.

The dentate gyrus (DG) of the human hippocampus is a complex and dynamic structure harboring mature and immature granular neurons in diverse proliferative states. While most mammals show persistent neurogenesis through adulthood, human neurogenesis is still under debate. We found nuclear alterations in granular cells in autopsied human brains, detected by immunohistochemistry. These alterations differ from those reported in pyramidal neurons of the hippocampal circuit. Aging and early AD chromatin were clearly differentiated by the increased epigenetic markers H3K9me3 (heterochromatin suppressive mark) and H3K4me3 (transcriptional euchromatin mark). At early AD stages, lamin B2 was redistributed to the nucleoplasm, indicating cell-cycle reactivation, probably induced by hippocampal nuclear pathology. At intermediate and late AD stages, higher lamin B2 immunopositivity in the perinucleus suggests fewer immature neurons, less neurogenesis, and fewer adaptation resources to environmental factors. In addition, senile samples showed increased nuclear Tau interacting with aged chromatin, likely favoring DNA repair and maintaining genomic stability. However, at late AD stages, the progressive disappearance of phosphorylated Tau forms in the nucleus, increased chromatin disorganization, and increased nuclear autophagy support a model of biphasic neurogenesis in AD. Therefore, designing therapies to alleviate the neuronal nuclear pathology might be the only pathway to a true rejuvenation of brain circuits.

Life-long arsenic exposure damages the microstructure of the rat hippocampus.

Epidemiological studies demonstrate that arsenic exposure is associated with cognitive dysfunction. Experimental arsenic exposure models showed learning and memory deficits and molecular changes resembling the functional and pathologic neurodegeneration features. The present work focuses on hippocampal pathological changes in Wistar rats induced by continuous arsenic exposure from in utero up to 12 months of age, evaluated by magnetic resonance imaging along with immunohistochemistry. Diffusion-weighted images revealed age-related lower fractional anisotropy and higher radial-axial and mean diffusivity at 6 and 12 months, indicating that arsenic exposure leads to hippocampal demyelination. These structural alterations were paralleled by immunohistochemical changes that showed a significant loss of myelin basic protein in CA1 and CA3 regions accompanied by increased glial fibrillary acidic protein expression at all time-points studied. Concomitantly, arsenic exposure induced an altered morphology of astrocytes at all studied ages, whereas increased synaptogenesis was only observed at two months of age. These results suggest that environmental arsenic exposure is linked to impaired hippocampal connectivity and perhaps early glial senescence, which together might resemble a premature aging phenomenon leading to cognitive deficits.

Cortical Synaptic Reorganization Under Chronic Arsenic Exposure.

There is solid epidemiological evidence that arsenic exposure leads to cognitive impairment, while experimental work supports the hypothesis that it also contributes to neurodegeneration. Energy deficit, oxidative stress, demyelination, and defective neurotransmission are demonstrated arsenic effects, but it remains unclear whether synaptic structure is also affected. Employing both a triple-transgenic Alzheimer's disease model and Wistar rats, the cortical microstructure and synapses were analyzed under chronic arsenic exposure. Male animals were studied at 2 and 4 months of age, after exposure to 3 ppm sodium arsenite in drinking water during gestation, lactation, and postnatal development. Through nuclear magnetic resonance, diffusion-weighted images were acquired and anisotropy (integrity; FA) and apparent diffusion coefficient (dispersion degree; ADC) metrics were derived. Postsynaptic density protein and synaptophysin were analyzed by means of immunoblot and immunohistochemistry, while dendritic spine density and morphology of cortical pyramidal neurons were quantified after Golgi staining. A structural reorganization of the cortex was evidenced through high-ADC and low-FA values in the exposed group. Similar changes in synaptic protein levels in the 2 models suggest a decreased synaptic connectivity at 4 months of age. An abnormal dendritic arborization was observed at 4 months of age, after increased spine density at 2 months. These findings demonstrate alterations of cortical synaptic connectivity and microstructure associated to arsenic exposure appearing in young rodents and adults, and these subtle and non-adaptive plastic changes in dendritic spines and in synaptic markers may further progress to the degeneration observed at older ages.

Demyelination associated with chronic arsenic exposure in Wistar rats.

Inorganic arsenic is among the major contaminants of groundwater in the world. Worldwide population-based studies demonstrate that chronic arsenic exposure is associated with poor cognitive performance among children and adults, while research in animal models confirms learning and memory deficits after arsenic exposure. The aim of this study was to investigate the long-term effects of environmentally relevant arsenic exposure in the myelination process of the prefrontal cortex (PFC) and corpus callosum (CC). A longitudinal study with repeated follow-up assessments was performed in male Wistar rats exposed to 3 ppm sodium arsenite in drinking water. Animals received the treatment from gestation until 2, 4, 6, or 12 months of postnatal age. The levels of myelin basic protein (MBP) were evaluated by immunohistochemistry/histology and immunoblotting from the PFC and CC. As plausible alterations associated with demyelination, we considered mitochondrial mass (VDAC) and two axonal damage markers: amyloid precursor protein (APP) level and phosphorylated neurofilaments. To analyze the microstructure of the CC in vivo, we acquired diffusion-weighted images at the same ages, from which we derived metrics using the tensor model. Significantly decreased levels of MBP were found in both regions together with significant increases of mitochondrial mass and slight axonal damage at 12 months in the PFC. Ultrastructural imaging demonstrated arsenic-associated decreases of white matter volume, water diffusion anisotropy, and increases in radial diffusivity. This study indicates that arsenic exposure is associated with a significant and persistent negative impact on microstructural features of white matter tracts.

Perinuclear Lamin A and Nucleoplasmic Lamin B2 Characterize Two Types of Hippocampal Neurons through Alzheimer's Disease Progression.

BACKGROUND: Recent reports point to a nuclear origin of Alzheimer's disease (AD). Aged postmitotic neurons try to repair their damaged DNA by entering the cell cycle. This aberrant cell cycle re-entry involves chromatin modifications where nuclear Tau and the nuclear lamin are involved. The purpose of this work was to elucidate their participation in the nuclear pathological transformation of neurons at early AD. METHODOLOGY: The study was performed in hippocampal paraffin embedded sections of adult, senile, and AD brains at I-VI Braak stages. We analyzed phospho-Tau, lamins A, B1, B2, and C, nucleophosmin (B23) and the epigenetic marker H4K20me3 by immunohistochemistry. RESULTS: Two neuronal populations were found across AD stages, one is characterized by a significant increase of Lamin A expression, reinforced perinuclear Lamin B2, elevated expression of H4K20me3 and nuclear Tau loss, while neurons with nucleoplasmic Lamin B2 constitute a second population. CONCLUSIONS: The abnormal cell cycle reentry in early AD implies a fundamental neuronal transformation. This implies the reorganization of the nucleo-cytoskeleton through the expression of the highly regulated Lamin A, heterochromatin repression and building of toxic neuronal tangles. This work demonstrates that nuclear Tau and lamin modifications in hippocampal neurons are crucial events in age-related neurodegeneration.

Arsenic Exposure Contributes to the Bioenergetic Damage in an Alzheimer's Disease Model.

Worldwide, every year there is an increase in the number of people exposed to inorganic arsenic (iAs) via drinking water. Human populations present impaired cognitive function as a result of prenatal and childhood iAs exposure, while studies in animal models demonstrate neurobehavioral deficits accompanied by neurotransmitter, protein, and enzyme alterations. Similar impairments have been observed in close association with Alzheimer's disease (AD). In order to determine whether iAs promotes the pathophysiological progress of AD, we used the 3xTgAD mouse model. Mice were exposed to iAs in drinking water from gestation until 6 months (As-3xTgAD group) and compared with control animals without arsenic (3xTgAD group). We investigated the behavior phenotype on a test battery (circadian rhythm, locomotor behavior, Morris water maze, and contextual fear conditioning). Adenosine triphosphate (ATP), reactive oxygen species, lipid peroxidation, and respiration rates of mitochondria were evaluated, antioxidant components were detected by immunoblots, and immunohistochemical studies were performed to reveal AD markers. As-3xTgAD displayed alterations in their circadian rhythm and exhibited longer freezing time and escape latencies compared to the control group. The bioenergetic profile revealed decreased ATP levels accompanied by the decline of complex I, and an oxidant state in the hippocampus. On the other hand, the cortex showed no changes of oxidant stress and complex I; however, the antioxidant response was increased. Higher immunopositivity to amyloid isoforms and to phosphorylated tau was observed in frontal cortex and hippocampus of exposed animals. In conclusion, mitochondrial dysfunction may be one of the triggering factors through which chronic iAs exposure exacerbates brain AD-like pathology.

Low levels of alpha-synuclein in peripheral tissues are related to clinical relapse in relapsing-remitting multiple sclerosis: a pilot cross-sectional study.

The protein alpha-synuclein (α-Syn) has been linked to neuroinflammatory conditions. We investigated whether the presence of α-Syn in peripheral tissues is a surrogate of brain inflammatory status in a small group of relapsing-remitting multiple sclerosis (RRMS) patients in a pilot cross-sectional study. Skin biopsies and peripheral blood were sampled from 34 healthy controls and 23 MS patients for measurement of α-Syn levels. Within the RRMS group 15 patients were in remission, and 8 patients were in the relapsing phase. The protein α-Syn was evaluated by means of immunohistochemistry and flow cytometry in skin and nucleated blood cells, respectively. In the skin, α-Syn levels were lower in relapsing MS than in the other groups, both in positive area (p = .021) and staining intensity (p = .004). In blood, the percentage of α-Syn-positive lymphocytes and monocytes were not statistically different between study groups. Moreover, the use of systemic steroids did not affect α-Syn positivity in MS-relapse patients. Finally, epidermic Langerhans cells did not stain positively for α-Syn. Overall, the levels of α-Syn positivity were lower in inflammatory relapse of RRMS patients when measured in peripheral tissues. We discuss the role of α-Syn levels in inflammation according to the obtained results.

Evaluation of cardiovascular responses to silver nanoparticles (AgNPs) in spontaneously hypertensive rats.

Silver nanoparticles (AgNPs) are used in the medical, pharmaceutical and food industry. Adverse effects and toxicity induced by AgNPs upon cardiac function related to nitric oxide (NO) and oxidative stress (OS) are described. AgNPs-toxicity may be influenced by cardiovascular pathologies such as hypertension. However, the molecules involved under pathophysiological conditions are not well studied. The aim of this work was to evaluate perfusion pressure (PP) and left ventricle pressure (LVP) as physiological parameters of cardiovascular function in response to AgNPs, using isolated perfused hearts from spontaneously hypertensive rats (SHR), and identify the role of NO and OS. The results suggest that AgNPs reduced NO derived from endothelial/inducible NO-synthase and increased OS, leading to increased and sustained vasoconstriction and myocardial contractility. Additionally, the hypertension condition alters phenylephrine (Phe) and acetylcholine (ACh) classic effects. These data suggest that hypertension intensified AgNPs-cardiotoxicity. Nevertheless, the precise mechanism of action is still under elucidation.

Tau Protein in Oral Mucosa and Cognitive State: A Cross-sectional Study.

Neurodegenerative diseases are characterized by the presence of abnormal aggregates of proteins in brain tissue. Among them, the presence of aggregates of phosphorylated Tau protein (p-Tau) is the hallmark of Alzheimer's disease (AD) and other major neurodegenerative disorders such as corticobasal degeneration and frontotemporal dementia among others. Although Tau protein has previously been assumed to be exclusive to the central nervous system, it is also found in peripheral tissues. The purpose of this study was to determine whether there is a differential Tau expression in oral mucosa cells according to cognitive impairment. Eighty-one subjects were enrolled in the study and classified per Mini-Mental State Examination test score into control, mild cognitive impairment (MCI), and severe cognitive impairment (SCI) groups. Immunocytochemistry and immunofluorescence revealed the presence of Tau and four p-Tau forms in the cytoplasm and nucleus of oral mucosa cells. More positivity was present in subjects with cognitive impairment than in control subjects, both in the nucleus and cytoplasm, in a speckle pattern. The mRNA expression of Tau by quantitative real-time polymerase chain reaction was higher in SCI as compared with the control group (P < 0.01). A significantly higher percentage of immunopositive cells in the SCI group was found via flow cytometry in comparison to controls and the MCI group (P < 0.01). These findings demonstrate the higher presence of p-Tau and Tau transcript in the oral mucosa of cognitively impaired subjects when compared with healthy subjects. The feasibility of p-Tau quantification by flow cytometry supports the prospective analysis of oral mucosa as a support tool for screening of proteinopathies in cognitively impaired patients.

Oxidative Stress Modifies the Levels and Phosphorylation State of Tau Protein in Human Fibroblasts.

Since the tau protein is closely involved in the physiopathology of Alzheimer's disease (AD), studying its behavior in cellular models might lead to new insights on understanding this devastating disease at molecular levels. In the present study, primary cultures of human fibroblasts were established and used to determine the expression and localization of the tau protein in distinct phosphorylation states in both untransfected and tau gene-transfected cells subjected to oxidative stress. Higher immunopositivity to phospho-tau was observed in cell nuclei in response to oxidative stress, while the levels of total tau in the cytosol remained unchanged. These findings were observed in both untransfected cells and those transfected with the tau gene. The present work represents a useful model for studying the physiopathology of AD at the cellular level in terms of tau protein implications.

The Presence of Alpha-Synuclein in Skin from Melanoma and Patients with Parkinson's Disease.

BACKGROUND: The misfolding and prion-like propagation of the protein α-synuclein (α-syn) is the leading molecular signature in Parkinson's disease (PD). There is a significant coincidence of PD and melanoma that may suggest a shared pathophysiology. This study compared the presence of α-syn in neural crest-derived tissues, such as nevi, melanoma, skin tags, and skin biopsies from patients with PD and healthy controls. METHODS: Biopsies from participants with PD were obtained from patients from a tertiary referral center for dermatology and neurology in Mexico and a private dermatopathology center in Florida between January 2015 and March 2016. Biopsies from 7 patients with melanoma, 15 with nevi, 9 with skin tags, 8 with PD, and 9 skin biopsies from healthy volunteers were analyzed for immunohistochemical determination of α-syn and tyrosinase. All analyses were performed by pathologists who were blinded with respect to the clinical diagnosis. RESULTS: In healthy controls, positive α-syn status was restricted to scattered cells in the basal layer of the epidermis and accounted for 1 ± 0.8% of the analyzed area. In patients with PD, there was increased staining for α-syn PD (3.3 ± 2.3%), with a higher percentage of positive cells in nevi (7.7 ± 5.5%) and melanoma (13.6 ± 3.5%). There was no increased staining in skin tags compared with healthy controls. CONCLUSION: Patients with PD and melanoma have increased staining for α-syn in their skin. The authors propose that neurons and melanocytes, both derived from neuroectodermal cells, may share protein synthesis and regulation pathways that become dysfunctional in PD and melanoma.

Parkinson disease and progressive supranuclear palsy: protein expression in skin.

OBJECTIVE: This study characterizes the expression of tau (p-tau) and α-synuclein (α-syn) by immunohistochemistry in the skin of three different populations: healthy control (HC), Parkinson disease (PD), and progressive supranuclear paralysis (PSP) subjects, with the purpose of finding a biomarker that could differentiate between subjects with PD and PSP. MATERIAL AND METHODS: We evaluated the presence of p-tau and α-syn in a pilot study in the skin of three distinct groups of patients: 17 healthy subjects, 17 patients with PD, and 10 patients with PSP. Four millimeters punch biopsies were obtained from the occipital area and analyzed by immunohistochemistry using antibodies against α-syn and phosphorylated species of tau. PHF (paired helical filaments) antibody identifies p-tau in both normal and pathological conditions and AT8 recognizes p-tau characteristic of pathological conditions. Differences between the three groups were assessed by quantification of immunopositive areas in the epidermis. RESULTS: The immunopositivity pattern of p-tau and α-syn was significantly different among the three groups. Healthy subjects showed minimal staining using AT8 and α-syn. The PD group showed significantly higher α-syn and AT8 immunopositivity, while the PSP group only expressed higher AT8 immunopositivity than HCs. CONCLUSION: These data suggest that the skin reflects brain pathology. Therefore, immunohistochemical analysis of p-tau and α-syn in the skin can be useful for further characterization of PD and PSP.

The coronary endothelium behaves as a functional diffusion barrier for intravascular Angiotensin II.

Diverse intracoronary hormones cause their cardiac effects solely via activation of their coronary endothelial luminal membrane (CELM) receptors. To test this hypothesis for Ang II, we synthesized: a) two large polymers of Ang II (Ang II-POL) and Losartan (Los-POL) which act only in the CELM's AT1R because they cannot cross the endothelial barrier and b) biotin-labeled Ang II (Ang II-Biotin) and Ang II-POL-Biotin to be identified by microscopy in tissues. Sustained coronary perfusion of Ang II (potentially diffusible) or Ang II-POL caused a positive inotropic effect (PIE) and an increase in coronary perfusion pressure (CPP) of equal magnitude that were blocked by Losartan and Los-POL. However, Ang II effects, in contrast to Ang II-POL effects, were transient due to desensitization and resulted in tachyphylaxis to a second administration of Ang II or Ang II-POL. Furthermore, if Ang II and Ang II-POL acted differently on the same receptor; a competition of effects would be expected. This was demonstrated by infusing simultaneously a molar ratio of Ang II:Ang II-POL. As this molar ratio decreased, Ang II-induced desensitization and tachyphylaxis decreased. Intravascularly-administered Ang II-Biotin and Ang II-POL-Biotin remained bound and confined to the endothelium. Our results support the hypothesis and indicate intravascular Ang II, not by mass exchange with the interstitium, but by an action restricted to the CELM's AT1R, causes release of endothelial chemical messengers that exert physiological effects and modulate the effects and metabolism of paracrine Ang II. Endocrine Ang II controls and communicates with its paracrine counterparts solely through endothelial receptors.

Cardiac ischemia and ischemia/reperfusion cause wide proteolysis of the coronary endothelial luminal membrane: possible dysfunctions.

BACKGROUND: Ischemia and ischemia-reperfusion (I/R) are common clinical insults that disrupt the molecular structure of coronary vascular endothelial luminal membrane (VELM) that result in diverse microvasculature dysfunctions. However, the knowledge of the associated biochemical changes is meager. We hypothesized that ischemia and I/R-induced structural and functional VELM alterations result from biochemical changes. First, these changes need to be described and later the mechanisms behind be identified. METHODS: During control conditions, in isolated perfused rat hearts VELM proteins were labeled with biotin. The groups of hearts were: control (C), no flow ischemia (I; 25 min), and I/R (I; 25 min, reperfusion 30 min). The biotinylated luminal endothelial membrane proteins in these three different groups were examined by 2-D electrophoresis and identified. But, it must be kept in mind the proteins were biotin-labeled during control. RESULTS: A comparative analysis of the protein profiles under the 3 conditions following 2D gel electrophoresis showed differences in the molecular weight distribution such that MW(C) > MW(I) > MW(I/R). Similar analysis for isoelectric points (pH(i)) showed a shift toward more acidic pHi under ischemic conditions. Of 100 % proteins identified during control 66% and 88% changed their MW-pH(i) during ischemia and I/R respectively. Among these lost proteins there were 9 proteins identified as adhesins and G-protein coupled receptors. GENERAL SIGNIFICANCE: I and I/R insults alter MW-pH(i) of most luminal glycocalyx proteins due to the activation of nonspecific hydrolizing mechanisms; suspect metalloproteases and glycanases. This makes necessary the identification of hydrolyzing enzymes reponsible of multiple microvascular dysfunctions in order to maintain the integrity of vascular endothelial membrane. VELM must become a target of future therapeutics.

Luminal endothelial lectins with affinity for N-acetylglucosamine determine flow-induced cardiac and vascular paracrine-dependent responses.

Coronary blood flow applied to the endothelial lumen modulates parenchymal functions via paracrine effectors, but the mechanism of flow sensation is unknown. We and others have demonstrated that coronary endothelial luminal membrane (CELM) oligosaccharides and lectins are involved in flow detection, and we proposed that cardiac effects of coronary flow result from a reversible flow-modulated lectin-oligosaccharide interaction. Recently, glycosylated and amiloride-sensitive Na(+)/Ca(++) channels (ENaCs) have been proposed to be involved in the flow-induced endothelial responses. Because N-acetylglucosamine (GlcNac) is one of the main components of glycocalyx oligosaccharides (i.e., hyaluronan [-4GlcUAbeta1-3GlcNAcbeta1-](n)), the aim of this article is to isolate and define CELM GlcNac-binding lectins and determine their role in cardiac and vascular flow-induced effects. For this purpose, we synthesized a 460-kDa GlcNac polymer (GlcNac-Pol) with high affinity toward GlcNac-recognizing lectins. In the heart, intracoronary administration of GlcNac-Pol upon binding to CELM diminishes the flow-dependent positive inotropic and dromotropic effects. Furthermore, GlcNac-Pol was used as an affinity probe to isolate CELM GlcNac-Pol-recognizing lectins and at least 35 individual lectinic peptides were identified, one of them the beta-ENaC channel. Some of these lectins could participate in flow sensing and in GlcNac-Pol-induced effects. We also adopted a flow-responsive and well-accepted model of endothelial-parenchymal paracrine interaction: isolated blood vessels perfused at controlled flow rates. We established that flow-induced vasodilatation (FIV) is blocked by endothelial luminal membrane (ELM) bound GlcNac-Pol, nitro-l-arginine methyl ester and indomethacin, amiloride, and hyaluronidase. The effect of hyaluronidase was reversed by infusion of soluble hyaluronan. These results indicate that GlcNac-Pol inhibits FIV by competing and displacing intrinsic hyaluronan bound to a lectinic structure such as the amiloride-sensitive ENaC. Nitric oxide and prostaglandins are the putative paracrine mediators of FIV.

Two dissimilar AT(1) agonists distinctively activate AT(1) receptors located on the luminal membrane of coronary endothelium.

Diverse intracoronary agonists cause cardiac effects while acting on coronary endothelial luminal membrane (CELM) receptor. Our data show: a) the presence of AT(1)R in isolated CELM and in all cardiac cell types and b) sustained intracoronary infusions of Ang II-POL, a large sized molecule (approximately 15,000 kDa) confined to the vessel lumen that can only act on CELM's AT(1)R or Ang II (approximately 1 kDa); both exert the same maximum positive inotropic (PIE) and coronary constriction (CPP). The effects of these two agonists are blocked by Losartan and by Sar-POL; a large size antagonist (approximately 15,000 kDa) that acts only on CELM. Ang II effects are transient due to desensitization and cause tachyphylaxis to Ang II and toward Ang II-POL suggesting that both Ang II and Ang II-POL act on the same receptor group. In contrast, Ang II-POL effects are sustained and do not cause tachyphylaxis. The results show that intravascular Ang II and Ang II-POL act differentially by an unknown mechanism on CELM's AT(1)R and suggest that intravascular Ang II and Ang II-POL cause PIE and CCP by activation limited to CELM's AT(1)R through an unknown mechanism that is space-confined to the CELM's AT(1)R.

Involvement of endothelial Man and Gal-binding lectins in sensing the flow in coronary arteries.

The coronary endothelial luminal membrane (CELM) glycocalyx has diverse molecules involved in blood flow signal transduction. Evidence suggests that some of these structures may be lectinic. To test this, we synthesized two monosaccharide polymers (Mon-Pols) made of Mannose (Man-Pol) or Galactose (Gal-Pol) covalently coupled to Dextran (70 kDa) and used them as lectin affinity probes. In situ intracoronary infusion of both polymers resulted in CELM-binding but only Man-Pol caused a reduction in flow-induced positive inotropism and dromotropism. To demonstrate that our lectinic probes could bind to CELM lectins, a representative CELM protein fraction was isolated via intracoronary infusion of a cationic silica colloid and either Mannose- or Galactose-binding lectins were purified from the CELM protein fraction using the corresponding Mon-Pol affinity chromatography resin. Resin-bound CELM proteins were eluted with the corresponding monosaccharide. 2D-SDS-PAGE (pH 4-7) revealed 9 Mannose- and approximately 100 Galactose-selective CELM lectins. In summary, the CELM glycocalyx contains Mannose- and Galactose-binding lectins that may be involved in translating coronary flow into a cardiac parenchymal response.