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Faculty

Pioneering Rehabilitation

Dianna E. Willis, Ph.D.

Director, Pain Research

Assistant Professor,  Neurology and Neuroscience

Weill Cornell Medical College

Phone:

(914) 368-3141

Research Focus

Localized protein synthesis provides a means for the distal processes of a neuron to rapidly and autonomously respond to its environment. Although the capability of dendrites to locally synthesize new proteins has been well studied, it had long been believed that mRNAs and protein translation machinery were actively excluded from axons. The prevailing dogma was that all axonal protein was derived from the cell body and that the axonal compartment was translationally incompetent. The majority of our early work focused on identifying transcripts that could localize into regenerating axons of sensory neurons and thus become available for local translation. These studies lead to the identification of a complex population of axonal mRNAs, hinting at a diversity of functional roles for locally synthesized axonal proteins.

Our more recent work looked to address how axonal protein synthesis could be altered in response to local environmental signals encountered by the axon. We have found that axons alter the transport of specific mRNAs into the axon in response to various axonal stimuli. These studies should significantly advance the field of RNA localization, since we have shown specificity of RNA transport at the levels of individual ligands, signaling pathways and mRNAs. We are currently completing a study that is the first to clearly link the role of a specific RNA binding protein to transport and translation of multiple mRNA cargos underlying axonal outgrowth in vitro and in vivo. Considering the complex population of proteins that is synthesized in axons, we suspect that axonally synthesized proteins play a role in the function of mature axons and contribute to activity-dependent processes.

We are now focusing our efforts on understanding how axonal transport and local protein synthesis contribute to activity-dependent alterations in sensory neuron pathophysiology. Our central hypothesis is that axonal mRNA transport and local protein synthesis in sensory axons is altered by activity and that this mechanism can modify the capacity for neurotransmission. We are focusing on changes in the axonal localization and availability of mRNAs encoding ion channel proteins and neuropeptides that have been implicated in chronic pain, with a particular emphasis on the signaling mechanisms and RNA binding proteins that drive axonal localization.

Biography

Education:

B.S., Biology
1990-1994        
University of Pittsburgh, Pittsburgh, PA               

Ph.D., Biology (Molecular Biology and Genetics)
1995-2002        
University of Delaware, Newark, DE

Postdoctoral Fellowship
2002-2007
Nemours Biomedical Research, Alfred I. duPont Hospital for Children, Wilmington, DE

Publications

Willis D, Parameswaran B, Shen W and Molloy GR (1999). Conditions providing enhanced transfection efficiency in rat pheochromocytoma PC12 cells permit analysis of the activity of the far-upstream and proximal promoter of the brain creatine kinase gene.  J. Neurosci. Meth. 92:3-13.

 Shen W, Willis D, Zhang Y, Schlattner U, Williams T and Molloy GR. (2002)  Expression of creatine kinase isozyme genes during postnatal development of the rat brain cerebellum:Evidence for transcriptional regulation.  Biochem. J. 15:369-380. 

Shen W, Willis D, Zhang Y, and Molloy GR. (2003). Expression of creatine kinase isoenzyme genes during postnatal development of rat brain cerebrum: evidence for post-transcriptional regulation.  Dev. Neurosci. 25:421-435. 

Shlomit H, Perlson E,  Willis D, Zheng JQ,  Massarwa R, Huerta JJ,  Koltzenburg M,  Kohler M , van-Minnen J, Twiss JL, and  Fainzilber M. (2003).  Axoplasmic importins enable retrograde injury signaling in lesioned nerve. Neuron 40:1095-1104.

Rajasekaran SA, Gopal J, Willis D, Espineda C, Twiss JL and Rajasekaran AK. (2004).  Na,K-ATPase β1-subunit increases the translation efficiency of the α1-subunit in MSV-MDCK cells.  Mol. Bio. Cell 15:3224-32.

Willis DE, Zhang Y, and Molloy GR. (2005). Transcription of brain creatine kinase in  U87-MG glioblastoma is modulated by factor AP2.  Biochim. Biophys. Acta. 1728:18-33.

Willis DE, Li K-W, Zheng J-Q, Kelly T, Smit A, Sylvester J, van Minnen J, and Twiss JL. (2005). Differential transport and local translation of cytoskeletal, injury-response, and neurodegeneration protein mRNAs in axons.  J. Neurosci. 25:778-791

Willis DE and Twiss JL (2006).  The evolving roles of axonally synthesized proteins in regeneration.  Curr. Opinion Neurobio. 16:111-118. 

Wang W, van Niekerk E, Willis DE, and Twiss JL (2007).  RNA transport and localized protein synthesis in neurological disorders.  Dev Neurobiol. 67:1166-1182.

van Niekerk E, Chang JH, Willis DE, Reumann K, Heise T, and Twiss JL (2007). Sumoylation in axons triggers retrograde transport of the RNA binding protein La.  PNAS 104:12913-12918.

Willis DE, van Niekerk E, Merianda TT, Williams GG, Kendall M, and Twiss JL.  Extracellular stimuli specifically regulate transport of individual neuronal mRNAs.  J Cell Biol 178:965-980.

Yudin D, Hanz S, Yoo S, Iavnilovitch E, Willis DE, Gradus T, Segal-Ruder Y, Ben-Yaakov K, Hieda M, Yoneda Y, Twiss JL, and Fainzilber M. (2008). Localized regulation of axonal RanGTPase controls retrograde injury signaling in peripheral nerve.  Neuron 59:241-252.

Merianda TT, Lin A, Lam J, Vuppalanchi D, Willis DE, Karin N, Holt CE, and Twiss JL. (2009).  A functional equivalent of endoplasmic reticulum and Golgi in axons for secretion of locally synthesized proteins.  Mol. Cell. Neurosci. 40:128-142.

Toth CC, Willis DE, Twiss JL, Walsh S, Martinez JA, Liu WQ, Midha R, and Zochodne DW. (2009). Locally synthesized calcitonin gene-related peptide has a critical role in peripheral nerve regeneration. J. Neuropathol. Exp. Neurol. 68:326-337. 

Vuppalanchi D, Willis DE, and Twiss JL. (2009). Regulation of mRNA transport and translation in axons.  Results Probl. Cell Differ. PMID: 19582411 

Rivieccio MA, Brochier C, Willis DE, Tolhurst M, McLaughlin K, Kozikowski AP, Twiss JL, Ratan RR, and Langley B.  HDAC6 is a target for neuroprotection and regeneration in the nervous system.  Manuscript accepted by PNAS.

Vuppalanchi D, Coleman J, Yoo S, Merianda TT, Yadhati AG, Hossain J, Blesch A, Willis DE, Twiss JL. (2010).  Conserved 3UTR sequences direct subcellular localization of chaperone protein mRNAs in neurons.  J. Biol. Chem. 285:18025-38.

Ma TC, Campana A, Lnage PS, Lee HH, Banerjee K, Bryson JB, Mahishi L, Alma S, Giger RJ, Barnes S, Morris Jr. SM, Willis DE, Twiss JL, Filbin MT, Ratan RR. (2010). A large-scale chemical screen for regulators of arginase 1 promoter identifies the soy isoflavone daidzein as a clinically approved small molecule that can promote neuronal protection or regeneration via a cAMP-independent pathway.  J. Neurosci. 30:739-48.

Willis DE and Twiss JL. (2010).  Regulation of protein levels in subcellular domains through mRNA transport and localized translation.  Mol. Cell. Proteomics. 9:952-62.

Gumy LF, Yeo GS, Loraine Tung YC, Zivraj KH, Willis D, Coppola G, Lam BY, Twiss JL, Holt CE, Fawcett JW (2010). Transcriptome analysis of embryonic and adult sensory axons reveals changes in mRNA repertoire localization. RNA. 17:85-98. 

Willis DE and Twiss JL. (2011). Profiling axonal mRNA transport.  Methods Mol. Biol. 714:335-352. 

Current Projects

Axonal transport and local translation in neuropathic pain
The capacity of the axon to locally synthesize proteins has now been shown to be critical for successful regeneration following injury.  Once regeneration has occurred, how does the axon return to a “normal” state (i.e., how does a newly regenerated axon alter the transport and local translation of mRNAs back to the pre-injured state)?  If this return to a pre-injured condition does not occur, what are the physiological consequences of this failure in terms of maladaptive plasticity and conditions such as neuropathic pain?  The central hypothesis of this study is that changes in local protein synthesis in sensory axons alter the neuron’s capacity for propagating noxious stimuli.  Our objective is to understand how axonal transport and local protein synthesis contribute to hyperexcitability exhibited by damaged neurons leading to neuropathic pain states. Current animal models of nerve trauma have provided some insights into the neuronal changes that occur in response to peripheral nerve damage - revealing a remarkable degree of plasticity in both the sensory neurons and spinal cord.  Understanding how axonal transport and local protein synthesis contribute to increased hyperexcitability of these damaged sensory neurons may point to alternative methods of treating pathological pain states.

Axonal mRNA transport and local translation in spinal muscular atrophy
Spinal muscular atrophy (SMA) is an autosomal disease caused by deletion or mutation(s) of the survival motor neuron 1 (SMN1) gene. A highly homologous gene, SMN2, is present in all patients but yields low levels of the full-length SMN protein. This low expression of the SMN protein results in selective death of spinal motor neurons and muscle paralysis. SMN is ubiquitously expressed and contributes to the assembly of ribonucleoprotein complexes, transcriptional regulation, neurite outgrowth, and cell survival. However, exactly why motor neurons selectively die in SMA remains unclear. Accumulated evidence indicates that SMN localizes into neuronal processes where it associates with proteins involved in RNA transport and translation. Reduced levels of SMN protein decrease axonal transport of b-actin mRNA, with a presumed decrease in localized b-actin translation and defects in neurite outgrowth. However, the effect of SMN reduction on localized translation of other mRNAs has not been tested and it is not clear if SMN plays a role in axonal mRNA localization, translation, or both. Furthermore, it is not clear whether decreases in axonal mRNA transport with SMN depletion are restricted to motor neurons or if other neuronal populations are affected.  In this project we are directly testing both of these possibilities. The overall objective is to determine if SMN affects neurite outgrowth by controlling axonal mRNA transport and localization to affect local protein synthesis. We hypothesize that SMN regulates neurite outgrowth by controlling local protein synthesis through directing the transport of specific mRNAs into the axonal compartment. 

Mechanisms of axonal RNA transport
The objective of this study is to determine how the capacity for localized protein synthesis in axons is altered by injury. In the peripheral nervous system, localized protein synthesis can be triggered by axotomy and lack of capacity for localized protein synthesis may contribute to failed regeneration of axons in the central nervous system (CNS). Here we are focusing on the role of RNA binding proteins (RBPs) in delivering mRNAs into axons.  From ongoing studies in our and other labs, it is now obvious that transport of mRNAs is regulated by both exogenous and endogenous mechanisms. We hope to determine whether injury changes the capacity for localization of axonal mRNAs through altered expression or altered activity of its RBPs. These studies will provide a unique molecular view of how the capability for axonal mRNA localization and localized translation contributes to axonal regeneration. These studies are 2 pronged. First, to identify the axonal RNA localization elements within the 3’UTR using conserved sequence and structural motif bioinformatic analyses. Second, to identify the proteins that bind to these cis elements, and test whether injury alters their levels or the ability to interact with their target mRNAs.

Laboratory

Lab Members:        

Wilfredo Mellado, Ph.D. —  Senior Scientist; Lab Manager
wim2007@med.cornell.edu 

Thong Ma, Ph.D. —  Postdoctoral Fellow
tcm2001@med.cornell.edu

Caitlin Cooney, B.S. — Graduate Student
cac2040@med.cornell.edu

Yael Oren — Graduate Student, Tel Aviv University
yao3001@med.cornell.edu

James Jones III, B.S. — Postbaccalaureate Fellow
jaj3001@med.cornell.edu
 

Funding

Dianna E. Willis (PI)
K99NR010797
Axonal transport and local translation in neuropathic pain.
National Institute of Nursing Research
10/01/2007-02/28/2010

Dianna E. Willis (PI)
R00NR010797
Axonal transport and local translation in neuropathic pain.
National Institute of Nursing Research        
08/18/2010 – 06/30/2013

Spotlight

Dianna E. Willis, Ph.D., studies the mechanisms that underlie how nerves grow and repair themselves and the chronic pain that can develop when these mechanisms go awry.  She joined the Burke faculty in 2009 and is the Director of Pain Research.  Along with John Cave, Ph.D., Dr. Willis co-directs BMRI’s Summer Student Research Program, which offers undergraduate students the opportunity to work in a lab and participate in cutting-edge research.  When she’s not busy running experiments or training the generation of scientists, Dr. Willis finds time to participate in mud runs and triathlons.

As an undergrad, did you know you wanted to be a scientist?
I really thought I was going to go to med school.  When I was an undergrad at the University of Pittsburgh, I worked in a hospital and also spent some time in a lab.  Over the course of doing both of those things, I realized I really much preferred the lab work rather than working with patients—which I think is a good time to figure that out and not two years into med school. 

Were you working in a neuroscience lab?
Most of my early work was on microbiology and immune work, actually.  I made antibodies and things like that.  Really as an undergrad in many ways you’re just a pair of hands and so I did a lot of different things—but I knew I enjoyed being at the bench and doing hands-on work.

You’re now the Director of Pain Research here at Burke.  How did you get into this area of research?
As a post-doc, I worked with Jeff Twiss at the Nemours Biomedical Research Institute at the Alfred I. DuPont Hospital for Children in Wilmington, DE and we were working on axonal regeneration.  Say, you damage your sciatic nerve, which enervates your leg and your foot.  As part of the peripheral nervous system, the sciatic nerve has the capacity to repair itself—unlike if you break your back and damage your spinal cord, or damage your brain, there’s only very limited capacity for repair [in the central nervous system].  I studied what intrinsic capacity of these neurons allows for their axons to repair. 

What we started to see were changes that happen after injury that facilitate regeneration. The problem is that once that repair has been successful, that neuron doesn’t go back to what it looked like before the injury—it doesn’t reboot.  Our question now is: are some of those changes that occur after injury, that are good for repair, at a certain point do they become maladaptive?  Do they lead to chronic neuropathic pain?  That’s one of the prime interests in our lab right now. 

What is neuropathic pain?
If I hit my toes with a hammer, that’s normal pain—called nociception.  It’s a good thing that tells us not to hit our toes with hammers. The problem is when pain becomes pathophysiological.  Now, instead of having pain when I step on a tack, I have pain when I draw the covers over my legs at night.  Neuropathic pain means painful response to stimulus that normally would never cause pain or in the absence of any external stimulus at all.

When did researchers first recognize neuropathic pain as something distinct from regular nociceptive pain?
Neuropathic pain has been an interest of labs for several decades but now we’re getting closer to the fundamental biology questions.  I think now both clinically and from a society standpoint people are understanding these are real syndromes and there’s actually biology behind it—it’s not all just in your head.  And that has helped to lead to greater funding and increased interest in research into neuropathic pain causes and treatments.

What are you trying to understand about neuropathic pain?
One of the first steps in neuropathic pain is what they call peripheral sensitization, which lowers the threshold at which peripheral neurons fire.  If I damage my sciatic nerve, those neurons can change to the point where they alter their firing points—the point at which they send signals to the brain.  But that’s not the only change that happens.  If peripheral sensitization is allowed to progress long enough, you get central changes as well.  The neurons in my leg talk to neurons in my spinal cord, which talk to neurons in my brain, and those neurons talk to other neurons that send signals back down.  It’s a loop system.  If the first part of the loop has been sensitized so long it can also change everything up the stream and down the stream.

There are so many steps to the repair process we have to figure out.  How fast do we have to fix the first part of the loop before it propagates the damage?  And if we aren’t fast enough, how can we fix the loop to return the system to normal?  Right now, my lab is primarily interested in how we repair the first neuron in the loop. 

Why is neuropathic pain so hard to treat?
Of the thirty-plus clinical trials for neuropathic pain, every one of them has largely failed.  In large part, it’s because it’s really such a systemic issue.  It’s also a heterogeneous patient population.  Neuropathic pain patient A may not be the same as neuropathic pain patient B and C—their pain syndromes result from a variety of reasons and may be fundamentally different.

The other thing is that pain is not a quantitative thing.  It’s not like measuring blood pressure.  It’s subjective.  In our mouse models we use measures of temperature and mechanical sensitivity.  So we poke their foot and see at what point they withdraw from the stimulus.  When you’ve done something to give them neuropathic pain, it’s a much lower threshold.  Of course you’ve gotta figure out how to translate a behavioral measurement like that in a rodent to a human population.

One focus of your research is local protein synthesis.  Can you explain why local protein synthesis is important in pain?
If you think about the distances covered by a single neuron of the peripheral nervous system, like the sciatic nerve—that’s a long distance between the cell body and the axon.  The dogma, for year and years, was that everything that the axon and the growth cone (the tip of the axon) requires comes from the cell body and gets trafficked out there.  So when that axon is growing and it encounters a signal of some kind and has to decide to go towards or away from it, the dogma would say, the axon has to send a signal back to the cell body and the cell body has to send a protein that the growing axon needs.  That never made any sense.  When encountering a dynamic situation it seems a lot more favorable for the axon to be able to respond autonomously. 

So the question we have is: Can the axon make some of its own proteins to respond to environmental cues?  We went about studying whether or not the templates and machinery required for protein synthesis are present in the axon.  And indeed they are, particularly after injury.  We’ve shown in several cases that to prevent these RNAs from getting out there and to prevent local protein synthesis means you attenuate regeneration.  It was really the first suggestion that local translation in the axon provides a fundamental role for repair.  I think the dogma is changing but it’s been a hard sell for a lot of people.

So what’s next?
In a perfectly functioning axon that hasn’t been damaged, what role might local translation play in that cell?  We don’t know that yet. 

Some people also question whether this is strictly the domain of the peripheral nerves—do other neurons of the body also do this?  We have an ongoing set of projects in our lab looking at axons in motor neurons particularly as it revolves around motor neuron diseases like ALS and spinal muscular atrophy.

Ten years from now, what do you hope we’ll know about neuropathic pain?
I hope we will have at least a more fundamental understanding of what causes neuropathic pain so that we can target therapeutics to non-nociceptive pain. And from a more clinical aspect, how can we identify what mechanism has driven the neuropathic pain in a specific patient?  If there are multiple mechanisms that drive neuropathic pain and multiple types of neuropathic pain patients, we better figure out how to line those up.  Remember those old tests in school where you had to draw the line from something in this column to something in that column?  We want to be able to do that—draw the line from what we understand about mechanism in the lab to a specific type of patient. 

You co-direct the Summer Student Research Program here at Burke.  How did that come about? 
Burke has always taken summer students, but on a lab-by-lab basis—there was no official program.  We decided before last summer to formalize the program.  We had four official students in the program last year, and expanded to ten this year.  Those ten students are really some of the cream of the crop in terms of their application packages, their letters of recommendation.  And we probably have another 10 to 15 students outside the program who also do summer research here.

What do you try to teach your summer students?
I think an important thing to do for these students is not just teach them how to pipette.  Fundamentally scientists are here to think, and so you can’t just tell them add this and this to the tube—you have to tell them why.  And beyond the science, we try to teach our students the process of being in a lab.  Why is it important to keep a good notebook?  Why is it important to be a good lab citizen?  Why is it not a good idea to ever use the last of something and not replace it?  So they understand that what they do have real ramifications for the lab.

We do a seminar series for the students where once a week we have a faculty member come in.  They talk about their research and things like that, but they also talk about how they got into science, what they like about it, what they don’t like about it.  It’s a job like anything else, so of course there are going to be things you don’t like about it.  Each faculty gives a different perspective to these students.  We all get here from different directions.

August 2014