Robert J Milner - US grants

This application proposes studies directed towards the identification and characterization of novel brain specific proteins. Rat brain cDNA clones were selected which correspond to mRNAs expressed in brain but not in liver or kidney. The nucleotide sequence of one such clone (plB236) has been determined, providing the amino acid sequence of the protein (1B236) encoded by the cloned mRNA. Antibodies raised against synthetic peptides corresponding to regions of this sequence detect immunoreactivity in a series of widely distributed neuronal structures in rat brain distinct from any other immunocytochemically defined neuronal system. In addition, the amino acid sequence of 1B236 has structural features in common with neuropeptide precursors, suggesting that the 1B236 protein may be cleaved to generate several novel neuropeptides. Preliminary evidence indicates that 1B236 immunoreactivity can be detected in rat brain extracts as both a 100,000 dalton glycoprotein and as processed peptides (15-40 amino acids). Synthetic peptides corresponding to 1B236 sequences have behavioral and electrophysiological activity. It is the aim of the proposed studies to: 1. Determine the predominant molecular forms of 1B236 immunoreactivity in rat brain and their relative amounts. 2. Isolate and characterize the 1B236 protein from rat brain. 3. Determine the exact forms of any peptides generated from the 1B236 protein in vivo and released in vitro from nerve terminals. 4. Deduce the sequence of steps involved in the post-translational processing of the 1B236 protein. 5. Determine the structure of the gene coding for the 1B236 protein. 6. Investigate the expression of the 1B236 protein and its products during development of the rat. 7. Test for similar molecules in other species. These studies will generate structural, genetic and developmental information on a novel brain specific protein which may represent a previously undetected neurotransmitter system. The data will provide the basis for functional testing of this protein and its peptide products and may provide a new avenue for the elucidation of neurologic disorders.

This application proposes to identify and characterize glial specific proteins. In a study designed to identify novel brain specific proteins, rat brain cDNA clones were selected which corresponded to mRNAs expressed in brain but not in other tissues. One such clone (plB208) hybridized to two highly abundant rat brain mRNAs, 3200 and 1600 nucleotides in length, which were also present with a similar high abundance in rat C6 glioma cells. The nucleotide sequence of the clone was determined, providing the partial amino acid sequence of the correspondence proteins. Antibodies raised against synthetic peptides corresponding to regions of this sequence detected a 50 kd protein in astrocytes distributed throughout the brain. Preliminary evidence indicates that the cDNA clone is derived from the larger RNA species and that this mRNA probably encodes a glial specific protein of unknown structure and function, glial fibrillary acidic protein (GFAP). It is the aim of the proposed studies to: 1. Determine the complete structure of the mRNAs and protein corresponding to clone p1B208 and rigorously establish their relationship to GFAP. 2. Determine the structure of the gene encoding 1B208 and investigate the transcription of the two mRNA molecules which hybridize to p1B208. 3. Determine the time course of expression of 1B208 mRNAs and protein during development. 4. Select and characterize further cDNA clones corresponding to astrocyte specific mRNAs. 5. Select and characterize cDNA clones corresponding to mRNAs specific for oligodendrocytes and Schwann cells. These studies will generate structural and genetic information on a highly abundant glial specific molecule which we have tentatively identified as GFAP's a major brain protein which has been difficult to study because of its physical properties. Characterization of this and other glial proteins will provide a basis for determining the functions of glia, a means for classifying glia and their subtypes, and ultimately will improve our ability to understand and identify the types of glia involved in neurological disease.

The rat brain protein 1B236 is a membrane-associated glycoprotein of approximately 100,000 daltons that was defined originally by molecular cloning of brain-enriched mRNAs. Previous studies have provided the complete amino acid sequence of 1B236 and have demonstrated several interesting features of this molecule: a) the amino terminal region consists of five, roughly equal-sized domains that are homologous to each other and to proteins of the immunoglobulin superfamily; b) peptide fragments of the carboxyl terminus, as well as the predominant 100,000 dalton form of the 1B236 protein, can be detected in brain extracts by antibodies against appropriate synthetic peptides; c) several forms of 1B236 mRNA are generated by differential exon splicing: one mRNA encodes a 1B236 protein with a variant carboxyl terminus; d) at 12-25 days after birth 1B236 and protein are expressed abundantly in oligodendrocytes, coincident with myelinogenesis, in contrast to the adult where 1B236 expression is largely neuronal; e) preliminary evidence suggests that the 1B236 protein is identical or very similar to the previously described protein, myelin associated glycoprotein. It is the aim of the proposed studies to: 1. Determine the relationship between myelin associated glycoprotein and the 1B236 molecules expressed in oligodendrocytes and neurons. 2. Investigate the expression in the brain of the differentially spliced forms of 1B236 mRNAs and their encoded proteins. 3. Determine the complete structure of the 1B236 gene and analyse its regulation. 4. Determine the subcellular localization of the 1B236 glycoprotein and peptide forms and investigate the mechanism of their biosynthesis. 5. Test for molecules that may interact with the 1B236 glycoprotein or peptides. 6. Search for related molecules expressed in the brains and other tissues of rates and other species. These studies will generate further structural and genetic information for the 1B236 protein, which may be identical to myelin associated glycoprotein. The data will provide the basis for functional testing of the 1B236 protein, which is expressed in a highly regulated fashion in particular subpopulations of both neurons and glia. The apparent involvement of the 1B236 protein in myelinogenesis suggest that the study of this molecule may also further our understanding of demyelinating diseases.

This application proposes to identify and characterize proteins that are specific to glia. Previous studies have focussed on the structure and genetics of proteolipid protein (PLP), a major component of CNS myelin that is expressed specifically in oligodendrocytes. We have isolated full-length cDNA clones for two rat pLp mRNAs and determined their nucleotide sequences. Characterization of cDNA clones for a second myelin proteolipid, DM-20, indicated that the sequence of DM-20 is identical to that of PLP, except for the absence of 35 amino acids from the major internal hydrophilic domain of PLP. The mRNAs encoding PLP and DM- 20 are derived from the single PLP gene by alternative RNA splicing. Studies of the dysmyelinating mouse mutant jimpy demonstrated that jimpy PLP mRNA contains a deletion of 74 nucleotides relative to wild type PLP mRNAs and that this defect is generated by aberrant RNA processing rather than by deletion of genomic sequences. We have also begun selection of clones corresponding to other glial-specific proteins, from cDNA libraries constructed from cultured astrocytes and from optic nerve. It is the aim of the proposed studies to: 1. Determine the structure of the PLP gene in the rat, analyse its regulation, and investigate the alternative splicing of PLP gene transcripts. 2. Characterize the mutation in the jimpy allele of the PLP gene. 3. Investigate the expression of the PLP gene in the dysmyelinating mutant mouse jimpy-msd and in the myelin deficient (md) rat. 4. Analyse the cellular expression of PLP and DM-20 in normal and mutant animals by in situ hybridization. 5. Determine the topological organization of the PLP polypeptide and the mechanism of its insertion in the oligodendrocyte membrane. 6. Select and characterize cDNA clones corresponding to further glial-specific or glial enriched mRNA expressed in oligodendroyctes and astrocytes. Analysis of the expression of the PLP gene in normal and mutant animals will lead to an understanding of the regulation of this gene, particularly during myelination. Characterization of PLP and other glial proteins will allow us to define the extent of genetic similarity between glial cells and provide a basis for determining the functions of glia, a means for classifying glia and their subtypes and ultimately may improve our understanding of the involvement of glia in neurological disease.

This application proposes to identify and characterize proteins that are specific to glia. Previous studies have focussed on the structure and genetics of proteolipid protein (PLP), a major component of CNS myelin that is expressed specifically in oligodendrocytes. We have isolated full-length cDNA clones for two rat pLp mRNAs and determined their nucleotide sequences. Characterization of cDNA clones for a second myelin proteolipid, DM-20, indicated that the sequence of DM-20 is identical to that of PLP, except for the absence of 35 amino acids from the major internal hydrophilic domain of PLP. The mRNAs encoding PLP and DM- 20 are derived from the single PLP gene by alternative RNA splicing. Studies of the dysmyelinating mouse mutant jimpy demonstrated that jimpy PLP mRNA contains a deletion of 74 nucleotides relative to wild type PLP mRNAs and that this defect is generated by aberrant RNA processing rather than by deletion of genomic sequences. We have also begun selection of clones corresponding to other glial-specific proteins, from cDNA libraries constructed from cultured astrocytes and from optic nerve. It is the aim of the proposed studies to: 1. Determine the structure of the PLP gene in the rat, analyse its regulation, and investigate the alternative splicing of PLP gene transcripts. 2. Characterize the mutation in the jimpy allele of the PLP gene. 3. Investigate the expression of the PLP gene in the dysmyelinating mutant mouse jimpy-msd and in the myelin deficient (md) rat. 4. Analyse the cellular expression of PLP and DM-20 in normal and mutant animals by in situ hybridization. 5. Determine the topological organization of the PLP polypeptide and the mechanism of its insertion in the oligodendrocyte membrane. 6. Select and characterize cDNA clones corresponding to further glial-specific or glial enriched mRNA expressed in oligodendroyctes and astrocytes. Analysis of the expression of the PLP gene in normal and mutant animals will lead to an understanding of the regulation of this gene, particularly during myelination. Characterization of PLP and other glial proteins will allow us to define the extent of genetic similarity between glial cells and provide a basis for determining the functions of glia, a means for classifying glia and their subtypes and ultimately may improve our understanding of the involvement of glia in neurological disease.

Myelin-associated glycoprotein (MAG) is a 100,000 dalton component of the myelin membrane. The molecule is similar in structure to neural cell adhesion molecules and it has been proposed that MAG may mediate cell-cell interactions during the initial stages of myelinogenesis. In previous studies, supported by this grant, we have characterized MAG in rat brain by molecular cloning, determined the structure of the rat MAG gene and analyzed the expression of MAG mRNAs and protein in the rodent nervous system. The studies proposed here will focus on the structure of the MAG gene, the mechanisms of its regulation during myelination and remylination, and attempts to understand the function of MAG by disrupting its expression. It is the aim of the proposed studies to: 1. Analyze the mechanisms of regulation of the MAG gene. 2. Generate transgenic mice expressing regulatory regions of the MAG gene and to analyse the expression of the MAG gene in vivo. 3. Analyse the expression of the MAG gene during remyelination following a demyelinating viral infection of the central nervous system. 4. Disrupt the expression of the MAG gene, in order to asses the functional consequences of decreased or absent MAG expression on myelinogenesis. 5. Investigate the genetic defects in the mouse mutant quivering and the relationship of this mutation to the MAG gene. MAG appears to play a critical role in the early stages of myelinogenesis by mediating the initial contact between the myelinating cell and the axon and it is likely that MAG plays a similar role in remyelination following a demyelinating disease. A detailed understanding of the factors that regulate the expression of MAG will be essential for designing therapies to encourage the formation of new myelin in diseases such as multiple sclerosis.

Myelin-associated glycoprotein (MAG) is a 100,000 dalton component of the myelin membrane. The molecule is similar in structure to neural cell adhesion molecules and it has been proposed that MAG may mediate cell-cell interactions during the initial stages of myelinogenesis. In previous studies, supported by this grant, we have characterized MAG in rat brain by molecular cloning, determined the structure of the rat MAG gene and analyzed the expression of MAG mRNAs and protein in the rodent nervous system. The studies proposed here will focus on the structure of the MAG gene, the mechanisms of its regulation during myelination and remylination, and attempts to understand the function of MAG by disrupting its expression. It is the aim of the proposed studies to: 1. Analyze the mechanisms of regulation of the MAG gene. 2. Generate transgenic mice expressing regulatory regions of the MAG gene and to analyse the expression of the MAG gene in vivo. 3. Analyse the expression of the MAG gene during remyelination following a demyelinating viral infection of the central nervous system. 4. Disrupt the expression of the MAG gene, in order to asses the functional consequences of decreased or absent MAG expression on myelinogenesis. 5. Investigate the genetic defects in the mouse mutant quivering and the relationship of this mutation to the MAG gene. MAG appears to play a critical role in the early stages of myelinogenesis by mediating the initial contact between the myelinating cell and the axon and it is likely that MAG plays a similar role in remyelination following a demyelinating disease. A detailed understanding of the factors that regulate the expression of MAG will be essential for designing therapies to encourage the formation of new myelin in diseases such as multiple sclerosis.

The unique collaboration between the University of Massachusetts Lowell and the University of Massachusetts Medical School has as its primary focus to quantify microinequities that affect the recruitment, retention and advancement of women in the STEM disciplines. Many advances have been made in addressing the most visible barriers to increased participation of women in STEM fields. However, to date, the focus on microinequities has relied on qualitative analysis of the subtle biases that prevent women STEM faculty from achieving their full potential. To date, little to no evidence has been provided on the creation or development of quantifiable metrics and tools for the assessment of subtle gender biases and other roots of microinequities in an effort to eliminate these subtle discriminations and, ultimately, make them negligible. A scientific understanding of and metrics for microinequities are expected to be relevant in refining existing techniques and designing new methods for addressing gender biases in the academy. This project, because of the nature of the partnership between these institutions, is expected to impact not only the campuses involved, but also multiple campuses within the Massachusetts public higher education system. The products that result from this project are also expected to benefit not only women, but also male faculty in all fields; and they will provide the foundation for addressing similar subtle biases in non-academic and/or international environments. Ultimately, with a robust dissemination plan, this project will transform the study of microinequities and how their impact on all underrepresented groups, both within and outside of academic arenas, is determined.