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Biology

Kowalski Research

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.    

Research Diagram

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).

Research Diagram 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. 

 Worming

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).

 

Research Diagram

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. 

Lab Members

Alex Jennings

Alex Jennings, B.S.

Research Assistant

Biology, Class of 2014

Current Research Students

 
Ally Munneke

Allyson Munneke                              

Biology major, Class of 2016

August 2013 - present

*2014 Butler Summer Institute participant

Kyle Cherry

Kyle Cherry

Biology major, Class of 2016

January 2013 - present

Amber Kline

Amber Kline

Biology major, Class of 2016

August 2013 - present

Lauryn Campagnoli

Lauryn Campagnoli

Biology major, Class of 2016

January 2015 - present

*2015-2016 USRP Fellow

Victoria Kreyden

Victoria Kreyden

Biology major, Class of 2017

January 2014 - present

*2015 Butler Summer Institute participant

Sierra Williams

Sierra Williams

Biology major, Class of 2017

February 2015 - present

 

Barry Wei 

High School Researcher

Spring 2015 - present

Former Research Students

JoAnne Babula, Biology, Class of 2014, Interdisciplinary PhD Program, Indiana University School of Medicine

Andrew Banks, Biology, Class of 2011, M.A., Bioethics & Medical Humanities, University of Louisville

Kyle Brumbaugh, Biology, Class of 2016

Erica Damler, Exercise Science, Class of 2014                                                 

Zachary Didier, Biology, Class of 2013, Dental School, University of Louisville

Hitesh Dube, Chemistry, Class of 2013, Medical School, Indiana University                            

Shataakshi Dube, Biology, Class of 2015, Neuroscience PhD Program, Duke University 

Rachael Essig, Biology, Class of 2013, Medical School, West Virginia University

Debra Goldsmith, B.S., Cytotechnology, IUPUI

Nicole Green, Biology and Chemistry, Class of 2015

Morgan Harrison, Biology major, Class of 2015                                      

Julie Kolnik, Biology major, Class of 2014

Daniel Lester, Biology, Class of 2015, Cancer Biology PhD Program, University of South Florida 

Logan Metzger, Biomedical Engineering, Class of 2013                                   

Mara Olson, Biology, Class of 2015                                      

Sarah Olofsson, STS, Class of 2014

Kristen Rush, Chemistry, Class of 2013, Medical School, Indiana University

Amy Wasilk, Biology, Class of 2013, PuLSE PhD Program, Purdue University

Julia Wang, (University High School, Class of 2015), Stanford University

Lab Photos

2015

 

Lab Members at ASCB 2015

After many hours in airports, Amber, Kyle, and Ally are ready for their first day at ASCB 2015! (San Diego, CA) 

Lab Members at ASCB 2015

Enjoying a walk along the San Diego Harbor after the meeting! (December 2015)

Lab Members at 2015 Indiana Academy of Science Meeting

Kowalski lab represents at the 2015 Indiana Academy of Science Meeting! (March 2015)

Lab Holiday Party

Kowalski lab holiday party (December 2015)

2014

 

Lab Member Presentation

Shataakshi and Dan present their posters at the 2014 ASCB Meeting in Philadelphia.

Lab Members with the Liberty Bell

We also enjoyed seeing the Liberty Bell and Independence Hall after the meeting! (December 2014)

JoAnne presents

JoAnne presents her poster at the 2014 Butler Undergraduate Research Conference (April 2014)

Lab Senior Lunch

The lab enjoys a farewell lunch in Broad Ripple in honor of our graduating seniors (May 2014)

2013

 

Lab Members at Celebrate Science

Morgan and Kristen showing off the worms (and getting their faces painted!) at Celebrate Science Indiana 2013 

End of summer gathering

End of summer gathering (August 2013)

Lab Members See Young Frankenstein

The lab makes a trip to see Kyle perform in Young Frankenstein at the Footlite Theater (July 2013)

Final Lab Lunch

Final Lab Lunch (May 2013)

Lab Members at the URC

Kowalski lab ready to present at the URC (Apr 2013)

 

2012

 

Lab Members Present at ASCB

Hitesh and Amy present their posters at the 2012 American Society for Cell Biology Meeting in San Francisco.

ASCB Siteseeing

They also enjoy some sightseeing at the Golden Gate bridge with Dr. Kowalski! (December 2012)

Lab Members Summer 2012

Kowalski Lab (Summer 2012)

Julie Presenting at URC

Julie and Rachael present results of their summer work at the 2012 BSI poster session (July 2012).

Rachael Presenting at URC

 

2011

 

Lab Members at Celebrating Science

Amy and Hitesh show off the worms at Celebrate Science Indiana Day  (October 2011)

Summer Lab Party

Logan, Hitesh, and Kristen at the summer lab party.

Lab Members Present

Hitesh and Dr. Kowalski present at the 2011 Indiana Academy of Sciences Meeting. (March 2011)

 

 

Lab News

December 2015

  • Kyle Cherry, Amber Kline, and Allyson Munneke presented posters at the 2015 ASCB Meeting in San Diego!

October 2015

  • Dr. Kowalski helped to organize at three-day inquiry-based workshop on C. elegans neurobiology with 6th grade honors biology students in Ms. Cori Neff's (Butler COE grad) class at Westlane Middle School. The 6th graders spent the final day of the workshop presenting posters on their work and exploring the science at Butler - thanks to Kowalski lab members and Butler Biological Sciences faculty and staff for all of the help!

September 2015

  • Dr. Kowalski spent a fantastic month of her sabbatical learning CRISPR genome editing in the Updike lab at MDIBL in Maine! Thanks Updike lab for all of your help and hospitality!

August 2015

  • Congratulations and good luck to lab alums, Shataakshi Dube, who began work towards her Ph.D. Neuroscience at Duke University, and Dan Lester, who began work towards his Ph.D. in cancer biology at University of South Florida!!
  • Welcome to Alex Jennings (2015 Butler Biology grad) who has joined the lab as our new Research Assistant!

May 2015

  • Congratulations to our graduating seniors, Shataakshi Dube and Dan Lester!!  Shataakshi also completed successfully completed her honors thesis.  Best wishes to you both and thanks for all of your hard work!

March 2015

  • Dr. Kowalski received promotion and tenure to Associate Professor!
  • Kyle Cherry, Victoria Kreyden, Dan Lester, and Allyson Munneke, presented posters and Shataakshi Dubegave a talk at the 2015 Indiana Academy of Sciences Meeting in Indianapolis!

February 2015

  • Welcome to our newest lab members: Lauryn Campagnoli (biology, class of 2017) and Sierra Williams (biology, class of 2017)!

December 2014

  • Shataakshi Dube and Dan Lester presented posters at the 2015 ASCB Meeting in San Diego!

August 2014

  • Congratulations and good luck to lab alums, Hitesh Dube and Kristen Rush, who began medical school at Indiana University!
  • Congratulations and good luck to lab alums, JoAnne Babula, who began work towards her Ph.D. in the Indiana University School of Medicine, and Julie Kolnik, who began her one-year Master's of Physiology at IUSM!!

May 2014

  • Welcome to Sarah Olofsson (2014 Butler STS grad) who has joined the lab as our new Research Assistant!
  • Congratulations to our graduating seniors, Julie Kolnik and JoAnne Babula!!  Julie also completed successfully completed her honors thesis.  Best wishes to you both and thanks for all of your hard work!

January 2014

  • Our lab's first paper is published in the Molecular Cellular Neuroscience with Hitesh Dube, Kristen Rush, and Zach Didier all as co-authors!
  • Welcome to our newest lab member: Victoria Kreyden (biology, class of 2017)!

September 2013

  • Nikki Green presented a poster on her collaborative work with lab alum, Hitesh Dube, investigating the use of ester-masked fluorophores as imaging tools in C. elegans (a collaborative project with Dr. Jeremy Johnson in the Butler University Chemistry Department) at the 2013 American Chemical Society national meeting in Indianapolis.
  • Thanks to Morgan Harrison and Kristen Rush who had fun volunteering their time to show off mutant C. elegans and other cool microorganisms to kids at the 2013 Celebrate Science Indiana event at the Indianapolis Motor Speedway on Sunday, September 9th!

August 2013

  • Congratulations and good luck to 2013 lab alum, Amy Wasilk, who began work towards her Ph.D. in the Purdue University Interdisciplinary Program in the Life Sciences (PULSe)!
  • Congratulations and good luck to 2013 lab alum, Rachael Essig, who began medical school at West Virginia University!
  • Welcome to our newest lab members: JoAnne Babula (biology, class of 2014), Camila Avello (STS, class of 2015), Shataakshi Dube (biology, class of 2015), Amber Kline (biology, class of 2016), Allyson Munneke(biology, class of 2015)!

May 2013

  • Welcome to Kristen Rush who has re-joined the lab in her new position as Research Assistant!
  • Congratulations to our four graduating seniors, all of whom also successfully completed honors theses:Hitesh Dube, Rachael Essig, Kristen Rush, and Amy Wasilk!!  Best wishes and thanks for all of your hard work!
  • Congratulations to Kristen Rush on her Excellence in Biology Award! and her outstanding performance on the Biology Honors Exam!

April 2013

  • Congratulations to Rachael Essig and Hitesh Dube on their selection as Butler Top 100 Students!
  • Congratulations to Julie Kolnik on her selection as a 2013-2014 Fairbanks Fellow (USRP award)!

March 2013

  • Hitesh Dube and Kristen Rush presented posters at the 2013 Indiana Academy of Sciences Meeting in Indianapolis.