Interactions of Ubiquitination and Adrenergic Signaling in

Neurotransmission
Mitchell C Krawczyk, Katherine M Myers, Anna Caputo, and Felix E Schweizer
Department of Neurobiology, David Geffen School of Medicine UCLA

Results
Isoproterenol

Normalized mini frequency

Normalized mini frequency

The ubiquitin signaling system may be one significant way that modulates
synaptic transmission. Ubiquitin is a small protein that is ligated to protein
substrates either leading to proteasomal degradation or potentially modified
functionality. Drugs that disrupt ubiquitination or deubiquitination show
marked differences in the properties of synaptic transmission. In particular,
the E1 inhibitor Ziram blocks ubiquitination, and subsequently mini potential
frequency and evoked response amplitude change in neuronal culture.

Timolol

Baseline

Control 10 min

100 ms

0
10

20

30

-5

40

10

20

30

40

Spont

0
0

Time (min)

Time (min)

Ub

Ub
Ub

Ub

Ub
Ub

E1

E1

Ub

Ub

Figure 1. The ubiquitin proteasome

system.

Ub

E2

E2

10

20

30

40

Time (min)

1.0

evoked

0.5

0.0
0

10

15

20

25

Time (min)
1

Ub Ub

Ub

E3

Ubiquitin is activated by E1, then E2

and E3 work together to ligate
ubiquitin (Ub) to a substrate (S).
Ubiquitinated substrate may either be
degraded by the proteasome, or have
ubiquitin removes via deubiquitinase
enzymes (DUBs). Ziram blocks E1
activation of ubiquitin.

-10

Ub

Ziram

Normalized mini frequency

Ub

TTX only

It is also known that signaling via adrenergic receptors can also alter synaptic
transmission. Here we are interested in exploring the possible intersection of
these two signaling pathways.

Ziram 20 min

Ziram 10 min

10

-5

Control 20 min

100 pA

A
Synaptic transmission is a fundamental process for signal transduction in the
central nervous system where neurons release signaling molecules called
neurotransmitters at specialized junctions, synapses, with adjoining cells.
Neurotransmitters then bind receptors that transduce the signal to the
postsynaptic cell. In order to accurately convey and process all of the
complex information in the central nervous system, synaptic transmission is
an appropriately dynamic and finely tuned process.

Results

Normalized amplitude

Introduction

E3

S
Ub

CAPTION

0
0

Ub

10

20

30

40

Time (min)

Ub

Ub Ub
Ub

Proteasome

Ub

CAPTION

Ub

DUB
Ub

Ub

Ub

TTX only
n=1

60

Isoproterenol
n=2

Conclusions

Timolol
n=2

The results support earlier findings that Ziram induces an

increase in mini potential frequency and a decrease in evoked
response amplitude

Determine whether there is an interaction between adrenergic (ant)agonists and Ziram

Measure mini potentials and evoked responses in
response to adrenergic drugs and Ziram
Confirm previous findings that Ziram decreases evoked
responses in neuronal culture

Methods

Normalized mini frequency

Goals

Neither agonists nor antagonists of -adrenergic receptors

alone modulate mini potential frequency on this time scale
Both -adrenergic (ant)agonists prolong the time course of the
Ziram-induced increase in mini potential frequency, particularly
the antagonist

40

Future Directions
20

Apply adrenergic drugs and Ziram to paired cells to measure

the effects on evoked response
Use drugs targeting other adrenergic receptor types

Hippocampal neurons from rats were cultured (Sippy et al, 2008) and then
recorded alone or in pairs with whole cell patch clamp.
In paired recordings, neurons were stimulated and recorded to measure
evoked response.
Both neurons were held at -75 mV and given
depolarizations to trigger a pair of action potentials. Induced currents in the
postsynaptic cell were recorded.
In single-cell recordings, neurons were held at -75 mV, and currents evoked
by the stochastic release of neurotransmitter (mini potentials) were
measured. Cells were immersed in artificial cerebrospinal fluid (ACSF) with
20 nM tetrodotoxin and various combinations of Ziram (10 M) and the adrenergic drugs timolol and isoproterenol (1 M).

Use drugs targeting other neuromodulator systems: serotonin,

dopamine

0
0

10

20

30

CAPTION
Time (min)

40

50

Examine the effects of neuromodulators in brain slices as

opposed to neuronal culture

Acknowledgements
Thank you for the financial support provided through the Amgen
Scholars Program, and thank you to the ODell lab at UCLA for
the use of their reagents.