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MECHANISMS OF VESICULAR MONOAMINE TRANSPORTER-2 DEGRADATION

Stachowski, Erin (2017) MECHANISMS OF VESICULAR MONOAMINE TRANSPORTER-2 DEGRADATION. Doctoral Dissertation, University of Pittsburgh.

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Abstract

The vesicular monoamine transporter-2 (VMAT2) packages monoamines into synaptic vesicles in the central nervous system. Not only vital for monoaminergic neurotransmission, VMAT2 protects neurons from cytosolic dopamine-related toxicity by sequestering dopamine into vesicles. This dissertation research is focused on determining the basic mechanisms of VMAT2 degradation—an unexplored aspect of VMAT2 regulation. The processes of protein synthesis and degradation balance to maintain proteostasis. As VMAT2 availability and function directly impact monoamine neurotransmission, its degradation is an important aspect of VMAT2 maintenance to study. While it has been proposed that VMAT2 is degraded by the lysosome, an acidic membrane-bound organelle, there is no direct evidence demonstrating this. In a PC12 cell model system stably expressing VMAT2-GFP, pharmacological tools were used to determine the impact of inhibiting pieces of cellular degradation machinery (the lysosome or the 26S proteasome) on VMAT2. Both mature and immature forms of VMAT2 accumulated following inhibition of the proteasome, but not the lysosome. In addition, at least a portion of the accumulated VMAT2 following proteasomal inhibition appears to be K48-linked polyubiquitinated—a post-translational modification associated with proteasomal degradation. Immature VMAT2 also accumulated following inhibition of endoplasmic reticulum-associated degradation (ERAD), a proteasome-dependent quality control mechanism for misfolded or damaged proteins undergoing processing in the ER. Demonstrated with immunocytochemistry and confocal imaging, the proportion of VMAT2 overlapping with an ER marker increased following proteasomal inhibition. These results indicate that immature VMAT2 is likely subject to ERAD as it undergoes processing in the ER. Unexpectedly, mature VMAT2 accumulated following proteasomal, but not lysosomal inhibition. While this result could be due to secondary effects of proteasomal inhibition or changes in VMAT2 synthesis, it is also possible that mature VMAT2 can be degraded by the proteasome. This would be a unique finding, as glycosylated, transmembrane proteins, such as VMAT2, must overcome a large energy barrier for retrotranslocation from a membrane. As these experiments were done under basal conditions, the results don’t exclude a role for lysosomal degradation under other circumstances. For example, there may be multiple pathways of VMAT2 degradation: proteasomal-dependent under basal conditions and lysosomal-dependent under conditions of synaptic activity.


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Details

Item Type: University of Pittsburgh ETD
Status: Published
Creators/Authors:
CreatorsEmailPitt UsernameORCID
Stachowski, Erineks30@pitt.edueks30
ETD Committee:
TitleMemberEmail AddressPitt UsernameORCID
Thesis AdvisorTorres, Gonzalogonzalotorres@ufl.edu
Committee ChairThiels, Eddathiels@pitt.edu
Committee MemberBrodsky, Jeffreyjbrodsky@pitt.edu
Committee MemberSorkin, Alexandersorkin@pitt.edu
Committee MemberHastings, Teresahasttx@upmc.edu
Committee MemberKhoshbouei, Habibehhabibeh@ufl.edu
Date: 2 February 2017
Date Type: Publication
Defense Date: 7 November 2017
Approval Date: 2 February 2017
Submission Date: 18 December 2016
Access Restriction: No restriction; Release the ETD for access worldwide immediately.
Number of Pages: 146
Institution: University of Pittsburgh
Schools and Programs: School of Medicine > Neurobiology
Degree: PhD - Doctor of Philosophy
Thesis Type: Doctoral Dissertation
Refereed: Yes
Uncontrolled Keywords: vesicular monoamine transporter-2; degradation; ubiquitin-proteasome system; lysosome; parkin
Date Deposited: 02 Feb 2017 16:11
Last Modified: 03 Feb 2017 06:15
URI: http://d-scholarship.pitt.edu/id/eprint/30603

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