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.

The primary somatosensory (S1) and primary motor cortex (M1) have roles in the learning of non-declarative memories. Previous studies suggest that this learning is subserved by long-term potentiation (LTP), which is in turn maintained by the persistently active kinase, PKMζ. Whereas the role of PKMζ in animal models of declarative knowledge is established, its involvement in the short and long term maintenance of procedural knowledge is not well understood. Here we show that injection of PKMζ pseudosubstrate inhibitor ZIP into the adult rat sensorimotor cortex disrupts both recently and developmentally acquired sensorimotor memories (> 1 year post-acquisition). Our data further suggests that these effects are due to a simultaneous disruption of sensory map borders and a net decrease in stimulus response amplitudes. Based on these findings we propose a model of permanent, ongoing maintenance of sensory map borders by LTPenabled center-surround inhibition. We present this model as a non-conflicting addendum to existing models which suggest a more temporary role of LTP in sensorimotor memory maintenance. Finally, our results represent the first demonstration that procedural, sensorimotor memories share the same underlying molecular mechanism for persistence as declarative memories.

Immune responses that are caused by seizures or that can contribute to epileptogenesis are poorly understood. Lymphocyte immune responses in brain after a single seizure are unknown. We developed a new murine model system to analyze early brain immune responses after a single generalized tonic clonic seizure. C57bl/6 mice (males, 6-8 wk old) received either sham (electrodes placed subcutaneously over frontal lobe but no current passed) or electrical stimulus (electrodes placed subcutaneously over frontal lobe, 15-30 mA, 100 Hz, 0.4ms burst duration, 1 sec train duration) that resulted in submaximal (no hind-limb extension) or maximal seizures (hind-limb extension). Pretreatment of mice with pentobarbital (i.p., 25mg/kg) or inhaled ethyl-chloride (for 20s) prevented convulsions. Mice were killed at 1-24 hr and 7 days, brains pooled, and single-cell suspensions prepared, after which cells were separated according to density (Percoll gradients) and cell numbers determined (flow cytometry). In some experiments, brain was dissected into neocortex, hippocampus, midbrain, and cerebellum before assay. This is the first report that a single maximal seizure induced by frontal electric stimulus results in increased lymphocyte numbers in brain. This effect was not obtained when similar frontal electric stimulus resulted in submaximal seizures, or when mice were pretreated with pentobarbital or ethyl-chloride. The increases in lymphocyte numbers after maximal seizure were predominantly in neocortex (CD4+ and CD8+ T and B cells) and in hippocampus (T cells). The T and B cell subsets also expressed high levels of the activation markers/adhesion receptors CD11a, CD29, and CD44, which are required for lymphocyte migration into brain. After seizure, these markers were upregulated on peripheral T and B cells before T and B cell numbers increased in brain, suggesting that the increases in these cells in brain are due to their entry from the periphery. CD4+ and CD8+ T cells in brain intracytoplasmically expressed IL-2, a cytokine that is a potent stimulator of lymphocyte proliferation and enhances the killing activity of cytotoxic T cells. CD4+ T cells also expressed IL-4, the cytokine required for IgE isotype switching by B cells. B cells in brain was IgG+ and IgE+, setting the stage for immune responses, and even allergic response in brain. CD8+ T cells expressed IFN-g and TNF-a, cytokines that activate microglia and brain macrophages. Taken together, the results indicate that there are early immune responses in brain after a single seizure, which may play a role in allergy/autoimmunity, synaptic remodeling, and in the development of epilepsy.

Multiple myeloma (MM), a bone marrow (BM) neoplasm of B-lymphocytes arrested in their maturation at the pre-plasma cell stage of differentiation, is the second most common hematologic malignancy in the U.S. Despite prolonged median survival with anti-myeloma strategies aimed at the tumor and its BM microenvironment, MM remains invariably fatal. Endothelial progenitor cells (EPCs) are CD133+/KDR+ cells that originate in the BM and play a key role in providing tumor neoangiogenesis, growth and MM progression. Using X-chromosome inactivation and RT-PCR analyses, our lab previously found EPCs from MM patients to be clonally restricted and genetically similar to tumor cells in approximately 60% of patients. Based on genetic similarity between EPCs and tumor cells in MM that we and other laboratories have demonstrated, my thesis explored the hypothesis that the clonal EPC population contains CD133+ MM precursor cells capable of self-renewal, and differentiation into a tumorigenic population. Results of my experiments strongly suggest that at least a subset of CD133+ cells qualify as MM progenitor cells evidenced by: (1) engraftment and differentiation of BM derived EPCs from MM patients in vivo in NOD/SCID mice; (2) in vitro and in vivo studies showing the differentiation of CD133+ cells from patients with MM into cells with a multiple myeloma cell phenotype (CD138/38+cells); (3) analysis of gene expression microarray studies showing upregulation of genes involved in stem cell function. Furthermore, I show the in vivo anti-myeloma effects of targeting Hsp70 whose gene expression is upregulated in MM. Taken together, my studies investigated MM pathogenesis through the in vivo characterization of progenitor cells as well as through genomic analyses, and targeting of a dysregulated pathway within EPCs and tumor cells which may be clinically effective.

Claustrum is a region of grey matter between the striatum and cerebral cortex that is among the most well-connected structures in the brain. It is hypothesized to function as a high-level coordinator of brain-wide activities like the integration of senses, attention, sleep, and consciousness. The exact anatomical boundaries of claustrum have been controversial and claustral subregions have not been well-defined. This may be in part due to its compact structure in rodents and other commonly studied species. In contrast, Seba’s short-tailed fruit (Carollia perspicillata) bat has a remarkably large claustrum, lending itself as a model and magnified view for investigating claustrum. We studied the distributions of the claustral marker latexin and the calcium-binding proteins calbindin, calretinin, and parvalbumin in claustrum. Using these markers, we defined clear claustral boundaries and several distinct subregions. The calcium-binding proteins (markers of different inhibitory neurons subtypes) were differentially distributed among subregions, suggesting that these regions are under the control of different inhibitory systems. In addition to having a large claustrum, Carollia is a relatively long-lived species, lending itself as a model for the neurobiology of aging and neurodegeneration. Two brain regions highly affected in the aging process are retrosplenial cortex (Brodmann areas 29 and 30) and hippocampus. In the course of this work, we found latexin was present in retrosplenial cortex, a region involved in memory and navigation, but only in Brodmann areas 29a and 29b. This distinct division of retrosplenial cortex differs from cytoarchitecturally-defined divisions but aligns with connectivity evidence that supports the separate grouping of areas 29a and 29b from areas 29c and 30. Finally, we found, several features of Carollia hippocampus including a compacted CA3 cell layer and a prosubiculum that are also present in primate but not rodent hippocampus. Due to these unique neuroanatomical features, Carollia may offer advantages in studying claustrum and other limbic cortical structures, especially in the context of aging, that are not present in more commonly studied model species.

Brain machine interfaces (BMIs) aim to restore lost motor functions by channeling movement-related brain signals to end-effectors skipping compromized parts of the central nervous system (CNS.) As there is increasing number of patients who can potentialy benefit from such help, there is a very urgent demand for optimizing and controlling the outputs of such devices. These BMI devices are currently is far from performing the every day and seemingly unintelligent acts of reaching, grasping, object manipulation, force-adaptation and motor learning. This is because these acts call on the CNS to make complex computations on its visual, proprioceptive and cutaneous inputs which artificial limbs lack, the main reason why even the best BMIs still give unstable movements. Efforts are now underway to create artificial proprioceptive and cutaneous inputs to BMIs. However, due to lack of thorough understanding of its effects on the brain and BMI movements, the presently accepted candidate for such a feedback channel, Microstimulation (MiSt) of the primary somatosensory cortex (S1), is not yet fully functional. Numerous critical questions have yet to be addressed, such as which S1 area and layer to target, what stimulus parameters to use, if unwanted side-effects, such as brain lesions, movement etc. should be major concerns, and many more. Moreover, we asked, why not even microstimulate other brain areas? Currently, there is little information on MiSt for somatosensation in other brain areas, such as the thalamus. This thesis aimed at: 1) Finding the discernable differences and similarities between the two inputs, natural and artificial (MiSt in S1 cortex and the VPL thalamus); 2) Finding the best region, cortex or VPL, where MiSt gives the most natural-looking response in S1 cortex; 3) Studying the dynamics of causal information transfer in thalamocortical networks in natural and MiSt for somatosensory neuroprosthesis to investigate possible behavioral consequences. MiSt was applied to S1 cortex and VPL thalamus of a monkey (multiple implants) and rats implanted with multielectrode and multiwire arrays respectively. The resulting spike and Local Field Potential (LFP) cortical and thalamic activities were recorded and analyzed per the goals of the thesis. The main findings indicate that cortical responses in both input conditions are comparable and that VPL MiSt may be a better candidate for somatosensory neuroprosthesis mainly because it causes reduced or no cortical inhibition. The results show that the different stimulation conditions have specific effects on the causal interaction in and between the two brain regions.

The neurophysiology of the somatosensory system has been under investigation for decades. The coding properties of neural populations and single units in cortical and subcortical regions have been characterized extensively using electrophysiology. Tactile stimulation produces evoked responses in the thalamus and cortical regions dedicated to processing of sensory information. The ability to evoke naturalistic neural responses as well as tactile perceptual experiences by means of microstimulation in the absence of real stimulus presented to the periphery is imperative for the development of a sensory neuroprosthesis. Three specific aims were pursued in the present study: 1. The development and validation of a novel assessment technique to quantify sensory evoked responses. 2. The investigation of effects of ketamine anesthesia on the cortical evoked responses. 3. The assessment of the feasibility of microstimulation of Ventroposterior Lateral (VPL) nucleus of the thalamus as a way to produce naturalistic responses in the primary somatosensory cortex. Brief tactile stimulation of various hand locations produces characteristic responses in both thalamic and cortical multi-neuron populations. These responses were used to create somatotopic maps of the hand regions in awake animals with chronically implanted electrode arrays in the SI cortex and in anesthetized animals during acute preparations. In the first Aim, method based on the analysis of correlation was developed to compare the firing recorded on individual electrodes during different stimuli presentations to determine which neural populations showed most activation during an evoked response. The procedure required relatively short duration of stimulation sessions. The effects of anesthesia on neural coding were studied in the second Aim using ketamine anesthetized monkeys. Ketamine anesthesia is easily induced, relatively short lasting, and generally safe. In awake chronically implanted monkeys properties of neural responses to tactile stimulation were assessed. Receptive fields (RF) corresponding to five digits were identified using recordings from an electrode array placed in area 1 of SI. Cortical stimulation fields (SF) representing signal flow from each receptive field were identified. Neural responses under ketamine anesthesia were reassessed using the same mapping techniques. The responses recorded during the maintenance phase of anesthesia were similar to responses evoked under awake condition. Most changes in the neural responses occurred during induction and recovery phases. Overall, ketamine produced responses of shorter duration compared to the awake condition. In the final Aim, isofluorane/fentanyl anesthetized monkeys had microelectrode arrays acutely implanted in the VPL thalamus and area1, 3b, and 2 of SI. Recordings were used to estimate the sizes of SFs in the thalamus and cortex. Thalamic neural representations were associated with RFs that typically spanned several digits, while cortical recordings were associated with smaller RFs. Microstimulation of the VPL (1ms single pulses, 50A) resulted in the activation of somatotopically appropriate cortical neural ensembles. The responses in the VPL and cortex were characterized by the presence of High Frequency Oscillations (HFO) (up to 800Hz). The presence of the HFOs was observed following both tactile and electrical stimulation. In summary, techniques for analyzing evoked multiunit responses were developed and used to explore cortical and thalamic responses to tactile stimulation, the effects of ketamine anesthesia on such responses, and parameters for thalamic stimulation to simulate tactile “sensation” in cortex.

Co-application of the convulsant 4-aminopyridine (4-AP) and the GABAB receptor antagonist CGP 55845 to adult guinea pig hippocampal slices elicits giant GABA-mediated postsynaptic potentials (GPSPs) and interictal-like epileptiform discharges. In the first part of the thesis, I tested the effects of the group I metabotropic glutamate receptor (mGluR) subtype-selective antagonists on this synchronous activity. Electrophysiological field recordings were performed in the CA3 region of hippocampal slices from adult guinea pigs. The mGlu5 receptor antagonists MPEP and MTEP increased GPSP rate, but the mGlu1 receptor antagonist LY 367385 did not. Simultaneous block of both subtypes of group I mGluR did not decrease pre-existing epileptiform activity and also did not prevent the emergence of epileptiform activity. Thus, in the 4-AP/CGP 55845 model, enough glutamate was released to activate group I mGluRs and affect GPSP rate via mGlu5 receptors; however, this group I mGluR activation was not required for the generation of the epileptiform activity. Application of 4-AP (100 μM) in a solution containing reduced divalent cation concentrations (0.6mM Mg2+ and 1.2 mM Ca2+) to hippocampal-entorhinal-perirhinal slices of adult rat brain induced ictal-like epileptiform activity in entorhinal and perirhinal cortices as revealed by electrophysiological field potential recordings. The most exciting finding of the second part of my thesis was that epileptiform activity, including ictal-like activity, persisted after washing out the 4-AP. This persistence indicated that a change had occurred in the slice so that it was now “epileptic” in the absence of the convulsant 4-AP. The maintenance of the persistent ictal-like epileptiform activity required ionotropic glutamate-mediated synaptic transmission but not group I mGluR activation. The presence of hippocampus was not required for the induction of persistent ictal-like events in entorhinal cortex. The induction of persistent ictal-like epileptiform activity was dependent upon both NMDA and AMPA/kainate ionotropic glutamate receptor activation but not upon mGluR activation. Application of the protein synthesis inhibitor cycloheximide did not prevent the induction of persistent ictal-like activity. In conclusion, I have demonstrated both interictal-like and ictal-like activity that is not dependent upon group I mGluR activation and have characterized a new in vitro model of epileptogenesis that is clearly distinct from the group I metabotropic glutamate-dependent model.

The microsomal triglyceride transfer protein (MTP) transfers lipids (triacylglycerols, cholesteryl esters, and phospholipids) in vitro and is required for the secretion of apoB-lipoproteins in vivo. Inhibitors of MTP lipid transfer activity significantly increase the degradation of apoB and reduce its secretion. Therefore, transfer of lipids to apoB by MTP is believed to be essential for the assembly of apoB-lipoproteins. In the following studies we explored the structures and activities of evolutionarily distinct MTP homologs in order to better understand how MTP might be assisting apoB-lipoprotein assembly. We describe specific and sensitive in vitro assays to measure MTP cholesteryl ester and phospholipid transfer activities using fluorescent lipids. These activities are dependent on MTP concentration and demonstrate saturation kinetics. In contrast to the human MTP that transfers all lipids, we observed that the Drosophila MTP is deficient in the transfer of triacylglycerols and cholesteryl esters but does transfer phospholipids. Similarly the zebrafish MTP, like the human MTP, transfers triacylglycerols while the C. elegans MTP lacks this activity. Despite differences in their abilities to transfer lipids, all MTP orthologs assisted in the assembly and secretion of human apoB48-lipoproteins. Furthermore, the amounts of apoB secreted with the assistance of different MTP proteins were augmented by increasing triacylglycerol synthesis (the addition of oleic acid or expression of acyl-CoA:monoacylglycerol acyltransferase and acyl-CoA:diacylglycerol acyltransferase proteins) and decreased by treating the cells with triacsin C, an inhibitor of triacylglycerol synthesis. We subsequently showed that the secondary and tertiary structures in the orthologs were highly conserved while their primary amino acid sequences were less conserved. Interestingly the bC domains as well as helices 4-6 in the a-helical domains demonstrated greater conservation in vertebrate MTPs than the other structural domains. Together these data reveal that the phospholipid transfer activity is the most ancient activity associated with MTP and is sufficient to generate an apoB-lipoprotein particle. Triacylglycerol transfer activity was acquired during evolution and is specific to vertebrate MTP proteins. This activity is associated with greater conservation in the bC domain and helices 4-6 in the a-helical domain. Therefore, we propose that the amino acid content contained in the bC domain as well as helices 4-6 might be critical for the robust triacylglycerol transfer activity in vertebrates.

To prevent sexual transmission of HIV, a successful vaccine must elicit protective immune responses and durable immunologic memory that can respond rapidly upon virus exposure at mucosal sites. Thus, replication competent vesicular stomatitis virus (VSV) vectors are being investigated for HIV vaccine delivery with particular focus on the effect of delivery route on immune responses. VSV is a practical vector candidate because it naturally enters through mucosal sites, infects multiple cell types, and is highly immunogenic. Moreover, VSV typically does not cause disease in humans and pre-existing immunity is rare. Attenuated, replication-competent VSV vectors have been designed to co-express HIV Envelope (Env) trimers (from HIV-1 subtype B, strain JRFL) and the natural VSV attachment protein (G) derived from the Indiana (VSV-Env-GIN) or New Jersey serotype (VSV-Env-GNJ). To optimize Env trimer expression, several insert designs have been tested utilizing VSV or CD5 (human T-cell surface glycoprotein) signal sequences and different combinations of Env and VSV-G transmembrane (TM) and intracellular domains. Improved Env expression and vector genetic stability was observed when the CD5 signal and VSV-G TM sequences were used. Infected Vero cells were used to analyze cell surface expression and the antibody-binding profile of the Env trimer immunogen. Infected cells that expressed VSV G on their surface also expressed Env which was recognized by Env-specific broadly neutralizing antibodies b12, 2F5, 2G12, PGV04 and PGT126. Furthermore, the cells produced Env in a conformation that bound the antibody PGT145, which preferentially binds conformational determinants on trimeric Env. These results demonstrate that VSV can express membrane-bound trimeric Env in a conformation containing epitopes recognized by known broadly neutralizing antibodies. To investigate cellular and humoral responses elicited by vectors expressing HIV Env trimers, a heterologous prime-boost regimen was conducted in Balb/c mice with plasmid DNA prime and rVSV boost. DNA plasmids expressing EnvG were administered by intramuscular (IM) injection and electroporation (EP) with plasmid expressing IL-12 (pIL-12) at week 0 and 3 followed by a VSV-Env-GIN infection at week 6. The effect of administering the VSV boost by different routes also was investigated. A second study based on VSV-Env-GIN prime/VSV-Env-GNJ boost with both vectors administered by intranasal (IN) or IM route in different combinations administered at weeks 0 and 3 was used to study the immunogenicity of the two vectors without a DNA prime. Immune responses were analyzed by flow cytometry, ELISA, and competition binding assay at various times after the immunizations. Both studies elicited strong Env-specific antibody responses in the serum. pDNA+pIL-12 prime/ rVSV boost elicited Env-specific CD4+ T cells in the spleen and lung for the IN route. In the same pDNA+pIL-12 prime/ rVSV boost study, Env-specific CD8+ T cells were detected in the lungs for the DNA and IN groups. Notably, the IN route was able to elicit b6 and PGV04-like antibodies. These findings are of great interest as they seem to underline the importance of the quality of the elicited antibody response, which cannot be readily ascertained by measurement of the quantity of Env-specific antibodies. The titer of Env-specific antibodies has been used as a widespread measurement of vaccine efficacy in the HIV vaccine field. The results from the current studies point to the need for the characterization of these antibody responses and perhaps the quality of the antibody response should be used as the standard measurement of vaccine efficacy. The viral prime and viral boost study also elicited high Env-specific antibody responses in the serum but had a different cell-mediated response profile in that both CD4+ and CD8+ T cells were detected in the lungs but none in the spleen. The IM-IN route was able to elicit b6-like antibodies. The outcome of these studies highlights that VSV vectors expressing EnvG can elicit Env-specific humoral and cell-mediated immune responses in two different vaccine regimens. Even more importantly, these VSV-EnvG vectors were able to elicit antibodies similar to known broadly neutralizing antibodies. These findings give tremendous support to the use of EnvG as an immunogen in a live-replicating vector platform for future HIV vaccines.