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ABSTRACT: The four Rab3 paralogs A-D are involved in exocytosis, but their mechanisms of action are hard to study due to functional redundancy. Here, we used a quadruple Rab3 knockout (KO) (rab3a, rab3b, rab3c, rab3d null, here denoted as ABCD(-/-) ) mouse line to investigate Rab3 function in embryonic mouse adrenal chromaffin cells by electron microscopy and electrophysiological measurements. We show that in cells from ABCD(-/-) animals large dense-core vesicles (LDCVs) are less abundant, while the number of morphologically docked granules is normal. By capacitance measurements, we show that deletion of Rab3s reduces the size of the releasable vesicle pools but does not alter their fusion kinetics, consistent with an altered function in vesicle priming. The sustained release component has a sigmoid shape in ABCD(-/-) cells when normalized to the releasable pool size, indicating that vesicle priming follows at a higher rate after an initial delay. Rescue experiments showed that short-term (4-6 h) overexpression of Rab3A or Rab3C suffices to rescue vesicle priming and secretion, but it does not restore the number of secretory vesicles. We conclude that Rab3 proteins play two distinct stimulating roles for LDCV fusion in embryonic chromaffin cells, by facilitating vesicle biogenesis and stabilizing the primed vesicle state.
Traffic 11/2010; 11(11):1415-28. · 4.92 Impact Factor
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ABSTRACT: Pathogenic aggregates of alpha-synuclein are thought to contribute to the development of Parkinson's disease. Inclusion bodies containing alpha-synuclein are present in Parkinson's disease and other neurodegenerative diseases, including Alzheimer's disease. Moreover, alpha-synuclein mutations are found in cases of familial Parkinson's disease, and transgenic overexpression of alpha-synuclein causes neurodegeneration in mice. The molecular mechanisms involved, however, remain incompletely understood. Here we show that, in transgenic mice, alpha-synuclein induced neurodegeneration involves activation of the ubiquitin/proteasome system, a massive increase in apolipoprotein E (ApoE) levels and accumulation of insoluble mouse Abeta. ApoE was not protective, but was injurious, as deletion of ApoE delayed the neurodegeneration caused by alpha-synuclein and suppressed the accumulation of Abeta. Our data reveal a molecular link between central pathogenic mechanisms implicated in Parkinson's disease and Alzheimer's disease and suggest that intracellular alpha-synuclein is pathogenic, at least in part, by activation of extracellular signaling pathways involving ApoE.
Nature Neuroscience 04/2008; 11(3):301-8. · 15.53 Impact Factor
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ABSTRACT: Presynaptic vesicle trafficking and priming are important steps in regulating synaptic transmission and plasticity. The four closely related small GTP-binding proteins Rab3A, Rab3B, Rab3C, and Rab3D are believed to be important for these steps. In mice, the complete absence of all Rab3s leads to perinatal lethality accompanied by a 30% reduction of probability of Ca2+-triggered synaptic release. This study examines the role of Rab3 during Ca2+-triggered release in more detail and identifies its impact on short-term plasticity. Using patch-clamp electrophysiology of autaptic neuronal cultures from Rab3-deficient mouse hippocampus, we show that excitatory Rab3-deficient neurons display unique time- and frequency-dependent short-term plasticity characteristics in response to spike trains. Analysis of vesicle release and repriming kinetics as well as Ca2+ sensitivity of release indicate that Rab3 acts on a subset of primed, fusion competent vesicles. They lower the amount of Ca2+ required for action potential-triggered release, which leads to a boosting of release probability, but their action also introduces a significant delay in the supply of these modified vesicles. As a result, Rab3-induced modifications to primed vesicles causes a transient increase in the transduction efficacy of synaptic action potential trains and optimizes the encoding of synaptic information at an intermediate spike frequency range.
Journal of Neuroscience 02/2006; 26(4):1239-46. · 7.11 Impact Factor
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ABSTRACT: Alpha-synuclein and cysteine-string protein-alpha (CSPalpha) are abundant synaptic vesicle proteins independently linked to neurodegeneration. Dominantly inherited mutations in alpha-synuclein cause Parkinson's disease, but the physiological role of alpha-synuclein remains unknown. Deletion of CSPalpha produces rapidly progressive neurodegeneration in mice, presumably because the cochaperone function of CSPalpha is essential for neuronal survival. Here, we report the surprising finding that transgenic expression of alpha-synuclein abolishes the lethality and neurodegeneration caused by deletion of CSPalpha. Conversely, ablation of endogenous synucleins exacerbates these phenotypes. Deletion of CSPalpha inhibits SNARE complex assembly; transgenic alpha-synuclein ameliorates this inhibition. In preventing neurodegeneration in CSPalpha-deficient mice, alpha-synuclein does not simply substitute for CSPalpha but acts by a downstream mechanism that requires phospholipid binding by alpha-synuclein. These observations reveal a powerful in vivo activity of alpha-synuclein in protecting nerve terminals against injury and suggest that this activity operates in conjunction with CSPalpha and SNARE proteins on the presynaptic membrane interface.
Cell 12/2005; 123(3):383-96. · 32.40 Impact Factor
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Francesco Fornai, Oliver M Schlüter,
Paola Lenzi,
Marco Gesi,
Riccardo Ruffoli,
Michela Ferrucci,
Gloria Lazzeri,
Carla L Busceti,
Fabrizio Pontarelli,
Giuseppe Battaglia,
Antonio Pellegrini,
Ferdinando Nicoletti,
Stefano Ruggieri,
Antonio Paparelli,
Thomas C Südhof
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ABSTRACT: In animals, sporadic injections of the mitochondrial toxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) selectively damage dopaminergic neurons but do not fully reproduce the features of human Parkinson's disease. We have now developed a mouse Parkinson's disease model that is based on continuous MPTP administration with an osmotic minipump and mimics many features of the human disease. Although both sporadic and continuous MPTP administration led to severe striatal dopamine depletion and nigral cell loss, we find that only continuous administration of MPTP produced progressive behavioral changes and triggered formation of nigral inclusions immunoreactive for ubiquitin and alpha-synuclein. Moreover, only continuous MPTP infusions caused long-lasting activation of glucose uptake and inhibition of the ubiquitin-proteasome system. In mice lacking alpha-synuclein, continuous MPTP delivery still induced metabolic activation, but induction of behavioral symptoms and neuronal cell death were almost completely alleviated. Furthermore, the inhibition of the ubiquitinproteasome system and the production of inclusion bodies were reduced. These data suggest that continuous low-level exposure of mice to MPTP causes a Parkinson-like syndrome in an alpha-synuclein-dependent manner.
Proceedings of the National Academy of Sciences 04/2005; 102(9):3413-8. · 9.68 Impact Factor
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Francesco Fornai, Oliver M. Schlüter,
Paola Lenzi,
Marco Gesi,
Riccardo Ruffoli,
Michela Ferrucci,
Gloria Lazzeri,
Carla L. Busceti,
Fabrizio Pontarelli,
Giuseppe Battaglia,
Antonio Pellegrini,
Ferdinando Nicoletti,
Stefano Ruggieri,
Antonio Paparelli,
Thomas C. Südhof
[show abstract]
[hide abstract]
ABSTRACT: In animals, sporadic injections of the mitochondrial toxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) selectively
damage dopaminergic neurons but do not fully reproduce the features of human Parkinson's disease. We have now developed a
mouse Parkinson's disease model that is based on continuous MPTP administration with an osmotic minipump and mimics many features
of the human disease. Although both sporadic and continuous MPTP administration led to severe striatal dopamine depletion
and nigral cell loss, we find that only continuous administration of MPTP produced progressive behavioral changes and triggered
formation of nigral inclusions immunoreactive for ubiquitin and α-synuclein. Moreover, only continuous MPTP infusions caused
long-lasting activation of glucose uptake and inhibition of the ubiquitin-proteasome system. In mice lacking α-synuclein,
continuous MPTP delivery still induced metabolic activation, but induction of behavioral symptoms and neuronal cell death
were almost completely alleviated. Furthermore, the inhibition of the ubiquitinproteasome system and the production of inclusion
bodies were reduced. These data suggest that continuous low-level exposure of mice to MPTP causes a Parkinson-like syndrome
in an α-synuclein-dependent manner.
Proceedings of the National Academy of Sciences 02/2005; 102(9):3413-3418. · 9.68 Impact Factor
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ABSTRACT: Rab3A, Rab3B, Rab3C, and Rab3D are closely related GTP-binding proteins of synaptic vesicles that may function in neurotransmitter release. We have produced knock-out (KO) mice for Rab3B and Rab3C and crossed them with previously generated Rab3A and 3D knock-out mice to generate double, triple, and quadruple Rab3 knock-out mice. We have found that all single and double Rab3 knock-out mice are viable and fertile. Most triple Rab3 knock-out mice perish whenever Rab3A is one of the three deleted proteins, whereas all triple knock-out mice that express Rab3A are viable and fertile. Finally, all quadruple knock-out mice die shortly after birth. Quadruple Rab3 KO mice initially develop normally and are born alive but succumb to respiratory failure. Rab3-deficient mice display no apparent changes in synapse structure or brain composition except for a loss of rabphilin, a Rab3-binding protein. Analysis of cultured hippocampal neurons from quadruple knock-out mice uncovered no significant change in spontaneous or sucrose-evoked release but an approximately 30% decrease in evoked responses. This decrease was caused by a decline in the synaptic and the vesicular release probabilities, suggesting that Rab3 proteins are essential for the normal regulation of Ca2+-triggered synaptic vesicle exocytosis but not for synaptic vesicle exocytosis as such. Our data show that Rab3 is required for survival in mice and that the four Rab3 proteins are functionally redundant in this role. Furthermore, our data demonstrate that Rab3 is not in itself essential for synaptic membrane traffic but functions to modulate the basic release machinery.
Journal of Neuroscience 08/2004; 24(29):6629-37. · 7.11 Impact Factor
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[show abstract]
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ABSTRACT: Rab3A, Rab3B, Rab3C, and Rab3D constitute a family of GTP-binding proteins that are implicated in regulated exocytosis. Various localizations and distinct functions have been proposed for different and occasionally even for the same Rab3 protein. This is exemplified by studies demonstrating that deletion of Rab3A in knock-out mice results in dysregulation of the final stages of exocytosis, whereas overexpression of Rab3A in neuroendocrine cells causes nearly complete inhibition of Ca(2+)-triggered exocytosis. We have now examined the properties of all Rab3 proteins in the same assays, with the long-term goal of identifying a common conceptual framework for their functions. Using quantitative immunoblotting, we found that all four Rab3 proteins were expressed in brain and endocrine tissues, although at widely different levels. Rab3A, Rab3B, and Rab3C co-localized to synaptic and secretory vesicles consistent with potential redundancy, whereas Rab3D was expressed at high levels only in the endocrine pituitary (where it was more abundant than Rab3A, Rab3B, and Rab3C combined), in exocrine glands, and in adipose tissue. In transfected PC12 cells, all four Rab3 proteins strongly inhibited Ca(2+)-triggered exocytosis. Except for a mutation that fixes Rab3 into a permanently GDP-bound state, all Rab3 mutations tested had no effect on this inhibition, including a mutation in the calmodulin-binding site that was described as inactivating (Coppola, T., Perret-Menoud, V., Lüthi, S., Farnsworth, C. C., Glomset, J. A., and Regazzi, R. (1999) EMBO J. 18, 5885-5891). Unexpectedly, overexpression of wild type Rab3A and permanently GTP-bound mutant Rab3A in PC12 cells caused a loss of secretory vesicles and an increase in constitutive, Ca(2+)-independent exocytosis that correlated with the inhibition of regulated Ca(2+)-triggered exocytosis. Our data indicate that overexpression of Rab3 in PC12 cells impairs the normal control of the final step in exocytosis, thereby converting the regulated secretory pathway into a constitutive pathway. These results offer an hypothesis that reconciles Rab3 transfection and knock-out studies by suggesting that Rab3 functions as a gatekeeper of a late stage in exocytosis.
Journal of Biological Chemistry 11/2002; 277(43):40919-29. · 4.77 Impact Factor
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[show abstract]
[hide abstract]
ABSTRACT: Rab3A, Rab3B, Rab3C, and Rab3D constitute a family of GTP-binding proteins that are implicated in regulated exocytosis. Various
localizations and distinct functions have been proposed for different and occasionally even for the same Rab3 protein. This
is exemplified by studies demonstrating that deletion of Rab3A in knock-out mice results in dysregulation of the final stages
of exocytosis, whereas overexpression of Rab3A in neuroendocrine cells causes nearly complete inhibition of Ca2+-triggered exocytosis. We have now examined the properties of all Rab3 proteins in the same assays, with the long-term goal
of identifying a common conceptual framework for their functions. Using quantitative immunoblotting, we found that all four
Rab3 proteins were expressed in brain and endocrine tissues, although at widely different levels. Rab3A, Rab3B, and Rab3C
co-localized to synaptic and secretory vesicles consistent with potential redundancy, whereas Rab3D was expressed at high
levels only in the endocrine pituitary (where it was more abundant than Rab3A, Rab3B, and Rab3C combined), in exocrine glands,
and in adipose tissue. In transfected PC12 cells, all four Rab3 proteins strongly inhibited Ca2+-triggered exocytosis. Except for a mutation that fixes Rab3 into a permanently GDP-bound state, all Rab3 mutations tested
had no effect on this inhibition, including a mutation in the calmodulin-binding site that was described as inactivating (Coppola,
T., Perret-Menoud, V., Lüthi, S., Farnsworth, C. C., Glomset, J. A., and Regazzi, R. (1999) EMBO J. 18, 5885–5891). Unexpectedly, overexpression of wild type Rab3A and permanently GTP-bound mutant Rab3A in PC12 cells caused
a loss of secretory vesicles and an increase in constitutive, Ca2+-independent exocytosis that correlated with the inhibition of regulated Ca2+-triggered exocytosis. Our data indicate that overexpression of Rab3 in PC12 cells impairs the normal control of the final
step in exocytosis, thereby converting the regulated secretory pathway into a constitutive pathway. These results offer an
hypothesis that reconciles Rab3 transfection and knock-out studies by suggesting that Rab3 functions as a gatekeeper of a
late stage in exocytosis.
Journal of Biological Chemistry 10/2002; 277(43):40919-40929. · 4.77 Impact Factor
