Araceli Espinosa-Jeffrey

Innate immunity and transcription of MGAT-III and Toll-like receptors in Alzheimer's disease patients are improved by bisdemethoxycurcumin.

We have tested a hypothesis that the natural product curcuminoids, which has epidemiologic and experimental rationale for use in AD, may improve the innate immune system and increase amyloid-β (Aβ) clearance from the brain of patients with sporadic Alzheimers disease (AD). Macrophages of a majority of AD patients do not transport Aβ into endosomes and lysosomes, and AD monocytes do not efficiently clear Aβ from the sections of AD brain, although they phagocytize bacteria. In contrast, macrophages of normal subjects transport Aβ to endosomes and lysosomes, and monocytes of these subjects clear Aβ in AD brain sections. Upon Aβ stimulation, mononuclear cells of normal subjects up-regulate the transcription of β-1,4-mannosyl-glycoprotein 4-β-N-acetylglucosaminyltransferase (MGAT3) (P < 0.001) and other genes, including Toll like receptors (TLRs), whereas mononuclear cells of AD patients generally down-regulate these genes. Defective phagocytosis of Aβ may be related to down-regulation of MGAT3, as suggested by inhibition of phagocytosis by using MGAT3 siRNA and correlation analysis. Transcription of TLR3, bditTLR4, TLR5, bditTLR7, TLR8, TLR9, and TLR10 upon Aβ stimulation is severely depressed in mononuclear cells of AD patients in comparison to those of control subjects. In mononuclear cells of some AD patients, the curcuminoid compound bisdemethoxycurcumin may enhance defective phagocytosis of Aβ, the transcription of MGAT3 and TLRs, and the translation of TLR2–4. Thus, bisdemethoxycurcumin may correct immune defects of AD patients and provide a previously uncharacterized approach to AD immunotherapy.

Prevention of gliotic scar formation by NeuroGel allows partial endogenous repair of transected cat spinal cord.

Spinal cords of adult cats were transected and subsequently reconnected with the biocompatible porous poly (N‐[2‐hydroxypropyl] methacrylamide) hydrogel, NeuroGel™. Tissue repair was examined at various time points from 6–21 months postreconstructive surgery. We examined two typical phenomena, astrogliosis and scar formation, in spines reconstructed with the gel and compared them to those from transected non‐reconstructed spines. Confocal examination with double immunostaining for glial fibrillary acidic protein (GFAP) and myelin basic protein (MBP) showed that the interface formed between the hydrogel and the spine stumps did prevent scar formation and only a moderate gliosis was observed. The gel implant provided an adequate environment for growth of myelinated fibers and we saw angiogenesis within the gel. Electron microscopy showed that regenerating axons were myelinated by Schwann cells rather than oligodendrocytes. Moreover, the presence of the gel implant lead to a considerable reduction in damage to distal caudal portions of the spine as assessed by the presence of more intact myelinated fibers and a reduction of myelin degradation. Neurologic assessments of hindlimb movement at various times confirmed that spinal cord reconstruction was not only structural but also functional. We conclude that NeuroGel lead to functional recovery by providing a favorable substrate for regeneration of transected spinal cord, reducing glial scar formation and allowing angiogenesis.

Alzheimer disease macrophages shuttle amyloid-beta from neurons to vessels, contributing to amyloid angiopathy

Neuronal accumulation of oligomeric amyloid-β (Αβ) is considered the proximal cause of neuronal demise in Alzheimer disease (AD) patients. Blood-borne macrophages might reduce Aβ stress to neurons by immigration into the brain and phagocytosis of Αβ. We tested migration and export across a blood-brain barrier model, and phagocytosis and clearance of Αβ by AD and normal subjects’ macrophages. Both AD and normal macrophages were inhibited in Αβ export across the blood-brain barrier due to adherence of Aβ-engorged macrophages to the endothelial layer. In comparison to normal subjects’ macrophages, AD macrophages ingested and cleared less Αβ, and underwent apoptosis upon exposure to soluble, protofibrillar, or fibrillar Αβ. Confocal microscopy of stained AD brain sections revealed oligomeric Aβ in neurons and apoptotic macrophages, which surrounded and infiltrated congophilic microvessels, and fibrillar Aβ in plaques and microvessel walls. After incubation with AD brain sections, normal subjects’ monocytes intruded into neurons and uploaded oligomeric Aβ. In conclusion, in patients with AD, macrophages appear to shuttle Aβ from neurons to vessels where their apoptosis may release fibrillar Aβ, contributing to cerebral amyloid angiopathy.

Stem/progenitor cells in the postnatal inner ear of the GFP-nestin transgenic mouse

Nestin promoter‐GFP (green fluorescent protein) transgenic mice were used to determine the presence of stem/progenitor cells in the mouse inner ear. We examined the inner ear of mice at the following postnatal days (P): P0, P4, P5, P15 and P60. Hair cells stereocilia were identified with the use of the histochemical marker phalloidin. Whole endorgans or cryosections were analyzed under epi‐fluorescent or confocal microscopy. From P0 to P5, GFP expressing cells were found in the vestibular sensory epithelia of the macula utricle, but not in the crista ampullaris. Cells within the stroma (tissue underneath the sensory epithelia), utricle, and crista were also GFP‐positive. Satellite cells in the vestibular ganglia were GFP‐positive, while vestibular ganglia neurons were not. In the organ of Corti, GFP signal was found in inner border and inner phalangeal cells that surround the inner hair cells (GFP‐negative), Dieters cells and cells in the great epithelial ridge. Outer hair cells were mildly positive for GFP. Satellite cells in the spiral ganglia were GFP‐positive, while spiral ganglia neurons were not. Similar GFP expression was found in the vestibule and cochlea of animals at P15, however, outer hair cells showed no GFP expression. The inner ear of P60 animals contained moderate GFP expression in the stroma of the crista ampullaris and utricle, but not within the sensory epithelia. In the organ of Corti, moderate GFP expression was found in a few Deiters cells. The present data indicates that the expression of nestin in the mouse inner ear is developmentally regulated; yet in the adult inner ear there are some nestin expressing cells, suggesting an intrinsic repair potential, although to a more limited extent than during early post‐natal life.

Selective specification of CNS stem cells into oligodendroglial or neuronal cell lineage: Cell culture and transplant studies

Neural stem cells (NSCs) were isolated from embryonic day 16 Sprague–Dawley rats and cultured in a novel serum‐free stem cell medium that selected for the growth of NSCs and against the growth of GFAP+ cells (astrocytes). NSCs maintained in culture for extended periods of time retained immunoreactivity for both nestin and PSA‐NCAM, two markers characteristic of the stem cell phenotype. Moreover, using an oligodendrocyte (OL) specification medium, NSCs differentiated into OL as evidenced by their morphology and expression of multiple oligodendrocyte/myelin‐specific markers. In addition, NSCs are capable of acquiring a neuronal phenotype as evidenced by expressing neuronal markers, such as neurofilament (NF) and NeuN when cultured in a defined medium for neurons indicating that these cells are also a good source of neuroblasts, which could be used to replace neuronal populations in the brain. We also showed successful propagation and differentiation of NSCs into OL after cryostorage, allowing for the later use of stored NSCs. The long‐term goal of culturing NSCs and committed oligodendrocyte progenitors (OLP) is to obtain homogeneous populations for transplantation with the goal of remyelinating the myelin‐deficient CNS. Our preliminary experiments carried out on normal and myelin deficient rats demonstrate that these cells survive and migrate extensively in both types of hosts. NSCs grafted as such, as well as cells derived from NSCs exposed to selective specification before grafting, are able to differentiate within the host brain. As expected, NSCs are capable of giving rise to astrocytes in a medium favoring this phenotype.

Tumor necrosis factor modulates transcription of myelin basic protein gene through nuclear factor kappa B in a human oligodendroglioma cell line.

Tumor necrosis factor‐α (TNF‐α) is a major mediator of inflammation and it is involved in many neurological disorders such as multiple sclerosis. Levels of TNF‐α and lymphotoxin‐α have been found elevated in plaques, bloods, and cerebral spinal fluids from multiple sclerosis patients. The expression of myelin basic protein (MBP), a major protein of the myelin sheath, is affected by cytokines secreted by activated immune cells. To determine the signal transduction pathway involving tumor necrosis factors action in myelination and demyelination, we have cloned and analyzed cis‐elements on promoters of the human and mouse MBP genes. There are two putative nuclear factors kappa‐B (NF‐κB) cis‐elements on the human and one on the mouse gene promoter. In an electrophoretic mobility shift assay, all three NF‐κB cis‐elements showed binding to a protein, which was recognized by an antibody against NF‐κB P65 component. The specificity of the binding was demonstrated in a competitive assay using NF‐κB consensus oligonucleotides. A two base pair site‐directed mutation on the mouse NF‐κB cis‐element abolished its binding activity. We created a DNA construct by linking the mouse MBP gene promoter containing the NF‐κB cis‐element to luciferase gene. Transfection of this construct into a human oligodendroglioma cell line showed TNF‐α increased the transgene expression. Furthermore the mutation of NF‐κB site abolished TNF‐α ‐induction of the transgene. The data demonstrate that NF‐κB is the mediator between tumor necrosis factors action and MBP gene expression. Elucidating the molecular mechanisms underlying TNF‐α regulation of MBP gene expression provides new scientific bases for the development of therapy against oligodendrocyte‐specific and myelin‐related disorders such as multiple sclerosis.

Transferrin Regulates Transcription of the MBP Gene and Its Action Synergizes with IGF-1 to Enhance Myelinogenesis in the md Rat

Myelin-deficient (md) rats and their unaffected littermates were injected at postnatal day 4 either with a single dose of transferrin (Tf) or insulin-like growth factor one (IGF-1) singly or combined. Two weeks later, their brains were perfused and coronal sections were analyzed for MBP by in situ hybridization and for transferrin and myelin basic protein (Tf and MBP) by double immunofluorescence. Each of the factors separately had an effect on mutant animals as seen by both increased OL maturation, and MBP mRNA and protein synthesis. The combination of factors resulted in a profound enhancement of the myelinogenic properties of oligodendrocytes (OL) with a consequent increase in the number of MBP-labeled fibers. The brains of unaffected littermates also responded to growth factor(s) injection either by increasing myelination in some brain areas or by regulating the synthesis of MBP in OL. Using rat OL cultures we studied the site of transferrin action for the expression of MBP gene. We found by run off transcription that the MBP mRNA was significantly increased at the nuclear level but the PLP message was unaffected. Thus, transferrin selectively regulates MBP at the transcriptional level and together with IGF-1 synergizes to increase both the maturation and myelinogenic properties of md and normal OL.

Voluntary exercise increases oligodendrogenesis in spinal cord.

ABSTRACT Exercise has been shown to increase hippocampal neurogenesis, but the effects of exercise on oligodendrocyte generation have not yet been reported. In this study, we evaluated the hypothesis that voluntary exercise may affect neurogenesis, and more in particular, oligodendrogenesis in the thoracic segment of the intact spinal cord of adult nestin–GFP transgenic mice. Voluntary exercise for 7 and 14 days increased nestin–GFP expression around the ependymal area. In addition, voluntary exercise for 7 days significantly increased nestin–GFP expression in both the white and gray matter of the thoracic segment of the intact spinal cord, whereas, 14-day exercise decreased nestin–GFP expression. Markers for immature oligodendrocytes (transferrin and CNPase) were significantly increased after 7 days of voluntary exercise. These results suggest that voluntary exercise positively influences oligodendrogenesis in the intact spinal cord, emphasizing the beneficial effects of voluntary exercise as a possible co-treatment for spinal cord injury.

Calcium receptor expression and function in oligodendrocyte commitment and lineage progression: Potential impact on reduced myelin basic protein in CaR-null mice

Oligodendrocytes develop from oligodendrocyte progenitor cells (OPCs), which in turn arise from a subset of neuroepithelial precursor cells during midneurogenesis. Development of the oligodendrocyte lineage involves a plethora of cell‐intrinsic and ‐extrinsic signals. A cell surface calcium‐sensing receptor (CaR) has been shown to be functionally expressed in immature oligodendrocytes. Here, we investigated the expression and function of the CaR during oligodendrocyte development. We show that the order of CaR mRNA expression as assessed by quantitative polymerase chain reaction is mature oligodendrocyte > neuron > astrocyte. We next determined the rank order of CaR expression on inducing specification of neural stem cells to the neuronal, oligodendroglial, or astrocytic lineages and found that the relative levels of CaR mRNA expression are OPC > neuron > astrocytes. CaR mRNA expression in cells at various stages of development along the oligodendrocyte lineage revealed that its expression is robustly up‐regulated during the OPC stage and remains high until the premyelinating stage, decreasing thereafter by severalfold in the mature oligodendrocyte. In OPCs, high Ca2+ acting via the CaR promotes cellular proliferation. We further observed that high Ca2+ stimulates the mRNA levels of myelin basic protein in preoligodendrocytes, which is also CaR mediated. Finally, myelin basic protein levels were significantly reduced in the cerebellum of CaR‐null mice during development. Our results show that CaR expression is up‐regulated when neural stem cells are specified to the oligodendrocyte lineage and that activation of the receptor results in OPC expansion and differentiation. We conclude that the CaR may be a novel regulator of oligodendroglial development and function.

Oxidative stress and the deleterious consequences to the rat cochlea after prenatal chronic mild exposure to carbon monoxide in air

Pregnant rats (starting on E5) were exposed chronically to carbon monoxide (CO) from gestational days 5-20. In the postnatal period, rat pups were grouped as follows: group A: prenatal exposure to CO only; group B: prenatal exposure to CO then exposed to CO from postnatal day (P) 5 to P20; group C, control (air without CO). Groups A and B showed similar deleterious effects after CO exposure. At P3, rat pup cochlea from group A showed a normal organization of the organ of Corti. There was no morphological deterioration, or loss of inner or outer hair cells. At P20, animals from group A and B showed vacuolization on the afferent terminals at the basal portion of the cochlea. We found synapsin-1 immunoreactivity (IR) to be decreased in efferent nerve terminals in CO-exposed pups at P3. From P12 to P20, synapsin-1-IR is low in efferent terminals. At P20, type I spiral ganglia neurons and afferent nerve fibers showed decreased neurofilament-IR in CO-exposed groups when compared with controls. Heme oxygenase-1 and superoxide dismutase-1-IR were elevated in the stria vascularis and blood vessels from CO-exposed rat pups at P12 and P20 in group B; in contrast group A showed a comparable expression to controls. Inducible nitric oxide synthase (iNOS) and nitrotyrosine-IR were increased in blood vessels of the cochlea in CO-exposed groups, from P3 to P20. iNOS up-regulation and the presence of nitrotyrosine in blood vessels of the cochlea indicated that CO exposure activates the production of nitric oxide via increased iNOS activity. Prenatal chronic CO exposure promotes oxidative stress in the cochlea blood vessels that in turn is reflected in damage to spiral ganglia neurons and inner hair cells, suggesting for the first time that prenatal exposure to CO at concentrations expected in poorly ventilated environments impairs the development of the inner ear.