Ujendra Kumar

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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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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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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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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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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