Dora Acuna

Mutations in the gene encoding filamin B disrupt vertebral segmentation, joint formation and skeletogenesis

The filamins are cytoplasmic proteins that regulate the structure and activity of the cytoskeleton by cross-linking actin into three-dimensional networks, linking the cell membrane to the cytoskeleton and serving as scaffolds on which intracellular signaling and protein trafficking pathways are organized (reviewed in refs. 1,2). We identified mutations in the gene encoding filamin B in four human skeletal disorders. We found homozygosity or compound heterozygosity with respect to stop-codon mutations in autosomal recessive spondylocarpotarsal syndrome (SCT, OMIM 272460) and missense mutations in individuals with autosomal dominant Larsen syndrome (OMIM 150250) and the perinatal lethal atelosteogenesis I and III phenotypes (AOI, OMIM 108720; AOIII, OMIM 108721). We found that filamin B is expressed in human growth plate chondrocytes and in the developing vertebral bodies in the mouse. These data indicate an unexpected role in vertebral segmentation, joint formation and endochondral ossification for this ubiquitously expressed cytoskeletal protein.

Immunolocalization of voltage-gated calcium channel α1 subunits in the chinchilla cochlea

The immunohistochemical localization of α1A, α1B, α1C, α1D, and α1E voltage-gated calcium channel subunits was investigated in the chinchilla organ of Corti and spiral ganglia with the use of specific antipeptide antibodies. The inner and outer hair cells were immunoreactive for α1A and α1D subunit antibodies. α1C immunoreactivity localized to the nerve terminals innervating inner hair cells and the basal pole of the outer hair cell. There was only non-specific staining to α1B and α1E. Supporting cells were non-immunoreactive. Spiral ganglia neurons were α1B, α1C, and α1D immunoreactive. A few spiral ganglia neurons were α1E immunoreactive. The importance of α1D, the pore-forming subunit of the L-type channel, in outer and inner hair cell function has been clearly demonstrated in electrophysiological, molecular biological, and knockout models. The presence of α1A, the pore-forming subunit of the P/Q type channels, has not previously been demonstrated in inner and outer hair cells, and its function in the cochlear hair cell is unknown.

Evidence for oxidative stress in the developing cerebellum of the rat after chronic mild carbon monoxide exposure (0.0025% in air)

BackgroundThe present study was designed to test the hypothesis that chronic very mild prenatal carbon monoxide (CO) exposure (25 parts per million) subverts the normal development of the rat cerebellar cortex. Studies at this chronic low CO exposure over the earliest periods of mammalian development have not been performed to date. Pregnant rats were exposed chronically to CO from gestational day E5 to E20. 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 5 (P5) to P20; group C: postnatal exposure only, from P5 to P20, and group D, controls (air without CO). At P20, immunocytochemical analyses of oxidative stress markers, and structural and functional proteins were assessed in the cerebellar cortex of the four groups. Quantitative real time PCR assays were performed for inducible (iNOS), neuronal (nNOS), and endothelial (eNOS) nitric oxide synthases.ResultsSuperoxide dismutase-1 (SOD1), SOD2, and hemeoxygenase-1 (HO-1) immunoreactivity increased in cells of the cerebellar cortex of CO-exposed pups. INOS and nitrotyrosine immunoreactivity also increased in blood vessels and Purkinje cells (PCs) of pups from group-A, B and C. By contrast, nNOS immunoreactivity decreased in PCs from group-B. Endothelial NOS immunoreactivity showed no changes in any CO-exposed group. The mRNA levels for iNOS were significantly up-regulated in the cerebellum of rats from group B; however, mRNA levels for nNOS and eNOS remained relatively unchanged in groups A, B and C. Ferritin-H immunoreactivity increased in group-B. Immunocytochemistry for neurofilaments (structural protein), synapsin-1 (functional protein), and glutamic acid decarboxylase (the enzyme responsible for the synthesis of the inhibitory neurotransmitter GABA), were decreased in groups A and B. Immunoreactivity for two calcium binding proteins, parvalbumin and calbindin, remained unchanged. The immunoreactivity of the astrocytic marker GFAP increased after prenatal exposure.ConclusionWe conclude that exogenously supplied CO during the prenatal period promotes oxidative stress as indicated by the up-regulation of SOD-1, SOD-2, HO-1, Ferritin-H, and iNOS with increased nitrotyrosine in the rat cerebella suggesting that deleterious and protective mechanisms were activated. These changes correlate with reductions of proteins important to cerebellar function: pre-synaptic terminals proteins (synapsin-1), proteins for the maintenance of neuronal size, shape and axonal quality (neurofilaments) and protein involved in GABAergic neurotransmission (GAD). Increased GFAP immunoreactivity after prenatal CO-exposure suggests a glial mediated response to the constant presence of CO. There were differential responses to prenatal vs. postnatal CO exposure: Prenatal exposure seems to be more damaging; a feature exemplified by the persistence of markers indicating oxidative stress in pups at P20, following prenatal only CO-exposure. The continuation of this cellular environment up to day 20 after CO exposure suggests the condition is chronic. Postnatal exposure without prenatal exposure shows the least impact, whereas prenatal followed by postnatal exposure exhibits the most pronounced outcome among the groups.

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.

Neuroglobin, cytoglobin, and transcriptional profiling of hypoxia-related genes in the rat cerebellum after prenatal chronic very mild carbon monoxide exposure (25 ppm)

The expression of neuroglobin (Ngb) and cytoglobin (Cygb), two recently discovered globins with a potential neuroprotective activity against hypoxia and oxidative stress, was investigated in the cerebellum of young rats (postnatal day 20) after being exposed to chronic mild carbon monoxide (CO) at 25 ppm during prenatal (group A), prenatal and postnatal (group B), the postnatal period only (group C), and air (group D). The expression of genes associated with hypoxia signaling pathways was also investigated in the rat cerebella by real-time RT-PCR after CO exposure. Ngb and Cygb mRNAs did not change in any CO-exposed group. Quantitative immunohistochemistry showed no significant change in Ngb protein; however, there was a significant increase of Cygb protein in rats from groups A, B, and C when compared with group D. In group B, genes related to the generation of reactive oxygen species (Nos2) and lipid metabolism (Apat2) were upregulated. In contrast, no changes were found in the expression of 8 genes typically upregulated by hypoxic conditions (Angptl4, Arnt2, Casp1, Crebbp, Hif1a, Hif3a, Mt3, or Vegfa) in any CO-exposed group, suggesting that hypoxia-related gene expression is not altered by this mild CO exposure. Cygb but not Ngb may protect cerebellar cells from the chronic presence of CO exposure during prenatal and postnatal development.

Subcellular immunolocalization of NMDA receptor subunit NR1, 2A, 2B in the rat vestibular periphery.

The immunohistochemical localization of the NMDA glutamate receptor subunits NR1, NR2A, and NR2B was investigated in the rat vestibular periphery at the light and electron microscopy level using specific antipeptide antibodies. The afferent calyceal terminals and nerve fibers innervating type I vestibular hair cells were strongly NR1, NR2A, and NR2B immunoreactive. Under electron microscopy, the basolateral type I hair cell membrane was NR1 immunoreactive. The type II hair cell and its afferent boutons were NR1, NR2A, and NR2B non-immunoreactive. Nearly all of Scarpas ganglion neurons were NR1 immunoreactive, but there was a subset of NR2A non-immunoreactive neurons. Additionally, the larger sized Scarpas ganglia neurons were NR2B immunoreactive, while the smaller neurons were non-immunoreactive. These findings are strong evidence for functional NMDA receptor mediation or modulation of afferent excitatory neurotransmission from type I but not type II vestibular hair cells to the primary afferent nerve. The receptor subtype(s) may be a combination of NR1/NR2A, NR1/NR2B, and/or NR1/NR2A/NR2B.

Mild carbon monoxide exposure diminishes selectively the integrity of the cochlea of the developing rat

Rat pups were chronically exposed to carbon monoxide (CO) concentrations (12 or 25 ppm) in air starting at day 8, through 22 days of age, to examine the changes in the peripheral auditory system. Gastrostomy‐reared rat pups, with or without CO exposure, were used and compared with mother‐reared pups. The organ of Corti and the neurons of the spiral ganglion were analyzed for their morphology by using immunochemical and histological techniques. The inner and outer hair cells in the organ of Corti of animals exposed to 12 and 25 ppm CO were not different from the controls. However, at 25 ppm CO exposure, the nerve terminals innervating the inner hair cells were swollen. The somata of neurons in the spiral ganglion showed mild changes in the cytoplasm, and signs of mild vacuolization were observed in myelin covering their central processes. Synaptophysin, a marker for synaptic vesicles, and choline acetyltransferase, a marker for cholinergic terminals, showed no difference in immunoreactivity in CO exposed animals at 12 and at 25 ppm when compared with their age‐matched controls. Also, Na+K+ ATPase immunoreactivity patterns were normal compared with controls. Three enzymes were significantly reduced at the 25 ppm CO exposure: Cytochrome oxidase, NADH‐TR, and calcium ATPase were decreased in both the organ of Corti and the neurons of the spiral ganglion, and decreased immunostaining for the neurofilament and myelin basic proteins was found. We conclude that components of the cochlea are selectively affected by mild chronic CO exposure during development.

Neuroglobin expression in the cochlea of rat pups exposed to chronic very mild carbon monoxide (25 ppm) in air during and after the prenatal period

The distribution of neuroglobin (Ngb) was investigated in the normal rat cochlea using immunohistochemistry and non-radioactive insitu hybridization. We also determined whether chronic, very mild CO exposure at 25ppm in air over the gestational and postnatal period alters the expression of Ngb. Pregnant rats were exposed chronically to CO from gestational days 5-20. Four groups were made as follows: prenatal exposure to CO only; prenatal exposure to CO followed by postnatal exposure from postnatal days (5) P5 to P20; rat pups were exposed to CO from P5 to P20; controls (air without CO). In normal adult rats and control group pups, Ngb was found in spiral ganglia neurons, fibrocytes of the spiral ligament, and supporting cells of the organ of Corti. Ngb was not present in the stria vascularis and the inner and outer hair cells. At P20 Ngb immunoreactivity and transcript expression decreased in spiral ganglia neurons and the spiral ligament in the prenatal and pre- and postnatal groups. This decrease was not observed in the postnatal group. Ngb-IR did not decrease in supporting cells in any CO group. Cytochrome-C immunoreactivity followed Ngb distribution in normal controls and CO treated groups. A decrease in Ngb in spiral ganglia neurons and rat spiral ligament, but not in supporting cells, following CO exposure supports the idea that chronic, mild exposure to CO may create a vulnerable cellular environment predisposed to adverse cochlear development.

Limiting iron availability confers neuroprotection from chronic mild carbon monoxide exposure in the developing auditory system of the rat

Iron deficiency and chronic mild carbon monoxide (CO) exposure are nutritional and environmental problems that can be experienced simultaneously. We examined the effects of chronic mild CO exposure and iron availability on auditory development in the rat. We propose that chronic mild CO exposure creates an oxidative stress condition that impairs the spiral ganglion neurons. The CO‐exposed rat pups had decreased neurofilament proteins and increased copper, zinc‐superoxide dismutase (SOD1) in the spiral ganglion neurons. We conclude that the increased amount of SOD1 causes an increase in hydrogen peroxide production that allows the Fenton reaction to occur. This reaction uses both iron and hydrogen peroxide to generate hydroxyl radicals and leads to the development of oxidative stress that impairs neuronal integrity. However, rat pups with decreased iron and CO exposure (ARIDCO) exhibited in their cochlea an up‐regulation of transferrin, whereas their expression of neurofilament proteins and SOD1 were similar to control. Consequently, reduced iron availability and the normal expression of SOD1 do not promote oxidative stress in the cochlea. By using basal c‐Fos expression as a marker for cellular activation we found a significant reduction in c‐Fos expression in the central nucleus of the inferior colliculus in iron‐adequate rat pups exposed to CO. By contrast, rather than being reduced, c‐Fos expression in the ARIDCO group is the same as for controls. We conclude that the cochlea of rat pups with normal iron availability is selectively affected by mild CO exposure, causing a chronic oxidative stress, whereas limiting iron availability ameliorates the effect caused by mild CO exposure by averting conditions that facilitate oxidative stress.

Multiphoton, confocal, and lifetime microscopy for molecular imaging in cartilage

It has recently been shown that mutations in Filamin A and B genes produce a large spectrum of skeletal disorders in developing fetuses. However, high-resolution optical microscopy in cartilage growth plate using fluorescent antibody assays, which should elucidate molecular aspects of these disorders, is extremely difficult due to the high level of autofluoresce in this tissue. We apply multiphoton, confocal, lifetime and spectral microscopy to (i) image and characterize autofluorophores in chondrocytes and subtract their contributions to obtain a corrected antibody-marker fluorescence signal, and (ii) measure the interaction between Filamin A and B proteins by detecting the fluorescence resonance energy transfer (FRET) between markers of the two proteins. Taking advantage of the different fluorescence spectra of the endogenous and exogenous markers, we can significantly reduce the autofluorescence background. Preliminary results of the FRET experiments suggest no interaction between Filamin A and B proteins. However, developing of new antibodies targeting the carboxy-terminal immunoglobulin-like domain may be necessary to confirm this result.