Biomedical subjects

R A Lazzarini

Publications and source records attributed to R A Lazzarini.

At least 37 records · Page 2Linked to original sources

PubMed1996

Functional dissection of the mouse Hox-a5 gene.

The Hox genes are clustered in evolutionarily conserved complexes and encode DNA binding proteins that determine positional identity. Ubiquitous expression of fly or mammalian Hox proteins in Drosophila embryos provides an assay for gene function, since different Hox genes induce characteristic homeotic transformations. Drosophila Sex combs reduced (Scr) and its murine cognate Hox-a5 produce identical transformations in transgenic flies. To study the contributions of domains conserved between the two proteins, truncated versions of mouse Hox-a5 were assayed for their ability to activate transcription in cultured cells and to induce homeotic transformation and activate target gene expression in transgenic embryos. The homeodomain is essential for protein function and/or nuclear targeting; the N-terminal region contributes to transcription activity and transformation potential in the embryo, but plays no role in determining functional specificity. The YPWM motif is essential for biological specificity, although it does not contribute to transcriptional activation potential. It was recently shown that the Hox-a5 YPWM motif is necessary for in vitro interactions with the co-factor Pbx1. Our results suggest that this type of protein-protein interaction may be essential for the biological activities of Hox-a5 and Scr.

Amino Acid Sequence↗

PubMed1996

Proteolipid/DM-20 proteins bearing the paralytic tremor mutation in peripheral nerves and transfected Cos-7 cells.

Paralytic tremor (Plp-pt) is a missense mutation of the myelin proteolipid gene (Plp) in rabbits. The myelin yield in the Plp-pt brain is reduced and the protein and lipid composition of central nervous system (CNS) myelin is abnormal. We studied the intracellular transport of the normal and Plp-pt mutant PLP and DM-20 in transiently transfected Cos-7 cells. While the mutant PLP accumulates in the rough endoplasmic reticulum and does not reach the plasma membrane, the spliced isoform of PLP, mutant DM-20, is normally transported to the cell surface and integrated into the membrane. Analysis of rabbit sciatic nerves revealed that concentration of peripheral nervous system (PNS) myelin proteins is normal in Plp-pt myelin. In the PNS like in the CNS, the level of Plp gene products is subnormal. But this does not affect myelination in the PNS where PLP, present in low concentration, is not a structural component of compact myelin. The normal level of Plp gene expression in Schwann cells is low and these results suggest that, in the Plp-pt PNS, Schwann cell function is not affected by the deficiency in PLP and/or the impairment of intracellular PLP transport.

Animals↗

PubMed1996

Age-dependent spatial memory deficits in transgenic mice expressing the human mid-sized neurofilament gene: I.

Previous studies have revealed that the transgenic mouse line expressing the human neurofilament-mid-sized (NF-M) gene evidences age-dependent and cell-specific pathological neurofibrillary accumulation in the central nerve system. In the current study, we investigated the learning and memory processes of NF-M transgenic mice at 3 and 8 months of age in a modified Morris water maze using a series of tasks including those primarily related to reference memory (i.e., spatial learning, reversal learning and probe trials) and to working memory (i.e., matching to sample tasks with or without delays). At 3 months of age, NF-M transgenic mice were indistinguishable from age- and litter-matched non-transgenic wild-type controls on any of the tests of reference and working memory. At 8 months of age, however, the NF-M transgenic mice exhibited significantly poorer performance than the age- and litter-matched wild-type control mice on both reference and working memory tasks. Immunohistological study of the brains of the 8-month-old NF-M transgenic mice revealed spherical and tangle-like neurofilamentous accumulation in their cerebral cortices. These results suggest that NF-M transgenic mice express both age-related histopathological changes and age-dependent learning and memory deficits. Whether NF-M transgenic mice exhibit even more severe behavioral impairments when they become aged is currently under study.

Aging↗

PubMed1996

A cellular mechanism governing the severity of Pelizaeus-Merzbacher disease.

Pelizaeus-Merzbacher disease (PMD) is a leukodystrophy linked to the proteolipid protein gene (PLP). We report a cellular basis for the distinction between two disease subtypes, classical and connatal, based on protein trafficking of the two PLP gene products (PLP and DM20). Classical PMD mutations correlate with accumulation of PLP in the ER of transfected COS-7 cells while the cognate DM20 traverses the secretory pathway to the cell surface. On the other hand, connatal PMD mutations lead to the accumulation of both mutant PLP and DM20 proteins in the ER of COS-7 cells with little of either isoform transported to the cell surface. Moreover, we show that transport-competent mutant DM20s facilitate trafficking of cognate PLPs and hence may influence disease severity.

Alleles↗

PubMed1995

Intraperikaryal neurofilamentous accumulations in a subset of retinal ganglion cells in aged mice that express a human neurofilament gene.

Neurofilamentous changes in select groups of neurons are associated with the degenerative changes of many human age-related neurodegenerative diseases. To examine the possible effects of aging on the neuronal cytoskeleton containing human proteins, the retinas of transgenic mice expressing the gene for the human middle-sized neurofilament triplet were investigated at 3 or 12 months of age. Transgenic mice developed tangle-like neurofilamentous accumulations in a subset of retinal ganglion cells at 12 months of age. These neurofilamentous accumulations, which also involved endogenous neurofilament proteins, were present in the perikarya and proximal processes of large ganglion cells and were predominantly located in peripheral retina. The presence of the human protein may thus confer vulnerability of the cytoskeleton to age-related alterations in this specific retinal cell type and may serve as a model for similar cellular changes associated with Alzheimer's disease and glaucoma.

Aging↗

PubMed1995

Overexpression of the human NFM subunit in transgenic mice modifies the level of endogenous NFL and the phosphorylation state of NFH subunits.

Neurofilaments (NFs), the major intermediate filaments of central nervous system (CNS) and peripheral nervous system (PNS) neurons, are heteropolymers formed from the high (NFH), middle (NFM), and low (NFL) molecular weight NF subunits. To gain insights into how the expression of NF subunit proteins is regulated in vivo, two transgenes harboring coding sequences for human NFM (hNFM) with or without the hNFM multiphosphorylation repeat domain were introduced into mice. Expression of both hNFM constructs was driven by the hNFM promoter and resulted in increased levels of hNFM subunits concomitant with an elevation in the levels of mouse NFL (mNFL) proteins in the CNS of both lines of transgenic mice. The increased levels of mNFL appear specific to NFM because previous studies of transgenic mice overexpressing either NFL or NFH did not result in increased expression of either of the other two NF subunits. Further, levels of the most heavily phosphorylated isoforms of mouse NFH (mNFH) were reduced in the brains of these transgenic mice, and electron microscopic studies showed a higher packing density of NFs in large-diameter CNS axons of transgenic versus wild-type mice. Thus, reduced phosphorylation of the mNFH carboxy terminal domain may be a compensatory response of CNS neurons to the increase in NFs, and reduced negative charges on mNFH sidearms may allow axons to accommodate more NFs by increasing their packing density. Taken together, these studies imply that NFM may play a dominant role in the in vivo regulation of the levels of NFL protein, the stoichiometry of NF subunits, and the phosphorylation state of NFH. NFM and NFH proteins may assume similar functions in regulation of NF packing density in vivo.

Aging↗

PubMed1994

Intracellular transport and sorting of the oligodendrocyte transmembrane proteolipid protein.

Delineating the properties and functions of the major central nervous system myelin proteins has been the focus of intensive research for decades. For PLP, this task has been confounded by its unusual properties, the complexity of the cellular membrane in which it resides, and the absence of a functional assay for the protein. The development of new experimental paradigms in which to study PLP may shed fresh light on the properties and functions of this intrinsic membrane protein. In the present communication we have used indirect, double label, immunofluorescence, and confocal microscopy to examine the distribution of PLP in Cos-7 cells transfected with an expression vector bearing the human PLP cDNA. Our results show that PLP is synthesized in the rough endoplasmic reticulum of transfected cells and passes through the Golgi apparatus to the cell surface. These results are consistent with previous studies showing PLP reaches the cell surface by transport through the secretory pathway. Levels of PLP at the cell surface are modest, most likely because protein deposited in this compartment can be endocytosed and subsequently transported to perinuclear lysosomes. Similar results are reported in the companion communication by Sinoway et al. (J Neurosci Res, 37:551-562, 1994). Using transfected HeLa cells they show that DM20 alone and PLP coexpressed with DM20 assume appropriate conformations for transport to the cell surface. The presence of PLP in subcellular compartments beyond the endoplasmic reticulum in Cos-7 cells indicates that the protein achieves a conformation appropriate for transport in the absence of other oligodendrocyte-specific factors; however, accumulation of large amounts of PLP in the cytoplasmic membrane compartment may require interactions with such glial-specific factors. Thus, the transfection paradigm described herein should prove a useful tool for investigating the folding and sorting of wild type and mutant forms of PLP as well as its membrane topology and posttranslational processing.

Animals↗

PubMed1994

Many naturally occurring mutations of myelin proteolipid protein impair its intracellular transport.

The primary structure of the proteolipid protein (PLP) from the central nervous system (CNS) myelin of mammals is highly conserved with only three amino acid differences between the mouse, rat, dog, bovine, and human proteins. Furthermore, within a particular species no polymorphisms in the protein have been identified. Recent interest has focused on the targeting of PLP in oligodendrocytes and the role that mutant forms of this protein play in generating dysmyelinating or hypomyelinating diseases. We previously expressed the human cDNA encoding PLP in transiently transfected Cos-7 cells and characterized the subcellular distribution of the protein in this simple heterologous system. In the current study we have used the same paradigm to examine the effect of five missense mutations in the PLP gene on processing of the encoded protein. The mutations chosen span the carboxy-terminal half of PLP and encompass that part of the protein in which most mutations have been identified. Our results show that transport of all mutations examined was arrested in the secretory pathway at an early stage, causing the mutant proteins to accumulate in the endoplasmic reticulum. Thus, a common mechanism of protein misfolding and failure of PLP to reach the cell surface of oligodendrocytes rather than the inability of the mutant protein to perform some crucial function at the cell surface may be responsible for the diseases caused by many PLP mutations. Our results, together with those of others, prompt us to speculate that the pathobiology observed in PLP mutants may result from oligodendrocyte cell death caused by the accumulation of misfolded protein in the endoplasmic reticulum. This speculation is consistent with the observations that oligodendrocytes bearing misfolded PLP, as in the jimpy mutant, proliferate but die rapidly while oligodendrocytes from PLP deletion survive and produce a myelin-like membrane which lacks PLP.

Animals↗

PubMed1994

Enhancer trapping by a human mid-sized neurofilament transgene reveals unexpected patterns of neuronal enhancer activity.

In ten transgenic lines, expression of a human mid-sized (M) neurofilament (NF) transgene was restricted to neurons in the central and peripheral nervous systems. However, no two lines gave identical expression patterns and none exactly matched the expression of mouse NF(M). These varied expression patterns within the neural compartment likely result from interactions of the transgene with enhancer elements located in the regions flanking the insertion site. Unexpected patterns of enhancer activity included an enhancer active in subsets of cerebellar basket cells as well as others preferentially active in subsets of motor or sensory neurons.

Amino Acid Sequence↗

PubMed1994

Age-associated and cell-type-specific neurofibrillary pathology in transgenic mice expressing the human midsized neurofilament subunit.

Alterations in neurofilaments are a common occurrence in neurons of the human nervous system during aging and diseases associated with aging. Such pathologic changes may be attributed to species-specific properties of human neurofilaments as well as cell-type-specific regulation of this element of the cytoskeleton. The development of transgenic animals containing human neurofilament subunits offers an opportunity to study the effects of aging and other experimental conditions on the human-specific form of these proteins in a rodent model. The present study shows that mice from the transgenic line NF(M)27, which express the human midsized neurofilament subunit at low levels (2-25% of the endogenous NF-M), develop neurofilamentous accumulations in specific subgroups of neurons that are age dependent, affecting 78% of transgenic mice over 12 months of age. Similar accumulations do not occur in age-matched, wild-type littermates or in 3-month-old transgenic mice. In 12-month-old transgenic mice, somatic neurofilament accumulations resembling neurofibrillary tangles were present predominantly in layers III and V of the neocortex, as well as in select subpopulations of subcortical neurons. Intraperikaryal, spherical neurofilamentous accumulations were particularly abundant in cell bodies in layer II of the neocortex, and neurofilament-containing distentions of Purkinje cell proximal axons occurred in the cerebellum. These pathological accumulations contained mouse as well as human NF subunits, but could be distinguished by their content of phosphorylation-dependent NF epitopes. These cytoskeletal alterations closely resemble the cell-type-specific alterations in neurofilaments that occur during normal human aging and in diseases associated with aging, indicating that these transgenic animals may serve as models of some aspects of the pathologic features of human neurodegenerative diseases.

Aging↗

PubMed1993

Intracellular distribution of transgenic bacterial beta-galactosidase in central nervous system neurons and neuroglia.

Bacterial beta-galactosidase is widely used as a marker for gene expression and in cell tracing experiments. In a survey of three transgenic mouse lines expressing beta-galactosidase in the central nervous system (CNS) under the control of different promoters, we find substantial variation in the intracellular distribution of the lacZ protein. In line M beta P5, transgene beta-galactosidase expression is driven by a promoter/enhancer fragment from the oligodendrocyte-specific myelin basic protein gene; however, electron microscopy of histochemically stained preparations reveals transgene expression not only in oligodendrocytes but also in some neurons. Immunofluorescence and immunoperoxidase staining show the beta-galactosidase protein distributed throughout the perikaryal cytoplasm of oligodendrocytes and in processes reaching to myelin sheaths. By contrast, immunoreactive protein appears restricted in neurons to one or a few small perikaryal immunoreactive granules. The granules are visible in the electron microscope as amorphous inclusion bodies of moderate electron density and lack a limiting membrane. Histochemical staining patterns with X-gal and Bluo-gal echoed the protein distribution: diffuse distribution of enzyme protein yielded cells filled with substrate, while punctate enzyme distribution yielded restricted or punctate histochemical staining. Examination of two other lines using different promoter/enhancers to drive expression in the CNS showed both diffuse and punctate beta-galactosidase immunolocalization and histochemical staining. The amount of protein synthesized or other properties, yet unidentified, intrinsic to the target cells may determine the intracellular distribution of beta-galactosidase. In retroviral marking studies, clone members have been identified as those cells filled with X-gal reaction product. This approach may underestimate both clone size and the minimum number of divisions separating the members of each clone.

Animals↗

PubMed1993

The mouse Hox-1.3 gene is functionally equivalent to the Drosophila Sex combs reduced gene.

To test whether the mouse Hox-1.3 gene is a cognate of the Drosophila Sex combs reduced (Scr) gene, we inserted a hsp 70-Hox-1.3 fusion gene into the Drosophila genome. Transgenic flies displayed Scr-like homeotic transformations after ectopic expression of Hox-1.3 induced by heat shock. In larvae, the thoracic segments T2 and T3 are transformed toward T1. In adults, head structures are dramatically disrupted, including transformation of antenna towards leg. Transformations are not the result of ectopic activation of the endogenous Scr gene. Rather, Hox-1.3 appears to directly regulate Scr target genes, as demonstrated by the ectopic activation of fork head by Hox-1.3. The results suggest that mouse Hox-1.3 cannot only substitute functionally for Drosophila Scr in the determination of external structures but also can participate in the regulatory hierarchy of insect organogenesis.

Amino Acid Sequence↗

PubMed1992

Targeting of Sp1 to a non-Sp1 site in the human neurofilament (H) promoter via an intermediary DNA-binding protein.

The human neurofilament (H) promoter contains multiple binding sites for nuclear proteins including a Proximal (Prox) site centered around the sequence GGTTGGACC and an adjacent pyrimidine (Pyr) tract site centered around the sequence CCCTCCTCCCC. Surprisingly binding to a probe containing the Prox/Pyr region of the NF(H) promoter was competed in gel shifts by an oligonucleotide containing only an Sp1 binding site (GGGGCGGGG). Supershift assays with a polyclonal anti-Sp1 antisera confirmed that Sp1 was part of the complex formed with the Prox/Pyr probe. However neither bacterially expressed Sp1 516C or vaccinia virus expressed full-length Sp1 778C bound to the Prox or Pyr sequences in DNase I footprints or gel shift assays. Gel shift competitions and supershift assays with probes containing either Prox or Pyr tract sites alone demonstrated targeting of Sp1 to the Prox binding site and identified a non-Sp1 containing complex which contains a Prox binding protein. Adding exogenous Sp1 to a HeLa nuclear extract enhanced the Sp1-containing complex but had no effect on the Prox complex. These studies show that Sp1 can be targeted to a non-Sp1 site in the human NF(H) promoter through protein/protein interactions with a distinct sequence specific DNA-binding protein.

Base Sequence↗

PubMed1992

Expression of human mid-sized neurofilament subunit in transgenic mice.

We have created transgenic mice which carry and express the gene encoding the human NF(M) subunit. RNAase protection assays reveal that the transgene is abundantly expressed in CNS and PNS but also, at very low levels in some non-neural tissues as well. Although the neurospecificity of transgene transcription was not absolute, we are able to detect the protein only in neurons with immunocytochemical techniques. Glial and endothelial cells do not contain immunoreactive materials. Interesting subtle differences in the relative level of the human transgene encoded and endogenous murine encoded NF(M) proteins were noted in different regions of the brain. Similar differences were found in the levels of transgene and endogenous gene mRNA suggesting that these differences may be traceable to differences in RNA transcription or stability. Our data demonstrate, within the sensitivity of the immunocytochemical techniques we used, that the human NF(M) protein is present only in the neurons of the transgenic mice and that it is present in the same neurons as the endogenous NF(M). Furthermore, immunoelectron-microscopic examination of isolated neurofilaments shows that the human NF(M) coassembles with the endogenous NF(M) during filament formation. Thus, although the human NF(M) possesses a much larger multiphosphorylation site in its carboxy terminus, it seems to be the functionally equivalent to the mouse protein, even in the murine neuron.

Animals↗

PubMed1992

Novel DNA binding proteins participate in the regulation of human neurofilament H gene expression.

By a combination of DNase I footprinting, methylation interference, and gel shift analyses we have identified multiple binding sites for nuclear proteins within the promoter region of the human neurofilament H gene. Two sites likely bind the transcription factor Sp1 while two others may be targets for previously unrecognized DNA binding proteins. One site, PAL, occurs within the 10 bp sequence GGGGAGGAGG. Two copies of the PAL sequence form an interrupted palindrome around one of the Sp1 sites. A second site, PROX, is found within the sequence GGTTGGACC. Nuclear extracts prepared from both neural and non-neural cell lines, mouse brain, and mouse liver contain proteins that recognize and bind to the PROX and PAL sequences indicating that proteins which bind to these target sequences are widespread. The appearance of these target sequences in the 5' upstream region of several neuron specific genes suggests that they play key roles in the transcription of neuron specific genes. The functional activity of these target DNA sequences was demonstrated by transfection assays using a reporter gene fused to nested deletions of the NF(H) promoter region. Interestingly, these assays revealed that maximal transient expression was obtained with DNA fusion genes containing the PAL, PROX and TATA sequences. Inclusion of the Sp1 sites into the fusion genes failed to enhance the expression of the reporter gene. To determine if the NF(H) promoter can be activated in a tissue specific manner during development transgenic mice containing the promoter region linked to a beta-galactosidase reporter gene were generated. In one line sporadic expression of the transgene occurred in the CNS and testis while in four other lines no expression occurred. Collectively these results suggest that the NF(H) gene promoter is active in a tissue specific manner only by interactions with regulatory elements that lie further upstream or downstream of the start site of initiation.

Animals↗

PubMed1992

Multiple nuclear factors interact with the promoter of the human neurofilament M gene.

In order to identify potential regulatory elements of the human mid-sized (M) neurofilament (NF) gene we preformed DNase I footprinting, gel mobility shift assays and methylation interference studies with probes from the NF(M) immediate 5' flanking region. These studies identified multiple sites for DNA-binding proteins including four Sp1 sites, and single sites each for members of the NF-1 and AP-1 families of DNA binding proteins. In addition a binding site within a pyrimidine tract likely binds a novel DNA-binding protein which also interacts with the human NF(H) gene promoter. Factors that bind to these sites are found in both neural and non-neural cells suggesting that the NF(M) promoter may not contain tissue specific regulatory signals. In transient assays, addition of these binding sites to an NF(M) minimal promoter containing only a TATA box lead to a greater than 40-fold activation of transcription over background. Progressive 5' deletions reduced expression in a step wise manner suggesting that all the factors likely act synergistically as positive regulators of transcription.

Base Sequence↗

PubMed1992

Myelin basic protein gene contains separate enhancers for oligodendrocyte and Schwann cell expression.

The DNA sequence between position +36 and -1907 of the murine myelin basic protein gene contains the enhancer and promoter elements necessary for abundant and cell specific expression in transgenic mice. Surprisingly, the pattern of expression promoted by this DNA fragment is a subset of that exhibited by the endogenous myelin basic protein (MBP) gene. Fusion genes prepared with this promoter/enhancer and a Lac Z reporter gene are expressed only in oligodendrocytes and not in Schwann cells, whereas the endogenous MBP gene is expressed in both cell types. The level of transgene expression measured by nuclear run-on experiments is very substantial and rivals that of the endogenous MBP gene. Furthermore, this 1.9-kb DNA fragment directs transcription on the same (or very similar) developmental schedule as the endogenous gene. These results indicate that the MBP promoter/enhancer sequences are at least tripartite: a core promoter, the oligodendrocyte enhancer elements, and a third component that either expands the specificity of the oligodendrocyte enhancer to include Schwann cells or acts independently to specifically stimulate transcription in Schwann cells.

Animals↗

PubMed1990

Virus-induced autoantibody response to a transgenic viral antigen.

The induction of autoantibodies and their possible role in the pathogenesis of autoimmune disease are poorly understood. Involvement of infectious agents has been suspected, but direct evidence is sparse. Whether immunological unresponsiveness to self by antibody-forming B cells is maintained by clonal abortion, clonal anergy or suppression, or how the scenario of interactions between helper T cells, B cells and antigen-presenting cells is distorted in autoantibody responses, is being analysed and widely debated. To evaluate tolerance of neutralizing B-cell responses we used transgenic mice expressing the cell membrane associated glycoprotein (G) of vesicular stomatitis virus (VSV) as self-antigen. We show that autoantibodies to VSV-G cannot be induced by VSV-G in adjuvant or by recombinant vaccinia virus expressing VSV-G, but are triggered by infection with wild-type VSV. The data show that helper T-cell tolerance is crucial in maintenance of B-cell non-reactivity and that cognate T-B recognition is necessary to break tolerance of self-reactive B cells. These results may help to understand mechanisms of virus-induced autoimmunity.

Animals↗