Early onset torsion dystonia is an autosomal dominant movement disorder of variable caused by a glutamic acid, i.e. ΔE, deletion in DYT1, encoding the protein torsinA. Genetic and structural data implicate basal ganglia dysfunction in dystonia. TorsinA, however, is diffusely expressed, and therefore the primary source of dysfunction may be obscured in pan-neuronal transgenic mouse models. We utilized the tyrosine hydroxylase (TH) promoter to direct transgene expression specifically to dopaminergic neurons of the midbrain to identify cell-autonomous abnormalities. Expression of both the human wild type (hTorsinA) and mutant (ΔE-hTorsinA) protein resulted in alterations of dopamine release as detected by microdialysis and fast cycle voltammetry. Motor abnormalities detected in these mice mimicked those noted in transgenic mice with pan-neuronal transgene expression. The locomotor response to cocaine in both TH-hTorsinA and TH-ΔE-hTorsinA, in the face of abnormal extracellular DA levels relative to non-transgenic mice, suggests compensatory, post-synaptic alterations in striatal DA transmission. This is the first cell-subtype-specific DYT1 transgenic mouse that can serve to differentiate between primary and secondary changes in dystonia, thereby helping to target disease therapies.

In colorectal cancer, the antitumorigenic guanylyl cyclase C (GCC) signalome is defective reflecting ligand deprivation from downregulation of endogenous hormone expression. Although the proximal intracellular mediators of that signal transduction system, including cyclic guanosine monophosphate (cGMP) and cGMP-dependent protein kinase (PKG), are well characterized, the functional significance of its distal effectors remain vague. Dysregulation of ligand-dependent GCC signaling through vasodilator-stimulated phosphoprotein (VASP), an actin-binding protein implicated in membrane protrusion dynamics, drastically reduced cGMP-dependent VASP phosphorylation levels in colorectal tumors from patients. Restoration of cGMP-dependent VASP phosphorylation by GCC agonists suppressed the number and length of locomotory (filopodia) and invasive (invadopodia) actin-based organelles in human colon cancer cells. Membrane organelle disassembly reflected specific phosphorylation of VASP Ser239, the cGMP/PKG preferred site, and rapid VASP removal from tumor cell protrusions. Importantly, VASP Ser239 phosphorylation inhibited the proteolytic function of invadopodia, reflected by suppression of the cancer cell ability to digest DQ-collagen IV embedded in Matrigel. These results demonstrate a previously unrecognized role for VASP Ser239 phosphorylation, a single intracellular biochemical reaction, as an effective mechanism which opposes tumor cell shape promoting colon cancer invasion and metastasis. Reconstitution of physiological cGMP circuitry through VASP, in turn, represents an attractive targeted approach for patients with colorectal cancer.

Mature striatal medium size spiny neurons express the dopamine and cyclic AMP-regulated phosphoprotein, 32 kDa (DARPP-32), but little is known about the mechanisms regulating its levels or the specification of fully differentiated neuronal subtypes. Cell extrinsic molecules that increase DARPP-32 mRNA and/or protein levels include brain-derived neurotrophic factor (BDNF), retinoic acid, and estrogen. DARPP-32 induction by BDNF in vitro requires phosphatidylinositide 3-kinase (PI3K), but inhibition of phosphorylation of protein kinase B/Akt does not entirely abolish expression of DARPP-32. Moreover, the requirement for Akt has not been established. Using pharmacologic inhibitors of PI3K, Akt, and cyclin-dependent kinase 5 (cdk5) and constitutively active and dominant negative PI3K, Akt, cdk5, and p35 viruses in cultured striatal neurons, we measured BDNF-induced levels of DARPP-32 protein and/or mRNA. We demonstrated that both the PI3K/Akt/mammalian target of rapamycin and the cdk5/p35 signal transduction pathways contribute to the induction of DARPP-32 protein levels by BDNF and that the effects are on both the transcriptional and translational levels. It also appears that PI3K is upstream of cdk5/p35, and its activation can lead to an increase in p35 protein levels. These data support the presence of multiple signal transduction pathways mediating expression of DARPP-32 in vitro, including a novel, important pathway via by which PI3K regulates the contribution of cdk5/p35. The medium size spiny neuron (MSN)2 is the primary projection neuron of the striatum (caudate and nucleus accumbens) and accounts for over 95% of the striatal neurons (1). All MSNs receive glutamatergic input from the cortex. In addition, they receive dopamine input from the substantia nigra or ventral tegmental area and are therefore dopaminoceptive, usually expressing only a single dopamine receptor subtype (2, 3). Cellspecific MSN gene transcription is altered during the pathogenesis and treatment of many neuropsychiatric diseases, including Huntington disease, Parkinson disease, drug addiction, affective disorders, attention deficit hyperactivity disorder, and schizophrenia (4 -7). Despite the obvious clinical significance of gene regulation in MSNs and the requirement for cell-specific transcription in the development of the nervous system, few details are available regarding how the MSN phenotype is specified at the molecular level. Indeed, little is known about the molecular mechanisms underlying the specification of most central nervous system neuronal subtypes. Delineation of these pathways presents a major challenge to current molecular neurobiology.DARPP-32 (dopamine and cyclic AMP-regulated phosphoprotein, 32 kDa) is a homologue of protein phosphatase inhibitor-1 that plays a key role in integrating incoming first messenger signals. A host of transmitters and neuropeptides converges upon DARPP-32 and its state of phosphorylation, and in turn, levels of phospho-DARPP-32 provide moment-to-moment control of the signaling "tone" of ov...

Matrix metalloproteinase-9 (MMP-9) produced by colorectal cancer cells is a critical determinant of metastatic disease progression and an attractive target for antimetastatic strategies to reduce colon cancer mortality. Cellular signaling by cyclic GMP (cGMP) regulates MMP-9 dynamics in various cell systems, and the bacterial enterotoxin receptor guanylyl cyclase C (GCC), the principle source of cGMP in colonocytes, which is overexpressed in colorectal cancers, inhibits tumor initiation and progression in the intestine. Here, we show that ligand-dependent GCC signaling through cGMP induces functional remodeling of cancer cell MMP-9 reflected by a compartmental redistribution of this gelatinase, in which intracellular retention resulted in reciprocal extracellular depletion. Functional remodeling of MMP-9 by GCC signaling reduced the ability of colon cancer cells to degrade matrix components, organize the actin cytoskeleton to form locomotory organelles and spread, and hematogenously seed distant organs. Of significance, GCC effects on cancer cell MMP-9 prevented establishment of metastatic colonies by colorectal cancer cells in the mouse peritoneum in vivo. Because endogenous hormones for GCC are uniformly deficient in intestinal tumors, reactivation of dormant GCC signaling with exogenous administration of GCC agonists may represent a specific intervention to target MMP-9 functions in colon cancer cells. The notion that GCC-mediated regulation of cancer cell MMP-9 disrupts metastasis, in turn, underscores the unexplored utility of GCC hormone replacement therapy in the chemoprevention of colorectal cancer progression.