Hung Caohuy
Department of Anatomy, Physiology and Genetics
Doctor of
Philosophy
2002
Major Advisor: Harvey Pollard, M.D., Ph.D., Department of Anatomy, Physiology and Genetics
Thesis Title: Membrane Fusion Protein Annexin 7: A Common Site of Action for Calcium, Guanosine Triphosphate, Protein Kinase C and Botulinum Toxin Type C in Regulated Exocytosis
ABSTRACT
Numerous studies have shown that regulated exocytosis is activated simultaneously by calcium, guanosine triphosphate (GTP) and protein kinase C (PKC), and that this process is specifically inhibited by botulinum toxins (BoNTs). Although phenomenologically well known, the specific sites of action for these agents in the late stage of exocytosis, membrane fusion, remain unknown.
In this research project, we combined both in vitro and in vivo approaches to directly test the effects of these agents on annexin 7. Annexin 7 (ANX7) is a calcium-dependent GTP-activated membrane fusion protein. In a reconstituted membrane fusion system using artificial liposomes, ANX7 membrane fusion activity is substantially increased by the combination of individually optimal concentration of guanine nucleotide and PKC. This increasing ANX7 activity can be distinguished by a simple additive model when comparing activation by either guanine nucleotide or PKC alone. In other in vitro assays, the binding of GTP and its non-hydrolyzable analogues to ANX7 significantly enhance PKC phosphorylation, and conversely PKC phosphorylation markedly potentiates the binding and hydrolysis of GTP by ANX7. While certain other kinases label ANX7 efficiently, they do not substitute for PKC in potentiating GTP binding or membrane fusion.
To correlate the in vitro data with exocytotic events in cells, we examined the biochemical profile of endogenous ANX7 in secreting adrenal chromaffin cells. In vivo, both the ratio of ANX7-bound GDP/GTP as well as ANX7 phosphorylation by PKC change in proportion to the extent of catecholamine release from stimulated chromaffin cells. Thus, the stimulatory actions of calcium, GTP and PKC appear to specifically converge on ANX7 to drive membrane fusion activity occurring during exocytosis. To further support such an inference, we have found that BoNT type C efficiently cleaves ANX7 both in vitro and in permeabilized chromaffin cells. This proteolytic activity is concurrent with BoNT/C-dependent inhibition of ANX7 membrane fusion activity in vitro, and with inhibition of catecholamine secretion in vivo.
We therefore conclude that the exocytotic machinery includes ANX7 as a common site of action for calcium, GTP, PKC and Botulinum toxin in the exocytotic membrane fusion process.
Hugh M. Dainer
Department of Anatomy, Physiology & Genetics
Doctor of
Philosophy
2000
Major Advisor: Rosemary C. Borke, Ph.D., Department of Anatomy, Physiology & Genetics
Thesis Title: Alterations in the Local Axonal Environment Influence Target Reinnervation and Neuronal Survival after Postnatal Axotomy
ABSTRACT
Following peripheral nerve injury in adult animals, Schwann cells (SC) proliferate and provide guidance in the local axonal environment by generating the infrastructure along which regenerating nerves grow. A portion of the SC in the peripheral nerves of postnatal rats undergo apoptosis during normal development, and this apoptosis is augmented at the injury site and the neuromuscular junction (NMJ) following sciatic nerve axotomy. The current work determined that SC apoptosis occurs in the distal but not the proximal nerve segment after postnatal transection of the hypoglossal nerve, suggesting that SC apoptosis is a general, age-related and location-specific response to peripheral nerve injury. Apoptotic SC were found in two strategic locations for guiding axonal outgrowth during peripheral nerve regeneration: a) capping the transected end adjacent to the injury site and b) just proximal to the bifurcation of the nerve into medial and lateral branches. Electron microscopic (EM) analysis identified apoptotic and mitotic SC in close proximity within the distal nerve segment, supporting apoptosis and mitosis as closely related phenomena.
Poor rates of neuronal survival and tongue musculature reinnervation following hypoglossal nerve transection in the postnatal rat may be related to the increased apoptosis of SC in the distal nerve segment after injury. Insulin-like growth factor I (IGF-I) and basic fibroblast growth factor (bFGF) are neurotrophic factors which influence SC apoptosis in vitro. In an attempt to reduce SC apoptosis after postnatal axotomy, the effects of in vivo IGF-I, bFGF and vehicle treatments were examined in the distal nerve segment after postnatal hypoglossal nerve transection. All three treatments rescued SC, with the inclusion of bovine serum albumin (BSA) in the vehicle solution accounting for some of the decrease in SC apoptosis. While SC apoptosis was reduced with growth factor application, IGF-I treatment was deleterious to tongue musculature reinnervation and neuronal survival compared with bFGF and placebo treatments. Long-term bFGF treatment reduced neuronal survival compared with placebo treatment. These results imply that while SC in developing animals can be rescued by growth factor treatment, SC and motoneurons have not yet established the proper interactive relationship to translate this rescue into improved target musculature reinnervation or long-teml motoneuron survival.
LTC Cheryl
D. DiCarlo, U.S.A.
Department of Anatomy, Physiology and Genetics
Doctor of
Philosophy
2001
Major Advisor: Diane Borst, Ph.D., Department of Anatomy, Physiology & Genetics
Thesis Title: Cataracts in the Fat Sand Rat: An Ocular Complication of Diabetes
ABSTRACT
Purpose: The fat sand rat, Psammomys obesus, a spontaneous model for Type 2 diabetes, develops cataracts within days of the onset of clinical diabetes. The purpose of this research was to determine the crystallin composition of the normal sand rat lens and determine the changes that occur during diabetic cataract formation.
Materials and Methods: Slit-lamp biomicroscopy was used for ocular examinations. Blood glucose, glycated hemoglobin (HbA1c), aqueous glucose, vitreous glucose, vitreous glycated protein and lens extract glycated protein and protein values were determined using standard techniques and colorimetric test kits. Location of glucose transporter (GLUT) transcripts was determined using reverse transcriptase polymerase chain reaction (rtPCR). Lens extracts were prepared then separated by gel filtration chromatography to study the crystallin classes by SDS-PAGE. Western blotting and mass spectrometry were used to verify the crystallin composition.
Results: Blood glucose, aqueous glucose, vitreous glucose and glycated hemoglobin (HbA1c) levels were all significantly elevated in the diabetic sand rats. Water-soluble protein levels were higher in the control sand rats. No significant differences were seen among glycated protein levels of the lens extracts. GLUT1, GLUT3 and GLUT4 transcripts were present in the anterior epithelial cells of the lens but only the GLUT 4 transcript was present in the lens fiber cells. SDS-PAGE of the diabetic animals revealed an increased banding pattern at 35 - 42 kDa and a band shift from 22.5 to 23.5 kDa. Western blotting and mass spectrometry confirmed "-A crystallin aggregates and $2B/$B3 crystallin band shifts, respectively.
Discussion: This is the first report of characterization of the normal and cataractous lens in the fat sand rat. The elevated glucose and HbA1c values in the blood of diabetic sand rats result in high levels of glucose within the eye. In the diabetics, there are "A crystallin aggregates and $B2/$B3 crystallin ratio shifts with cataracts. The diabetic cataracts seen on the slit-lamp exam are similar to those seen in humans. The data reported here provides the groundwork for medical therapies aimed at reversal or prevention of diabetic cataracts instead of surgical alleviation of this common cause of blindness. The sand rat is an excellent choice for such diabetes research.
Steven Kenneth
Kim
Department of Anatomy, Physiology & Genetics
Doctor of
Philosophy
2002
Major Advisor: Meera Srivastava, Ph.D., Department of Surgery
Thesis Title: the Regulation of Nucleolin Expression in Prostate Epithelial Cells; Possible Involvement of Myc
ABSTRACT
Note: both abstracts for the two papers that comprise this thesis manuscript are located with the papers; that is, abstract 1 is included with paper 1, and abstract 2 is included with paper 2.
Paul Milward
Lea IV, M.S.
Department of Anatomy, Physiology and Genetics
Doctor of
Philosophy
2000
Major Advisor: Howard J. Bryant, Ph.D., Department of Anatomy, Physiology & Genetics
Thesis Title: Modulation of Long-Term Potentiation and Epileptiform Activity in the Rat Dentate Gyrus by the Group II Metabotropic Glutamate Receptor Subtype mGluR3
ABSTRACT
We are the first to report involvement of group n metabotropic glutamate receptors (mGluRs; specifically mGluR3) in modulation of epileptiform activity and long-term potentiation (LTP) in the hippocampal dentate gyrus. By stimulation and/or inhibition of group n mGluRs in rat hippocampal slices, we discovered that: (1) N-acetylaspartylglutamate (NAAG; 50 and 200 :M) blocked LTP of extracellular excitatory post-synaptic potentials (EPSPs) after high-frequency stimulation (100Hz; 2s) of the medial perforant path, (2) the beta-isomer of NAAG ($-NAAG) and ethyl glutamate (100:; group II mGluR antagonist) prevented this blockade, and (3) $-NAAG did not affect EPSPs recorded in a paired-pulse paradigm which argues against a presynaptic effect. These data are the first to indicate competitive effects between $-NAAG and NAAG on mGluR3 receptors. $-NAAG's effects were characterized using cultured cells. In cerebellar granule cells, we found that: (1) $-NAAG did not affect inositol phosphate production stimulated by mGluR group I agonists glutamate, L-CCG- I, and quisqualate, (2) $-NAAG reversed decreases in forskolin-stimulated cAMP caused by the mGluR group II agonist DCG-IV, and (3) $-NAAG did not reverse decreases in forskolin-stimulated cAMP caused by the mGluR group ill agonist L-AP4. These results ruled out group I and ill mGluRs as effectors of $-NAAG. We used cells stably transfected with mGluR2 or mGluR3 to determine that $-NAAG blocked forskolin- stimulated cAMP responses to glutamate, NAAG, the nonspecific group I, II agonist trans-ACPD, and the group II agonist DCG-IV via mGluR3, but not mGluR2. We conclude that $-NAAG is a specific antagonist of mGluR3. We used NAAG and $- NAAG to investigate the mGluR3 receptor contribution to epileptiform activity induced in hippocampal granule cells (considered 'gating' cells for the spread of neuronal activity) using a high-potassium, low-calcium perfusate [Schweitzer, Patrylo, Dudek. J.Neurophys 68:2016]. We found that: (1) burst frequency is decreased by ethyl glutamate and $-NAAG, and (2) NAAG, after induction of spontaneous bursting, has no effect on burst frequency. We conclude that: (1) group II mGluRs modulate epileptiform bursting of granule cells, (2) mGluR3 rather than mGluR2 mediates this modulation, and (3) specific antagonists to mGluR3 may have potential therapeutic effects.
David A. Zemo
Department of Anatomy, Physiology and Genetics
Doctor of
Philosophy
2000
Major Advisor: Joseph T. McCabe, Ph.D., Department of Anatomy, Physiology and Genetics
Thesis Title: Osmotic Stress Induces Transcriptional Changes in Vasopressin and Vasopressin 1b Receptor Gene Expression
Abstract
Arginine vasopressin (A VP) plays a critical role in the regulation of mammalian salt and water homeostasis. To further define central nervous system adaptation to osmotic challenges, transcription of A VP and vasopressin 1b receptor (V1BR) genes by magnocellular neurons of the hypothalamus and epithelial cells of the choroid plexus was studied using in situ hybridization. Compared to animals given a single injection of normal saline, animals that received a single injection of hypertonic saline had increases in AVP heteronuclear RNA (hnRNA) after 15 and 30 minutes. Animals given an injection of hypertonic saline followed by a second injection of hypertonic saline (H-H) had an increase in AVP hnRNA levels that were equivalent to the response seen after a single hypertonic saline injection. Levels of AVP hnRNA after H-H were greater than the levels detected in animals given an injection of normal saline followed by a second injection of hypertonic saline (N-H). This is the first study to show repeated exposure to hypertonic saline causes an immediate, robust, and reproducible increase in vasopressin gene transcription. These results suggest there is a correlation between increased neuronal firing rate and vasopressin gene transcription. We also studied long-term-adaptation to an osmotic challenge. Compared to rats maintained on tap water, salt-drinking rats had increased levels of AVP and V1BR mRNAs in the supraoptic and paraventricular nuclei, and in the choroid plexus. The increase in AVP and V1BR mRNAs in the SON and PVN as a result of plasma hyperosmolality may indicate the intranuclear release of AVP has a role in the autoregulation of magnocellular neuron activity. The role of AVP in cerebrospinal fluid formation remains to be elucidated. However, the increase of AVP and V1BR mRNA in the choroid plexus suggests the involvement of AVP in the regulation if brain water content and cerebral edema. Taken together, these results demonstrate coordinated transcriptional control in hypothalamic neurons and epithelial cells of the choroid plexus. Changes in gene regulation for a receptor and its binding peptide occurred in two structures that have crucial, complementary roles in water homeostasis for the periphery and the central nervous system.