Mark F. Mehler - US grants

Alpha-glucosidase deficiency (acid maltase deficiency-AMD) is a clinically and biochemically heterogeneous lysosomal storage disease existing in a generalized infantile and late-onset neuromuscular froms. Both clinical states are characterized by a catalytically active microsomal Alpha-glucosidase of unknown function. Studies of intracellular transport of lysosomal hydrolases and evolving models of glycoprotein biosynthesis suggest a dual role for the microsomal enzyme: as a biosynthetic precursor of the lysosomal enzyme and as a glycoprotein-processing enzyme in the post-translational assembly of macromolecular species. Recently two human neutral Alpha-glucosidases, with diverse biochemical properties, have been characterized and mapped to separate chromosomal loci distinct from the lysosomal Alpha-glucosidase. Similarly, two microsomal "processing" Alpha-glucosidases were subsequently identified and found to exhibit partial catalytic overlap with the neutral Alpha-glucosidase activity. In addition, we have identified a tissue-specific Alpha-glucosidase activity in renal tissue, which has partial electrophoretic identity with the lysosomal enzyme and is present in normal amounts in AMD renal samples. We had previously demonstrated reduced levels of catalytically-active lysosomal Alpha-glucosidase only in tissues from late-onset AMD patients, while recent studies have shown reduced rates of synthesis of a biosynthetic form of the lysosomal enzyme in these mutant cells, with lack of immunoprecipitable material in the infantile AMD cells. The finding of heterogeneous morphological alterations in AMD tissues coupled with the evolving complexity of lysosomal enzyme biosynthesis and pathology suggested to us that graded cellular alterations could occur as a consequence of a selective mutant locus. As such, we conducted preliminary studies which revealed major alterations in whole cell glycoprotein biosynthesis and membrane assembly. We believe a program combining study of glycoprotein biochemistry and molecular biology will best allow us to: characterize the complex interrelationships between lysosomal and microsomal Alpha-glucosidases, determine the molecular basis of functional differences between infantile and late-onset AMD, and study the consequences of a selective mutant locus on post-translational cellular processing events. These studies will involve: comparative hydrolase assays using putative natural saccharide substrates on purified neutral Alpha-glucosidases, detailed structural analysis of glycoprotein biosynthetic intermediates and mature membrane glycoproteins, in vitro cell-free translation and biosynthetic labeling, and recombinant DNA techniques. These studies are crucial to our understanding of molecular mechanisms in lysosomal and general cellular pathology and to refinement in techniques important to gene and enzyme replacement therapy in neurodegenerative diseases.

Alpha-glucosidase deficiency (acid maltase deficiency-AMD) is a clinically and biochemically heterogeneous lysosomal storage disease existing in a generalized infantile and late-onset neuromuscular froms. Both clinical states are characterized by a catalytically active microsomal Alpha-glucosidase of unknown function. Studies of intracellular transport of lysosomal hydrolases and evolving models of glycoprotein biosynthesis suggest a dual role for the microsomal enzyme: as a biosynthetic precursor of the lysosomal enzyme and as a glycoprotein-processing enzyme in the post-translational assembly of macromolecular species. Recently two human neutral Alpha-glucosidases, with diverse biochemical properties, have been characterized and mapped to separate chromosomal loci distinct from the lysosomal Alpha-glucosidase. Similarly, two microsomal "processing" Alpha-glucosidases were subsequently identified and found to exhibit partial catalytic overlap with the neutral Alpha-glucosidase activity. In addition, we have identified a tissue-specific Alpha-glucosidase activity in renal tissue, which has partial electrophoretic identity with the lysosomal enzyme and is present in normal amounts in AMD renal samples. We had previously demonstrated reduced levels of catalytically-active lysosomal Alpha-glucosidase only in tissues from late-onset AMD patients, while recent studies have shown reduced rates of synthesis of a biosynthetic form of the lysosomal enzyme in these mutant cells, with lack of immunoprecipitable material in the infantile AMD cells. The finding of heterogeneous morphological alterations in AMD tissues coupled with the evolving complexity of lysosomal enzyme biosynthesis and pathology suggested to us that graded cellular alterations could occur as a consequence of a selective mutant locus. As such, we conducted preliminary studies which revealed major alterations in whole cell glycoprotein biosynthesis and membrane assembly. We believe a program combining study of glycoprotein biochemistry and molecular biology will best allow us to: characterize the complex interrelationships between lysosomal and microsomal Alpha-glucosidases, determine the molecular basis of functional differences between infantile and late-onset AMD, and study the consequences of a selective mutant locus on post-translational cellular processing events. These studies will involve: comparative hydrolase assays using putative natural saccharide substrates on purified neutral Alpha-glucosidases, detailed structural analysis of glycoprotein biosynthetic intermediates and mature membrane glycoproteins, in vitro cell-free translation and biosynthetic labeling, and recombinant DNA techniques. These studies are crucial to our understanding of molecular mechanisms in lysosomal and general cellular pathology and to refinement in techniques important to gene and enzyme replacement therapy in neurodegenerative diseases.

DESCRIPTION: Using epidermal growth factor (EGF)-derived embryonic subventricular zone (SVZ) multipotent neural progenitor cells, we have identified a subclass of the transforming growth factor b (TGFb) superfamily, the bone morphogenetic proteins (BMPs), that mediate the selective, dose-dependent elaboration of astrocytes, with concurrent suppression of neuronal and oligodendroglial lineage development. Transcripts and proteins for the BMP ligands and BMP type I and II receptor subunits are present in brain and SVZ progenitor cells at the appropriate times to mediate these trophic actions. In concert with activation of the LIFb receptor, BMPs and bFGF potentiate the early expression of radial glia, with later enhancement (BMP) or inhibition (bFGF) of the astroglial phenotype. These observations suggest that the BMPs represent a new class of signaling molecules that may regulate astroglial lineage commitment and may interact with distinct early signaling molecules. The general hypothesis underlying this proposal is that the BMPs are instructive signals that regulate astroglial lineage commitment, and that regulation of BMP receptor subunits and/or BMP ligands determine the pattern of astrocyte lineage elaboration during brain development: A. Clonal and single cell analysis will be utilized to determine whether the BMP astroglial-promoting effects constitute instructive cellular signals, whether different BMPs suppress the neuronal and oligodendroglial lineages and whether progenitor cell responsiveness to the BMPs in vitro correlates with changes in BMP receptor expression. B. Isolated EGF-responsive progenitor cells and tissue sections from sequential stages of gliogenesis will be examined to define changes in the degree of BMP-progenitor cell responsiveness, the profile of astroglial lineage species, and alterations in the spatiotemporal expression of BMP receptors and ligands to establish in vitro-in vivo correlations. C. The role of LIF and bFGF in modulating the BMP-mediated expression and cellular responsiveness of radial glial populations will be studied to determine whether these cellular changes reflect opposing effects on BMP receptor expression. These studies will further our understanding of the epigenetic signals and cellular mechanisms that regulate the development of the astroglial lineage. Because many of the cellular processes present during astroglial development may be recapitulated during pathological states, these findings may also have important implications for our understanding of glial-mediated responses in traumatic injury, demyelinating disorders and mammalian central nervous system cellular transformation.

We have isolated multipotent progenitor cells from the cerebral cortex independent of periventricular generative zones during the peak period of early postnatal gliogenesis. These progenitor species undergo cellular expansion and self-renewal in vitro in response to epidermal growth factor and can generate neurons and glia, including myelin protein-expressing oligodendrocytes. Glial progenitors derived from these multipotent progenitors express the neurotrophin-3 receptor, trkC, and application of neurotrophin-3 selectively promotes the expansion of oligodendrocyte progenitors that require additional environmental signals (ciliary neurotrophic factor, CNTF) for oligodendrocyte differentiation. Neurotrophin-3 can also induce the expression of the CNTFalpha receptor on glial progenitors derived from cortical multipotent cells, while bone morphogenetic proteins promote the generation of astrocytes and induce the expression of trkC on these progenitor species. In preliminary in vivo studies, we have also shown that transplanted cortical multipotent cells can undergo cellular expansion and give rise to glial and neuronal progeny. In addition, glial progenitors isolated from these multipotent cells proliferate in vivo and give rise to oligodendrocytes and astrocytes. Cortical injury enhances progenitor cell expansion and differentiation. In vitro analysis: 1. To define the cellular properties of epidermal growth factor-responsive cortical multipotent progenitors and their progeny: A. Do these progenitors undergo long-term self-renewal? B. What is the composition of neural lineage species derived from these multipotent cells? C. What are the cellular actions of neurotrophin-3 on oligodendroglial and astroglial development from glial progenitors derived from these multipotent cells? In vivo analysis: 2. To define the presence of appropriate early postnatal microenvironmental signals for cortical progenitor cell development: A. Are the cellular profiles of progenitor expansion, lineage restriction and differentiation equivalent in vitro and in vivo? B. Are glial-restricted progeny bipotent in vivo? C. Do different areas of the CNS neuraxis promote distinct progenitor cell response profiles? These studies will further our understanding of early progenitor cell regulatory events in neural lineage development during normal mammalian cerebral cortical maturation, identify pathologic mechanisms underlying a range of genetic and acquired neurologic disorders, and promote the development of novel regenerative strategies.