Functional enhancement and protection of dopaminergic terminals by RAB3B overexpression - PubMed (original) (raw)
Functional enhancement and protection of dopaminergic terminals by RAB3B overexpression
Chee Yeun Chung et al. Proc Natl Acad Sci U S A. 2009.
Abstract
In Parkinson's disease (PD), dopaminergic (DA) neurons in the substantia nigra (SN, A9) are particularly vulnerable, compared to adjacent DA neurons within the ventral tegmental area (VTA, A10). Here, we show that in rat and human, one RAB3 isoform, RAB3B, has higher expression levels in A10 compared to A9 neurons. RAB3 is a monomeric GTPase protein that is highly enriched in synaptic vesicles and is involved in synaptic vesicle trafficking and synaptic transmission, disturbances of which have been implicated in several neurodegenerative diseases, including PD. These findings prompted us to further investigate the biology and neuroprotective capacity of RAB3B both in vitro and in vivo. RAB3B overexpression in human dopaminergic BE (2)-M17 cells increased neurotransmitter content, [(3)H] dopamine uptake, and levels of presynaptic proteins. AAV-mediated RAB3B overexpression in A9 DA neurons of the rat SN increased striatal dopamine content, number and size of synaptic vesicles, and levels of the presynaptic proteins, confirming in vitro findings. Measurement of extracellular DOPAC, a dopamine metabolite, following l-DOPA injection supported a role for RAB3B in enhancing the dopamine storage capacity of synaptic terminals. RAB3B overexpression in BE (2)-M17 cells was protective against toxins that simulate aspects of PD in vitro, including an oxidative stressor 6-hydroxydopamine (6-OHDA) and a proteasome inhibitor MG-132. Furthermore, RAB3B overexpression in rat SN both protected A9 DA neurons and resulted in behavioral improvement in a 6-OHDA retrograde lesion model of PD. These results suggest that RAB3B improves dopamine handling and storage capacity at presynaptic terminals, and confers protection to vulnerable DA neurons.
Conflict of interest statement
The authors declare no conflict of interest.
Figures
Fig. 1.
RAB3B expression is elevated in VTA (A10) DA terminals in rat and RAB3B mRNA is elevated in VTA (A10) DA neurons in human. In rat striatum, RAB3A (red) was evenly distributed throughout the striatum including TH (green) positive fibers (A and D). In contrast, RAB3B (red) was enriched in the VTA (A10) DA projection area, including the ventromedial striatum (B and E) and the septum (F). Unlike RAB3A, a RAB3B expression pattern was exclusive to TH-positive terminals in a punctate manner (B–F). Colocalization of RAB3B and TH was confirmed by the z-stack confocal image (C). (G–J) SN (A9) and VTA (A10) DA neurons were collected using LCM. DA neurons were labeled using the quick TH-staining method described (3) (G). The TH-positive cells were targeted for laser capture with a 7.5-μm laser diameter (H). Captured cells on the thermoplastic film were visualized before processing for RNA extraction (I). Quantitative PCR results using unamplified LCM RNA samples demonstrated that among isoforms of RAB3, only RAB3B mRNA was highly elevated in VTA (A10) VTA (A10) compared to SN (A9) DA neurons in human (J). Data are shown as mRNA ratios of A9/A10 DA neurons ± SEM (n = 4 for male human and n = 4 for female human).
Fig. 2.
RAB3B overexpression in vivo increases striatal dopamine tissue content and prevents an extracellular DOPAC surge after
l
-DOPA injection in the striatum. AAV2 RAB3Bc-myc injection into the SN resulted in very efficient transduction of DA neurons (A–C) and their projection target, striatum (E) detected by an antibody against c-myc. The z-stack image of the perforated square in (C) confirmed colocalization of TH/c-myc (D). Three weeks after injection, GFP or RAB3B overexpressing striata were dissected for HPLC analysis. A significant increase in dopamine content was measured in the RAB3B overexpressing striatum compared to the GFP expressing striatum. Ratios of dopamine metabolites to dopamine, however, remain unchanged in the RAB3B overexpressing striatum (F). Data are shown as means ± SEM (AAV GFP, n = 8; AAV RAB3Bc-myc, n = 8; *, P < 0.05 two-tailed t test). Extracellular dopamine and DOPAC levels were measured in the striatum of GFP or RAB3B overexpressing rats before and after 50 mg/kg
l
-DOPA administration using microdialysis.
l
-DOPA administration at this dose did not alter dopamine levels (G). DOPAC levels were dramatically increased after
l
-DOPA injection in GFP overexpressing striatum whereas they remain unaltered in the RAB3B overexpressing striatum (H). Data are shown as mean ± SEM (GFP, n = 6; RAB3B, n = 5; *, P < 0.05 two-tailed t test).
Fig. 3.
RAB3B overexpression in vivo increases the number and size of synaptic vesicles in DA presynaptic terminals. The number and size of synaptic vesicles were quantifed in GFP or c-myc (RAB3B)-positive presynaptic terminals in the striatum identified by ImmunoGold technique (A and B). Vesicle number was determined by counting all vesicles contained in the individual presynaptic terminal (73 terminals for GFP and 85 terminals for RAB3B-expressing conditions). RAB3B-positive terminals possessed a greater number of synaptic vesicles than GFP-positive terminals when averaged (C). RAB3B-positive terminals had a tendency to contain a higher number of synaptic vesicles, when sorted by total number of synaptic veiscle per terminal (D). Average vesicle size of a single presynaptic terminal was determined using fractionator and nucleator function in Stereo Inveistigator software (Microbrightfield). RAB3B-positive terminals contained significantly larger vesicles compared to GFP-positive terminals (E).
Fig. 4.
RAB3B overexpression in vivo increases levels of presynaptic proteins in the striatum. Western blot analysis revealed that TH and VAMT2 levels were significantly reduced whereas calmodulin, synaptophysin, and SNAP-25 levels were increased (A and B). Optical densities of the individual bands were quantified using NIH image. Optical densities of RAB3B-overexpressing conditions were normalized by the averaged value of GFP expressing condition. Data are shown as mean ± SEM (GFP, n = 4; RAB3B, n = 4; *, P < 0.05 two-tailed t test).
Fig. 5.
RAB3B overexpression protects DA neurons from a retrograde 6-OHDA lesion in rat. 6-OHDA was injected into the striatum 3 weeks after AAV GFP or RAB3Bc-myc injection. Paw reaching test results showed that RAB3B overexpression improved the behavioral asymmetry caused by the 6-OHDA lesion (A). Data are shown as means ± SEM (*; two-tailed t test). TH-positive neurons in the SN were stained using DAB immunohistochemistry (C–E) and double immunofluorescent (GFP/TH or c-myc/TH; F–L) technique. Stereological counting demonstrated that more TH-positive neurons remained in the AAV RAB3Bc-myc injected SN compared to the AAV GFP injected SN (Data are shown as means ± SEM.*; two-tailed t test). Most of the remaining TH-positive neurons in AAV RAB3Bc-myc injected midbrain were strongly c-myc-positive (I–K). Colocalization was confirmed by a z-stack image of the perforated square in K (L). HPLC analysis in the striatum after the 6-OHDA lesion, demonstrated that a significant increase in DA tissue content was measured in the RAB3B-overexpressing striatum compared to the GFP expressing striatum (M). Ratios of dopamine metabolites to dopamine were reduced in the RAB3B overexpressing striatum (M). Data are shown as means ± SEM (AAV GFP, n = 12; AAV RAB3Bc-myc, n = 12; *, P < 0.05 two-tailed t test).
Fig. 6.
Putative synaptic vesicle dynamics in RAB3B overexpressing DA presynaptic terminals. Potential synaptic vesicle dynamics in DA terminals overexpressing GFP (control) or RAB3B were described. RAB3B overexpressing terminals contain more and larger synaptic vesicles, which increases vesicular dopamine content. These changes potentially provide improved dopamine handling and storage capacity to RAB3B overexpressing terminals. Due to the increase in the dopamine content in these terminals, synaptic vesicles contain less VMAT2 levels as a compensatory response. Following
l
-DOPA injection, in control DA terminals,
l
-DOPA is metabolized to dopamine by aromatic amino acid decarboxylase (AADC), the levels of which RAB3B did not alter. DOPAC levels were dramatically increased in the control GFP expressing striatum due to the instant metabolism of the increased cytosolic dopamine to DOPAC by monoamine oxidase (MAO) in the cells, which would diffuse out to the extracellular space and be detected by microdialysis probes. In contrast, the increase in DOPAC levels after
l
-DOPA injection is abolished in the RAB3B overexpressing striatum potentially because the increased cytosolic dopamine followed by
l
-DOPA injection was rapidly taken up in synaptic vesicles due to increased dopamine storage capacity (increased number and size of synaptic vesicles), avoiding metabolism by MAO.
References
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