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
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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 ... read moreshown 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
- Component ID:
- tufts:20469
- To Cite:
- TARC Citation Guide EndNote
