I am interested in how molecular signaling cascades impact our control of motor behavior. Our ability to initiate specific actions and simultaneously inhibit inappropriate motor movements is dependent on signaling through the basal ganglia, a group of brain nuclei that integrate and focus cortical information. Cortical input to the basal ganglia is via the striatum, a large subcortical structure that receives excitatory inputs from all areas of the cortex. The striatum is enriched in various neurochemicals, such as dopamine, acetylcholine, opioids and endocannabinoids, that signal through cell-surface receptors to modulate cortical inputs and control motor behaviors. Such neuromodulation is thought to enhance our ability to learn specific sequences of motor behaviors that lead to reward. However, disruption of signaling through basal ganglia nuclei can have both motoric and psychological consequences. For example, degeneration of the dopaminergic system, as occurs in Parkinson’s disease, leads to a loss of voluntary movement and increased risk of depression. By contrast, drugs of abuse stimulate parts of the striatum and elevate both mood and motor behaviors. In Huntington’s disease, loss of medium spiny neurons in the striatum is correlated with uncontrollable motor movements and mood disturbances.

To understand molecular mechanisms underlying the control of motor behaviors, I have focused on studies of signaling molecules that are enriched in the striatum. My studies are part of a collaboration between the laboratories of Profs. Ann Graybiel and David Housman, thus providing a multi-disciplinary approach, ranging from molecular biology to neuroanatomy and behavioral analyses, to understanding motor control. We have focused on two families of signaling molecules, termed CalDAG-GEFs (aka RasGRPs) and cAMP-GEFs (aka EPACs). By generating and studying mice that lack these signaling proteins, we have discovered how these proteins contribute to normal biological functions. We are particularly focused on how these molecules are changed in post-mortem samples from individuals with movement disorders, and whether manipulation of these signaling molecules can ameliorate symptoms in models of Huntington’s disease, drug addiction, repetitive movement disorders such as autism and OCD, and dyskinesias associated with Parkinson’s disease therapy.

We have discovered that CalDAG-GEFI (calcium- and diacylglycerol-regulated guanine nucleotide exchange factor I) is required for calcium-dependent adhesion in platelets, a hematopoietic cell type that normally serves to arrest bleeding following an injury. This discovery led to the development of a genetic test for CalDAG-GEFI mutations, which are now known to cause bleeding disorders in dogs, cattle and horses. Whether CalDAG-GEFI mutations are associated with diseases in humans remains unknown. We have, however, found that CalDAG-GEFI expression levels are low in post-mortem striatal tissue from individuals with Huntington’s disease and in mouse models of Huntington’s disease. Most importantly, down-regulation of CalDAG-GEFI in a rat brain slice model of Huntington’s disease is neuroprotective. Based on these results, we hypothesize that the down-regulation of CalDAG-GEFI in Huntington’s disease is a compensatory mechanism to protect against neurotoxicity caused by mutant Huntingtin protein. This could be related to the fact that CalDAG-GEFI is specifically enriched in the medium spiny neurons of the striatum and that this neuronal subtype undergoes preferential degeneration in Huntington’s disease. As is observed in most diseases caused by expansions of nucleotide triplets (ie CAG), the expression of the disease-causing allele is wide-spread but neurodegeneration is somehow restricted to a subset of neuronal cell types. Thus, down-regulation of genes that are enriched in these vulnerable cell types might be beneficial to resisting neurotoxic effects of the disease-causing mutation.

Aberrant signaling in medium spiny neurons of the striatum contributes to another serious movement disorder, termed L-DOPA-induced dyskinesia. L-DOPA-induced dyskinesias are severe and unwanted motor movements that occur in response to L-DOPA therapy for Parkinson’s disease. To identify molecular changes that might contribute to this disorder, we measured gene expression in striatal neurons from a rat model of L-DOPA-induced dyskinesia, relative to controls that were treated only with L-DOPA or only with dopamine-depleting neurotoxins. We discovered that striking changes occurred in the expression of CalDAG-GEFs in the rats that developed dyskinesia: CalDAG-GEFI was down-regulated whereas CalDAG-GEFII was up-regulated, at both the mRNA and protein levels. This observation is interesting in regards to CalDAG-GEFI function, because it represents a second motor disorder (in addition to Huntington’s disease), in which there is a correlation between excessive movement and low CalDAG-GEFI. The possibility that loss of CalDAG-GEFI contributes to hyperkinesia is supported by our studies of mice lacking CalDAG-GEFI, which exhibit exaggerated motoric responses to psychomotor stimulants. This hyper-responsivity phenotype fits with reports that CalDAG-GEFI transduces signaling from acetylcholine receptors in cultured cells. In PC12D cells, the M1 muscarinic acetylcholine receptor stimulates the ERK MAP kinase pathway via CalDAG-GEFI activation. Data from numerous laboratories implicate the M1 muscarinic receptor and the ERK MAP kinases in motor control and responses to indirect dopamine receptor agonists such as L-DOPA and psychomotor stimulants. The cholinergic system is in a delicate balance with the dopaminergic system to control movement and both of these systems are exploited for therapeutic benefit in movement disorders. Nevertheless, the need for more targeted therapies remains, owing to serious side-effects associated with current medications. We are therefore testing if manipulation of CalDAG-GEFI or CalDAG-GEFII expression levels may provide some relief of symptoms in mouse models of L-DOPA-induced dyskinesias and also how these gene manipulations impact compulsive and pathologically repetitive behaviors.

The finding that CalDAG-GEFI and CalDAG-GEFII are oppositely dysregulated in models of L-DOPA-induced dyskinesia is intriguing for its potential consequences on compartmentalized striatal signaling. The dorsal striatum is comprised of two distinct compartments, termed striosomes (or patch) and matrix. CalDAG-GEFI and CalDAG-GEFII are oppositely enriched in these two compartments: CalDAG-GEFI expression is primarily in medium spiny neurons of the matrix whereas CalDAG-GEFII is more highly expressed in striosomal medium spiny neurons. The striosome/matrix categorization of striatal neurons is distinct from the direct/indirect pathway classification, and is much less well understood. In the classic but surely over-simplified view of striatal circuitry, the direct pathway projects to the internal globus pallidus and the substantia nigra pars reticulata, which are the output nuclei of the basal ganglia that control movement through their projections to brainstem motor nuclei and, via the thalamus, to the primary motor cortex. The indirect pathway projects first to the external globus pallidus and subthalamic nucleus before finally reaching the basal ganglia output nuclei. A balance of activity between these two pathways is thought to activate specific motor movements and simultaneously inhibit competing ones. Both striosomes and matrix contain striatal neurons that target the direct and indirect pathway nuclei, however, the matrix makes up most of the dorsal striatum and thus contains the majority of the direct and indirect pathway neurons. The striosomes are intermingled throughout the matrix and contain a special class of striatal neurons that project to the dopamine-containing neurons of the substantia nigra pars compacta. Furthermore, in brain slice studies, the striosome and matrix compartments have been found to exert differential presynaptic control of dopamine release. Thus, the striosomes are in a position to modulate striatal signaling by both local and global effects on dopamine release. An imbalance in striosome versus matrix activity has been associated with several movement disorders, including L-DOPA-induced dyskinesias, Huntington's disease, dystonia and drug addiction. These diseases share features of abnormally repetitive behaviors and mood disturbances. Some of this compartmentalized signaling imbalance may be related to disease-related dysregulation of the CalDAG-GEFs and other compartmentally-enriched molecules. We are working to identify genes that are dysregulated in these movement disorders and whether selective modulation of striosome or matrix compartment signaling can ameliorate symptoms in animal models. 

PUBLICATIONS

Crittenden, J. R., Sauvage, M., Burguierem E. Yim, M. J., Cepeda, C., Andre, V., Costa, C., Martella, G., Liu, T., Verma, S., Harlan, P., Levine, M., Calabresi, P., Housman, D. E., and Graybiel, A. M. Caldag-GEFI modulates behavioral sensitization to psycho-motor stimulants and is required for cortico-striatal long-term potentiation. (In preparation)

Crittenden, J. R. and Graybiel, A. M. Basal ganglia disorders associated with imbalances in the striatal striosome and matrix compartments. Frontiers in Neuroanatomy, 5:59 (2011).

Cantuti-Castelvetri I, Hernandez LF, Keller-McGandy CE, Kett LR, Landy A, Hollingsworth ZR, Saka E, Crittenden J. R., Nillni EA, Young AB, Standaert DG, Graybiel AM. Levodopa-induced dyskinesia is associated with increased thyrotropin releasing hormone in the dorsal striatum of hemi-parkinsonian rats. PLoS One. 5(11): e13861 (2010).

Crittenden J. R., Dunn DE, Merali FI, Woodman B, Yim M, Borkowska AE, Frosch MP, Bates GP, Housman DE, Lo DC, Graybiel AM. (2010) CalDAG-GEFI down-regulation in the striatum as a neuroprotective change in Huntington's disease. Hum Mol Genet. 19(9): 1756-65 (2010).

Crittenden, J. R., Cantuti-Castelvetri, I., Saka, E., Keller-McGandy, C., Fernandez-Hernandez, L., Kett, L. R., Young, A. B., Standaert, D. G., and Graybiel, A. M. Dysregulation CalDAG-GEFI and CalDAG-GEFII predicts the severity of motor side-effects induced by anti-Parkinsonian therapy. Proc Natl Acad Sci USA 106, 2892-6 (2009).

Bergmeier W., Goerge T., Wang H. W., Crittenden J. R., Baldwin A. C., Cifuni S. M., Housman D. E., Graybiel A. M., and Wagner D. D. Mice lacking the signaling molecule CalDAG-GEFI represent a model for leukocyte adhesion deficiency type III. J Clin Invest 117, 1699-707 (2007).

Crittenden, J. R., Heidersbach, A. and McManus, M. T. Lentiviral strategies for RNAi knockdown of neuronal genes. Current Protocols in Neuroscience (2007).

Bernadi, B., Guidetti, G. F., Campus, F. Crittenden, J. R., Graybiel, A. M., Balduini, C. and Torti. M. The small GTPase Rap1b regulates the cross-talk between platelet integrin a2b1 and integrin aIIbb3. Blood 107, 2728-2735 (2006).

Crittenden, J. R., Bergmeier*, W., Zhang, Y., Piffath, C. L., Liang, Y., Wagner, D. D., Housman, D. E. and Graybiel, A. M. CalDAG-GEFI integrates signaling for platelet aggregation and thrombus formation. Nature Medicine 10, 982-986 (2004).

Crittenden, J. R. RNA Modalities in Huntington’s Disease Therapy, HDF Workshop Report (2002).

Crittenden, J. R., Skoulakis, E. M. C., Han, K. A., Kalderon, D. and Davis, R. L. Tripartite mushroom body architecture revealed by antigenic markers. Learning and Memory 5, 38-51 (1998).

Harrington, L. A., Hull, C. M., Crittenden, J. R. and Greider, C. W. Gel shift and UV cross-linking analysis of Tetrahymena telomerase. J Biol Chem 270, 8893-8901 (1995).

Zolan, M. E., Crittenden, J. R., Heyler, N. K. and Seitz, L. C. Efficient isolation and mapping of rad genes of the fungus Coprinus cinereus using chromosome-specific libraries. Nucleic Acids Research 20, 3993-3999 (1992).

SCIENTIFIC CONFERENCE POSTER PRESENTATIONS

Crittenden, J. R., Sauvage, M., Burguiere, E., Yim, M. J., Costa,C., Martella, G., Ghiglieri, V., Zhang, H., Pescatore, K. A., Liu, T., Unterwald, E. M., Picconi, B., Sulzer, D., Calabresi P. and Graybiel, A. M. CalDAG-GEFI is required for normal striatum-based egocentric maze learning in mice and the balance between cholinergic and dopaminergic signaling in the striatum. Society for Neuroscience Annual Meeting, San Diego, CA 2010.

Crittenden, J. R., Dunn, D., Merali, F. I., Woodman, B., Bates, G. P., Housman, D. E., Lo, D. C. and Graybiel, A. M. Down-regulation of the striatum-enriched signaling molecule, CalDAG-GEF1/RasGRP2, is protective in a model of mutant Htt-induced neurodegeneration. The Hereditary Disease Foundation Milton Wexler Celebration Meeting, Cambridge, MA 2008.

Crittenden, J. R., Sauvage, M., Cepeda, C., Andre, V., Costa, C., Martella, G., Liu, T., Verma, S., Levine, M., Calabresi, P., Housman D.E., and Graybiel, A. M. CalDAG-GEFI modulates behavioral sensitization to psychomotor stimulants and is required for cortico-striatal long-term potentiation. The American College of Neuropsychopharmacology Annual Meeting, Boca Raton, FL 2007.

Crittenden, J. R., Cantuti-Castelvetri, I., Saka, E., Fernandez-Hernandez, L., Keller-McGandy, C., Harlan, P., Kett, L. R., Housman, D. E., Standaert, D. G., Young, A. B., and Graybiel, A. M. The striatal signaling molecule, CalDAG-GEF2, is up-regulated in the dopamine-depleted striatum of hemi-parkinsonian rats. Society for Neuroscience Annual Meeting, San Diego, CA 2007.

Crittenden, J. R., Picconi, B., Ghiglieri, V., Calabresi, P., Harlan, P., Housman D. E., and Graybiel A. M. CalDAG-GEFI is required for sensitization to amphetamine-induced stereotypy and cortico-striatal LTP, but not for locomotor sensitization and LTD. Society for Neuroscience Annual Meeting, Washington, D.C. 2006.

Crittenden, J. R., Sauvage, M., Picconi, B., Costa, C., Martella, M., Andre, V., Cepeda, C., Levine, M., Calabresi, P. Housman D. E., and Graybiel, A. M. The striatum-enriched signaling molecule, CalDAG-GEFI, is essential for cortico-striatal LTP and sensitization of drug-induced stereotypies. Cellular and Molecular Treatments of Neurological Diseases Meeting, Cambridge, MA 2006.

Crittenden, J. R., Sauvage, M., Picconi, B., Costa, C., Martella, M., Andre, V., Cepeda, C., Levine, M., Calabresi, P. Housman D. E., and Graybiel, A. M. The striatum-enriched signaling molecule, CalDAG-GEFI, is essential for cortico-striatal LTP and sensitization of drug-induced stereotypies. The Hereditary Disease Foundation HD (CAG)n Meeting, Cambridge, MA 2006.

Crittenden, J. R., C. Costa, G., Martella, G., Calabresi, P., Harlan, P., Sauvage, M., Housman D. E., and Graybiel, A. M. CalDAG-GEFI is critical for striatal plasticity. Society for Neuroscience Annual Meeting, Washington, D.C. 2005.

Crittenden, J. R., Bergmeier, W., Zhang, Y., Liang, Y., Wagner, D. D., Housman, D. E. and Graybiel, A. M. Calcium and diacylglycerol signal through CalDAG-GEFI to control platelet aggregation. Gordon Research Conference, Newport, RI, 2004.

Crittenden, J. R. and Davis, R.L. The role of D-mef2 in developing mushroom bodies. 40th Annual Drosophila Research Conference, Bellvue, WA, 1999.