Protein Regulators of Phosphoinositides as Promoters of Cancer Metastasis

Metastasis, the spread of cancer cells from a primary tumor to a distal organ, represents the predominant cause of mortality in patients with solid tumors. However, the molecular mechanisms underpinning this multistep progression are poorly defined. Successful metastasis requires a cancer cell to acquire multiple capabilities, including the ability to migrate, invade, and co-opt the microenvironment in the distal organ for colonization. Many of these phenotypes require the actions of a family of lipids called phosphoinositides (PIs). Although structurally small molecules and minor components of the cellular lipidome, PIs are critical mediators of many cellular processes through their localization, abundance, and recruitment of effector proteins. The actions of PIs are orchestrated by PI regulator proteins that bind to or act upon each PI. Identifying and characterizing PI regulator proteins that promote metastasis could elucidate novel cellular pathways and enable the development of therapeutic approaches targeting mechanisms unique to metastatic disease. In this thesis, I describe work delineating the molecular mechanisms of three PI regulator proteins in promoting breast cancer metastasis. These proteins were initially identified as putative targets of the metastasis suppressor miRNAs, miR-126 and miR- 335. We identify PITPNC1 as amplified in nearly half human breast cancers, and overexpressed in metastatic melanoma, breast, and colon cancer. Biochemical and cellbiological experiments reveal that PITPNC1 binds to phosphatidyl inositol 4-phosphate (PI4P) in the Golgi. Through this binding, PITPNC1 recruits RAB1B to the Golgi, which in turn recruits GOLPH3. GOLPH3 facilitates elongation of the Golgi structure, enhancing vesicular release. Through this pathway, PITPNC1 increases the secretion of a set of pro-angiogenic and pro-invasive proteins including ADAM10, FAM3C, HTRA1, MMP1, and PDGFA. In the second half of this thesis, I characterize the molecular mechanism of PTPRN2 and PLCβ1 in driving breast cancer metastasis by enhancing cellular migration. I find that increased expression of PTPRN2 and PLCβ1 associates with human metastatic relapse. PTPRN2 and PLCβ1 enzymatically reduce plasma membrane phosphatidyl inositol 4,5-bisphosphate (PI(4,5)P2) through two independent mechanisms. Reduction of plasma membrane PI(4,5)P2 abundance releases the PI(4,5)P2-binding protein cofilin from its membrane-bound inactive state into the cytoplasm. Cytoplasmic cofilin binds and severs actin, generating free barbed ends in actin filaments and inducing actin polymerization. PTPRN2 and PLCβ1-mediated actin remodeling dynamics increasecellular migration, a key metastatic phenotype

Effects of neonatal gonadal steroids on adult CA3 pyramidal neuron dendritic morphology and spatial memory in rats

The hippocampus is implicated in spatial cognition, which is sexually dimorphic and developmentally sensitive to gonadal steroids. Previously we have shown a sex difference in CA3 pyramidal cell layer volume and neuronal soma size that was reversible with neonatal castration in males or prenatal treatment of females with either testosterone propionate (TP) or a nonaromatizable androgen, dihydrotestosterone propionate, but not estradiol benzoate, all of which correlated with adult water maze navigation. The present study further investigates developmental androgen sensitivity of CA3 pyramidal neurons by measuring dendritic morphology and its relation to adult spatial ability. Female rats were injected with TP on postnatal day (P) 3 and P5 or ovariectomized (OVX) on P2, and male rats were castrated on P2, with or without testosterone replacement (Cas+T). Sham surgery controls were also included. Animals were tested on a water maze in adulthood, sacrificed, and CA3 pyramidal neurons were Golgi-stained and reconstructed in three dimensions using a computer-interfaced morphometry system. High-androgen groups (control males, Cas+T, TP females) performed better in spatial navigation and exhibited CA3 neurons with longer dendrites, a larger number of dendritic branches, and volumes of influence compared to low-androgen groups (control females, castrated males, OVX). Collectively, these findings indicate that the critical time period for organizational effects of androgens on the CA3 pyramidal neurons includes both prenatal and postnatal life, during which time androgens regulate developmental events such as somal growth and neuronal differentiation, all of which significantly contribute to establishing the sex difference in adult spatial navigation. © 2003 Wiley Periodicals, Inc. J Neurobiol 55: 179–190, 2003Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/34483/1/10200_ftp.pd

Inhibition of cPLA2 has neuroprotective effects on motoneuron and muscle atrophy following spinal cord injury

Surviving motoneurons undergo dendritic atrophy after spinal cord injury (SCI), suggesting an important therapeutic target for neuroprotective strategies to improve recovery of function after SCI. Our previous studies showed that phospholipase A2 (PLA2) may play an important role in the pathogenesis of SCI. In the present study, we investigated whether blocking cPLA2 pharmacologically with arachidonyl trifluoromethyl ketone (ATK) or genetically using cPLA2 knockout (KO) mice attenuates motoneuron atrophy following SCI. C57BL/6 mice received either sham or contusive SCI at the T10 level. At 30 min after SCI, mice were treated with ATK or vehicle. Four weeks later, motoneurons innervating the vastus lateralis muscle of the quadriceps were labeled with cholera toxin-conjugated horseradish peroxidase, and dendritic arbors were reconstructed in three dimensions. Soma volume, motoneuron number, lesion volume, and tissue sparing were also assessed, as were muscle weight, fiber cross-sectional area, and motor endplate size and density. ATK administration reduced percent lesion volume and increased percent volume of spared white matter compared to the vehicle-treated control animals. SCI with or without ATK treatment had no effect on the number or soma volume of quadriceps motoneurons. However, SCI resulted in a decrease in dendritic length of quadriceps motoneurons in untreated animals, and this decrease was completely prevented by treatment with ATK. Similarly, the vastus lateralis muscle weights of untreated SCI animals were smaller than those of sham-surgery controls, and these reductions were prevented by ATK treatment. No effects on fiber cross-sectional areas, motor endplate area or density were observed across treatment groups. Remarkably, genetically deleting cPLA2 in cPLA2 KO mice attenuated dendritic atrophy after SCI. These findings suggest that after SCI, cord tissue damage and regressive changes in motoneuron and muscle morphology can be reduced by inhibition of cPLA2, further supporting a role for cPLA2 as a neurotherapeutic target for SCI treatment

Characterization of dendritic morphology and neurotransmitter phenotype of thoracic descending propriospinal neurons after complete spinal cord transection and GDNF treatment

After spinal cord injury (SCI), poor regeneration of damaged axons of the central nervous system (CNS) causes limited functional recovery. This limited spontaneous functional recovery has been attributed, to a large extent, to the plasticity of propriospinal neurons, especially the descending propriospinal neurons (dPSNs). Compared with the supraspinal counterparts, dPSNs have displayed significantly greater regenerative capacity, which can be further enhanced by glial cell line-derived neurotrophic factor (GDNF). In the present study, we applied a G-mutated rabies virus (G-Rabies) co-expressing green fluorescence protein (GFP) to reveal Golgi-like dendritic morphology of dPSNs. We also investigated the neurotransmitters expressed by dPSNs after labeling with a retrograde tracer Fluoro-Gold (FG). dPSNs were examined in animals with sham injuries or complete spinal transections with or without GDNF treatment. Bilateral injections of G-Rabies and FG were made into the 2nd lumbar (L2) spinal cord at 3 days prior to a spinal cord transection performed at the 11th thoracic level (T11). The lesion gap was filled with Gelfoam containing either saline or GDNF in the injury groups. Four days post-injury, the rats were sacrificed for analysis. For those animals receiving G-rabies injection, the GFP signal in the T7-9 spinal cord was visualized via 2-photon microscopy. Dendritic morphology from stack images was traced and analyzed using a Neurolucida software. We found that dPSNs in sham injured animals had a predominantly dorsal-ventral distribution of dendrites. Transection injury resulted in alterations in the dendritic distribution with dorsal-ventral retraction and lateral-medial extension. Treatment with GDNF significantly increased the terminal dendritic length of dPSNs. The density of spine-like structures was increased after injury, and treatment with GDNF enhanced this effect. For the group receiving FG injections, immunohistochemistry for glutamate, choline acetyltransferase (ChAT), glycine, and GABA was performed in the T7-9 spinal cord. We show that the majority of FG retrogradely-labeled dPSNs were located in the Rexed Lamina VII. Over 90% of FG-labeled neurons were glutamatergic, with the other three neurotransmitters contributing less than 10% of the total. To our knowledge this is the first report describing the morphologic characteristics of dPSNs and their neurotransmitter expressions, as well as the dendritic response of dPSNs after transection injury and GDNF treatment

Nuclear expression of PG-21, SRC-1, and pCREB in regions of the lumbosacral spinal cord involved in pelvic innervation in young adult and aged rats

In rats, ageing results in dysfunctional patterns of micturition and diminished sexual reflexes that may reflect degenerative changes within spinal circuitry. In both sexes the dorsal lateral nucleus and the spinal nucleus of the bulbospongiosus, which lie in the L5-S1 spinal segments, contain motor neurons that innervate perineal muscles, and the external anal and urethral sphincters. Neurons in the sacral parasympathetic nucleus of these segments provide autonomic control of the bladder, cervix and penis and other lower urinary tract structures. Interneurons in the dorsal gray commissure and dorsal horn have also been implicated in lower urinary tract function. This study investigates the cellular localisation of PG-21 androgen receptors, steroid receptor co-activator one (SRC-1) and the phosphorylated form of c-AMP response element binding protein (pCREB) within these spinal nuclei. These are components of signalling pathways that mediate cellular responses to steroid hormones and neurotrophins. Nuclear expression of PG-21 androgen receptors, SRC-1 and pCREB in young and aged rats was quantified using immunohistochemistry. There was a reduction in the number of spinal neurons expressing these molecules in the aged males while in aged females, SRC-1 and pCREB expression was largely unchanged. This suggests that the observed age-related changes may be linked to declining testosterone levels. Acute testosterone therapy restored expression of PG-21 androgen receptor in aged and orchidectomised male rats, however levels of re-expression varied within different nuclei suggesting a more prolonged period of hormone replacement may be required for full restoratio

Zebrafish dou yan mutation causes patterning defects and extensive cell death in the retina

The size of an organ is largely determined by the number of cells it contains, which in turn is regulated by two opposing processes, cell proliferation and cell death, however, it is generally not clear how cell proliferation and cell death are coordinated during development. Here, we characterize the zebrafish dou yan mi234 mutation that results in a dramatic reduction of retinal size and a disruption of retinal differentiation and lamination. The retinal size reduction is caused by increased retinal cell death in a non–cell-autonomous manner during early development. The phenotypic defect in dou yan mi234 arises coincident with the onset of retinal neurogenesis and differentiation. Interestingly, unlike many other small eye mutations, the mutation does not increase the level of cell death in the brain, suggesting that the brain and retina use different mechanisms to maintain cell survival. Identification and further study of the dou yan gene will enhance our understanding of the molecular mechanisms regulating retinal cellular homeostasis, i.e., the balance between cell proliferation and cell death. Developmental Dynamics 236:1295–1306, 2007. © 2007 Wiley-Liss, Inc.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/56024/1/21148_ftp.pd

Retinal Ganglion Cell Topography in Teleosts: A Comparison Between Nissl-Stained Material and Retrograde Labelling From the Optic Nerve

The retinal topography of cells within the ganglion cell layer of three teleost species is examined in Nissl-stained material in which all neuronal elements containing Nissl substance in the cytoplasm are counted. A topographic comparison is made with retrogradely labelled ganglion cells to differentiate the proportion of nonganglion cells not possessing an axon joining the optic nerve. In the three species studied 92%, 80%, and 66% were found to be the maximum proportion of true ganglion cells in the area centralis, horizontal streak, and periphery, respectively. The proportion of nonganglion cells in the total population of cells counted was 24%. The major contribution to this discrepancy is from peripheral nonspecialized regions of the retina. There is little difference in both topography and peak densities of retinal ganglion cells between the two techniques. The soma areas of both populations are analysed, with the homogeneous nonganglion cell population possessing cells between 5 and 15 um2 and the heterogeneous ganglion cell soma between 5 and 68 um2, increasing in size with eccentricity