The CaM kinase, CMK-1, mediates a negative feedback mechanism coupling the C. elegans AMPA receptor, GLR-1, with its own transcription.
Moss, Benjamin.
2017
-
Abstract: Chronic changes in synaptic activity result in compensatory alterations in AMPA-type glutamate receptors (AMPARs), but the mechanisms underlying this process have not been fully elucidated. In this thesis, I investigate a feedback mechanism that bidirectionally regulates transcription of the C. elegans AMPAR GLR-1 in response to chronic changes in synaptic activity. It was previously sho... read morewn that GLR-1 trafficking mutants with decreased synaptic GLR-1, such as animals with mutations in the kinesin klp-4 or the deubiquitinating enzyme usp-46, exhibit increased levels of glr-1 transcript. In Chapter 2, I show that this increase in glr-1 mRNA is due in part to increased glr-1 promoter (Pglr-1) activity assayed with a glr-1 transcriptional reporter (Pglr-1::NLS-GFP::LACZ). glr-1 null mutants exhibit similar increases in glr-1 transcription, suggesting that decreased synaptic GLR-1 is sufficient to trigger the feedback pathway. Increased glr-1 transcriptional activity could be due to decreased synaptic GLR-1 protein levels or decreased glutamatergic transmission. To assay whether synaptic activity regulates glr-1 transcription, I measured the glr-1 transcriptional reporter in animals with mutations in the presynaptic vesicular glutamate transporter eat-4/VGLUT. In eat-4 mutants, which have reduced presynaptic glutamate release, I found similar increases in the glr-1 transcriptional reporter. These data suggest that chronic reduction of synaptic activity is sufficient to trigger the feedback pathway. To test whether acute reductions in synaptic activity can trigger the feedback pathway, I utilized exogenous expression of a histamine-gated chloride channel to reduce activity specifically and acutely in glr-1-expressing cells. I found increased glr-1 transcriptional reporter fluorescence after one and four hours of activity suppression, suggesting that decreased GLR-1 activity can repress glr-1 transcription. The feedback mechanism is bidirectional, as unc-11/AP180 clathrin adaptin endocytic mutants, which accumulate synaptic GLR-1, exhibit decreased glr-1 transcription and animals with increased GLR-1 signaling, such as animals expressing a dominant-active version of the receptor (GLR-1(A/T), also exhibit decreased glr-1 transcription. These findings suggest that the feedback mechanism responds to both decreases and increases in activity. In Chapter 3, investigation of signaling pathways mediating the synapse-to-nucleus feedback pathway revealed that the CMK-1/CaM kinase I pathway normally functions to repress glr-1 transcription. Analysis of cmk-1 loss-of function;glr-1 and cmk-1 gain-of-function;glr-1 double mutants suggests that CMK-1 functions in the same pathway as decreased synaptic activity to regulate glr-1 transcription. In support of this, we found that the subcellular distribution of GFP-tagged CMK-1 shifts from the nucleus to the cytoplasm in glr-1 mutants and from the cytoplasm to the nucleus in unc-11 mutants. In Chapter 4, the establishment of a yeast-one-hybrid (Y1H) system to screen for transcription factors necessary for the feedback mechanism is presented, along with several possible hits for glr-1 transcription factors. Together, our results reveal a bidirectional homeostatic feedback mechanism where changes in synaptic activity trigger CMK-1 translocation between the nucleus and cytoplasm to regulate glr-1 transcription. The establishment of a Y1H to probe for transcription factors required for glr-1 expression, both basally and in the feedback mechanism, provides the basis for identification of novel AMPAR regulatory factors.
Thesis (Ph.D.)--Tufts University, 2017.
Submitted to the Dept. of Neuroscience.
Advisor: Peter Juo.
Committee: Larry Feig, Michele Jacob, and Michael Francis.
Keyword: Neurosciences.read less - ID:
- vm40z391d
- To Cite:
- DCA Citation Guide EndNote
