Results Through Rehabilitation & Research
Assistant Professor, Neurology and Neuroscience
Weill Cornell Medical College
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.
B.S., Biology, May 1994
University of Pittsburgh, Pittsburgh, Pa.
Ph.D., Biology (Molecular Biology and Genetics), August 2002
University of Delaware, Newark, Del.
Nemours Biomedical Research, Alfred I. duPont Hospital for Children, Wilmington, Del.
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 3’UTR 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.
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.
Wilfredo Mellado, Ph.D. (Senior Scientist; Lab Manager)
Thong Ma, Ph.D. (Postdoctoral Fellow)
Caitlin Cooney, B.S. (Postbaccalaureate Fellow)
James Jones III (Undergraduate student researcher)
Dianna E. Willis (PI)
Axonal transport and local translation in neuropathic pain.
National Institute of Nursing Research
Dianna E. Willis (PI)
Axonal transport and local translation in neuropathic pain.
National Institute of Nursing Research
08/18/2010 – 06/30/2013