RSS 1.0Downstate School of Graduate Studies Theses and Dissertations
Recent Submissions
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Antibiotic-mediated gut microbiota depletion affects behavioral and neuronal correlates of spatial cognitionTrillions of microbes —comprising communities of bacteria, viruses, fungi, and other microorganisms— inhabit the gastrointestinal tract. Known as gut microbiota, these microorganisms are central to human health. Growing evidence connects imbalances of microbial communities with several neurological and psychiatric conditions, and signals to the modulating role of gut bacteria on brain function, including learning and memory. However, how the microbiota influences cognitive behavior remains unclear. Here, we investigated whether gut microbiota depletion disrupts the behavioral and neuronal correlates of spatial cognition. We depleted gut bacteria in mice by giving a cocktail of non-absorbable antibiotics and characterized 1) learning and memory performance in modalities of a place avoidance task with different cognitive demands and 2) Arc and c-Fos-expressing neuronal ensembles in the supra and infrapyramidal blades of the dentate gyrus. We found impaired learning and memory performance in place avoidance tasks that require high, but not low, cognitive demand and blade-specific alterations in Arc and c-Fos-expressing granule cell ensembles with microbiota depletion. Our results suggest that gut microbial signaling may regulate the expression of neural mechanisms underlying cognitive control.
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Age-dependent and multifaceted effects of methylphenidate in pre- and peri-adolescent rats.Attention deficit hyperactivity disorder (ADHD) is a developmental disorder affecting approximately 9% of school-aged children. A number of therapeutic options exist to treat ADHD. One, methylphenidate (MPD), improves behavior of patients with ADHD in real life, as well as their performance in laboratory settings. Recent evidence indicates that MPD affects healthy humans in a manner similar to that in ADHD patients. However, data regarding the mechanisms and effects of MPD are inconsistent, particularly with respect to age groups (i.e., children, adolescents, adults). Using the rat as a model, we conducted several studies to clarify the effects of oral MPD on cognitive function, activity and anxiety during pre- and peri-adolescence in animals. A preliminary study revealed apparent age-dependent and multifaceted effects of oral MPD in pre- and peri-adolescent rats. There, we found that daily oral administration of MPD at 3 mg/kg resulted in: (1) improved performance from postnatal day (PND) 22 to 24 but impaired performance from PND 32-34; (2) increased locomotor activity and reduced anxiety-like behaviors at PND 22-23; and (3) improved performance in an attentional task. Moreover, we found that responses to oral MPD at 3 mg/kg varied among rats, specifically in locomotor activity and performance in the attention task. Here, we examined the following three hypotheses stemming from these preliminary data: (1) the effects of oral MPD at clinically-relevant doses on performance in cognitive tasks are age-dependent; (2) oral MPD alters anxiety-related behaviors and locomotor activity in an age-dependent manner, both of which contribute to the effects of MPD on performance in cognitive tasks; (3) individual responses to novelty (i.e., new environments) in rats predict individual behavioral responses to oral MPD, including cognitive performance, anxiety-like behaviors, and locomotor activity. To test our hypotheses, we tested drug-naive pre- and peri-adolescent rats over three age ranges (PND 23 to 28, PND 29 to 34 and PND 36 to 41) for (1) the effects of oral MPD in a radial arm maze (RAM) task; (2) the effects of oral MPD on anxiety-related behaviors and locomotor activity on the elevated plus-maze; and (3) interaction effects between treatment conditions and individual responses to novelty on measures from the behavioral tests. Plasma and brain levels of MPD were also measured to examine the absorption and metabolism of the drug across these age ranges. We found that (1) oral MPD at 3 mg/kg improved RAM performance during PND 36-41 but not during PND 23-28 and PND 29-34; (2) oral MPD at 3 mg/kg reduced anxiety-related behaviors across all ages and increased locomotor activity only on PND 24 and 37; and (3) the MPD-induced increase in locomotor activity correlated with individual response to novelty. Brain and plasma levels of MPD were highest on PND 24, lower on PND 30, and lowest on PND 37. In addition, the higher dose of oral MPD improved RAM performance significantly during PND 36-37. These results suggest that, during pre- and peri-adolescence: (1) oral MPD at a clinically-relevant dose produces multifaceted effects that change across ages, which together contribute to the improved performance on cognitive tasks; (2) these effects, according to known brain circuit information, are likely through independent circuits; and (3) the effects of oral MPD on cognitive performance vary according to the testing procedures and the state of maturation.
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Early thrombopoietin intervention in stroke: Requirement for thrombopoietin receptor for brain and behavioral protection.Cerebral ischemia results in brain injury. The brain inflammatory response to ischemia contributes significantly to stroke-induced ischemic injury. Thrombopoietin (TPO), a hematopoietic growth factor that regulates platelet production, has recently been reported to protect the rat from ischemic stroke brain injury and associated behavioral deficits. My research has focused on early post-stroke TPO intervention using a mouse stroke model. Here I present data that addresses TPOs brain- and behavioral-protective effects in mouse ischemic stroke. I found that TPO treatment in WT mice produces significant protection from ischemic hemisphere swelling (57.4 ± 6.5%), hemisphere infarction (43.2 ± 5.2%), neurological deficits (36.9 ± 12.6%) and provides significant preservation of post-stroke cognitive functioning. No TPO protection was observed in TPOR KO mice. IHC data indicates that TPO significantly reduced peri-infarct up-regulation of TNF(40.4 ± 10.2%) and MMP-9 (27.1 ± 11.1%) in microvascular endothelium. Furthermore, peri-infarct vascular neutrophil infiltration into brain (25.1 ± 3.4%) and microgliosis (35.4 ± 6.5%) were inhibited by TPO. In addition to reducing brain infarction and swelling, TPO also acts to reduce microvascular inflammation and thereby to preserve the blood brain barrier. In TPOR KO mice, TPO treatment neither attenuated increased microvascular TNFor MMP-9, nor inhibited neutrophil infiltration or microgliosis. Thus, I have demonstrated that the TPOR is required for TPO brain protection from stroke-induced injury and behavioral deficits. Mechanistically, TPO treatment reduces the microvascular and parenchymal brain inflammatory response to stroke resulting in protection of the brain and blood brain barrier resulting in preservation of functioning on all levels examined.
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Control of GABAA Receptor α4 Subunit Expression In a Human Neuroblastoma Cell LineThe GABAA receptor (GABAR) is a pentameric ligand-gated Cl- channel, normally composed of two α, two β and one γ subunit. α4 expression has been shown to increase in response to extended periods of in vivo administration of positive modulators of the GABAR, such as neurosteroids, benzodiazepines (BZs) and ethanol. However, the mechanism of α4 upregulation is not clear. Therefore, the purpose of this study was to establish an in vitro model to study neurosteroid regulation of α4 expression. I proposed two potential initial triggers for α4 upregulation: i.) increased current produced by a positive GABA modulator or ii.) allosteric events produced by ligand-receptor interactions. To this end, I used the human neuroblastoma IMR-32 cell line to demonstrate increases in α4 expression by 48 h administration of the GABA-modulatory steroid 3α,5β-THP (3α -OH-5β -pregnan-20-one) following differentiation by nerve growth factor (NGF) in serum-free medium. Functional expression of α4βγ2 GABAR was validated by the unique pharmacology characteristic of this GABAR isoform, which include a near complete insensitivity to BZ agonists, but potent positive modulation by the BZ antagonist flumazenil and the BZ partial inverse agonist RO15-4513. THP induction of α4 expression was a result of its enhancement of GABA-gated current because co-application of the GABAR blocker picrotoxin prevented this effect. Moreover, 48 h administration of compounds which decrease GABA-gated current, such as submaximal concentrations of the GABAR open channel blocker penicillin G or the competitive GABAR antagonist gabazine, decreased α4 expression, as did the BZ inverse agonist DMCM. However, 48 h exposure to the BZ antagonist flumazenil, which allosterically binds to the GABAR but has no effect on GABA-gated current, did not change α4 expression. These findings suggest that α4 expression is correlated with the change in GABA-gated current. The results from my study may have implications for alterations in α4 expression across naturally occurring fluctuations in endogenous steroids.
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Identification of MicroRNAs Regulating Apolipoprotein Secretion.A major risk factor for cardiovascular disease (CVD) is plasma cholesterol that is carried in plasma by two lipoproteins LDL and HDL. Currently available therapies lower LDL and attempts are undergoing to enhance HDL levels for therapeutic purposes. With advances in microRNAs (miRs), small non-coding RNAs that affect multiple pathways, we hypothesize that there might be miRs that regulate plasma lipoproteins. The major proteins in LDL and HDL are apoB and apoA1. To identify miRs that regulate both LDL and HDL, I took two different approaches. In the first approach, I searched for miRs that could potentially modulate apoB and apoA1 expression. From TargetScan, I found that human apoB mRNA contained two predicted targeting sites for miR-548p. In both Huh-7 and HepG2 cells, overexpression of miR-548p significantly decreased intracellular and secreted medium apoB without affecting apoAI. Mechanistic studies revealed that miR-548p decreased apoB expression levels by destabilizing its mRNA. Luciferase reporter assay indicated that miR-548p interacts with apoB 3'-UTR. Site-directed mutagenesis revealed that both seed and supplementary sequences on Site II, not Site I, were required for the targeting of miR-548p on apoB 3'-UTR. Additionally, the cholesterol and fatty acid syntheses rates were decreased in miR-548p overexpressing Huh-7 cells. We also found that Hmgcr and ACSL4, two important enzymes in cholesterol and fatty acid synthesis process, were predicted targets of miR-548p. Indeed, the mRNA and protein levels of Hmgcr and ACSL4 were significantly lowered by miR-548p. The direct targeting of miR-548p on the 3'-UTR of Hmgcr and ACSL4 was studied by luciferase reporter assay. In summary, a novel miR-548p decreased apoB secretion from Huh-7 cells by targeting apoB 3'-UTR and promoting its posttranscriptional degradation. MiR-548p also reduces cholesterol and fatty acid synthesis by down-regulating Hmgcr and ACSL4 expression levels. In the second approach, I took an unbiased global approach to identify miRs that individually and simultaneously affect apoB and apoA1. I transfected human hepatoma Huh7 cells in duplicate with a library of 1237 miRs and measured apoB and apoA1 in the media. This resulted in the identification of 13 and 28 miRs that increased and decreased apoB secretion. In addition, I identified 11 and 23 miRs that increased and decreased apoA1 secretion. Most interestingly, I identified 3 miRs that simultaneously increased apoB and reduced apoA1 levels. I further characterized miR-1200 and explained mechanisms how it regulates apoB and apoA1 secretion. Mechanistic studies showed that miR-1200 reduced apoB secretion by lowering the mRNA levels of apoB. In addition, post-transcriptional degradation of apoB was promoted in miR-1200 transfected cells. Bioinformatic analysis revealed apoB as a predicted target of miR-1200. Luciferase reporter assay and site-directed mutagenesis were used to confirm the direct miR-mRNA interaction. In contrast, miR-1200 increased apoAI protein and mRNA levels by increasing its de novo transcription. The mechanistic studies resulted in the identification of a novel transcription repressor of apoAI, BCL11B. Therefore, miR-1200 increases the transcription of apoAI by reducing the expression levels of BCL11B, and promotes the secretion of apoAI. I also observed that the overexpression of miR-1200 in Huh-7 cells decreased lipid synthesis, which might avoid the lipid accumulation in the liver. In summary, these studies identified a novel human microRNA-1200 that could potentially reduce apoB-containing lipoproteins and increase HDL production from the liver, as well as decreasing lipid synthesis in human hepatoma cell line. These three different roles of miR-1200 in lipid metabolism regulation suggest that it may have protective function against atherosclerosis. In short, I have identified two novel miRs-548p and 1200 as potent regulators of apolipoprotein and lipid metabolism in hepatoma cells. Both have the potential to be developed as therapeutic agents for the treatment of dyslipidemia and atherosclerosis while avoiding hepatic steatosis. As these are primate specific miRs, the physiological effect of overexpressing or inhibiting endogenous miR-548p or miR-1200 could be tested in humanized mice or non-human primate models.
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ComplementC3 regulated cell death in myocardial ischemia/reperfusion injuryIschemic heart disease is a life-threatening condition and requires immediate treatment by unblocking the occluded blood vessel. However, the restoration of blood flow to the ischemic area causes additional damage, named ischemia/reperfusion injury (I/R injury).Currently, there is no clinically approved therapy available to reduce I/R injury. Basic research suggests that myocardial cell death, i.e., necrosis and apoptosis, are key events in I/R injury. Our group previously found that complement and ROS were involved in different stages of necrosis development during the early phase of I/R injury in a murine myocardial model. In particular, SOD1 overexpression significantly reduced necrosis at 1 hour of reperfusion, while catalase overexpression reduced necrosis at 3 hours’ reperfusion. The inhibition of I/R injury by SOD1 and catalase was transient, and I/R injury appeared to be restored at 24 hours’ reperfusion. Meanwhile, complement C3 deposition became significant at 3 hours’ reperfusion and reached the peak at 24 hours of reperfusion. Therefore, we hypothesized that SOD1 and catalase are important in regulation of ROS mediated I/R injury during the first 3 hours of reperfusion, while complement may contribute to I/R injury thereafter. This thesis investigated how complement C3 regulates cell death during I/R injury after 3 hours of reperfusion. In Aim 1 of this study using the I/R mouse model, we found that after 1 hour ischemia/ 24 hours reperfusion, the level of necrosis in C3-/- mice was significantly lower than that in WT mice, while the level of apoptosis in C3-/- mice was significantly higher than that in WT mice. Furthermore, we found that 4 weeks after the initial 1 hour of ischemia, C3-/- mice had significantly less cardiac fibrosis and better cardiac function than WT mice. Our comparative proteomics analyses showed that Cyt c, a key factor in the intrinsic apoptotic pathway, was preferentially present in the C3-binding complexes in WT mice after 3 hours of reperfusion. These results indicate that C3 may promote necrosis and inhibit apoptosis in myocardial I/R injury. In Aim 2 of our study, we further explored the mechanism of C3-mediated apoptosis using an in vitro system of AC16 human ventricular cardiomyocyte cell line. We found that although AC16 cells do not express C3, they can uptake exogenous C3 when exposed to it at 37°C. The uptake of C3 was not significant at low temperature(4°C), suggesting a receptor-mediated uptake of C3. However, AC16 cells do not express the known complement receptors 1, 2, 3 and 4. Thus, C3 uptake in AC16cells is likely through unknown receptor(s). Incubation of exogenous C3 could significantly reduce H2O2-induced-apoptosis in AC16cells. In a cell free apoptosis system, we found C3 could significantly reduce the intrinsic pathway apoptosis, possibly through interaction with factor(s)downstream of Cyt c. In a cell free pull-down assay, pro-C3 was able to bind with the apoptotic factor pro-caspase 3. In summary, our results showed that 1)At early stage of I/R injury, complement C3 can promote necrosis and apoptosis. At the late stage of I/R injury, C3 promotes cardiac fibrosis and causes worse cardiac function. 2) Human cardiomyocytes (AC16 cells), which did not expressC3, readily uptake the exogenous C3 from extracellular milieu. The uptake of C3into AC16 cells is likely through a receptor-mediated endocytosis, although the identity of the receptor is still unknown. 3) The inhibition of oxidative-related apoptosis by exogenous C3 in AC16 cells is very likely through the binding with apoptotic factor(s) in the intrinsic apoptosis pathway.
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Determinants for Alzheimer’s Disease: An Exploration of Metabolic and Clinical Risk Factors.Alzheimer’s Disease (AD) is characterized by the pathological deposition of amyloid plaques or tau tangles in the brain. Currently, most therapeutic treatments for AD focus on arresting and reversing amyloid and tau pathologies. To date, unfortunately, clinical trials for drugs targeting amyloid or tau depositions have proven ineffective. Genome-wide and transcriptome-wide association studies have identified various genes and genetic variants in lipid metabolism that are associated with AD. Particularly, dysregulation of sphingomyelin (SM) metabolism has been implicated in AD, leading to the speculation that modulators of the SM pathway could emerge as candidate targets for therapeutic AD treatment. However, the effectiveness of modulators of the SM pathway remains understudied. This study tested whether the SM pathway activator Fingolimod (FTY720), a Sphingosine-1-Phosphate (S1P) signaling modulator, can rescue the behavioral and functional deficits associated with the pathology observed in mouse models of AD. We examined two mouse models, one expressing the amyloid pathology (APP/PS1) and the other the tau pathology (Tg4510), which are known to exhibit severe cognitive and synaptic plasticity impairments. We found that after two months of exposure to Fingolimod in drinking water, the mice showed recovered cognitive function measured in novel object recognition and Barnes maze memory tasks and rescued expression of synaptic plasticity measured in long-term synaptic potentiation in the entorhinal cortex and hippocampal synapses. Our findings suggest that upregulation of the SM pathway via modulation of S1P signaling may have protective effects in AD.
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The role of hepatic phospholipid transfer protein in lipoprotein metabolism.Atherosclerosis is the leading cause of death in western countries. Lipoprotein metabolism is closely related with the disease. ApoB-containing lipoproteins (BLp) are atherogenic particles, while, high density lipoproteins (HDL) are anti-atherogenic. Phospholipids Transfer Protein (PLTP) activity has important impacts on BLp and HDL homeostasis. Animal studies, including general knock out (KO) and transgenic approaches; have shown pro-atherogenic properties for PLTP. However, the tissue specific function of PLTP remains mostly unknown. The liver is one of the critical organs for lipoprotein metabolism and is also a major source for PLTP expression. To address the impact of liver-specific PLTP on lipoprotein metabolism, we created two mouse models which either express PLTP specifically in the liver on a PLTP-null background or lack PLTP in the liver (liver-specific PLTP KO) in a wild type background. In both models, our findings show that the liver has a 25% contribution into total plasma PLTP activity and liver-specific PLTP is highly involved in BLp and HDL production by hepatocytes. We found that hepatic ablation of PLTP leads to a significant decrease in plasma PLTP activity, HDL-cholesterol, HDL-phospholipids, non-HDL-cholesterol, non-HDL-phospholipids, apoA-I, and apoB levels. In addition, nuclear magnetic resonance (NMR) examination of lipoproteins showed that hepatic PLTP deficiency decreases HDL and apoB-containing particle numbers, as well as the VLDL particle size which was confirmed by electron microscopy. However, liver-specific PLTP expression results in a remarkable increase in plasma levels of BLp, as well as a slight increase in HDL levels. In order to unravel the mechanism, we evaluated the apoB and triglyceride production after blocking the clearance of the particles. We found that liver-specific PLTP increases apoB (both apo48 and apoB100) and triglyceride (TG) secretion. To investigate the role of liver PLTP in HDL production, we cultured primary hepatocytes from our knockout and wild type mice and tested their ability to form nascent HDL particles in the presence and absence of exogenous PLTP. We found that PLTP activity, as opposed to heat inactivated PLTP, promotes nascent HDL production through ABCA1-mediated pathway since ablation of ABCA1 tremendously diminished HDL production and the presence of active PLTP had no effect. We also showed that PLTP closely interact with ABCA1 in order to enhance HDL formation. Taken together, our results indicate that liver-generated PLTP promotes both BLp and HDL production by hepatocytes. In addition, the liver makes a 25% contribution to the total plasma PLTP activity, and thus ablation of liver PLTP may influence lipoprotein metabolism in the plasma.
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Regional Variations in Microstructure and Biomechanical Properties of the Human Vertebral Endplate and Trabecular Bone.Vertebral compression fractures represent one of the most common injuries resulting from osteoporosis with an incidence rate of 700,000 per year in the United States. Spinal osteoporosis affects the anterior part of the vertebrae, leading to its collapse and kyphosis. However, there is limited data on the regional differences in the microstructure of the vertebral endplate and cancellous bone and their relationships with degenerative disc diseases and vertebral fractures. Does bone resorption occur at a higher rate in the anterior than in the lateral region of the vertebra? We conducted shear testing experiments on isolated cylindrical bone cores of thoracic vertebrae. Our data showed a 5-fold increase in mechanical strength from the anterior to the lateral regions of both endplate and cancellous bone (p=0.04). Material and ash density measurements correlated positively with shear strength. We further studied the regional variation in biomechanical properties inside human and bovine thoracic vertebrae using surgical anterior self-tapping cancellous screws. Our results showed a trend for increased insertion torque, pullout strength, and stiffness from divergent (laterally oriented) to convergent (centrally oriented) screws. Radiological image analyses from Faxitron X-ray, CT scan, and microCT confirmed that density was higher in the central than in the lateral regions of the cancellous bone. However, we observed the opposite relationship at the cervical and lumbar levels where the divergent screw configuration was mechanically stronger than the convergent one. According to our histological analyses, the density of microvessels at the vertebral endplate decreased from central to lateral regions by 15%. The ratio bone volume over total volume (BV/TV) increased by 31% from superior to inferior bony endplates. There was a decrease in the ratio osteoid matrix surface/bone surface (OS/BS) by 20% from superior to inferior bony endplates. We also observed that older individuals with increased degenerative disc diseases (DDD) had lower cancellous bone density, but higher bone remodeling activity than younger ones. The biomechanical properties of both endplate and cancellous bone vary considerably within the human spinal column. The lateral region of the vertebral body (EP and cancellous) is denser and stronger at the cervical and lumbar levels. The opposite relationship is observed in the thoracic segment. Newer implants could prioritize a lateral or central loading accordingly; thus, reducing the likelihood of screw loosening and the subsidence of disc replacement devices. Moreover, the superior endplate is weaker than the inferior, but more porous, and probably more important for the diffusion of nutrients to the nucleus pulposus. Therefore, the likelihood of disc degeneration might increase with superior endplate injuries.
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Inhibition of Connective Tissue Growth Factor prevents increase in extracellular matrix molecules in a rodent model of Diabetic Retinopathy.Connective tissue growth factor (CTGF) is a profibrotic factor that induces extracellular matrix (ECM) production and angiogenesis, two processes involved in diabetic retinopathy (DR). In this study, we examined whether insulin therapy or a CTGF specific siRNA administered to diabetic rats decreased the levels of CTGF and of selected putative downstream genes in retina. Rats with streptozotocin-induced diabetes were used for these studies. Animals received either no treatment or were administered constant insulin therapy for 12 weeks. mRNA and protein levels of CTGF and select ECM genes were determined using Real-Time PCR and western blotting of retina. Localization of CTGF, GFAP (Glial Fibrillary Acidic Protein), a marker of astrocytes and activated Müller cells, and Vascular Endothelial Growth Factor, (VEGF), a well studied growth factor in the pathologies of DR, were visualized in the retina using immunohistochemistry. CTGF mRNA and protein significantly increased in retina of diabetic rats at 8 and 12 weeks of hyperglycemia. There was also an increase CTGF, GFAP and VEGF immunostaining during hyperglycemia. Double label immunohistochemistry indicated that retinal Müller cells of diabetic rats expressed CTGF. Hyperglycemia also upregulated mRNA levels of fibronectin, laminin ß1, collagen IVa3 and VEGF, and this increase was prevented by insulin therapy. Treatment of diabetic rats with CTGF siRNA decreased laminin ß1, collagen IVa3 mRNAs and CTGF mRNA and protein but did not affect fibronectin or VEGF mRNA levels. Since insulin therapy is associated with oscillations in blood glucose levels, we sought to develop a supplementary gene therapy to inhibit CTGF in DR. Prior to its use in vivo, we sought to determine whether a gene therapy tool could inhibit CTGF in a rat cell culture model that expresses CTGF. First, we tested the efficiency of an adenovirus encoding for a CTGF antisense oligonucleotide and three CTGF siRNA's in vitro to decrease the levels of Transforming Growth Factor-β (TGF-β)-induced CTGF expression in Rat 2 fibroblasts. The adenovirus and siRNA both decreased CTGF in Rat 2 fibroblasts. To circumvent any possible immune responses caused by the adenovirus we chose to use the siRNA for our in vivo studies to inhibit CTGF in the diabetic retina. A subset of diabetic rats received a single intravitreal injection of CTGF siRNA in one eye and a scrambled, control, siRNA in the contralateral eye. Retinas were examined three and ten days after the injection. CTGF siRNA significantly inhibited CTGF mRNA and protein 3 days after injection. CTGF siRNA also induced a significant decrease in laminin β1, collagen IV α3 and GFAP mRNA but had no effect on fibronectin mRNA levels. The level of CTGF mRNA was similar in retina of control and CTGF siRNA injected animals 10 days after injection In conclusion, these studies indicate that CTGF and ECM genes are regulated with insulin therapy. Importantly, CTGF siRNA regulates changes in ECM molecules and reduced GFAP expression suggesting that the treatment decreased the level of glial cell activation and reactivity in DR. Taken together, our results indicate that CTGF is an important target for the treatment of DR.
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Interaction of eukaryotic initiation factors eIF1A and eIF4G with the ribosome and assembly of ribosomal initiation complexes on Picornavirus IRESs.Ribosomes serve as the platform for decoding the genetic information carried in mRNA and translating it into polypeptides; they are a major target for natural and synthetic antibiotics. Although fundamental to gene expression, many aspects of translation in eukaryotes are still not well understood, particularly translation initiation. The goal of my thesis was to yield insights of the interplay of mRNA, tRNA, cellular factors with the ribosome that are critical for the initiation of polypeptide synthesis. Specifically, my work focused on the interactions of translation initiation factors eIF1A and eIF4G with the 40S ribosomal subunit and the interactions of picornavirus RNAs with cellular factors that help them to bypass the regulation of canonical translation initiation. Initiation is the key rate-limiting stage in the eukaryotic translation process and more than 12 eukaryotic initiation factors (eIFs) are involved in this step. Most eukaryotic cellular mRNAs utilize the cap-dependent initiation pathway, in which the ribosomal preinitiation complex first binds to the ‘cap’ structure of the mRNA and then scans to select the AUG start codon to initiate the translation process. eIF4G and eIF1A are two factors that are required for steps in the canonical translation initiation process and they have distinct functions: eIF4G promotes attachment of mRNA to the ribosome and facilitates scanning by ribosomal complexes on the mRNA. eIF1A is involved in tRNA recruitment, scanning, start codon selection and subunit joining. Instead of eIF4F binding to the 5’-end of capped mRNA, some viral RNAs contain an internal ribosomal entry site (IRES) in their 5’UTR; this structure allows the viral RNA to recruit the 40S ribosomal subunit to an internal site on the mRNA independently of the cap and of the 5’-end of the mRNA. Four types of IRESs that have been identified in viral mRNAs can bypass one or all steps of the cap-dependent initiation pathway. Type 1 IRESs are found in picornaviruses such as poliovirus; type 2 IRESs are located in the 5’ UTRs of picornaviruses of the Cardiovirus and Aphthovirus genera, such as Encephalomyocarditis virus (EMCV) and Foot-and-mouth disease virus (FMDV). These two IRESs bind directly to eIF4G and eIF4A subunits of eIF4F, don’t need eIF4E, and can bypass the scanning step in initiation. Although they do share other steps with and use many of the same canonical factors as the cap-dependent initiation pathway, these IRESs also need IRES trans-acting factors (ITAFs) to fulfill their function. Different IRESs in the same family have different requirements for the ITAFs. The reason for this, and the mechanism by which ITAFs promote IRES function have both been obscure. To answer these questions, the specific aims of my thesis are: (1) To locate the position of eIF4G on the 40S ribosomal subunit; (2) To characterize the mechanism of action of eIF1A and to determine its location on the 40S ribosomal subunit; (3) To characterize aspects of the mechanism of translation initiation on Type 1 and Type 2 picornavirus IRESs. By using the technique of directed hydroxyl radical cleavage, we determined the positions of eIF4G/eIF4A on the ribosome, near the exit (E) site of the mRNA-binding channel, suggesting that these subunits of eIF4F are situated at the trailing edge of the ribosome and that eIF4A may unwind secondary structure in the cap-proximal region of the 5’UTR to promote “landing” on them by ribosomal preinitiation complexes. We positioned all domains of eIF1A on the 40S subunit and correlated these positions with the functions of eIF1A in promoting recruitment of initiator tRNA to the ribosomal peptidyl (P) site, preventing premature binding of elongator tRNA to the aminoacyl (A) site, coordinating sequential adoption by the 40S subunit of the ‘open’ conformation (to permit binding of mRNA) and the ‘closed’ conformation (on initiation codon recognition), and in contributing to the stability of ribosomal complexes during scanning. We determined that Type 2 IRESs bound to eIF4G adopt a compact side-by-side orientation, and that ITAFs promote this orientation. We also observed that eIF4G binds with similar orientations to domain V of Type 1 IRESs and to the J-K domain of Type 2 IRESs, suggesting that this factor plays a similar mechanistic role in initiation on Type 1 and Type 2 IRESs. Together, these results have yielded insights into how canonical initiation factors interact with the ribosome to promote the recruitment of mRNA, how they promote ribosomal scanning and how IRESs interact with ITAFs, a subset of initiation factors and ribosomal complexes to bypass early steps in the canonical eukaryotic initiation pathway.
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Identification of Functional Domains in Sphingomyelin Synthase (SMS) and Further Investigation into the Role of Phospholipid Transfer Protein (PLTP) in Lipid Metabolism and AtherosclerosisLipid metabolism is closely related to the development of atherosclerosis. The work presented in this thesis involves two genes which are important for lipid metabolism and atherosclerosis: sphingomyelin synthase (SMS) and phospholipid transfer protein (PLTP). Sphingomyelin synthase (SMS) utilizes ceramide and phosphatidylcholine as substrates to produce sphingomyelin (SM) and diacylglycerol, thereby regulating important lipid messengers. There are two isoforms of the enzyme, SMS1 and SMS2. It has been speculated that the active site of both SMS1 and SMS2, containing two evolutionary conserved histidines and one aspartic acid, is identical to that of lipid phosphate phosphatase (LPP). We systematically mutated these amino acids by site-directed mutagenesis and found that each point mutation abolished SMS activity without altering cellular distribution. Despite sharing 77% sequence homology to each other, SMS1 is located in the trans-Golgi while SMS2 is located in the plasma membrane as well as the Golgi. This prompted us to explore what targeting signals exist in SMS1 and SMS2. Conventional motifs responsible for protein targeting to the plasma membrane or Golgi are either not present in, or unique to, SMS1 and SMS2. Therefore, we determined the targeting signals in SMS1 and SMS2 through a combination of creating truncations of non-homologous amino acids from SMS1 and SMS2 and creating SMS chimeras by swapping non-homologous amino acid sequences between SMS1 and SMS2. We found that SMS1 and SMS2 transit through the classical secretory pathway. Furthermore, SMS1 contains a C-terminal Golgi targeting signal and SMS2 contains a C-terminal plasma membrane targeting domain. (PLTP) is an ubiquitously expressed, secreted protein which functions in the blood and in tissues. A high saturated fat diet induces free cholesterol and phospholipid accumulation in the plasma of phospholipid transfer protein (Pltp) deficient mice. We examined the atherogenic consequence of this phenomenon and investigated the possible mechanism(s). To study atherosclerosis, we bred Pltp KO mice with Apoe KO mice, a mouse model of atherosclerosis. Pltp KO/Apoe KO mice, fed a coconut oil-enriched high-fat diet (COD) for 7 weeks, had higher plasma free cholesterol (149%), phospholipids (15%), and sphingomyelin (54%) than Apoe KO controls. In contrast to chow-fed animals, COD-fed Pltp KO/Apoe KO mice had the same atherosclerotic lesion size as that in Apoe KO mice. Similar to Pltp KO mice, plasma from COD fed Pltp KO/Apoe KO mice contained VLDL/LDL sized lamellar particles. Bile measurement indicated that COD-fed PltpKO mice have 33% less hepatic cholesterol output than controls. In conclusion, COD-fed Pltp deficient mice are no longer protected from atherosclerosis and have impaired biliary lipid secretion associated with free cholesterol and phospholipid accumulation.
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Mechanism of Action of Novel Anti-Cancer Peptide, PNC-27, that Selectively Kills Cancer Cells.In vivo and in vitro studies have shown the efficiency of anti-cancer peptides PNC-27 and PNC-28 in killing cancer cells by necrosis without affecting normal cells (1, 2 and 5). These peptides were derived from the hdm2-binding domain of p53 (PNC-27: aa12-26 and PNC-28: aa17-26) and linked to a membrane residency peptide (MRP). Here I provide morphological and biochemical evidence of the effect of PNC-27 on a wide range of cancer cells including primary cancer cells, and the absence of any effect on untransformed cells. I also demonstrate that PNC-27-induced cytotoxicity is time, dose and temperature dependent. Moreover, I demonstrate the role of HDM2 as a possible tumor marker and a target for PNC-27 in the tumor cell’s plasma membrane which is confirmed by: immunofluorescence, exogenous expression of HDM2 in normal cell plasma membrane, competition experiments and immunoblot of isolated plasma membrane. I also evaluate sequence of cellular events following the exposure of tumor cells to PNC-27 using live cell imaging spinning disc confocal microscopy. Furthermore, using Immuno-gold TEM and SEM I show that gold-conjugated antibodies to HDM2 and PNC-27 decorated ring-like pore structures in the plasma membrane of tumor cells exposed to PNC-27, further confirming the co-localization of HDM2 and PNC-27 in tumor cell membrane. These results strongly suggest that the highly amphipatic PNC-27 inserts itself into the plasma membrane of only cancer cells upon binding to HDM2, where, by random movement, the HDM2-PNC-27 complexes assemble into oligomers and eventually into membrane pores. Influx of free PNC-27 molecules through the newly formed membrane pores into the cell’s cytoplasm leads to the assembly of pores in mitochondrial membranes and rapid cell death.
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Regulation of Na,K-ATPase α1 and β1 subunit gene expression by low external K in neonatal rat cardiac myocytesNa,K-ATPase (the Na,K-pump) is an intrinsic plasma membrane enzyme that plays a critical role in many fundamental processes, such as resting membrane potentials, cell volume control and basal energy utilization. Na,K-ATPase consists of two subunits ? and ?. The ? subunit is responsible for the catalytic and transport properties, whereas the ? subunit is crucial for the correct insertion of the ? subunit in the plasma membrane. Regulation of Na,K-ATPase expression is of vital importance for basic cellular functions and reduced Na,K-ATPase function is associated with several diseases such as essential hypertension, congestive heart failure and diabetes. Decreased extracellular K is a well-established model for the study of the regulation of Na,K-ATPase expression. Low external K initially inhibits Na,K-ATPase activity due to substrate limitation; however, prolonged inhibition up-regulates ?1 and ?1 subunit gene transcription and Na,K-ATPase activity. In this study, we investigate the signal transduction pathway for up-regulation of Na,K-ATPase ?1 and ?1 subunit gene transcription by low external K using transient transfection with luciferase plasmids containing the 5’ flanking region of ?1 and ?1 subunit genes. Our results showed that the low K stimulatory effect on Na,K-ATPase ?1 and ?1 subunit gene transcription is dependent on intracellular Ca and calcineurin. The augmentation of ?1 subunit gene transcription by low K is also dependent on ERK1/2 MAPK, PKA and histone deacetylase, whereas the up-regulation of ?1 subunit gene transcription by low K is dependent on PKC, p38 and JNK MAPK. These results suggest that overlapping and distinct signal transduction pathways are involved in the low K stimulatory effect on Na,K-ATPase ?1 and ?1 subunit gene transcription. We also demonstrated that the -102 to -61 base pair region is required for the low K stimulatory effect on Na,K-ATPase ?1 subunit gene transcription. Site-directed mutation studies showed that the ATF/CRE site at -70 to -63 and the adjacent GC box at -57 to -48 in the proximal promoter region of Na,K-ATPase ?1 subunit gene are both essential for the low K response. Moreover, utilizing electrophoretic mobility shift assays, we demonstrated that transcription factors Sp1, Sp3, CREB/ATF-1 and phosphorylated CREB/phosphorylated ATF-1 bind to the low K response region and binding to the GC box and the ATF/CRE site were enhanced in response to low K. Finally, by using Western Blotting we showed that exposure of cardiac myocytes to low K increased nuclear content of Sp1, phosphorylated Sp1, Sp3 and CREB. Based on the above results, we conclude that low K-mediated up-regulation of Na,K-ATPase ?1 subunit gene transcription is dependent, in part, on ATF/CRE- and GC box-binding transcription factors.
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Structure-Function Analysis of Microsomal Triglyceride Transfer Protein using Human Mutations.Microsomal triglyceride transfer protein (MTP) is an evolutionarily conserved endoplasmic reticulum (ER) resident protein that is required for apolipoprotein B (apoB)-containing lipoprotein assembly and secretion. MTP physically interacts with apolipoprotein B and transfers lipids (triglycerides, phospholipids, choelsteryl esters, sphingomyelin, and ceramide) to nascent apoB. MTP is a heterodimer composed of the catalytically active M subunit and protein disulfide isomerase (PDI). The M subunit is divided into 3 domains: N-terminal -barrel, middle -helical, and C-terminal. Computational, biochemical, and mutagenesis studies of missense mutations in all three domains of the M subunit identified critical amino acids for MTP function and lent insight into MTP’s structure-function. Loss of function mutations in MTP lead to Abetalipoproteinemia (ABL), which is characterized by the absence of plasma apoB-containing lipoproteins. We identified three ABL patients, who each had a novel homozygous missense mutation in MTP: R46G, D169V, or D361Y. To determine if any of these missense mutations were detrimental to MTP’s function, we created these mutations via site-directed mutagenesis, expressed them in Cos-7 cells, and investigated their ability to support apoB secretion. R46G, a polymorphism, had no effect on MTP’s ability to support apoB secretion or transfer triglycerides, suggesting that it is not the basis for the ABL phenotype in this patient. D169V and D361Y, on the other hand, were both located in the ER and able to bind apoB17, but were not able to bind PDI, transfer lipids, or support apoB secretion, suggesting that they were the cause of the ABL phenotype. D361E, which has a similar helix forming potential and charge as D361, is able to restore PDI binding, lipid transfer, and apoB secretion. Therefore, we postulate that D361Y may disrupt the tightly packed middle -helical domain, and consequently alter the PDI binding site in this domain. Computational modeling of D169 revealed that it may be part of a crucial internal salt bridge in the N-terminal -barrel. Computational modeling suggested that D169 could form an internal salt bridge with K187 and K189. Mutagenesis of these lysines to leucines abolished PDI heterodimerization, lipid transfer, and apoB secretion without affecting apoB binding or subcellular location. Further, mutants with preserved charges (D169E, K187R, K189R) rescued these activities, suggesting that these three residues form a salt bridge and a PDI binding site in the N-terminal region. This binding site is independent of the apoB binding site, suggesting that the N-terminal domain is actually divided into two microdomains. Next, we investigated whether MTP missense mutations (H297Q, D384A, G661A) reported in a previously identified patient with hypobetalipoproteinemia (HBL), which is characterized by plasma apoB in the bottom 5th percentile. We hypothesized that missense mutations from this patient would modulate, but not abolish, MTP’s ability to support apoB secretion. We created these mutants individually and in combination and investigated their ability to transfer lipids and support apoB secretion. However, none of these mutations affected MTP’s ability to support apoB secretion or transfer triglycerides, suggesting that these mutants function normally and may be polymorphisms. Therefore, the HBL in this patient may be caused by confounding factors. In summary, these studies characterized 12 missense mutations in MTP and identified 4 critical amino acids for MTP’s function. D361Y destroys the PDI binding site in the middle -helical region. The other three residues, D169, K187, and K189 form a salt bridge in the N-terminal domain, which stabilizes the PDI binding site in this region. They were also the first detrimental missense mutations in the Nterminal domain, demonstrating that this domain is also important for MTP activity. Further mutagenesis studies may help refine the tertiary structure of MTP.
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Role of laminins containing beta2 and gamma3 chains in regulating retinal neurogenesis and morphogenesis.Establishment and maintenance of cell polarity plays a critical role in the development, organization, and function of tissues. Disruptions of cell polarity and tissue organization lead to pathophysiology in many organ systems including epithelial gland cancers. A key element in establishing cell polarity and the maintenance of tissue organization is the attachment to laminin-rich basement membrane that underlies all epithelial derived tissues. Arising from the ectoderm, the CNS shares an epithelial lineage and indeed laminins containing the b2 and g3 chains have been isolated from both the brain and retina. In fact, laminin b2 chain is a major constituent of the basement membrane of the retina – the inner limiting membrane (ILM). In this study, we have specifically addressed questions to understand the contribution of laminins containing the b2 and g3 chains in retinal development. Specifically, we have investigated how laminins regulate proliferation and differentiation of the retinal progenitor cell (RPC), their involvement in the process of neuronal maturation and in regulation of structural and functional properties of Müller cells (MC), glial cells that are critical for retinal homeostasis. Therefore, experiments in this study were focused on three cell types that are adherent to the ILM – the RPCs, the MC (terminal progeny of RPC) and retinal ganglion cells (RGCs), the only output neuron of the retina. Deletion of laminin b2 and g3 chains results in a specific disruption of the ILM that leads to the loss of adhesion of the RPCs to the ILM, thereby disrupting the establishment of polarity in RPCs. The loss of polarity in the RPCs lead to changes in the rate of proliferation and affects photoreceptor production. Our data also suggests that altered proliferation due to laminin deletion is mediated by a decrease in Notch effectors. These studies provide novel insights into the role of laminin b2 and g3 chains in regulating proliferation of RPCs and additionally in suppressing the pool of photoreceptors in vivo. Further, loss of polarity in the MCs causes cytoskeleton changes and disruption in the spatial orientation of the water and ions channels, resulting in MC dysfunction leading to pathophysiological response in the retina. Finally, we analyzed the effects of laminin deletion on RGC development showed that RGC spacing, and dendritic elongations are disrupted along with more minor perturbations in intra-retinal axonal guidance. Moreover, our in vivo data that laminin in the ILM promote RGC survival. Taken together, these studies suggest a critical role of laminins in retinal development and the maintenance of retinal structural integrity. The insights gained from these studies will contribute to the development of rationally designed biomimetic niches that can be used in ocular tissue regeneration. Furthermore, the insights from the MC studies demonstrate the importance of MC adhesion to the ILM in maintenance of retinal architecture and will contribute to our understanding of retinal pathologies such as retinal detachment, proliferative vitreoretinopathy and ILM stripping. The insights gained from the role of laminin in RGC development may well be important in understanding CNS congenital dysgenesis as well as being relevant to CNS pathologies including glaucoma, neurodegeneration and the ischemia.
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ENVolution: Immunoselection of Novel HIV Envelope Immunogens for HIV Vaccine Development.A major challenge for human immunodeficiency virus (HIV) vaccine development is the design of HIV envelope (Env) immunogens that elicit broadly neutralizing antibodies (BnAbs). Most of the characterized BnAbs against HIV-1 block the infectivity of a wide spectrum of isolates by targeting conserved regions located on the functional Env trimer such as the CD4-binding site. Vesicular stomatitis virus (VSV), the prototypic member of the Rhabdoviridae family of negative-strand RNA viruses, is a promising vaccine delivery vector because it can infect human cells, but is not a natural human pathogen, has low seroprevalence in most human populations, propagates robustly in culture, induces strong immune responses in vivo and tolerates foreign gene insertion including HIV Env. As with most RNA viruses, the VSV RNA-dependent RNA polymerase lacks proof-reading function and the genome randomly incorporates mutations during replication. Viral variants accumulate during propagation, and evolution of the viral population can be directed by applying selective pressure. For example, recombinant VSV (rVSV) vectors encoding HIV-1 Env in place of its natural attachment and entry protein, VSV G, are under selective pressure to maintain the foreign gene and express functional trimers that specifically infect CD4/CCR5(+) cells. We hypothesized that the application of BnAb-specific selective pressure during serial passage of rVSV-HIV Env vectors could be exploited to generate novel functional Env trimer immunogens. To explore the use of VSV’s evolutionary potential to generate novel HIV-1 Env immunogens, rVSV vectors expressing clade B and clade C Env were constructed and methods to apply selective pressure were developed. First, as proof of concept for the ability of rVSV to rapidly adapt in the presence of a BnAb-directed selective pressure, rVSV viruses expressing HIV-1 Env proteins in place of VSV G were passaged on CD4/CCR5(+) cells in the presence of neutralizing concentrations of b12, a BnAb targeting an epitope that overlaps with the CD4 binding site (CD4s). Four b12-escape variants containing substitutions in the CD4bs of HIV-1 Env were identified: P369L, P369L_V372A, G366D and G366S_T278A. All four variants exhibited decreased binding to b12 and retained CD4-dependent entry despite the presence of substitutions of CD4 contact residues on the Env protein. Only one of the four variants, G366D, was completely resistant to b12 neutralization. The P369L_V372A and G366D variants were 2-fold more resistant to neutralization by the CD4bs-directed BnAb PGV04, but only the G366D variant showed decreased binding to PGV04. Surprisingly, all four variants exhibited an increase in both binding and neutralization by a glycan-specific BnAb, PGT126, which targets a quaternary epitope located on the V3 loop of gp120. Most importantly, these studies for the first time showed the neutralization phenotype of viruses expressing HIV-1 Env with substitutions at residue G366, a residue that is 99% conserved among all circulating HIV-1 isolates and is critical for HIV-1 viability. Second, a positive selection methodology coupled with serial passage was developed to generate novel Env immunogens that potentially have higher binding affinities for BnAbs. The BnAb b12 was used to select for viral variants that maintain binding to b12 under stringent conditions. Two Env variants, N280S and Q652R, were isolated and contained substitutions of residues that are 99% conserved among circulating HIV-1 isolates. The N280S variant contained a substitution in gp120 that is a contact point for b12 and CD4. The Q652R variant contained a substitution in gp41. Both variants retained CD4-dependent entry. b12 binding and neutralization to the N280S variant was unaltered compared to WT. While b12 binding to the Q652R variant was unaltered, this variant was slightly resistant to neutralization by b12. Although both N280S and Q652R variants were more resistant to neutralization by the CD4bs-directed BnAbs VRC01 and PGV04, only variant N280S demonstrated decreased binding to VRC01 and PGV04. Importantly, these studies showed for the first time the neutralization phenotype of viruses expressing HIV-1 Env substitutions at residue N280, a residue that is reported to be critical for HIV-1 viability. These studies show that novel HIV-1 Env proteins can be rapidly and specifically generated by directing the evolution of VSV-HIV EnvG vectors via the application of BnAb-specific selective pressure. By employing this biological system with a panel of BnAbs, the strategies we describe here can complement standard approaches to identify BnAb epitopes, to determine structural conformations required for epitope exposure and provide an improved understanding of BnAb binding and neutralization properties that may lead to the design of improved immunogens. Ultimately, we envision that this system may be used to generate novel Env immunogens as promising candidates for HIV vaccine trials and can be broadly applied to generate novel proteins as promising candidates for other vaccine immunogens or as novel therapeutical agents against disease.
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PKMζ Regulation of Na/K ATPase: A Potential Endogenous Neuro-protective Mechanism of Ischemic Preconditioning.In ischemic preconditioning, a sublethal ischemic insult protects neurons from subsequent ischemia. We studied ischemic preconditioning in organotypic hippocampal slice cultures, in which a preconditioning 5-minute hypoxia-hypoglycemia treatment protected against a 10-minute experimental ischemic (EI) treatment of hypoxia-hypoglycemia. Whereas the preconditioning treatment protected against EI given 24 hours later, it did not prevent neuronal loss when EI given 2 hours later. This model was used to identify two regulators of ischemic preconditioning; the atypical PKC isoform PKMζ and the Na/K ATPase. Two hours following preconditioning, when there was no neuroprotection, Na/K ATPase activity was unchanged from basal level. In contrast, Na/K ATPase activity as measured by 86Rb uptake was significantly increased 24 hours after the preconditioning treatment. Elevated Na/K ATPase activity 24 hours following preconditioning was accompanied by increased surface expression of the Na/K ATPase α 1 and α 2 isoforms. Similarly, levels of active PKM ζ were increased at 24 hours, but not 2 hours, after preconditioning. To examine the possibility of PKMζ regulation of Na/K ATPase, pharmacological occlusion experiments were performed, using marinobufagenin to inhibit α1, dihydroouabain to inhibit α2/3 and a ζ-pseudosubstrate peptide to inhibit PKMζ. These experiments showed that PKMζ regulated both the activity and surface expression of the α1 isoform of the Na/K ATPase. Finally, we used marinobufagenin, dihydroouabain and ζ-pseudosubstrate peptide to determine if PKMζ or the α1 and α2 Na/K ATPase isoforms protected neurons. All three compounds blocked neuroprotection following ischemic preconditioning. These data indicate key roles of PKMζ and Na/K ATPase in neuroprotection following ischemic preconditioning.
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PKMζ Maintains Postsynaptic Structures in Dissociated Hippocampal Neurons: A Mechanism for Memory Maintenance.Protein kinase Mζ (PKMζ), the brain-specific, constitutively active PKC atypical isoform, has been shown to maintain long-term potentiation (LTP) and long-memory storage. Inhibition of PKMζ activity by the selective ζ-inhibitory peptide (ZIP) disrupts LTP maintenance. Similarly, infusion of ZIP into the brain appears to permanently erase established memory without preventing future learning. These findings indicate that PKMζ plays a pivotal role in memory storage, and PKMζ activity is required for maintenance of memory. To explore the underlying mechanisms of PKMζ in synaptic plasticity, I investigated its effect on spine morphology, thought to be involved in memory storage. It has been previously established that LTP can induce increases in dendritic spine size, which corresponds proportionally to the strength of the synapse. In addition, larger spines are more stable. Immunolabeling showed that PKMζ protein increased with forskolin, an agent known to induce “chemical LTP”. PKMζ inhibition with 10 μM ZIP or 10 μM chelerythrine, another PKMζ-selective inhibitor, reversed the chemical LTP induced increase in spine size. Overexpression of PKMζ increased dendritic spine size. PKMζ inhibition also decreased spine size of control neurons, thereby making PKMζ both necessary and sufficient for maintenance of spine size. Immunostaining for postsynaptic density-95 (PSD-95), the major component of the postsynaptic density, decreased with PKMζ inhibition, and PKMζ overexpression increased, PSD-95 clustering. Similarly, inhibition of PKMζ significantly reduced postsynaptic alpha-amino-3-hydroxy-5-methylisoxazole-4-propionate receptor (AMPARs) subunit GluR2, the main postsynaptic receptors mediating fast excitatory transmission. Furthermore, overexpression of PKMζ increased postsynaptic GluR2 receptors. Blocking palmitoylation reverses PKMζ-mediated PSD-95 aggregation and synaptic GluR2 localization. Therefore, PKMζ plays a role in maintaining postsynaptic structures in dendritic spines.
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Structural and Biochemical Characterization of the p27-cyclin D-cdk4 complex.The cell cycle is controlled by the cyclin-cdks, which phosphorylate substrates required for progression. These kinases are regulated in part by their interaction with the stoichiometric inhibitor p27Kip1. We demonstrated for the first time that p27 could exist as a cyclin D-cdk4 bound-inhibitor and a bound, non-inhibitor, dependent on the growth state of the cell. In asynchronously growing cells (A), p27’s interaction with cyclin D-cdk4 was non-inhibitory, while in contact arrested cells (G0) p27 bound and inhibited cyclin D-cdk4 activity. The p27-cyclin D-cdk4 complex was assembled under both A and G0 conditions, as shown by immunoprecipitation-immunoblot analysis. However, the outcome of p27’s association with cyclin D-cdk4 was different. p27 levels in contact arrested cells, suggested that an increase in the level of p27 might lead to the inhibition of the cyclin D-cdk4 complex in G0 cells. Using a tetracycline repressible p27 induction system (Tetp27), we showed that overexpression of p27 did not inhibit cyclin D-cdk4 complexes, suggesting that p27’s ability to inhibit or alternatively not inhibit was not concentration dependent. We demonstrated p27’s ability to switch from a cyclin D-cdk4 bound inhibitor to a bound non-inhibitor was due to the absence or presence of tyrosine phosphorylation in the 3-10 helix of p27, respectively. We demonstrated that the association of non-tyrosine phosphorylated p27 with cyclin D-cdk4 prevented the required, activating phosphorylation of cdk4’s T-loop by cyclin H-cdk7 (CAK). Tyrosine phosphorylation of p27, which occurs preferentially in proliferating cells, permitted cdk7’s phosphorylation of cdk4. In vitro, the tyrosine kinase Abl was capable of phosphorylating pre-formed p27-cyclin D-cdk4 complexes, permitting both cdk7 phosphorylation of the T-loop and direct access to the cdk4 active site. Our data suggested that activation of cdk4 in vivo may occur in a temporal manner with p27 becoming phosphorylated by a tyrosine kinase in order for cyclin H-cdk7 to gain access to the T-loop. We additionally studied the p27-cyclin D-cdk4 interaction with a series of p27 peptide fragments: Tet p27 (25-106) and Tet p27 (25-73). In vitro, it was previously shown that peptide fragment p27 (22-73) bound without inhibiting cdk4, but bound and inhibited cdk2 activity. In vivo, peptide fragment p27 (25-73) continued to associate with cdk4 in a non-inhibitory manner but now was unable to bind to cdk2. This demonstrated that the interaction of p27 with cdk4 and cdk2 was different both in vivo and in vitro. Our findings help to elucidate the interaction domains of p27 with cyclin D-cdk4.
