Ujendra Kumar


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Graduate Student Supervision

Doctoral Student Supervision

Dissertations completed in 2010 or later are listed below. Please note that there is a 6-12 month delay to add the latest dissertations.

Crosstalk between somatostatin receptor subtypes and cannabinoid receptor 1 in excitotoxicity (2018)

Both somatostatin (SST) and cannabinoid receptor 1 (CB1R) are critical components modulating neurotransmission in the central nervous system (CNS), with sharing functional properties in various neurological activities. To explore their potential crosstalk, first we investigated the expression of SST and CB1R in rat brain hypothalamus and hippocampus. The distributional patterns and colocalization of CB1R and SST were selective and region specific. Neuronal population expressing either SST or CB1R alone, as well as colocalization were seen in various intensities in different regions, suggesting a possible interaction.SST exerts its biological effects via binding to somatostatin receptors (SSTRs). Both SSTRs and CB1R belong to G-protein coupled receptor family that are known to function as oligomers. Accordingly, we investigated the colocalization of CB1R and SSTR5 in rat brain and HEK-293 cells cotransfected with hCB1R and hSSTR5. Our results showed that CB1R and SSTR5 colocalized in rat brain regions. In cotransfected HEK-293 cells, SSTR5 and CB1R existed in a constitutive heteromeric complex under basal condition. Agonist treatments lead to the disruption of CB1R/SSTR5 heterodimer, along with preferential formation of SSTR5 homodimer and dissociation of CB1R homodimer. cAMP and ERK1/2 signaling was modulated in a SSTR5-dominant manner in co-transfected cells. To explore pathological significance of such interaction, we further expanded our study in HD transgenic mice and Huntingtin (Htt) knock-in striatal neuronal cells. We observed significant loss of neuronal subpopulation displaying colocalization between SSTRs and CB1R with selective sparing of SSTR positive neurons in cortex and hippocampus but not in striatum of 11-week-old R6/2 mice, in comparison to wild-type and 7-week-old R6/2 mice. Using mHtt knock-in (STHdhQ¹¹¹/¹¹¹) and wild-type (STHdhQ⁷/⁷) striatal cells, we discovered that STHdhQ¹¹¹/¹¹¹ cells were more vulnerable to QUIN and displayed suppressed cell survival signalings. Receptor-specific agonist protected cells against QUIN-induced toxicity and selectively activated ERK1/2 in both STHdh cells. Co-activation of SSTR subtypes and CB1R resulted in diminished protective effects, delayed ERK1/2 phosphorylation and altered receptor complex composition, with more pronounced effects in STHdhQ¹¹¹/¹¹¹ cells than STHdhQ⁷/⁷ cells. Taken together, our results provide evidence for functional interaction between SSTR and CB1R, emphasizing its therapeutic potentials in excitotoxicity and associated neurological disorders.

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The neuroprotective role of somatostatin against beta amyloid induced toxicity in in vitro models of Alzheimer's disease (2018)

Alzheimer’s disease (AD) is a chronic neurodegenerative disease affecting more than 60 million people worldwide. This debilitating disease harbors toxic environment to the brain causing neuronal cell death and causes general impairment of the cognitive function. In our laboratory, we have been studying the effect of somatostatin (SST) in serving neuroprotective role against various disease models including hyperinflammation, Huntington’s disease and AD. In the present study, we aim to study the mechanisms involved in SST mediated neuroprotection against beta amyloid induced toxicity in blood brain barrier and in neurons. In AD, the impaired clearance of β-amyloid peptide (Aβ) due to disrupted tight junction and transporter proteins is the prominent cause of disease progression. We demonstrate that SST prevents Aβ induced blood brain barrier permeability by regulating low density lipoprotein receptor-related protein and receptor for advanced glycation end products expression and improving the disrupted tight junction proteins. Furthermore, SST abrogates Aβ induced c-JUN NH2-terminal kinase phosphorylation and expression of matrix metalloproteinase. Next, as the neurites are often the initial point of damage upon accumulation of Aβ, we examined the role of SST in all-trans retinoic acid (RA) induced progression of neurite outgrowth in SH-SY5Y cells. We also determined the morphological changes in prominent intracellular markers of neurite growth including microtubule-associated protein 2, Tuj1 and Tau. Here, we present evidence that SST is a molecular determinant in regulating the transition of SH-SY5Y cells from non-neuronal entity to neuronal phenotype in response to RA. Lastly, to elucidate the mechanism involved in SST mediated protection against Aβ-induced toxicity in neurons, phosphorylation level of collapsing response mediator 2 (CRMP2), a well-established regulator of neurite homeostasis hyperphosphorylated in AD was monitored. We demonstrate that SST effectively inhibits the hyperphosphorylation of CRMP2 as Ser522, which plays a critical role in priming the phosphorylation of subsequent sites. Furthermore, we identified the underlying mechanism involved in the regulation of CRMP2 phosphorylation by monitoring the SST mediated regulation of calcium influx.Taken together, results presented here suggest that SST might serve as a therapeutic intervention in AD via targeting multiple pathways responsible for neurotoxicity, impaired BBB function and disease progression.

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Functional elucidation of human somatostatin receptor-3: Implications in breast tumor biology (2013)

Somatostatin (SST) inhibits cell proliferation through five SST receptors(SSTR1-5). Amongst all SSTR subtypes, SSTR2 and SSTR3 are the prominentreceptor subtypes which exert antiproliferative effects in cells of different origin.SSTR2-mediated inhibition of cell proliferation is largely cytostatic, whereas SSTR3is cytotoxic. Whether SSTR2/SSTR3 display synergistic antiproliferation than singlereceptor is not well understood. To ascertain the role of SSTR3, the present studywas first conducted in HEK-293 cells which lack endogenous SSTRs expression.Cells were stably transfected with wt-SSTR3, treated with agonist and studied fordimerization, cAMP, receptor trafficking and signaling molecules. Since receptorsignaling properties are confined in C-tail, cells expressing C-tail deleted SSTR3were also studied for comparative analysis. wt-SSTR3 exists as preformedhomodimer at cell surface and displays agonist-mediated cytotoxic effects. The cellsurface expression, homodimerization and agonist-induced internalization of SSTR3were independent of C-tail, whereas agonist-mediated apoptosis was lost upon Ctaildeletion.Next, HEK-293 cells cotransfected with SSTR2/SSTR3 were examined forheterodimerization and signaling molecules governing cell proliferation. Pb-FRET/CO-IP analyses suggest SSTR2/SSTR3 heterodimerization. The decreasedcAMP upon agonist activation of SSTR2/SSTR3 suggests that this heterodimer isfunctional. Agonist-mediated SSTR2/SSTR3 antiproliferation was Gi-dependent, andinvolved apoptosis and cell-cycle arrest. iiiTo derive direct pathological significance of the observations from heterologous system, additional experiments were conducted in two breast cancer cell lines MCF-7 and MDA-MB-231, which differ in origin and biochemical features including presence or absence of ERα. Breast tumor cell lines overexpressing SSTR3 were studied for cell proliferation and downstream signaling molecules. EGF served as an index of positive cell proliferation. SSTR3 overexpression in MCF-7 (R3-MCF-7) and MDA-MB-231 (R3-MB-231) cells displayed inhibition of EGF-induced proliferation and enhanced antiproliferative effect of SSTR3-specific agonist in comparison to non-transfected cells. SSTR3 overexpression in R3-MCF-7 cells constitutively enhanced TUNEL staining, PARP-1 and p27Kip1 expression suggesting apoptosis and cell-cycle arrest. Conversely, in R3-MB-231 cells, SSTR3 overexpression exerted cytostatic but not cytotoxic effects.These results provide compelling evidence for antiproliferative role of SSTR3 in breast cancer cell lines. The constitutive activation of cytotoxic signaling in R3-MCF-7 but not R3-MB-231 cells reveals a distinct cell-specific role for SSTR3 in breast tumor biology.

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Characterization of somatostatin receptor in rat/mice brain: implication in Huntington's disease (2012)

Somatostatin (SST) is a multifunctional peptide present in most brain regions as well as inperipheral organs. In QUIN/NMDA-induced excitotoxicity, an experimental model ofHuntington’s disease (HD), SST positive interneuron coexpressing NPY/NADPH-d/bNOSare selectively spared whereas, projection neurons expressing NMDA receptors and DARPP-32 are vulnerable. SST plays neuroprotective role in excitotoxicity, however, which SSTRsubtypes mediate the neuroprotective role is not known. Accordingly, as a first step, wedescribe the colocaliztion of SSTR subtypes with DARPP-32 to determine the percentage ofreceptor subtypes in projection neurons. We further extended our study and compared HDtransgenic mice (R6/2) with SSTR1 and 5 double knock out mice. In both strains wecompared the expression pattern of NMDARs, DARPP-32, SST, bNOS and SSTRs and keydownstream signaling pathways linked to the neuronal loss in HD such as PI3K, ERK1/2PKC-α, synapsin-IIa, enkephalin and calpain. Our data shows that SSTR1/5 double knockout mice mimic the neurochemical changes of HD transgenic mice indicating a keyneuroprotective role of SSTR1 and 5 in HD. To derive direct physiological implications andmechanistic explanations for the role of SSTR subtypes in excitotoxicity we used striatalbrain slices and determined the effect of SSTR1 and 5 agonist, alone or in combination withNMDA on key proteins such as DARPP-32, calpain, PSD-95 and signaling pathwaysassociated with NMDA induced neurotoxicity. Our results here show significant decrease inNMDA currents and dissociation of NMDARs heterodimerization upon treatment withSSTR1 and 5-specific agonist. Our data further demonstrates significant decrease inNMDARs expression and upregulation of SSTR1 and 5 upon agonist treatment. UnlikeNMDA, activation of SSTR1 or 5 in striatal slices induced DARPP-32 phosphorylation atThr34 and Thr75 enhanced CREB phosphorylation and inhibits expression of calpain andPSD-95. The data presented in this thesis provides a new insight for the role of SSTRsubtypes in excitotoxicity with relevance to neurological disorders.

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Role of somatostatin receptors in EGF mediated EGFR signaling in human breast cancer and human embryonic kidney-293 cells (2012)

Epidermal growth factor receptor (ErbB1) and somatostatin receptors (SSTRs) exert opposing effects on tumor promoting signaling pathways. Whether SSTRs functionally interact with ErbB1 and modulate tumor-promoting signaling is currently unknown. For this reason, the specific emphasis of this thesis is to examine the role of SSTRs in ErbB1 mediated signaling in breast cancer cells and human embryonic kidney (HEK) 293 cells. First, I determined the mRNA and protein expression of SSTR1, SSTR5 and ErbB1 in human breast cancer cell lines namely MCF-7 and MDA-MB231. I next demonstrated that SSTR1 or 5 exist as pre-formed heterodimers with ErbB1, which dissociated in an agonist dependent manner. Somatostatin (SST) modulated epidermal growth factor (EGF) mediated MAPK in a time and agonist dependent manner. Furthermore, SST and/or EGF treatment altered the expression of key adapter proteins including Grb2, SOS, Shc, SH-PTP1 and SH-PTP2, which are known to play a role in MAPK activation. Since breast cancer cells endogenously express SSTR and ErbB subtypes, this study was further extended in HEK-293 to gain insight into the effect of individual SSTR on ErbB1 activated signaling. We demonstrated that HEK-293 cells transfected with SSTR1, SSTR5 or SSTR1/5 negatively regulate EGF mediated effects attributed to the inhibition of ErbB1 phosphorylation, MAPKs and PI3K/AKT pathways. Moreover, SST effects were significantly enhanced in cells when ErbB1 was knocked down using small interference ribonucleic acid (siRNA) or treated with selective ErbB1 antagonist (AG1478). The presence of SSTRs, in addition to modulating signaling pathways, led to the dissociation of constitutive and EGF induced heteromeric complex of ErbB1/ErbB2. Most significantly, cells co-transfected with SSTR1/5 display pronounced effects of SST on the signaling and dissociation of the ErbB1/ErbB2 heteromeric complex than cells expressing either SSTR1 or 5 alone. The findings of this study discovered a new mechanism and potential role of SSTRs in attenuation of ErbB1 mediated signaling pathways via dissociation of ErbB1/ErbB2 heteromeric complex. In conclusion, the results presented in this thesis suggest that formulating novel drugs that activate SSTRs along with inhibition of ErbB1 might likely serve as an important therapeutic approach in the treatment of ErbBs positive tumors.

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Master's Student Supervision

Theses completed in 2010 or later are listed below. Please note that there is a 6-12 month delay to add the latest theses.

Role of somatostatin and muscarinic -acetylcholine system in 3NP-induced neurotoxicity in mutant Htt knock-in cultured striatal neuronal cells (2020)

Huntington’s disease (HD), is an inherited neurological disease with severe neuronal loss in the striatum. Previous studies have shown the selective sparing of somatostatin (SST) positive medium-sized aspiny interneurons and large cholinergic interneurons expressing choline acetyltransferase (ChAT) in the striatum. This suggests a crucial role of somatostatinergic and cholinergic neurotransmission in the pathophysiology of HD. The biological effects of SST in the central and peripheral systems are mediated by five different somatostatin receptors (SSTR1 - 5); whereas five metabotropic muscarinic receptors (M1R - M5R) mediate acetylcholine (ACh) functions. Whether SSTR and MR subtypes work in concert in HD is not known. In the present study, using STHdhQ7/7 (wt) cells and STHdhQ111/111 (mutant) cells and 3-Nitro propionic acid (3NP)-induced model of toxicity, first the expression levels of SSTR and MR subtypes were determined using the immunocytochemistry and Western blot analysis, second cell viability study and apoptosis were performed using MTT assay and Hoechst 33258 dye, third we determined the status of downstream signaling pathways including ERK1/2 and Akt upon treatment with SST and Carbachol (Carb) alone and in combination. STHdhQ7/7 and STHdhQ111/111 cells display moderate to strong expression and colocalization between M1R/M4R and SSTR2/4. Both STHdhQ7/7 and STHdhQ111/111 cells display significant changes in internalization and cell surface-expression of M1R/M4R and SSTR2/4 in a receptor- and treatment-specific manner. STHdhQ111/111 cells exhibit a slow proliferation rate than STHdhQ7/7 cells, and exhibit concentration- and time-dependent effect on the cell viability in the presence of 3NP. STHdhQ111/111 cells are more susceptible to 3NP-induced toxicity and apoptosis when compared to STHdhQ7/7 cells. In cell viability assay, agonist-treatment before and after 3NP-toxicity affords better protection than co-treatment. Consistent with receptor expression, cell proliferation and cell viability, time- and concentration-dependent regulation of signal transduction pathways- ERK1/2 and Akt attest the neuroprotective role of Carb and SST against 3NP-induced neurotoxicity. In conclusion, the results presented here shed a new light on the neuroprotective role of Carb and SST, and further enhance our understanding of functional interaction between MRs - SSTRs in an in vitro model of HD.

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Crosstalk and modulation of signaling between somatostatin and growth factor receptors in human breast cancer cell lines (2017)

The molecular mechanisms of breast cancer are poorly understood, which present serious therapeutic problems and complicates drug design. Cell surface receptors belonging to G-protein-coupled receptor (GPCR) and receptor tyrosine kinase (RTK) families are potential drug targets relevant to pathological conditions, and have attracted great interest from pharmaceutical industry. Recent studies have suggested that somatostatin (SST) receptors (SSTR1-5) belonging to GPCR family may interact with human epidermal growth factor (EGF) receptors (ErbB 1-4) from RTK family in pathophysiological conditions, exerting antiproliferative effects that may be useful in the treatment of breast cancers. An understanding of molecular mechanisms responsible for these effects reveal new approaches to the design of efficient breast cancer therapies that would significantly improve the lives of patients. The work presented in this thesis was conducted to investigate crosstalk between SSTR and ErbB proteins in BT-474 and SK-BR-3 breast cancer cell lines upon SST and/or EGF treatment, to clarify the underlying molecular mechanisms, and to explore their implications for cancer therapy. Several pairs of SSTR and ErbB proteins exhibited strong membrane coexpression and crosstalk in the presence of the tested ligands, including SSTR2/ErbB1, SSTR3/ErbB2, and SSTR5/ErbB3 in BT-474 cells, and SSTR5/ErbB1 in SK-BR-3 cells. New crosstalk processes between SSTR and ErbB subtypes were observed in both cell lines. In BT-474 cells, there were substantial reductions in the membrane expression of ErbB1 (degradation and termination of signaling) and ErbB2, as well as moderately reduced expression of ErbB3 and greatly enhanced activation of SSTR1 and SSTR4. Similarly, SK-BR-3 cells exhibited strong reductions in the expression of ErbB1 (degradation), ErbB3, and ErbB2 expression (partial degradation), while enhanced activation of SSTR1 and SSTR2 expression at the cell surface. The activated SSTRs were shown to antagonize ErbB-mediated MAPK signaling and tumor-promoting signaling pathways, resulting in pronounced antiproliferative effects. In BT-474 cells, they inhibited ERK1/2, p38 and PI3K, and enhanced PTEN pathways, while in SK-BR-3 cells they promoted ERK1/2 and p38, inhibited PI3K and maintained PTEN pathways. These results show that the activated SSTRs exert antiproliferative effects in breast cancer cells via mechanisms that resemble those determined for drugs modulating cancer-related signaling pathways.

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The role of NMDA receptors in excitotoxicity (2013)

NMDA receptors are glutamate-gated cation channels named after their prototypical selective agonist NMDA. The channels occur as multiple subtypes, which are formed from interactions between different receptor subunits. NMDA receptor subunits are classified into three families: NR1, NR2A-D, and NR3A, B. NMDA receptors are implicated in HD pathology. During HD, a subset of medium-sized aspiny interneurons in the striatum that co-localize SST, NPY, and the enzyme NOS are selectively spared. In contrast, medium-sized spiny cells that constitute 80 % of all striatal neurons undergo selective neurodegeneration. While it was suggested that the interneurons survive because they lack NMDA receptors, studies including from our lab have shown the presence of NR1 in SST-positive striatal neurons. The finding of NR1 expression and co-localization with SST-positive neurons indicates that NMDA receptor-induced toxicity may be regulated in a receptor-specific manner. Therefore, the present study was conducted to investigate whether NMDA application leads to toxicity that is receptor-specific in HEK293 cells stably transfected with NR1, NR2A, or NR2B. The main findings of this study indicate that NMDA application causes cell death, which varies in intensity and nature, depending upon the NMDA concentration applied, and the receptor-type expressed by the cells. Cells expressing NR1 were found to undergo apoptosis but not necrosis, while cells expressing NR2A/NR2B underwent both apoptosis and necrosis in a receptor-specific manner. In cells expressing NR2A/NR2B, exposure to low concentrations of NMDA resulted in cell death that was predominantly apoptotic. In contrast, exposure to high concentrations of NMDA produced mostly necrosis. In cells expressing NR1, NMDA application caused apoptosis, which exhibited a gradual increase in response to greater concentrations of NMDA. In addition, cell death through apoptosis and/or necrosis was determined to be the greatest at all NMDA concentrations in cells expressing NR2B, followed by those expressing NR2A, and then NR1. Taken together, these results indicate that the activation of receptors formed by NR1, NR2A, or NR2B have different toxic consequences. Thus, the selective neurodegeneration observed during HD may be due to the variation in expression levels of NR1, NR2A, and NR2B between medium-sized aspiny interneurons and medium-sized spiny projection neurons.

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Peripheral mechanisms of masseter muscle nociceptor sensitization by Nerve Growth Factor (NGF) (2010)

No abstract available.


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