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Michael E Selzer

Publications and source records attributed to Michael E Selzer.

At least 19 recordsLinked to original sources
PubMed2026

Cloning of human DING: Developmental expression and downregulation by EtOH in-utero.

INTRODUCTION: An estimated 15-20% of women consume alcohol (EtOH) during pregnancy. Women with alcohol use in early pregnancy are likely to have a child with fetal alcohol spectrum disorders (FASD). Recently, we reported neuroprotective effects of human DING (a member of the DING family of phosphatases) against EtOH-mediated toxicity in rats and in human fetal cortical neurons in vitro. Now, we report the sequencing and developmental expression patterns of endogenous DING in human fetal brain. METHODS: DING cDNA was cloned from human U87MG astrocytoma cells with primers specific to the plant DING gene and known prokaryotic DING genes. This cDNA was used to prepare antibodies. The full-length human DING gene p38hu (1095 nucleotide bases) is flanked by the first initiating codon, ATG, and the last, stop codon, TAA. Post-mortem fetal tissues and maternal blood were collected during pregnancy between 8 and 37 weeks' gestation. The developmental, spatial, and temporal expression of DING protein in fetal brain tissue was analyzed by immunohistochemistry. Developmental expression of DING in fetal brain and placenta was quantified by qWestern blots. DING promoter expression was assayed by ddPCR. Statistical analysis included ANOVA. RESULTS: Sequencing revealed different-sized genomic DNA clones. The anti-DING antibody detected proteins ranging in size from 35 to 40 kDa, and high molecular weight precursor protein in fetal brain and placenta. DING protein was present in fetal brain at early stages and its level was increased at later gestational ages. The DING promoter was expressed in fetal brain, neurospheres, and fetal brain-derived exosomes. DING levels were reduced in samples exposed to maternally consumed alcohol. CONCLUSIONS: Because DING is neuroprotective, its reduced expression in fetuses exposed to alcohol may suggest a mechanism that contributes to the pathogenesis of FASD, which could lead to the development of therapeutic tools aimed at preventing, ameliorating or reversing this prevalent group of syndromes that are implicated in as many as 5% of births world-wide.

DING gene cloning↗
PubMed2006

Semaphorins and their receptors in lamprey CNS: Cloning, phylogenetic analysis, and developmental changes during metamorphosis.

The large, conserved semaphorin gene family encodes axon guidance molecules in both invertebrates and vertebrates. The primitive vertebrate lamprey diverged near the time of vertebrate origins and is useful for understanding the gene duplication events that led to the increased complexity of the vertebrate genome. We characterized the sequence and expression pattern of semaphorins and their receptors genes in the sea lamprey, Petromyzon marinus. We uncovered two members of the semaphorin family in sea lamprey. The first encodes a diffusible class 3 type semaphorin protein that is most similar to the human and mouse Sema3F (71% amino acid identity). The second encodes a transmembrane class 4 type semaphorin that is most similar to mouse Sema4D and human Sema4G, with 38% amino acid identity within the Sema domain. We also identified in lamprey two members of the semaphorin receptor family, lamprey Plexin A1 and Plexin A2. Phylogenetic analysis indicates that lamprey Sema3 and Sema4 represent precursor genes existing prior to the origin of the vertebrate Sema3A-G and Sema4A-G subfamilies. Therefore, the gene duplication event that gave rise to those subfamilies must have occurred after the divergence of jawed vertebrates from jawless fish. These semaphorins and plexins are expressed in unique and dynamic patterns in lamprey spinal cord and brain during development.

Animals↗
PubMed2006

Developmental regulation of sensory axon regeneration in the absence of growth cones.

The actin filament (F-actin) cytoskeleton is thought to be required for normal axon extension during embryonic development. Whether this is true of axon regeneration in the mature nervous system is not known, but a progressive simplification of growth cones during development has been described and where specifically investigated, mature spinal cord axons appear to regenerate without growth cones. We have studied the cytoskeletal mechanisms of axon regeneration in developmentally early and late chicken sensory neurons, at embryonic day (E) 7 and 14 respectively. Depletion of F-actin blocked the regeneration of E7 but not E14 sensory axons in vitro. The differential sensitivity of axon regeneration to the loss of F-actin and growth cones correlated with endogenous levels of F-actin and growth cone morphology. The growth cones of E7 axons contained more F-actin and were more elaborate than those of E14 axons. The ability of E14 axons to regenerate in the absence of F-actin and growth cones was dependent on microtubule tip polymerization. Importantly, while the regeneration of E7 axons was strictly dependent on F-actin, regeneration of E14 axons was more dependent on microtubule tip polymerization. Furthermore, E14 axons exhibited altered microtubule polymerization relative to E7, as determined by imaging of microtubule tip polymerization in living neurons. These data indicate that the mechanism of axon regeneration undergoes a developmental switch between E7 and E14 from strict dependence on F-actin to a greater dependence on microtubule polymerization. Collectively, these experiments indicate that microtubule polymerization may be a therapeutic target for promoting regeneration of mature neurons.

Actins↗
PubMed2005

Lamprey neurofilaments contain a previously unreported 50-kDa protein.

We have previously hypothesized that regeneration of axons after spinal cord injury in the lamprey may involve assembly and transport of neurofilaments (NFs) into the growing tip. A single NF, NF-180, has been cloned in this laboratory and until now was thought to be the only NF subunit in lamprey nervous system. However, homopolymerization of NF-180 has not been observed either in experiments on transfected cells or in self-assembly tests in vitro. Forty-three monoclonal antibodies designated as LCM series were generated previously against cytoskeletal proteins of the lamprey nervous system. Seven LCMs were NF specific, and five were keratin specific, as demonstrated by immunohistochemistry. In the present study, one antibody, LCM40, selectively labeled axons in immunohistochemical sections and recognized a single 50-kDa protein in Western blots. Other neuron-specific LCMs and anti-NF antibodies, e.g., LCM39, recognized a known NF subunit, NF-180. Two-dimensional (2-D) gel electrophoresis was employed to separate otherwise indistinguishable individual cytoskeletal proteins. Western blot analysis with an antibody (IFA) that selectively labels all known intermediate filaments indicated that this 50-kDa protein is an intermediate filament (IF). The new protein was incorporated into IF polymers in vitro. Immunoelectron microscopy confirmed that neuronal IFs contain this novel protein. These results suggest that the 50-kDa protein is a previously unrecognized neuronal IF subunit in the lamprey.

Animals↗
PubMed2005

Fate of immortalized human neuronal progenitor cells transplanted in rat spinal cord.

BACKGROUND: Replacement of neurons and glia by transplantation has been proposed as a therapy for neurodegenerative diseases, including amyotrophic lateral sclerosis. This strategy requires using human motor neuronal progenitor cells or xenografts of animal cells, but there is little evidence that xenografted neuronal cells can survive in spinal cord despite immunosuppression. OBJECTIVE: To clarify the mechanisms responsible for the death of xenografted neurons in spinal cord. METHODS: Cells from an immortalized, neuronally committed, human embryonic spinal cord-derived cell line (HSP1) that expresses motor neuronal properties in vitro were transplanted into adult rat spinal cord. The rats were killed at intervals up to 8 weeks and serial sections through the graft sites were processed for immunofluorescence using primary antibodies against human nuclear and mitochondrial antigens, microtubule-associated protein 2, TUJ1, CD5, natural killer cells, and activated microglia-macrophages, caspase-3 and caspase-9. RESULTS: Grafted cells did not migrate and underwent partial differentiation along a neuronal pathway. They were rejected after 4 weeks despite cyclosporine immunosuppression. Cells died by apoptosis via the cytochrome c/caspase-9/caspase-3 pathway. The host response included natural killer cells and activated microglia-macrophages but few T cells. CONCLUSIONS: Intraspinal neuronal xenotransplantation failed because of apoptotic cell death. Neither T cells nor the spinal cord environment, which favors gliogenesis, are likely to have been responsible, but natural killer cells may have been involved.

Animals↗
PubMed2005

Live imaging of regenerating lamprey spinal axons.

BACKGROUND: The sea lamprey has been used as a model for the study of axonal regeneration after spinal cord injury. Although the growing tips of developing axons in lamprey have not been described, in all species studied, growth cones are complex in shape, consisting of a lamellipodium and filopodia, rich in F-actin and lacking neurofilaments (NF). By contrast, static immunohistochemical and electron microscopic observations of fixed tissue suggested that the tips of regenerating lamprey spinal axons are simple in shape, densely packed with NF, but contain very little F-actin. Thus, it has been proposed that regeneration of axons in the CNS of mature animals is not based on the canonical pulling mechanism of growth cones but involves an internal protrusive force, perhaps generated by the transport and assembly of NF. To eliminate the possibility that these histological features are due to fixation artifact, fluorescently labeled regenerating axon tips were imaged live. METHODS: Spinal cords were transected, and after 0 to 10 weeks, the CNS was isolated in lamprey Ringer at 5 degrees C to 12 degrees C and the large reticulospinal axons were microinjected with fluorescent tracers. The proximal axon tips were imaged with a fluorescence dissecting microscope repeatedly over 2 to 5 days and photographed with confocal microscopy. Experiments were also performed through a dorsal incision in the living animal. Axon tips were microinjected as above or retrogradely labeled with tracer applied to the transection site and photographed through the fluorescence dissecting scope or with two-photon microscopy. The spinal cords were then fixed and processed for wholemount NF immunohistochemistry. RESULTS: The living axon tips were simple in shape, not significantly different from those in fixed spinal cords, and filled with NF. In isolated CNS preparations, very little axon retraction and no regeneration was observed. In the living animal, rapid retraction, up to 3 mm/day, was seen during the 1st few days posttransection. At more than 2 weeks posttransection, some fibers showed regeneration of up to 35 microm/day. CONCLUSIONS: 1) The tips of regenerating lamprey axons are simple in shape and filled with NF. 2) Both axon retraction and axon extension are active processes, requiring factors present in the living animal that are missing in the isolated CNS.

Actins↗
PubMed2004

The single neurofilament subunit of lamprey may need another element for filament assembly.

Regenerating axon tips in transected lamprey spinal cord contain dense accumulations of neurofilaments (NFs), suggesting that NFs may play a role in the mechanism of axonal regeneration. Compared with heteropolymeric assemblies of NF triplet proteins in mammals, NF in lampreys has been thought to contain only a single subunit (NF180). This would imply that NF180 self-assembles, which would be important for manipulating its expression in studies of axonal regeneration. In order to study the possible role of NF in process outgrowth and to determine whether NF180 can self-assemble, its gene was transfected into mammalian and fish cell lines that either contain or lack vimentin. In transfected NIH3T3 cells, NF180 was poorly phosphorylated and its expression did not alter the length or number of cell processes. Nor did it appear to form typical intermediate filaments, suggesting that it may not self-assemble. NF180 also did not form typical filaments in SW13cl cells that either possessed or lacked vimentin, nor in transfected fish cells that were cultured at 18 degrees C. In vitro, NF180 could not self-assemble but interacted with NF-L to interrupt its self-assembly. When cotransfected with rat NF-L into SW13c1.2vim(-) cells, NF180 did form thick, rod-like filamentous structures on immunofluorescence. More typical NFs were observed when NF180 was cotransfected with both NF-L and NF-M. Thus, NF180 cannot self-assemble but appears to require one or more additional elements for incorporation into NFs.

Animals↗
PubMed2004

Cell proliferation in the lamprey central nervous system.

After spinal cord transection, axons regenerate both in larval and adult lampreys. It is not known to what degree cells proliferate, even in the uninjured animal. Therefore, we have determined the prevalence of mitosis in the lamprey central nervous system (CNS). Bromodeoxyuridine (BrdU) was injected and incorporated for 4 hours into 2- to 5-year-old larvae, animals undergoing metamorphosis, and young adults. Labeled cells were counted in the rhombencephalon (where most supraspinal projecting neurons are located) and spinal cord. A mitotic index (MI) was calculated as the percentage of nuclei that were labeled. There was a seasonal variation in mitotic activity, with higher MIs occurring in summer. Within the summer, there was an additional transient spike in mitosis, especially in the rhombencephalon. There was no correlation between age and MI within the range of developmental stages examined. Baseline MIs in the rhombencephalon and spinal cord were approximately 0.15% and 0.20%, respectively. In most animals, the highest mitotic rates in both the rhombencephalon and spinal cord were seen in the ependyma, but many labeled cells were found in nonependymal regions as well. During the summer spike, almost all of the additional mitosis in the rhombencephalon was in the ependyma, but this finding was not true in the spinal cord. Many BrdU-labeled cells in the spinal cord and rhombencephalon were also stained by monoclonal antibodies specific for lamprey glial keratin but were never labeled by anti-neurofilament antibodies. These results suggest that (1) neurogenesis is uncommon in the lamprey CNS; (2) during most of the year, baseline gliogenesis occurs mainly in the ependyma with substantial contribution by nonependymal areas. During the summer, a spike of mitotic activity occurs in the ependyma of the rhombencephalon and throughout the spinal cord.

Animals↗
PubMed2003

Promotion of axonal regeneration in the injured CNS.

Molecules that are found in the extracellular environment at a CNS lesion site, or that are associated with myelin, inhibit axon growth. In addition, neuronal changes--such as an age-dependent reduction in concentrations of cyclic AMP--render the neuron less able to respond to axotomy by a rapid, forward, actin-dependent movement. An alternative mechanism, based on the protrusive forces generated by microtubule elongation or the anterograde transport of cytoskeletal elements, may underlie a slower form of axon elongation that happens during regeneration in the mature CNS. Therapeutic approaches that restore the extracellular CNS environment or the neuron's characteristics back to a more embryonic state increase axon regeneration and improve functional recovery after injury. These advances in the understanding of regeneration in the CNS have major implications for neurorehabilitation and for the use of axonal regeneration as a therapeutic approach to disorders of the CNS such as spinal-cord injury.

Animals↗
PubMed2002

Magnetic resonance microimaging of intraaxonal water diffusion in live excised lamprey spinal cord.

Anisotropy of water diffusion in axon tracts, as determined by diffusion-weighted MRI, has been assumed to reflect the restriction of water diffusion across axon membranes. Reduction in this anisotropy has been interpreted as degeneration of axons. These interpretations are based primarily on a priori reasoning that has had little empirical validation. We used the experimental advantages of the sea lamprey spinal cord, which contains several very large axons, to determine whether intraaxonal diffusion is isotropic and whether anisotropy is attributable to restriction of water mobility by axon surface membranes. Through the application of magnetic resonance microimaging, we were able to measure the purely intraaxonal diffusion characteristics of the giant reticulospinal axons (20-40 microm in diameter). The intraaxonal apparent diffusion coefficients of water parallel (longitudinal ADC, l-ADC) and perpendicular (transverse ADC, t-ADC) to the long axis were 0.98 +/- 0.06 (10(-3) mm2 sec) and 0.97 +/- 0.11 (10(-3) mm2 sec), respectively. In white matter regions that included multiple axons, l-ADCs were almost identical regardless of axon density in the sampled axon tract. By comparison, t-ADCs were reduced and varied inversely with the number of axons (and thus axolemmas) in a fixed cross-sectional area. Thus, diffusion was found to be isotropic when measured entirely within a single axon and anisotropic when measured in regions that included multiple axons. These findings support the hypothesis that the cell membrane is the primary source of diffusion anisotropy in fiber tracts of the central nervous system.

Animals↗
PubMed2002

Visualization of individual axons in excised lamprey spinal cord by magnetic resonance microscopy.

The direct visualization of axons within their native tissue environment by magnetic resonance (MR) microscopy is presented for the first time in the excised larval sea lamprey spinal cord. A home-built transverse radio frequency coil of 1.5 mm diameter was used in conjunction with a commercial 400 MHz MR microscopy system, implementing both 2-D and 3-D imaging pulse sequences. Images having nominal voxel sizes of 9x9x500 and 9x9x125 microm(3), respectively, in the lamprey spinal cord were obtained, resolving individual Mauthner and Müller axons. Furthermore, architectural changes associated with axonal degeneration were visualized in the spinal cord of one animal, excised 8 weeks after hemisection of the cord. Although the lamprey previously has not been the subject of MR microscopy investigations, these results demonstrate the method's potential for imaging this axon system, which is an important model of spinal cord injury and regeneration.

Animals↗