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Biomedical subjects

Michael Brenner

Publications and source records attributed to Michael Brenner.

At least 19 recordsLinked to original sources
PubMed2006

Efficient large-scale synthesis of BILN 2061, a potent HCV protease inhibitor, by a convergent approach based on ring-closing metathesis.

A multistep scalable synthesis of the clinically important hepatitis C virus (HCV) protease inhibitor BILN 2061 (1) is described. The synthesis is highly convergent and consists of two amide bond formations, one etherification, and one ring-closing metathesis (RCM) step, using readily available building blocks 2-5. The optimization of each step is described at length. The main focus of the paper is the study of the RCM step and the description of the main problems faced when scaling up to pilot scale this highly powerful but very challenging synthetic operation. Eventually, the RCM reaction was smoothly scaled up to produce >400 kg of cyclized product.

Antiviral Agents↗
PubMed2006

The Alexander disease-causing glial fibrillary acidic protein mutant, R416W, accumulates into Rosenthal fibers by a pathway that involves filament aggregation and the association of alpha B-crystallin and HSP27.

Here, we describe the early events in the disease pathogenesis of Alexander disease. This is a rare and usually fatal neurodegenerative disorder whose pathological hallmark is the abundance of protein aggregates in astrocytes. These aggregates, termed "Rosenthal fibers," contain the protein chaperones alpha B-crystallin and HSP27 as well as glial fibrillary acidic protein (GFAP), an intermediate filament (IF) protein found almost exclusively in astrocytes. Heterozygous, missense GFAP mutations that usually arise spontaneously during spermatogenesis have recently been found in the majority of patients with Alexander disease. In this study, we show that one of the more frequently observed mutations, R416W, significantly perturbs in vitro filament assembly. The filamentous structures formed resemble assembly intermediates but aggregate more strongly. Consistent with the heterozygosity of the mutation, this effect is dominant over wild-type GFAP in coassembly experiments. Transient transfection studies demonstrate that R416W GFAP induces the formation of GFAP-containing cytoplasmic aggregates in a wide range of different cell types, including astrocytes. The aggregates have several important features in common with Rosenthal fibers, including the association of alpha B-crystallin and HSP27. This association occurs simultaneously with the formation of protein aggregates containing R416W GFAP and is also specific, since HSP70 does not partition with them. Monoclonal antibodies specific for R416W GFAP reveal, for the first time for any IF-based disease, the presence of the mutant protein in the characteristic histopathological feature of the disease, namely Rosenthal fibers. Collectively, these data confirm that the effects of the R416W GFAP are dominant, changing the assembly process in a way that encourages aberrant filament-filament interactions that then lead to protein aggregation and chaperone sequestration as early events in Alexander disease.

Alexander Disease↗
PubMed2006

Astrocyte heterogeneity revealed by expression of a GFAP-LacZ transgene.

Glial fibrillary acidic protein (GFAP) is an intermediate filament protein present primarily in astrocytes. The gene is first expressed as astrocytes mature, and in the adult is strongly upregulated in response to CNS damage. Thus, in addition to its astrocyte specificity, transcriptional regulation of the GFAP gene is of interest as a reporter of CNS signaling during development and injury. Several laboratories have shown that approximately 2 kb of 5'-flanking DNA of the human or mouse GFAP gene is sufficient to direct transgene expression to astrocytes and to confer developmental and injury-induced regulation. Enhancer regions have been identified adjacent to the basal promoter and about 1500 bp upstream of the RNA start site. Juxtaposition of these two segments yielded a 447 bp promoter, gfa28, which strongly drove reporter activity in transfected glioma cells. We report here that in mice a gfa28-lacZ transgene expresses in only certain brain regions, revealing an unexpected heterogeneity among astrocytes. The restricted pattern of expression is present early in development, is not altered by injury, and is preserved in cultured astrocytes. However, astrocytes cultured from an inactive region strongly express a transiently transfected gfa28-lacZ construct, and activity of the embedded gfa28-lacZ transgene is partially restored by treatment with a histone deacetylase inhibitor. These results indicate that the absence of gfa28-lacZ expression in specific brain regions results from a developmental failure to remodel GFAP chromatin to an open structure. Thus, expression of the gfa28-lacZ transgene appears to serendipitously mark a distinct set of astrocyte precursors.

Animals↗
PubMed2006

Increased glia-specific transgene expression with glial fibrillary acidic protein promoters containing multiple enhancer elements.

The ability to direct transgene expression to astrocytes has become increasingly important as the roles for these cells continue to expand. Promoters consisting of the 5'-flanking region of the human or mouse glial fibrillary acidic protein (GFAP) gene have generally proved satisfactory. However, a more powerful promoter would be advantageous for several applications, such as expression of dominant negative RNAs or proteins, or for gene therapy. We investigated the possibility of increasing the transcriptional activity of the human GFAP promoter by inserting into it one or three additional copies of putative GFAP enhancer regions. The promoters enhanced with three additional copies gave 75-fold higher LacZ expression levels upon plasmid transfection into GFAP-expressing U251 cells than the parental gfa2 promoter. Surprisingly, in a transgenic mouse model, the enhanced promoters resulted in no or only very low expression of marker genes, probably caused by toxicity. When various cell lines were infected with replication-deficient adenoviral vectors, the enhanced promoters gave LacZ expression levels that were approximately 10-fold higher than those with the parental gfa2 promoter, while retaining specificity for GFAP-expressing cells. Injection of the adenoviral vectors carrying the enhanced promoters into nude mouse brain showed that LacZ expression was limited to GFAP-positive cells. We conclude that gfa2 enhanced promoters are useful for production of short-term, glia-specific, high expression levels of genes in an adenoviral context. Adenoviral vectors containing these enhanced promoters may be useful in glioma gene therapy.

Adenoviridae↗
PubMed2006

Enhanced hippocampal neurodegeneration after traumatic or kainate excitotoxicity in GFAP-null mice.

Astrocytes perform a variety of functions in the adult central nervous system. Recent evidence suggests that the upregulation of glial fibrillary acidic protein (GFAP), an astrocyte-specific intermediate filament component, is a biological marker of neurotoxicity after cerebral injury. We herein compared the response to traumatic brain injury or kainic acid (KA)-induced neurotoxicity in GFAP knockout (GFAP-KO) and wild-type (WT) mice. Seventy-two hours after injury, all GFAP-KO mice showed hippocampal CA3 neurodegeneration, whereas WT mice did not show neurodegeneration. Seventy-two hours after KA administration, GFAP-KO mice were more susceptible to KA-induced seizures and had an increased number of pyknotic damaged CA3 neurons than did WT mice. These results indicate that GFAP plays a crucial role in pyramidal neuronal survival after injury or KA-induced neurotoxicity.

Animals↗
PubMed2005

Propensity for paternal inheritance of de novo mutations in Alexander disease.

De novo dominant mutations in the GFAP gene have recently been associated with nearly all cases of Alexander disease, a rare but devastating neurological disorder. These heterozygous mutations must occur very early in development and be present in nearly all cells in order to be detected by the sequencing methods used. To investigate whether the mutations may have arisen in the parental germ lines, we determined the parental chromosome bearing the mutations for 28 independent Alexander disease cases. These cases included 17 different missense mutations and one insertion mutation. To enable assignment of the chromosomal origin of the mutations, six new single nucleotide polymorphisms in the GFAP gene were identified, bringing the known total to 26. In 24 of the 28 cases analyzed, the paternal chromosome carried the GFAP mutation (P < 0.001), suggesting that they predominantly arose in the parental germ line, with most occurring during spermatogenesis. No effect of paternal age was observed. There has been considerable debate about the magnitude of the male to female germ line mutation rate; our ratio of 6:1 is consistent with indirect estimates based on the rate of evolution of the sex chromosome relative to the autosomic chromosomes.

Adult↗
PubMed2005

Alexander-disease mutation of GFAP causes filament disorganization and decreased solubility of GFAP.

Alexander disease is a fatal neurological illness characterized by white-matter degeneration and the formation of astrocytic cytoplasmic inclusions called Rosenthal fibers, which contain the intermediate filament glial fibrillary acidic protein (GFAP), the small heat-shock proteins HSP27 and alphaB-crystallin, and ubiquitin. Many Alexander-disease patients are heterozygous for one of a set of point mutations in the GFAP gene, all of which result in amino acid substitutions. The biological effects of the most common alteration, R239C, were tested by expressing the mutated protein in cultured cells by transient transfection. In primary rat astrocytes and Cos-7 cells, the mutant GFAP was incorporated into filament networks along with the endogenous GFAP and vimentin, respectively. In SW13Vim(-) cells, which have no endogenous cytoplasmic intermediate filaments, wild-type human GFAP frequently formed filamentous bundles, whereas the R239C GFAP formed 'diffuse' and irregular patterns. Filamentous bundles of R239C GFAP were sometimes formed in SW13Vim(-) cells when wild-type GFAP was co-transfected. Although the presence of a suitable coassembly partner (vimentin or GFAP) reduced the potential negative effects of the R239C mutation on GFAP network formation, the mutation affected the stability of GFAP in cells in a dominant fashion. Extraction of transfected SW13Vim(-) cells with Triton-X-100-containing buffers showed that the mutant GFAP was more resistant to solubilization at elevated KCl concentrations. Both wild-type and R239C GFAP assembled into 10 nm filaments with similar morphology in vitro. Thus, although the R239C mutation does not appear to affect filament formation per se, the mutation alters the normal solubility and organization of GFAP networks.

Actin Cytoskeleton↗
PubMed2005

Unusual variants of Alexander's disease.

The purpose of this study was to describe unusual variants of Alexander's disease. We studied 10 patients who did not meet previously established magnetic resonance imaging (MRI) criteria for Alexander's disease, but for whom this diagnosis was considered because of Rosenthal fibers at histological examination or presence of some MRI features suggestive of Alexander's disease. Sequence analysis of the GFAP gene was performed. In eight patients, MRI results showed predominantly posterior fossa lesions, especially multiple tumor-like brainstem lesions. One patient had asymmetrical frontal white matter abnormalities and basal ganglia abnormalities. One patient (Patient 10) developed degeneration of the frontal white matter. In nine patients, a mutation was found that was concluded to be pathogenic, because the mutation was de novo (five patients), a known mutation was found (two patients), or assembly of the glial fibrillary acidic protein was abnormal in cultured cells (two patients). In Patient 10, a DNA variation was found that was also present in the patient's clinically unaffected father and was concluded to be a polymorphism. In conclusion, DNA diagnostics is warranted in patients who display MRI features suggestive of Alexander's disease, even if they do not meet the full set of previously established MRI criteria.

Adolescent↗
PubMed2005

Glial fibrillary acidic protein mutations in infantile, juvenile, and adult forms of Alexander disease.

Alexander disease is a progressive, usually fatal neurological disorder defined by the widespread and abundant presence in astrocytes of protein aggregates called Rosenthal fibers. The disease most often occurs in infants younger than 2 years and has been labeled a leukodystrophy because of an accompanying severe myelin deficit in the frontal lobes. Later onset forms have also been recognized based on the presence of abundant Rosenthal fibers. In these cases, clinical signs and pathology can be quite different from the infantile form, raising the question whether they share the same underlying cause. Recently, we and others have found pathogenic, de novo missense mutations in the glial fibrillary acidic protein gene in most infantile patients examined and in a few later onset patients. To obtain further information about the role of glial fibrillary acidic protein mutations in Alexander disease, we analyzed 41 new patients and another 3 previously described clinically, including 18 later onset patients. Our results show that dominant missense glial fibrillary acidic protein mutations account for nearly all forms of this disorder. They also significantly expand the catalog of responsible mutations, verify the value of magnetic resonance imaging diagnosis, indicate an unexpected male predominance for the juvenile form, and provide insights into phenotype-genotype relations.

Adolescent↗
PubMed2004

Prevalence of thyroid dysfunction in patients with Ménière's disease.

OBJECTIVE: To estimate the prevalence of hypothyroidism in a population of patients with Ménière's disease (MD). DESIGN: Retrospective case-control study comparing the use of thyroid hormone supplements between patients with MD and controls. SETTING: Outpatient neurology clinic specializing in the management of patients experiencing dizziness. PATIENTS: Fifty patients who met the 1995 American Academy of Otolaryngology criteria for MD and 50 controls matched for age and sex also experiencing dizziness. RESULTS: The 50 patients who met the criteria for MD were identified from a database of more than 2000 patients with dizziness seen over a 5-year period. Of these, 16 (32%) were taking thyroid hormone supplements in contrast to 2 (4%) of the 50 matched controls (P<.001). The median age of the patients with MD was 60 years, and 5 (19%) of the 26 patients younger than 60 years were taking thyroid hormone supplements, compared with 11 (46%) of 24 patients 60 years or older (P<.05). There were no statistically significant differences in the severity of hearing loss, pattern of hearing loss, or prevalence of bilateral hearing loss between patients with MD who were taking thyroid hormone supplements and patients with MD who were not. CONCLUSION: Ménière's disease is associated with corrected hypothyroidism.

Case-Control Studies↗
PubMed2004

Expression specificity of GFAP transgenes.

Glial fibrillary acidic protein (GFAP) is an intermediate filament protein found predominantly in astrocytes. This specificity has recommended the GFAP gene promoter for targeting transgene expression to astrocytes. Although both we [Brenner et al. J. Neurosci. 14:1030-1037, (1994)] and others [Mucke et al. New Biol. 3:465-474, (1991)] have reported astrocyte specificity for GFAP promoters, we demonstrate here that these DNA sequences can also direct activity in neurons. The pattern of neuronal activity varied with both the nature of the expressed sequence and the transgene insertion site. Specifically, neuronal expression was very high for a protective protein/cathepsin A minigene, moderate for lacZ and undetectable for GFP. These findings, coupled with a survey of the literature, recommend that investigators using GFAP-driven transgenes verify specificity for each line studied, using a detection system whose sensitivity is sufficient to detect a compromising level of misexpression.

Adenosine Diphosphate↗
PubMed2004

Expression of glial fibrillary acidic protein in primary cultures of human Müller cells.

Glial fibrillary acidic protein (GFAP) is an intermediate filament protein which is primarily found in astrocytes. However, in eye diseases or when eye injuries occur, GFAP is expressed in large quantities in retinal Müller cells. The mechanism for this altered expression is unknown, but presumably involves injury-dependent signaling. The purpose of this study was to investigate regulation of GFAP gene expression in human Müller cells in vitro. Immunofluorescence, western blot, RT-PCR and Northern blot analyses were used to demonstrate the expression of GFAP in cultured Müller cells. Plasmids bearing various segments of the human GFAP promoter fused to a CAT reporter gene were used to transfect primary cultures of human Müller cells as well as the non-glial cell lines 293T and HeLa. Transcription directed by the GFAP promoter was found to be more than 50-times stronger in the Müller cells than in either of the non-glial cell lines, consistent with the data for endogenous GFAP expression. To our knowledge, this is the first study demonstrating GFAP gene regulation in human Müller cells. By examining the transcriptional activity of various segments from the human GFAP promoter, it can be concluded that the GFAP gene is differently regulated in Müller cells compared to glioma cell lines from the central nervous system (CNS).

Cell Line↗
PubMed2004

Effects of external beam radiation in the rat tibial nerve after crush, transection and repair, or nerve isograft paradigms.

INTRODUCTION: In head and neck surgery, radiation therapy is often administered to an injured nerve. Previous studies have examined the effects of either preoperative or postoperative radiation on nerve regeneration in rodents. In these studies, histomorphometric analysis was performed up to 8 month postoperatively. Given the exceptional neuroregenerative capacity of rodents, significant differences in nerve regeneration may go undetected if nerves are evaluated at such distant postoperative time points. This study is designed with a more appropriate model and investigates the effects of radiation after three common nerve injury paradigms. METHODS: Sixty-four Lewis rates were randomized to 8 groups corresponding to uninjured, tibial nerve crush, transection and repair, or reconstruction with isografts. Half of the animals in each of these paradigms (n = 8 per group) were treated with 10 Gy of external beam radiation to the site of nerve injury at 7 days postoperatively. On postoperative day 28, functional recovery and histomorphometric assessment was performed. RESULTS: For a given paradigm of nerve injury, no significant differences in nerve fiber number, neural density, neural debris, or fiber width were noted between the control and radiated groups, and radiation did not affect functional recovery. CONCLUSION: Radiation had no discernible effect on nerve regeneration or functional recovery in the rodent nerve injury models studied. All assessments were made at time points suitable for detecting differences in nerve regeneration between groups. These findings suggest that administration of radiation to fields containing injured peripheral nerve is unlikely to adversely affect functional outcomes.

Animals↗
PubMed2003

Low prevalence of antibodies to glucose-6-phosphate isomerase in patients with rheumatoid arthritis and a spectrum of other chronic autoimmune disorders.

OBJECTIVE: Arthritis in the K/BxN mouse model results from pathogenic immunoglobulins that recognize glucose-6-phosphate isomerase (GPI), a glycolytic enzyme residing in the cytoplasm of all cells. Antibodies directed against GPI can, alone, transfer arthritis to healthy recipients. Previous experiments have revealed significant titers of anti-GPI antibodies in the serum of many patients with rheumatoid arthritis (RA). We evaluated the generality of these observations in cohorts of patients with 12 different arthritic and chronic autoimmune diseases and in population-matched healthy control subjects. METHODS: Anti-GPI antibodies were assayed in 811 individual serum samples by enzyme-linked immunosorbent assay with 2 forms of GPI, recombinant and native. Results were confirmed by immunoblotting. RESULTS: Several patients had significantly elevated anti-GPI antibody titers, but without the prevalence or the specificity reported previously. Only 15% of RA patients had anti-GPI antibodies (range 12-29% in different cohorts), with a higher prevalence in patients with active disease. Psoriatic arthritis, undifferentiated arthritis, and spondylarthropathy patients also displayed anti-GPI antibodies at similar frequencies (12-25%). Similar titers were detected in a proportion (5-10%) of control subjects or patients with Crohn's disease or sarcoidosis. Very high titers were found in rare cases of RA and systemic lupus erythematosus. CONCLUSION: No disease-specific pattern of antibody positivity to GPI was apparent. While the antibody-mediated mechanism at play in the mouse model may exemplify a generic mechanism for some forms of arthritis in humans, GPI itself does not appear to be a target common to the majority of RA patients.

Adult↗
PubMed2003

GFAP: functional implications gleaned from studies of genetically engineered mice.

GFAP is the major intermediate filament of mature astrocytes, and its relatively specific expression in these cells suggests an important function. To study the role of the GFAP gene, mice have been created carrying null alleles (no protein), modified alleles (altered protein), or added wild type alleles (elevated protein). Surprisingly, absence of GFAP has relatively subtle effects on development. On the other hand, over-expression can be lethal, and led to the discovery that GFAP coding mutations are responsible for most cases of Alexander disease, a devastating neurodegenerative disorder. Here we review what the various GFAP mouse models reveal about GFAP and astrocyte function.

Alexander Disease↗
PubMed2003

Alexander's disease: clinical, pathologic, and genetic features.

Alexander's disease, a rare and fatal disorder of the central nervous system, most commonly affects infants and young children but can also occur in older children and sometimes adults. In infants and young children, it causes developmental delay, psychomotor retardation, paraparesis, feeding problems, usually megalencephaly, often seizures, and sometimes hydrocephalus. Juvenile cases often do not have megalencephaly and tend to have predominant pseudobulbar and bulbar signs. In both groups, characteristic magnetic resonance imaging findings have been described. In adult cases, the signs are variable, can resemble multiple sclerosis, and might include palatal myoclonus. In all cases, the examination of brain tissue shows the presence of widely distributed Rosenthal fibers. Almost all cases have recently been found to have a heterozygous, missense, point mutation in the gene for glial fibrillary acidic protein, which provides a new diagnostic tool. In most cases, the mutation appears to occur de novo, not being present in either parent, but some adult cases are familial.

Age of Onset↗