Research in the Kowalski Lab
Regulation of Neuronal Communication (a.k.a., Synaptic
Transmission) by the Ubiquitin and SUMO systems in C. elegans
Overview: The ability of neurons
to communicate with one another through specialized cell-to-cell
junctions known as synapses is critical for information processing
and storage in the brain. Aberrant neuronal signaling
contributes to a number of neurological disorders, including
epilepsy and Alzheimer 's disease. Changes in the strength of
synaptic signaling involve alterations in the function or abundance
of synaptic proteins and occur during processes such as learning
and memory. Current work in the Kowalski lab is focused on
determining the role of two related and highly conserved enzymatic
pathways in regulating synaptic transmission. Enzymes
belonging to these two pathways - the ubiquitin pathway and the
SUMO (small ubiquitin-like modifier)
pathway - are used by neurons and other cells to control protein
abundance and activity. Hundreds of enzymes exist with the
ability to either add or remove ubiquitin and SUMO tags from target
proteins; however, their functions and relevant substrates in
neurons and at synapses are only beginning to be elucidated.
We hope that by obtaining a better understanding of the precise
mechanisms by which neurons use the ubiquitin and SUMO pathways to
control the localization, function, and abundance of synaptic
proteins, we will gain valuable insights into how synaptic
transmission is regulated in both normal and disease states while
expanding our basic knowledge of these fundamental cellular
signaling pathways.
Current Research
Questions:
(1) What are the ubiquitin and SUMO pathway
enzymes that regulate synaptic transmission in the roundworm,
C. elegans?
(2) What are the substrates and
regulators of these enzymes that are relevant to their effects
on synaptic signaling?
Synaptic
Transmission: Neurons are highly specialized cells
whose functions include storing, processing, and transmitting
information throughout the nervous system. They do this by
relaying chemical and electrical signals at specialized cellular
junctions called synapses. During synaptic transmission,
chemical neurotransmitters are released from synaptic vesicles in
the presynaptic ("sending") cell and diffuse through the synaptic
cleft to bind specific receptors on the surface of the postsynaptic
("receiving") cell (Figure 1). A single
neuron from the mammalian hippocampus can make thousands of
different synaptic connections, utilizing any one of a wide variety
of different neurotransmitter receptors at each synapse.
Synaptic signaling may be either excitatory or inhibitory,
depending on the nature of the receptor and the response that is
elicited by its activation in the postsynaptic cell.
Proper nervous system function requires a carefully regulated
balance of excitatory and inhibitory synaptic transmission, and
several human neurological diseases - including seizure syndromes,
mental retardation, schizophrenia, and neurodegenerative diseases
such as Alzheimer's and Parkinson's Diseases - result from an
imbalance in these two types of signals. Regardless of the nature
of signaling at a given synapse, alterations in the abundance
and/or activity of the hundreds of proteins required for either
synaptic vesicle release or postsynaptic receptor signaling can
affect the strength of synaptic transmission, which in turn impacts
overall nervous system function.
Figure 1.
Schematic diagram of two neurons communicating through a
synaptic connection. (Inset) Close-up view of the synapse.
The axon terminus of the presynaptic "sending cell"
releases synaptic vesicles filled with chemical neurotransmitters
into the synaptic cleft. The neurotransmitters diffuse
through the cleft to bind to specific receptors that reside in the
plasma membrane of the postsynaptic "receiving cell". This
interaction initiates signaling that may be either excitatory
(shown) or inhibitory (not pictured) in the postsynaptic
cell.
The Ubiquitin Signaling
System: Covalent modification by small
molecules or polypeptides is the central mechanism by which cells
of all types control the amount, activity, and localization of
proteins they use to carry out their diverse physiological
processes. Protein phosphorylation, for example, has been
known for decades to regulate everything from the enzymatic
activity of proteins to their binding partners and stability.
The addition of small polypeptide tags, called ubiquitin and SUMO
(small ubiquitin-like modifier), is
another important mechanism by which cells can alter protein
function and abundance. Special enzymes, called ligases,
promote the attachment of ubiquitin or SUMO tags to lysine amino
acids. These ligases recognize particular amino acid motifs
in their target proteins, providing specificity to the modification
reactions. Proteases that specifically remove ubiquitin and
SUMO moieties counterbalance the activity of the ligases, thus
serving as "off" switches for these pathways (Figure
2).
Figure 2:
The ubiquitin signaling system is composed of hundreds of
ubiquitin ligase enzymes that recognize specific target proteins to
which they covalently attach ubiquitin polypeptides.
Ubiquitin specific proteases, also called deubiquitinating enzymes
(DUBs), recognize and remove ubiquitin from target proteins.
One common outcome of protein ubiquitination is degradation.
Monoubiquitination of many transmembrane proteins
targets them for degradation in the lysosome, while
polyubiquitination of cytoplasmic proteins leads to their
destruction in the proteasome. In other instances,
ubiquitination causes changes in protein activity rather than
degradation. Addition of the ubiquitin-like molecule SUMO to
proteins also generally leads to changes in their
activity/interactions. SUMO addition to proteins is governed
by families of ligases and proteases related to ubiquitin ligases
and DUBs.
Numerous synaptic proteins are ubiquitinated or SUMOylated, and
alterations in synaptic activity lead to changes in their ubiquitin
and SUMO status. Roles for several ubiquitin ligases in both
vertebrate and C. elegans neurons have been identified;
however, there are hundreds of ubiquitin and SUMO ligases and
proteases encoded in the worm and human genomes. Many of these have
broad expression patterns that include the nervous system, but
their functions in neurons and their target proteins, are largely
unknown. There is still much to be learned about the
regulation and relevant substrates of these enzymes and their
potential control of synaptic transmission.
Why
C. elegans?
Caenorhabditis elegans (C. elegans) (Figure
3) is an excellent model organism for the genetic analysis
of diverse biological questions and is a particularly tractable
system for molecular neuroscience research. These tiny
roundworms have a simple nervous system consisting of a 302 neurons
whose developmental fates and 7000 chemical synapses are all known,
making them particularly useful for studying synapse biology.
In addition, simple behavioral assays can be used to measure the
amount of synaptic transmission at specific synapses. Studies
in C. elegans are highly relevant to mammalian
neurobiology since most synaptic genes in C. elegans are
conserved in mammals and several genes critical for synaptic
transmission in mammals were first identified in the worm.
Finally, a number of shared tools that facilitate screening
approaches and genetic analyses are available through the C.
elegans research community. These resources include a
complete and annotated genome sequence, a genome-wide RNA
interference (RNAi) library (which can be used to reduce the
expression of any C. elegans gene), an extensive protein
interaction map, and large collections of previously generated
mutants.
Figure 3. Wild type C.
elegans (Larval stage 4, L4) crawling on a lawn of their food
source, E. coli bacteria.
Synaptic Transmission at the C. elegans
Neuromuscular Junction
To study the role of the ubiquitin and SUMO systems in
regulating synaptic transmission in the worm, we are currently
focusing on one well-characterized synapse, the neuromuscular
junction (NMJ). The worm NMJ, like that in humans, uses the
neurotransmitter, acetylcholine (ACh) to signal for muscle
contraction. The action of ACh, which is released by
excitatory motor neurons, is counterbalanced by the inhibitory
neurotransmitter, GABA, released from inhibitory motor neurons
(Figure 4A). A balance between the muscle
contraction caused by excitatory cholinergic signaling and the
relaxation induced by inhibitory GABAergic transmission is
essential for the ability of the animal to move in its typical
sinusoidal crawling pattern. Similarly, a tightly regulated
balance of excitatory and inhibitory synaptic signaling is critical
for proper functioning of our own nervous systems (see
above)!
At the worm NMJ, the net amount of excitatory signaling into the
postsynaptic muscle can be quantified using a simple behavioral
assay, called the aldicarb assay. Aldicarb (an acetylcholine
esterase inhibitor) indirectly increases the amount of ACh in the
synaptic cleft, resulting in muscle hypercontraction and paralysis
over time (Figure 4B). Wild type worms
paralyze at a given rate in the presence of aldicarb; mutants that
cause increased cholinergic transmission paralyze more quickly
(aldicarb hypersensitive) and those causing decreased transmission
paralyze more slowly (aldicarb resistant).
Figure 4.
(A) Schematic of the C. elegans
neuromuscular junction, which is regulated by a balance between
excitatory cholinergic (ACh) signals and inhibitory GABAergic
signals that are released from separate classes of presynaptic
motor neurons and bind to ACh or GABA-specific receptors located in
the postsynaptic muscle. The drug aldicarb promotes a
build-up of ACh in the synaptic cleft where it continues to bind to
ACh receptors and signals for continued muscle contraction.
This muscle hyper- contraction leads to eventual paralysis of the
worm. (B) Possible aldicarb assay
outcomes. Aldicarb hypersensitivity in mutant animals
may be caused by either too much cholinergic or too little
GABAergic signaling, whereas aldicarb resistance could be due to
either of the opposite situations.
Identification & Characterization of Ubiquitin Pathway
Enzymes and their Substrates that Regulate NMJ Synaptic
Transmission
We are currently using the aldicarb assay, along with other
pharmacological assays that assess different aspects of NMJ
function, to identify and characterize ubiquitin and SUMO family
enzymes involved in regulating either excitatory and/or inhibitory
synaptic signaling at the NMJ. To do this, we
are testing the effects of loss of function of these enzymes using
both RNA interference screening and candidate genetic mutant
analyses. Following the identification of an enzyme involved
in regulating NMJ transmission, we are using a combination of
genetic (e.g., double mutant epistasis experiments), cell
biological (e.g., imaging, cell type specific rescue experiments)
and biochemical approaches (e.g., immunoprecipitations, Western
blots) to identify and characterize relevant substrates of those
enzymes that mediate the effects on NMJ signaling. Our
long term goals for this research are to contribute to the
general understanding of ubiquitin enzyme biology and to provide
new information about how the activity of ubiquitin and related
pathways impacts nervous system function.
Research Students
Current Research Students
Erica Damler
Biology major, Class of
2012
November 2009 - July 2010
*2010 Butler Summer Institute
participant
Hitesh Dube
Chemistry major, Class of 2013
November 2009 - present
*2010 Butler Summer Institute
participant
Amy Wasilk
Biology major, Class of
2013
November 2009 - present
(Photos to come):
Andrew Banks
Biology major, Class of
2011
August 2010 - present
Debra Goldsmith
Biology major, Class of
2011
August 2010 - present
Logan Metzger
Biomedical Engineering major, Class of
2013
August 2010 - present