Abstract. It has been known for a number of years that glycosyl-phosphatidylinositol (GPI)-anchored proteins, in contrast to many transmembrane proteins, are insoluble at 4°C in nonionic detergents such as Triton X-100. Recently, it has been proposed that this behavior reflects the incorporation of GPI-linked proteins into large aggregates that are rich in sphingolipids and cholesterol, as well as in cytoplasmic signaling molecules such as heterotrimeric G proteins and src-family tyrosine kinases. It has been suggested that these lipidprotein complexes are derived from caveolae, non-clathrin-coated invaginations of the plasmalemma that are abundant in endothelial cells, smooth muscle, and lung. Caveolin, a proposed coat protein of caveolae, has been hypothesized to be essential for formation of the complexes. To further investigate the relationship between the detergent-resistant complexes and caveolae, we have characterized the behavior of GPIanchored proteins in lysates of N2a neuroblastoma cells, which lack morphologically identifiable caveolae, and which do not express caveolin (Shyng, S.-L., J. E. Heuser, and D. A. Harris. 1994. J. Cell Biol. 125:1239-1250. We report here that the complexes prepared from N2a cells display the large size and low buoyant density characteristic of complexes isolated from sources that are rich in caveolae, and contain the same major constituents, including multiple GPIanchored proteins, ot and fl subunits of heterotrimeric G proteins, and the tyrosine kinases fyn and yes. Our results argue strongly that detergent-resistant complexes are not equivalent to caveolae in all cell types, and that in neuronal cells caveolin is not essential for the integrity of these complexes. diverse group of proteins is attached to the plasma membrane by a glycosyl-phosphatidylinositol (GPI) a anchor (reviewed by Cross, 1990;Englund, 1993). This group includes lymphocyte and trypanosome surface antigens, adhesion molecules, exofacial enzymes, and receptors. The core structure of the anchor consists of a phosphoethanolamine residue amide-linked to the COOH-terminal amino acid of the protein, three mannose residues, an unacetylated glucosamine residue, and a phosphatidylinositol molecule that is embedded in the outer leaflet of the lipid bilayer. This core is transferred en bloc to the polypeptide chain in the endoplasmic reticulum, following cleavage of a hydrophobic COOH-terminal domain that serves as a signal for anchor addition.

GDNF, neurturin, and persephin are transforming growth factor ␤-related neurotrophic factors known collectively as the GDNF family (GF). GDNF and neurturin signal through a multicomponent receptor complex containing a signaling component (the Ret receptor tyrosine kinase) and either of two glycosyl-phosphatidylinositol-linked binding components (GDNF family receptor ␣ components 1 and 2, GFR␣1 or GFR␣2), whereas the receptor for persephin is unknown. Herein we describe a third member of the GF coreceptor family called GFR␣3 that is encoded by a gene located on human chromosome 5q31.2-32. GFR␣3 is not expressed in the central nervous system of the developing or adult animal but is highly expressed in several developing and adult sensory and sympathetic ganglia of the peripheral nervous system. GFR␣3 is also expressed at high levels in developing, but not adult, peripheral nerve. GFR␣3 is a glycoprotein that is glycosyl-phosphatidylinositol-linked to the cell surface like GFR␣1 and GFR␣2. Fibroblasts expressing Ret and GFR␣3 do not respond to any of the known members of the GDNF family, suggesting that GFR␣3 interacts with an unknown ligand or requires a different or additional signaling protein to function.The GDNF family (GF) of neurotrophic factors denotes a subfamily of proteins within the transforming growth factor ␤ superfamily that currently contains three members: glial cell line-derived neurotrophic factor (GDNF), discovered by its ability to maintain the survival of dopaminergic neurons of the embryonic ventral midbrain (1); neurturin (NTN), identified because of its survival-promoting properties on superior cervical ganglion neurons in culture (2); and persephin (PSP), discovered as the result of its homology to GDNF and NTN (3).The sites of action of the GF ligands are broad and include neurons of the central and peripheral nervous system (CNS and PNS) and the developing kidney. In the CNS, sites of GF ligand action include dopaminergic midbrain neurons (1, 3-7), spinal and facial motor neurons (3, 8-10), Purkinje cells (11), and noradrenergic neurons of the locus ceruleus (12). Many sensory and autonomic ganglia of the PNS are also responsive to GDNF and NTN action, including neurons of dorsal root ganglion (DRG), superior cervical ganglion, trigeminal ganglion, and nodose ganglion (2, 13-15). Developing enteric neurons also respond to GDNF and NTN (R. Heuckeroth, personal communication). Finally, recent investigation has revealed that GF members also can act on ureteric bud branching in vitro (3,16,17). The intense study of GF ligand activities has revealed two generalized features: (i) GDNF and NTN qualitatively share activity on all responsive peripheral and central populations tested (2, 7); (ii) PSP shares only a subset of these activities, namely, those in the CNS (dopaminergic and motor neurons) and in the developing kidney (3).The multicomponent GF receptor system recently characterized accounts for the overlap observed in GDNF and NTN action. The signaling component is the Ret recepto...

Multiple mutations have been described in the human GBA1 gene, which encodes the lysosomal enzyme beta-glucocerebrosidase (GCase) that degrades glucosylceramide and is pivotal in glycosphingolipid substrate metabolism. Depletion of GCase, typically by homozygous mutations in GBA1, is linked to the lysosomal storage disorder Gaucher’s disease (GD) and distinct or heterozygous mutations in GBA1 are associated with increased Parkinson’s disease (PD) risk. While numerous genes have been linked to heritable PD, GBA1 mutations in aggregate are the single greatest risk factor for development of idiopathic PD. The importance of GCase in PD necessitates preclinical models in which to study GCase-related mechanisms and novel therapeutic approaches, as well as to elucidate the molecular mechanisms leading to enhanced PD risk in GBA1 mutation carriers. The aim of this study was to develop and characterize a novel GBA1 mouse model and to facilitate wide accessibility of the model with phenotypic data. Herein we describe the results of molecular, biochemical, histological, and behavioral phenotyping analyses in a GBA1 D409V knock-in (KI) mouse. This mouse model exhibited significantly decreased GCase activity in liver and brain, with substantial increases in glycosphingolipid substrates in the liver. While no changes in the number of dopamine neurons in the substantia nigra were noted, subtle changes in striatal neurotransmitters were observed in GBA1 D409V KI mice. Alpha-synuclein pathology and inflammation were not observed in the nigrostriatal system of this model. In summary, the GBA1 D409V KI mouse model provides an ideal model for studies aimed at pharmacodynamic assessments of potential therapies aiming to restore GCase.

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Parkinson disease (PD) is the second leading neurodegenerative disease in the US. As there is no known cause or cure for PD, researchers continue to investigate disease mechanisms and potential new therapies in cell culture and in animal models of PD. In PD, one of the most profoundly affected neuronal populations is the tyrosine hydroxylase (TH)-expressing dopaminergic (DA) neurons of the substantia nigra pars compacta (SNpc). These DA-producing neurons undergo degeneration while neighboring DA-producing cells of the ventral tegmental area (VTA) are largely spared. To aid in these studies, The Michael J. Fox Foundation (MJFF) partnered with Thomas Jefferson University and Taconic Inc. to generate new transgenic rat lines carrying the human TH gene promoter driving EGFP using a 11 kb construct used previously to create a hTH-GFP mouse reporter line. Of the five rat founder lines that were generated, three exhibited high level specific GFP fluorescence in DA brain structures (ie. SN, VTA, striatum, olfactory bulb, hypothalamus). As with the hTH-GFP mouse, none of the rat lines exhibit reporter expression in adrenergic structures like the adrenal gland. Line 12141, with its high levels of GFP in adult DA brain structures and minimal ectopic GFP expression in non-DA structures, was characterized in detail. We show here that this line allows for anatomical visualization and microdissection of the rat midbrain into SNpc and/or VTA, enabling detailed analysis of midbrain DA neurons and axonal projections after toxin treatment in vivo. Moreover, we further show that embryonic SNpc and/or VTA neurons, enriched by microdissection or FACS, can be used in culture or transplant studies of PD. Thus, the hTH-GFP reporter rat should be a valuable tool for Parkinson's disease research.