Biomedical subjects

A Messing

Publications and source records attributed to A Messing.

At least 37 records · Page 2Linked to original sources

PubMed1999

Studies in transgenic mice indicate a loss of connexin32 function in X-linked Charcot-Marie-Tooth disease.

X-linked Charcot-Marie-Tooth disease (CMTX) is an inherited demyelinating neuropathy caused by mutations in the gene encoding the gap junction protein connexin32 (Cx32). Despite the identification of over 160 different mutations in the Cx32 coding sequence, it is not known whether the mutations cause the disease manifestations through a loss of Cx32 function or through toxic effects on peripheral nerve. We created transgenic mice with a frameshift mutation at codon 175 (175fs), identified in a large CMTX pedigree. Light microscopic examination of the peripheral nerves from adult transgenic animals showed no pathological features. Western blotting did not show transgenic Cx32 protein in any of the 26 lines, although expression of transgenic messenger RNA was detected by reverse-transcriptase polymerase chain reaction and by ribonuclease protection assay. Our findings indicate that the 175fs mutation results in a loss of Cx32 function, without additional toxic effects.

Animals↗

PubMed1999

Nonlethal detection of transgene expression in the CNS of founder mice.

Determining whether transgenes targeted to the central nervous system (CNS) are expressed has previously required sacrificing the animal; thus, if a line was to be maintained, it was necessary to generate offspring from the original founder mouse before assay. We describe a method for assaying for the expression of a variety of CNS-targeted transgenes that allows for the survival of the founder mice. Many CNS genes are expressed in the retina, which can be obtained from a mouse by simple survival surgery. We demonstrate that reverse transcription PCR of RNA isolated from a single eye can detect the mRNAs for glial fibrillary acidic protein (GFAP), vimentin, neurofilament light chain (NF-68) and Thy-1, all of whose promoters have been used to direct transgene expression in the CNS. We also show that this method readily detects expression of an astrocyte-specific GFAP-driven lacZ transgene and a neuron-specific L7-driven lacZ transgene.

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PubMed1998

Temperature-sensitive neuromuscular transmission in Kv1.1 null mice: role of potassium channels under the myelin sheath in young nerves.

In mammalian myelinated nerves, the internodal axon that is normally concealed by the myelin sheath expresses a rich repertoire of K channel subtypes thought to be important in modulating action potential propagation. The function of myelin-covered K channels at transition zones, however, has remained unexplored. Here we show that deleting the voltage-sensitive potassium channel Kv1.1 from mice confers a marked temperature-sensitivity to neuromuscular transmission in postnatal day 14 (P14)-P21 mice. Using immunofluorescence and electrophysiology, we examined contributions of four regions of the peripheral nervous system to the mutant phenotype: the nerve trunk, the myelinated segment preceding the terminal, the presynaptic terminal membrane itself, and the muscle. We conclude that the temperature-sensitive neuromuscular transmission is accounted for solely by a deficiency in Kv1.1 normally concealed in the myelinated segments just preceding the terminal. This paper demonstrates that under certain situations of physiological stress, the functional role of myelin-covered K channels is dramatically enhanced as the transition zone at the neuromuscular junction is approached.

Age Factors↗

PubMed1998

Astrocytes cultured from transgenic mice carrying the added human glial fibrillary acidic protein gene contain Rosenthal fibers.

Mice carrying copies of the human glial fibrillary acidic protein (hGFAP) gene driven by its own promoter have been generated that express the human transgene at different levels (Messing et al.: 152:391-398, 1998). Lines that expressed high levels of the gene died shortly after birth. Astrocyte cultures prepared from a low overexpressor (Tg73.2) exhibited abnormal cytoplasmic inclusions identical to those seen in vivo in the high overexpressors. Astrocytes in the Tg73.2 cultures appear odd-shaped and enlarged, express increased levels of GFAP (both human and mouse), and express alphaB crystallin protein, Hsp27 protein, and vimentin protein. At the light microscopic level, the Tg73.2 astrocytes are filled with eosinophilic deposits surrounded by positive GFAP immunostain. Ultrastructurally, the Tg73.2 astrocytes contain osmophilic deposits on a bed of intermediate filaments identical to Rosenthal fibers found in the brain in Alexander's disease. It seems that Tg73.2 mouse astrocytes in culture do not require additional stress from external sources or contact with other neuroectodermal cells to produce Rosenthal fibers. This suggests that the added hGFAP gene is sufficient to induce Rosenthal fibers and that an excess of GFAP in astrocytes may be detrimental to normal function. We hypothesize that the normal mechanism for GFAP turnover may be insufficient to handle the excess GFAP, thus causing an accumulation of stress proteins. The increased amounts of stress proteins and GFAP results in the formation of Rosenthal fibers, similar to those found in Alexander's disease.

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PubMed1998

Mice lacking GFAP are hypersensitive to traumatic cerebrospinal injury.

Glial fibrillary acidic protein (GFAP) is an intermediate filament protein expressed primarily in astrocytes. We have tested whether GFAP protects against mechanical stress by inducing percussive head injury in GFAP-null mice with a weight drop device. When mice were positioned on a foam bed which allowed head movement at impact, all 14 wild-type mice tested survived, but 12 of 15 GFAP-null mice died within a few minutes. The cause of death appeared to be upper cervical spinal cord injury resulting in respiratory arrest. When the foam bed was replaced by a firm support, both GFAP-null and wild-type mice survived. These results indicate that mice lacking GFAP are hypersensitive to cervical spinal cord injury caused by sudden acceleration of the head.

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PubMed1998

A minimal human MBP promoter-lacZ transgene is appropriately regulated in developing brain and after optic enucleation, but not in shiverer mutant mice.

Previous studies, both in vitro and in vivo, suggest that small portions of the mouse myelin basic protein (MBP) promoter are sufficient to activate regulated expression of MBP. To confirm our previous in vitro studies, we prepared transgenic mice with short regions of the human MBP promoter fused to the lacZ reporter gene. We found that 750 nucleotides of the proximal human MBP promoter is sufficient to activate oligodendrocyte-specific, developmentally regulated expression of lacZ in three independent lines. This promoter, however, does not activate expression of lacZ in Schwann cells in peripheral nerve or in adult mouse brain. The relative levels of beta-galactosidase specific activity, mRNA, and transcription parallel those of MBP mRNA during myelinogenesis. Thus, we exploited this transgene as a quantitative tool to evaluate the response to stimuli known to affect myelination. Transgene expression is reduced 75 % after optic enucleation, as previously reported for levels of MBP mRNA, indicating that axons signal to this portion of the proximal MBP promoter to fully activate MBP expression during myelinogenesis. Instead, in adult shiverer mice, another setting in which MBP transcription is modulated, transgene expression is not increased, in contrast to the increased transcriptional activation of MBP previously reported in these mice. These data suggest that the regulatory region that mediates transcriptional activation of the MBP gene is modular, since discrete subregions are required for activation in Schwann cells, during myelinogenesis in oligodendrocytes, during maintenance myelination in adult brain, and in the dysmyelinating mutant shiverer mouse.

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PubMed1998

Deletion of the K(V)1.1 potassium channel causes epilepsy in mice.

Mice lacking the voltage-gated potassium channel alpha subunit, K(V)1.1, display frequent spontaneous seizures throughout adult life. In hippocampal slices from homozygous K(V)1.1 null animals, intrinsic passive properties of CA3 pyramidal cells are normal. However, antidromic action potentials are recruited at lower thresholds in K(V)1.1 null slices. Furthermore, in a subset of slices, mossy fiber stimulation triggers synaptically mediated long-latency epileptiform burst discharges. These data indicate that loss of K(V)1.1 from its normal localization in axons and terminals of the CA3 region results in increased excitability in the CA3 recurrent axon collateral system, perhaps contributing to the limbic and tonic-clonic components of the observed epileptic phenotype. Axonal action potential conduction was altered as well in the sciatic nerve--a deficit potentially related to the pathophysiology of episodic ataxia/myokymia, a disease associated with missense mutations of the human K(V)1.1 gene.

Action Potentials↗

PubMed1998

Metabolic engineering as therapy for inborn errors of metabolism--development of mice with phenylalanine hydroxylase expression in muscle.

Treatment of many inherited liver enzyme deficiencies requires the removal of toxic intermediate metabolites from the blood of affected individuals. We propose that circulating toxins can be adequately cleared and disease phenotype influenced by enzyme expressed in tissues other than the liver. Phenylalanine hydroxylase (PAH) activity was constitutively expressed in skeletal and cardiac muscle of transgenic mice which carried the PAH cDNA under the transcriptional control of the mouse muscle creatine kinase promoter. Muscle PAH-expressing mice were bred to liver PAH-deficient, hyperphenylalaninemic mice to yield progeny that lack PAH activity in liver but express PAH in muscle. These mice exhibited hyperphenylalaninemia at baseline, but serum phenylalanine levels decreased significantly when the mice were supplemented with tetrahydrobiopterin (BH4), a required cofactor for PAH. This is the first demonstration that a liver-specific enzyme, when expressed in a heterologous tissue and supplied with necessary cofactors, can effectively clear toxic metabolites from the circulation of individuals with inherited enzyme deficiency. This result suggests that gene therapy targeted to heterologous tissues, such as muscle, will be effective in the treatment of selected inborn errors of metabolism.

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PubMed1998

Fatal encephalopathy with astrocyte inclusions in GFAP transgenic mice.

Increased expression of glial fibrillary acidic protein (GFAP) is a hallmark of gliosis, the astrocytic hypertrophy that occurs during a wide variety of diseases of the central nervous system. To determine whether this increase in GFAP expression per se alters astrocyte function, we generated transgenic mice that carry copies of the human GFAP gene driven by its own promoter. Astrocytes of these mice are hypertrophic, up-regulate small heat-shock proteins, and contain inclusion bodies identical histologically and antigenically to the Rosenthal fibers of Alexander's disease. Mice in the highest expressing lines die by the second postnatal week. The results support the notion that Alexander's disease is a disorder of astrocytes, and provide an animal model for studying the causes and consequences of inclusion body disease.

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PubMed1998

Transgenic studies of peripheral and central glia.

Transgenic manipulation of gene expression in the nervous system has proven immensely useful for the study of glia. This review focuses on studies of Schwann cell and astrocyte biology and pathology. These studies began with promoter mapping for glial-specific genes (P0 and GFAP), and then progressed to oncogene-induced transformation and toxin-induced cell ablation of glia. For GFAP, an intermediate filament of astrocytes, we have investigated the effects of alterations in gene dosage, both in terms of deficiency or excess of this structural protein. Finally, the utility of green fluorescent protein as a marker for live astrocytes is described.

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PubMed1997

Sodium channel distribution in axons of hypomyelinated and MAG null mutant mice.

Na+ channel organization was studied with immunofluorescence in the peripheral nervous system of mice genetically altered to produce abnormal myelin. In two of these strains, transcription of inserted transgenes was targeted to myelinating Schwann cells through linkage to a promoter for the myelin protein P0. Adults of both of these strains had hindlimb paralysis and a tremor on lifting by the tail. In one case, Schwann cells were eliminated via expression of the diphtheria toxin A chain (DT-A). During postnatal days 3-7, Na+ channel clustering at forming nodes was dramatically reduced compared with that of normal animals. At 1-3 months of age, Na+ channel immunofluorescence was often found spread over long stretches of the axolemma, instead of being confined to nodal gaps. In the second P0-linked transgenic model, Schwann cell expression of the large T antigen tsA-1609 resulted in cell cycle dysfunction. Adult axons had regions of diffuse Na+ channel labeling. Focal clusters were rare within these zones, which were characterized by a series of cells of myelinating phenotype tightly apposed to the axon. Previous studies suggested that Schwann cells had to reach the stage of ensheathment characterized by periaxonal myelin associated glycoprotein (MAG) expression in order to induce Na+ channel clustering. However, in MAG-deficient mice, Na+ channel labeling patterns within sciatic nerves were normal.

Aging↗

PubMed1997

Grafts of EGF-responsive neural stem cells derived from GFAP-hNGF transgenic mice: trophic and tropic effects in a rodent model of Huntington's disease.

The present study examined whether implants of epidermal growth factor (EGF)-responsive stems cells derived from transgenic mice in which the glial fibrillary acid protein (GFAP) promoter directs the expression of human nerve growth factor (hNGF) could prevent the degeneration of striatal neurons in a rodent model of Huntington's disease (HD). Rats received intrastriatal transplants of GFAP-hNGF stem cells or control stem cells followed 9 days later by an intrastriatal injection of quinolinic acid (QA). Nissl stains revealed large striatal lesions in rats receiving control grafts, which, on average, encompassed 12.78 mm3. The size of the lesion was significantly reduced (1.92 mm3) in rats receiving lesions and GFAP-hNGF transplants. Rats receiving QA lesions and GFAP-hNGF-secreting grafts stem cell grafts displayed a sparing of striatal neurons immunoreactive (ir) for glutamic acid decarboxylase, choline acetyltransferase, and neurons histochemically positive for nicotinamide adenosine diphosphate. Intrastriatal GFAP-hNGF-secreting implants also induced a robust sprouting of cholinergic fibers from subjacent basal forebrain neurons. The lesioned striatum in control-grafted animals displayed numerous p75 neurotrophin-ir (p75NTR) astrocytes, which enveloped host vasculature. In rats receiving GFAP-hNGF-secreting stem cell grafts, the astroglial staining pattern was absent. By using a mouse-specific probe, stem cells were identified in all animals. These data indicate that cellular delivery of hNGF by genetic modification of stem cells can prevent the degeneration of vulnerable striatal neural populations, including those destined to die in a rodent model of HD, and supports the emerging concept that this technology may be a valuable therapeutic strategy for patients suffering from this disease.

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PubMed1997

Live astrocytes visualized by green fluorescent protein in transgenic mice.

Green fluorescent protein (hGFP-S65T) was expressed in transgenic mice under the control of the astrocyte-specific glial fibrillary acidic protein (GFAP) promoter. Tissues from two independent transgenic lines were characterized by Northern blot analysis and by confocal microscopy. The expression pattern in these two lines was identical in all tissues examined, and similar to that found previously with a lacZ transgene driven by the same promoter. Bright fluorescence was observed in the cell bodies and processes of unfixed or fixed astrocytes, using both whole mount and brain slice preparations, from multiple areas of the central nervous system. However, in contrast to GFAP-lacZ transgenics, retinal Müller cells expressed the GFP transgene in response to degeneration of neighboring photoreceptors. These data indicate that the 2.2-kb hGFAP promoter contains sufficient regulatory elements to direct expression in Müller cells, and that GFP is a suitable reporter gene for use in living preparations of the mammalian nervous system. Such mice should prove useful for studies of dynamic changes in astrocyte morphology during development, and in response to physiological and pathological conditions.

Animals↗

PubMed1997

Generation and transplantation of EGF-responsive neural stem cells derived from GFAP-hNGF transgenic mice.

EGF-responsive neural stem cells isolated from murine striatum have the capacity to differentiate into both neurons and glia in vitro. Genetic modification of these cells is hindered by a number of problems such as gene stability and transfection efficiency. To circumvent these problems we generated transgenic mice in which the human GFAP promoter directs the expression of human NGF. Neural stem cells isolated from the forebrain of these transgenic animals proliferate and form clusters, which appear identical to stem cells generated from control animals. Upon differentiation in vitro, the transgenic stem cell-derived astrocytes express and secrete bioactive hNGF. Undifferentiated GFAP-hNGF or control stem cells were transplanted into the striatum of adult rats. One and 3 weeks after transplantation, hNGF was detected immunocytochemically in an halo around the transplant sites. In GFAP-hNGF-grafted animals, intrinsic striatal neurons proximal to the graft appear to have taken up hNGF secreted by the grafted cells. Ipsilateral to implants of GFAP-hNGF-secreting cells, choline acetyltransferase-immunoreactive neurons within the striatum were hypertrophied relative to the contralateral side or control-grafted animals. Further, GFAP-hNGF-grafted rats displayed a robust sprouting of p75 neurotrophin receptor-positive fibers emanating from the underlying basal forebrain. These studies indicate that EGF-responsive stem cells which secrete hNGF under the direction of the GFAP promoter display in vitro and in vivo properties similar to that seen following other methods of NGF delivery and this source of cells may provide an excellent avenue for delivery of neurotrophins such as NGF to the central nervous system.

Animals↗

PubMed1997

Axonal and nonneuronal cell responses to spinal cord injury in mice lacking glial fibrillary acidic protein.

We have examined the regeneration of corticospinal tract fibers and expression of various extracellular matrix (ECM) molecules and intermediate filaments [vimentin and glial fibrillary acidic protein (GFAP)] after dorsal hemisection of the spinal cord of adult GFAP-null and wild-type littermate control mice. The expression of these molecules was also examined in the uninjured spinal cord. There was no increase in axon sprouting or long distance regeneration in GFAP-/- mice compared to the wild type. In the uninjured spinal cord (i) GFAP was expressed in the wild type but not the mutant mice, while vimentin was expressed in astrocytes in the white matter of both types of mice; (ii) laminin and fibronectin immunoreactivity was localized to blood vessels and meninges; (iii) tenascin and chondroitin sulfate proteoglycan (CSPG) labeling was detected in astrocytes and the nodes of Ranvier in the white matter; and (iv) in addition, CSPG labeling which was generally less intense in the gray matter of mutant mice. Ten days after hemisection there was a large increase in vimentin+ cells at the lesion site in both groups of mice. These include astrocytes as well as meningeal cells that migrate into the wound. The center of these lesions was filled by laminin+/fibronectin+ cells. Discrete strands of tenascin-like immunoreactivity were seen in the core of the lesion and lining its walls. Marked increases in CSPG labeling was observed in the CNS parenchyma on either side of the lesion. These results indicate that the absence of GFAP in reactive astrocytes does not alter axonal sprouting or regeneration. In addition, except for CSPG, the expression of various ECM molecules appears unaltered in GFAP-/- mice.

Animals↗

PubMed1997

Proline-rich-protein promoters direct LacZ expression to the granular convoluted tubular cells of the submandibular gland in adult transgenic mice.

The ability of two mouse PRP gene promoters to direct the expression of the bacterial lacZ reporter gene was tested in transgenic mice. Transgenes A1-lacZ and C1-lacZ consisted of 8.2 kb A1 and 7.8 kb C1 PRP promoters respectively fused to the lacZ coding sequence. A1 and C1 are two A-type PRP genes isolated from the inbred SWR mice, which show the same gene structure and similar sequence to the closely related MP2 and M14 PRP genes previously cloned from outbred CD-1 mice. We here show that both A1-lacZ and C1-lacZ transgenes have very similar expression patterns: (1) they expressed the lacZ gene in all 14 established transgenic lines under normal (non-stimulated) conditions; (2) the expression was restricted to the granular convoluted tubular cells of the submandibular glands; (3) the expression was developmentally regulated beginning at sexual maturation and lasting to at least 1.5 years of age; and (4) expression in some lines was probably influenced by sex hormones, since higher expression was found in males than in females. A1-lacZ and C1-lacZ are the first transgenes derived from the PRP/GRP (glutamine/glutamic acid-rich protein) gene superfamily to be expressed in the granular convoluted tubular cells (with known endocrine functions), rather than in the acinar cells (with mainly exocrine functions) of the submandibular glands.

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PubMed1996

Absence of the beta subunit (cchb1) of the skeletal muscle dihydropyridine receptor alters expression of the alpha 1 subunit and eliminates excitation-contraction coupling.

The multisubunit (alpha 1s, alpha 2/delta, beta 1, and gamma) skeletal muscle dihydropyridine receptor transduces transverse tubule membrane depolarization into release of Ca2+ from the sarcoplasmic reticulum, and also acts as an L-type Ca2+ channel. The alpha 1s subunit contains the voltage sensor and channel pore, the kinetics of which are modified by the other subunits. To determine the role of the beta 1 subunit in channel activity and excitation-contraction coupling we have used gene targeting to inactivate the beta 1 gene. beta 1-null mice die at birth from asphyxia. Electrical stimulation of beta 1-null muscle fails to induce twitches, however, contractures are induced by caffeine. In isolated beta 1-null myotubes, action potentials are normal, but fail to elicit a Ca2+ transient. L-type Ca2+ current is decreased 10- to 20-fold in the beta 1-null cells compared with littermate controls. Immunohistochemistry of cultured myotubes shows that not only is the beta 1 subunit absent, but the amount of alpha 1s in the membrane also is undetectable. In contrast, the beta 1 subunit is localized appropriately in dysgenic, mdg/mdg, (alpha 1s-null) cells. Therefore, the beta 1 subunit may not only play an important role in the transport/insertion of the alpha 1s subunit into the membrane, but may be vital for the targeting of the muscle dihydropyridine receptor complex to the transverse tubule/sarcoplasmic reticulum junction.

Action Potentials↗

PubMed1996

Conditional ablation of cerebellar astrocytes in postnatal transgenic mice.

Astrocytes have been proposed to have multiple roles in the development and maintenance of the vertebrate CNS. To facilitate documentation of these roles, we designed a transgene to enable their ablation at selectable times. The transgene consists of the coding region for the herpes simplex virus-thymidine kinase (HSV-TK) under the control of the human glial fibrillary acidic protein gene promoter. The HSV-TK is innocuous but converts the antiherpetic agent ganciclovir (GCV) to a toxic product that interferes with DNA replication in proliferating cells. In a developmental study, transgenic mice were treated with GCV during the first postnatal week, with evaluation at P19. Treated mice displayed severe ataxia. Histological examination revealed disrupted astrocyte development, particularly in the cerebellum, with marked secondary effects on other cell types. Cerebellar defects included a loss in the numbers of astrocytes and an overall reduction in cerebellar size and disruption of the normally well defined cellular layers. Radial glia were disordered, Purkinje cells were ectopically distributed and displayed abnormal dendritic trees, and granule cells were markedly depleted. These effects were more severe in animals treated on postnatal day 1 versus treatment at day 5. A major factor causing granule cell death was excitotoxicity attributable to activation of NMDA receptors. These results suggest a critical role for astrocytes in cerebellar development.

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