visualization-endosome-dynamics-living-nerve-terminals-with-four

Visualization of Endosome Dynamics in Living Nerve Terminals with Four-dimensional Fluorescence Imaging

Four-dimensional (4D) light imaging has been used to study behavior of small structures within motor nerve terminals of the thin transversus abdominis ...

Washington University School of Medicine

The cellular mechanisms by which prions cause neurological dysfunction are poorly understood. To address this issue, we have been using cultured cells to analyze the localization, biosynthesis, and metabolism of PrP molecules carrying mutations associated with familial prion diseases. We report here that mutant PrP molecules are delayed in their maturation to an endoglycosidase H-resistant form after biosynthetic labeling, suggesting that they are impaired in their exit from the endoplasmic reticulum (ER). However, we find that proteasome inhibitors have no effect on the maturation or turnover of either mutant or wild-type PrP molecules. Thus, in contrast to recent studies from other laboratories, our work indicates that PrP is not subject to retrotranslocation from the ER into the cytoplasm prior to degradation by the proteasome. We find that in transfected cells, but not in cultured neurons, proteasome inhibitors cause accumulation of an unglycosylated, signal peptide-bearing form of PrP on the cytoplasmic face of the ER membrane. Thus, under conditions of elevated expression, a small fraction of PrP chains is not translocated into the ER lumen during synthesis, and is rapidly degraded in the cytoplasm by the proteasome. Finally, we report a previously unappreciated artifact caused by treatment of cells with proteasome inhibitors: an increase in PrP mRNA level and synthetic rate when the protein is expressed from a vector containing a viral promoter. We suggest that this phenomenon may explain some of the dramatic effects of proteasome inhibitors observed in other studies. Our results clarify the role of the proteasome in the cell biology of PrP, and suggest reasonable hypotheses for the molecular pathology of inherited prion diseases.

Mutant PrP is delayed in its exit from the endoplasmic reticulum, but neither wild-type nor mutant PrP undergoes retrotranslocation prior to proteasomal degradation.

The prion protein (PrP) can adopt multiple membrane topologies, including a fully translocated form (SecPrP), two transmembrane forms (NtmPrP and CtmPrP), and a cytosolic form. It is important to understand the factors that influence production of these species, because two of them, CtmPrP and cytosolic PrP, have been proposed to be key neurotoxic intermediates in certain prion diseases. In this paper, we perform a mutational analysis of PrP synthesized using an in vitro translation system in order to further define sequence elements that influence the formation of CtmPrP. We find that substitution of charged residues in the hydrophobic core of the signal peptide increases synthesis of CtmPrP and also reduces the efficiency of translocation into microsomes. Combining these mutations with substitutions in the transmembrane domain causes the protein to be synthesized exclusively with the CtmPrP topology. Reducing the spacing between the signal peptide and the transmembrane domain also increases CtmPrP. In contrast, topology is not altered by mutations that prevent signal peptide cleavage or by deletion of the C-terminal signal for glycosylphosphatidylinositol anchor addition. Removal of the signal peptide completely blocks translocation. Taken together, our results are consistent with a model in which the signal peptide and transmembrane domain function in distinct ways as determinants of PrP topology. We also present characterization of an antibody that selectively recognizes CtmPrP and cytosolic PrP by virtue of their uncleaved signal peptides. By using this antibody, as well as the distinctive gel mobility of CtmPrP and cytosolic PrP, we show that the amounts of these two forms in cultured cells and rodent brain are not altered by infection with scrapie prions. We conclude that CtmPrP and cytosolic PrP are unlikely to be obligate neurotoxic intermediates in familial or infectiously acquired prion diseases.

Mutational analysis of topological determinants in prion protein (PrP) and measurement of transmembrane and cytosolic PrP during prion infection.

A synthetic peptide homologous to region 106-126 of the prion protein (PrP) is toxic to cells expressing PrP, but not to PrP knockout neurons, arguing for a specific role of PrP in mediating the peptide's activity. Whether this is related to a gain of toxicity or a loss of function of PrP is not clear. We explored the possibility that PrP106-126 triggered formation of PrP(Sc) or other neurotoxic PrP species. We found that PrP106-126 did not induce detergent-insoluble and protease-resistant PrP, nor did it alter its membrane topology or cellular distribution. We also found that neurons expressing endogenous or higher level of either wild-type PrP or a nine-octapeptide insertional mutant were equally susceptible to PrP106-126, and that sub-physiological PrP expression was sufficient to restore vulnerability to the peptide. These results indicate that PrP106-126 interferes with a PrP function that requires only low protein levels, and is not impaired by a pathogenic insertion in the octapeptide region.

The neurotoxicity of prion protein (PrP) peptide 106-126 is independent of the expression level of PrP and is not mediated by abnormal PrP species.

Inherited prion diseases are linked to mutations in the prion protein (PrP) gene, which favor conversion of PrP into a conformationally altered, pathogenic isoform. The cellular mechanism by which this process causes neurological dysfunction is unknown. It has been proposed that neuronal death can be triggered by accumulation of PrP in the cytosol because of impairment of proteasomal degradation of misfolded PrP molecules retrotranslocated from the endoplasmic reticulum (Ma, J., Wollmann, R., and Lindquist, S. (2002) Science 298, 1781-1785). To test whether this neurotoxic mechanism is operative in inherited prion diseases, we evaluated the effect of proteasome inhibitors on the viability of transfected N2a cells and primary neurons expressing mouse PrP homologues of the D178N and nine octapeptide mutations. We found that the inhibitors caused accumulation of an unglycosylated, aggregated form of PrP exclusively in transfected N2a expressing PrP from the cytomegalovirus promoter. This form contained an uncleaved signal peptide, indicating that it represented polypeptide chains that had failed to translocate into the ER lumen during synthesis, rather than retrogradely translocated PrP. Quantification of N2a viability in the presence of proteasome inhibitors demonstrated that accumulation of this form was not toxic. No evidence of cytosolic PrP was found in cerebellar granule neurons from transgenic mice expressing wild-type or mutant PrPs from the endogenous promoter, nor were these neurons more susceptible to proteasome inhibitor toxicity than neurons from PrP knock-out mice. Our analysis fails to confirm the previous observation that mislocation of PrP in the cytosol is neurotoxic, and argues against the hypothesis that perturbation of PrP metabolism through the proteasomal pathway plays a pathogenic role in prion diseases.

Cytosolic prion protein (PrP) is not toxic in N2a cells and primary neurons expressing pathogenic PrP mutations.

(Ctm)PrP is a transmembrane version of the prion protein that has been proposed to be a neurotoxic intermediate underlying prion-induced pathogenesis. In previous studies, we found that PrP molecules carrying mutations in the N-terminal signal peptide (L9R) and the transmembrane domain (3AV) were synthesized exclusively in the (Ctm)PrP form in transfected cell lines. To characterize the properties of (Ctm)PrP in a neuronal setting, we have utilized cerebellar granule neurons cultured from Tg(L9R-3AV) mice that developed a fatal neurodegenerative illness. We found that about half of the L9R-3AV PrP synthesized in these neurons represents (Ctm)PrP, with the rest being (Sec)PrP, the glycolipid anchored form that does not span the membrane. Both forms contained an uncleaved signal peptide, and they are differentially glycosylated. (Sec)PrP was localized on the surface of neuronal processes. Most surprisingly, (Ctm)PrP was concentrated in the Golgi apparatus, rather in the endoplasmic reticulum as it is in transfected cell lines. Our study is the first to analyze the properties of (Ctm)PrP in a neuronal context, and our results suggest new hypotheses about how this form may exert its neurotoxic effects.

A transmembrane form of the prion protein is localized in the Golgi apparatus of neurons.

Although PrP(Sc) is thought to be the infectious form of the prion protein, it may not be the form that is responsible for neuronal cell death in prion diseases. (Ctm)PrP is a transmembrane version of the prion protein that has been proposed to be a neurotoxic intermediate underlying prion-induced pathogenesis. To investigate this hypothesis, we have constructed transgenic mice that express L9R-3AV PrP, a mutant prion protein that is synthesized exclusively in the (Ctm)PrP form in transfected cells. These mice develop a fatal neurological illness characterized by ataxia and marked neuronal loss in the cerebellum and hippocampus. (Ctm)PrP in neurons cultured from transgenic mice is localized to the Golgi apparatus, rather than to the endoplasmic reticulum as in transfected cell lines. Surprisingly, development of the neurodegenerative phenotype is strongly dependent on coexpression of endogenous, wild-type PrP. Our results provide new insights into the cell biology of (Ctm)PrP, the mechanism by which it induces neurodegeneration, and possible cellular activities of PrP(C).

Neurodegenerative illness in transgenic mice expressing a transmembrane form of the prion protein.

In prion diseases, the infectious isoform of the prion protein (PrP(Sc)) may subvert a normal, physiological activity of the cellular isoform (PrP(C)). A deletion mutant of the prion protein (Delta105-125) that produces a neonatal lethal phenotype when expressed in transgenic mice provides a window into the normal function of PrP(C) and how it can be corrupted to produce neurotoxic effects. We report here the surprising and unexpected observation that cells expressing Delta105-125 PrP and related mutants are hypersensitive to the toxic effects of two classes of antibiotics (aminoglycosides and bleomycin analogues) that are commonly used for selection of stably transfected cell lines. This unusual phenomenon mimics several essential features of Delta105-125 PrP toxicity seen in transgenic mice, including rescue by co-expression of wild type PrP. Cells expressing Delta105-125 PrP are susceptible to drug toxicity within minutes, suggesting that the mutant protein enhances cellular accumulation of these cationic compounds. Our results establish a screenable cellular phenotype for the activity of neurotoxic forms of PrP, and they suggest possible mechanisms by which these molecules could produce their pathological effects in vivo.

A novel, drug-based, cellular assay for the activity of neurotoxic mutants of the prion protein.

Activity at the vertebrate nerve-muscle synapse creates large macroendosomes (MEs) via bulk membrane infolding. Visualized with the endocytic probe FM1-43, most (94%) of the ∼25 MEs/terminal created by brief (30-Hz, 18-second) stimulation dissipate rapidly (∼1 minute) into vesicles. Others, however, remain for hours. Here we study these "late" MEs by using 4D live imaging over a period of ∼1 hour after stimulation. We find that some (51/398 or 13%) disappear spontaneously via exocytosis, releasing their contents into the extracellular milieu. Others (at least 15/1,960 or 1%) fuse or closely associate with a second class of endosomes that take up acidophilic dyes (acidic endosomes [AEs]). AEs are plentiful (∼47/terminal) and exist independent of stimulation. Unlike MEs, which exhibit Brownian motion, AEs exhibit directed motion (average, 83 nm/sec) on microtubules within and among terminal boutons. AEs populate the axon as well, where movement is predominantly retrograde. They share biochemical and immunohistochemical markers (e.g., lysosomal-associated membrane protein [LAMP-1]) with lysosomes. Fusion/association of MEs with AEs suggests a sorting/degradation pathway in nerve terminals wherein the role of AEs is similar to that of lysosomes. Based on our data, we propose that MEs serve as sorting endosomes. Thus their contents, which include plasma membrane proteins, vesicle proteins, and extracellular levels of Ca(2+) , can be targeted either toward the reformation and budding of synaptic vesicles, toward secretion via exocytosis, or toward a degradation process that utilizes AEs either for lysis within the terminal or for transport toward the cell body.

Article	Year
Mutant PrP is delayed in its exit from the endoplasmic reticulum, but neither wild-type nor mutant PrP undergoes retrotranslocation prior to proteasomal degradation. The Journal of biological chemistry\| PubMed ID: 12663673	2003
Mutational analysis of topological determinants in prion protein (PrP) and measurement of transmembrane and cytosolic PrP during prion infection. The Journal of biological chemistry\| PubMed ID: 12933795	2003
The neurotoxicity of prion protein (PrP) peptide 106-126 is independent of the expression level of PrP and is not mediated by abnormal PrP species. Molecular and cellular neurosciences\| PubMed ID: 15607951	2005
Cytosolic prion protein (PrP) is not toxic in N2a cells and primary neurons expressing pathogenic PrP mutations. The Journal of biological chemistry\| PubMed ID: 15632159	2005
A transmembrane form of the prion protein is localized in the Golgi apparatus of neurons. The Journal of biological chemistry\| PubMed ID: 15671025	2005
Neurodegenerative illness in transgenic mice expressing a transmembrane form of the prion protein. The Journal of neuroscience : the official journal of the Society for Neuroscience\| PubMed ID: 15800202	2005
A novel, drug-based, cellular assay for the activity of neurotoxic mutants of the prion protein. The Journal of biological chemistry\| PubMed ID: 19940127	2010
"Late" macroendosomes and acidic endosomes in vertebrate motor nerve terminals. The Journal of comparative neurology\| PubMed ID: 22740045	2012

Richard S. Stewart

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