Kinase-dependent signaling mechanisms in neurons. Fast axonal transport regulation in health and disease

Gerardo Morfini

The mission of our laboratory is to elucidate molecular mechanisms for the regulation of intracellular trafficking in neurons. Toward this end, we use a variety of experimental approaches including microscopic, biochemical, and cell biological assays.

The polarized distribution of materials within neuronal cells ultimately underlies their ability to receive, process, and transmit information precisely. Precise targeting and delivery of cellular components to specific, discrete subcellular compartments in neurons is largely achieved via fast axonal transport (FAT) mechanisms (5). Consequently, basic neuronal functions including growth, information processing and trophic factor-dependent survival all rely upon appropriate FAT function. Supporting this idea, genetic evidence has recently demonstrated that human neuropathologies can indeed result from compromised FAT (3,4,13).

Kinesin-1 and cytoplasmic dynein (CDyn) are the major microtubule (MT)-based molecular motors responsible for the execution of FAT. Studies on the function and regulation of these motors first showed phosphorylation as a major regulatory mechanism (13). Moreover, specific kinases involved in the regulation of both kinesin-1 and cytoplasmic dynein have been recently identified (3,8,10,11). These kinases modify specific MT-based molecular motors subunits, and differentially affect their function.

Significantly, alterations in the activity of some of these kinases have been widely documented in several human neurodegenerative conditions, including Alzheimer’s, Huntington’s, Kennedy’s and Parkinson’s disease. This led us to hypothesize that FAT might represent a critical event in the pathogenesis of these diseases. Accordingly, several pathogenic mutant proteins which causing familiar forms of various neurodegenerative diseases were found to affect FAT. These include pathogenic versions of huntingtin (8), Androgen Receptor (3,8), presenilin-1 (9), and alpha-synuclein (1). Moreover, each mutant polypeptide affected FAT in a unique fashion, through selective activation of specific kinase-dependent signaling pathways (3,10).

Current research in our laboratory addresses basic questions on the regulatory mechanisms of kinesin-1 and cytoplasmic dynein-based motilities. Below is a list of major research projects currently undergoing.

Effects of kinase-related signaling pathways on axonal function in health and disease.
Abnormal patterns of protein phosphorylation have been extensively documented in Huntington’s, Parkinson’s and Alzheimer’s diseases, among others, suggesting that kinase deregulation might represent an important pathogenic mechanism (4, 12). However, relevant pathogenic targets for these kinase activities remain unknown. The high degree of structural organization characteristic of neuronal cells implies novel functions for kinases unrelated to those established in non-neuronal cells. Stress-activated protein kinases for example, are typically described as critical regulators of transcription factors involved in apoptosis. However, local activation of JNK in axons at distances far away (up to a meter) from cell bodies, and the unique biochemical composition of axons suggested the existence of novel axonal targets. Consistent with these observations, our studies of SAPKs function in axons demonstrated a role of these kinases in the regulation of kinesin-1 (3). Similar studies are currently being extended to other kinases associated to neuronal degeneration. These studies benefit from the use of the isolated squid axoplasm model, a unique experimental paradigm that proved instrumental in the original discovery of kinesin-1, and novel pathways for FAT regulation. In addition, cellular and animal models of human neurodegenerative diseases are being used to help identifying relevant axonal targets.

Identification and characterization of physiologically relevant kinases affecting microtubule-based motor function.
Novel phosphorylation sites for kinesin-1 and cytoplasmic dynein are being identified using various biochemical approaches, including metabolic labeling, phosphopeptide mapping, and mass spectrometry studies. We are also characterizing functional consequences for these phosphorylation events using biochemical assays with recombinant proteins, and various cell biological approaches.

Elucidating the molecular basis of kinase activation by mutant pathogenic proteins.
Pathogenic mutations in huntingtin (htt) and alpha-synuclein are associated to familiar forms of Huntington’s and Parkinson’s disease, respectively. Recent experiments show these pathogenic polypeptides can activate specific kinases in axons, in a transcription-independent manner (1,3,8). The molecular basis underlying kinase activation by pathogenic htt and alpha-synuclein is currently being studied in our laboratory. Experimental approaches addressing this subject include primary cultured neurons, cell transfection, immunocytochemistry, and molecular biology techniques. In addition, effects of pathogenic mutations on the structures of huntingtin, and alpha-synuclein are being determined by using fluorescence spectroscopy and cutting-edge biochemical approaches.

Selected References

1- Morfini G, Pigino G, Opalach K, Serulle Y, Moreira JE, Sugimori M, Llinas R and Brady ST (2006) MPP+-induced activation of cytoplasmic dynein-based motility: Implications for “dying back” neurodegeneration (2006) Proc Nat'l Acad Sci USA In press.

2- Morfini G, Pigino G, Mizuno N, Kikkawa M and Brady ST (2006) Tau binding to MTs does not directly affect microtubule-based vesicle motility. Journal of Neurosci Research In press.

3- Morfini G, Pigino G, Szebenyi G, You Y, Pollema S, Brady ST (2006). JNK Kinase Activity Mediates Pathogenic Effects of Polyglutamine-expanded Androgen Receptor on Fast Axonal Transport. Nat Neurosci (7):907-916.

4- Morfini G, Pigino G, Brady ST (2005) Polyglutamine expansion diseases: Faling to deliver. Trends Molec Med 11(2):64-70.

5- Morfini G, Stenoien DL, Brady ST (2005) “Axonal Transport” chapter in Basic Neurochemistry seventh edition (eds. G. Siegel, R.W. Albers, S.T. Brady and D. Price) Elsevier Press, New York.

6- Lazarov O, Morfini G, Lee B, Farah M, Szodorai A, Koliatsos V, Kins5 S, Lee VMY, Wong P, Price D, Brady ST, Sisodia SS. (2005) Axonal Transport, APP, Kinesin and the Processing Apparatus: Revisited. J Neuroscience 25(9):2386-2395.

7- Morfini G, Szebenyi G, Brown H, Pant H, Pigino G, deBoer S, Beffert U, Brady ST (2004) A novel CDK5-dependent pathway for regulation of GSK-3 activity and kinesin-driven motility in neurons. EMBO J. 23:2235-2245.

8- Szebenyi G, Morfini G, Babcock A, Gould M, Selkoe K, Stenoien DL, Young M, Faber PW, MacDonald ME, McPhaul MJ, Brady ST. (2003) Neuropathogenic forms of huntingtin and androgen receptor inhibit fast axonal transport. Neuron 40(1):41-52.

9- Pigino G, Morfini G, Pelsman A, Mattson MP, Brady ST, Busciglio J. (2003) Alzheimer's presenilin 1 mutations impair kinesin-based axonal transport. J Neuroscience 23(11):4499-4508.

10- Morfini G, Szebenyi G, Ellurru R, Ratner N, and Brady ST. (2002) Glycogen Synthase Kinase 3b phosphorylates kinesin light chains and negatively regulates kinesin-based motility. EMBO J. 21:281-293.

11- Donelan M, Morfini G, Julyan R, , Sommers S, Hays L, Kajio H, Briaud I., Easom RA, Molkentin JD, Brady ST, Easom R, and Rodhes C. (2002) Ca 2+-Dependent dephosphorylation of Kinesin Heavy Chain on ß-granules in Pancreatic ß-cells. Implications for regulated ß-granule transport and Insulin exocytosis. J. Biol. Chem. 277(27):24232-24242.

12- Morfini G, Pigino G, Beffert U, Busciglio J, Brady ST. (2002) Fast axonal transport misregulation and Alzheimer's disease. Neuromolecular Med. 2(2):89-99.

13- Morfini G, Szebenyi G, Richards B, and Brady ST. (2001) Regulation of kinesin: Implications for neuronal development. Dev. Neurosci. 23:364-376.


Assistant Professor Anatomy & Cell Biology
Ph.D., National University of Cordoba, 1997;
S., National University of Cordoba, 1994

Gerardo Morfini, Ph.D.