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Multiple myeloma (MM) is a hematological malignancy characterized by the neoplastic proliferation of the plasma cells. MM is a relatively rare cancer that accounts for about 1.8% of all cancers and is the second most common hematologic malignancies, and despite the advancement from untreatable to treatable malignancy, it is yet incurable. Calcium ions (Ca2+) play an important role as second messengers in regulating a plethora of physiological and pathological processes, hence cytoplasmic Ca2+ is tightly regulated with strict spatial and temporal control to initiate, maintain, and terminate appropriate signaling pathways and phenotypes including cellular proliferation, cell cycle control, migration, gene expression, muscle contraction, metabolism, and cell death. Multiple Ca2+ pumps and channels exist in the cell to tightly regulate cytoplasmic Ca2+ levels. The plasma membrane Ca2+ transport ATPase (PMCA), and the Na+/Ca2+ exchanger (NCX) are present on the plasma membrane, the mitochondrial Ca2+ uniporter (MCU) are located on the mitochondrial membrane, and the sarcoplasmic reticulum/endoplasmic reticulum Ca2+-ATPase (SERCA) pump, the 1,4,5-triphosphate receptor (IP3R) and ryanodine receptor (RyR) channels are expressed on SE/ER membranes, all these channels work seamlessly to regulate and redistribute Ca2+ levels among various cellular compartments. The major Ca2+ regulatory pathway in non-excitable cells is the Store-Operated Ca2+ Entry Pathway (SOCE) and one its major contributors is TRPC1. TRPC1 is a member of the TRP protein superfamily and a potential modulator of store-operated Ca2+ entry (SOCE) pathways. While TRPC1 is ubiquitously expressed in most tissues, its dysregulated activity may contribute to the hallmarks of various types of cancers, including breast cancer, pancreatic cancer, glioblastoma multiforme, lung cancer, hepatic cancer, multiple myeloma and thyroid cancer. Our group has developed a novel cyclic peptide referred to as MTI-101 for the treatment of MM. MTI-101 is derived from a 10 D-amino acids peptide HYD1, which was discovered to block cancer cell adhesion to extracellular matrix. It was previously reported that acquired resistance to HYD-1 correlated with repression of genes involved in store operated Ca2+ entry (SOCE): PLCβ, SERCA, ITPR3, and TRPC1 expression. We sought to determine the role of TRPC1 heteromers in mediating MTI-101 induced cationic flux. Our data indicate that consistent with activation of TRPC heteromers, MTI-101 treatment induced Ca2+ and Na+ influx. However, replacing extracellular Na+ with NMDG did not reduce MTI-101-induced cell death. In contrast, decreasing extracellular Ca2+ reduced both MTI-101-induced Ca2+ influx as well as cell death. The causative role of TRPC heteromers was established by suppressing STIM1, TRPC1, TRPC4 or TRPC5 function both pharmacologically and by siRNA, resulting in a reduction in MTI-101-induced Ca2+ influx. Mechanistically MTI-101 treatment induces trafficking of TRPC1 to the membrane and co-immunoprecipitation studies indicate that MTI-101 treatment induces a TRPC1-STIM1 complex. Moreover, treatment with calpeptin inhibited MTI-101-induced Ca2+ influx and cell death indicating a role of calpain in the mechanism of MTI-101-induced cytotoxicity. Finally, components of the SOCE pathway were found to be poor prognostic indicators among MM patients, suggesting that this pathway is attractive for the treatment of MM.

The vertebrate retina supports two distinct functions: image-forming vision, which is concerned with the detection of spatial patterns and objects in visual scenes, and non-image-forming vision, which detects the overall intensity of ambient illumination. Non-image-forming vision influences mood, sleep and body temperature, entrains circadian rhythms to the solar day, and drives light-evoked behaviors. The primary sensory neurons that enable non-image-forming vision are a population of ganglion cells in the retina that express the photopigment melanopsin and are therefore called intrinsically photosensitive retinal ganglion cells (ipRGCs). IpRGCs come in multiple subtypes, distinguished by differences in their morphology, circuit connectivity, cellular physiology, and projection targets in the brain. Analogous to other RGCs, adult ipRGCs receive synaptic inputs from the retinal circuits that convey inputs from rod and cone photoreceptors, but are also uniquely capable of signaling light intensity to the brain in the absence of rod and cone inputs, such as in disease states that cause rod and cone degeneration or disruption of retinal circuits. Interestingly, ipRGCs signal light intensity during early development, prior to the developmental maturation of the synapses that relay information from rods and cones to RGCs .The focus of this dissertation is to understand how the cellular specializations and circuit connectivity of ipRGC subtypes contribute to the encoding of light intensity in the developing retina and drive a specific non-image-forming function, an innate light avoidance behavior exhibited by newborn mammals. In Chapter 1, I summarize the current understanding of the neural implementation of non-image-forming-vision, with an emphasis on developing mammals. In Chapter 2, I use calcium imaging, an unsupervised clustering analysis, and genetic and pharmacological manipulations of gap junction coupling in the retina to investigate how the neonatal retina encodes light stimuli and whether cell-intrinsic properties or circuit connectivity determine the functional output of ipRGCs. I find that populations of ipRGCs and other retinal neurons in the developing mouse retina encode light stimuli as six functional groups that are mixtures of ipRGC subtypes and other retinal neurons. I also demonstrate that ipRGCs are anatomically and functionally gap junction coupled to one another and to other retinal neurons in a circuit arrangement that likely leads to functional mixing of subtypes and allows modulation of gap junction coupling to dramatically regulate the population light response. In Chapter 3, I investigate the neural basis of light avoidance behavior in neonatal mice using an automated assay of light avoidance behavior coupled with genetic and pharmacological manipulations of ipRGC phototransduction and gap junction circuitry. I find that a specific ipRGC subtype is necessary and sufficient for the behavior. Together, these results reveal the relative contributions of cell-intrinsic properties and gap junction circuitry of ipRGC subtypes in the developing retina to the encoding of light intensity and to light-evoked behavior.

Increased production of amyloid beta (A-beta) peptide, via altered proteolytic cleavage of amyloid protein precursor (APP), and abnormalities in neuronal calcium homeostasis play central roles in the pathogenesis of Alzheimer's disease (AD). Notch1, a membrane receptor that controls cell fate decisions during development of the nervous system, has been linked to AD because it is a substrate for the gamma-secretase protein complex in which mutations cause early-onset inherited AD. Numb is an evolutionarily conserved endocytic adapter involved in the internalization of transmembrane receptors. Mammals produce four Numb isoforms that differ in two functional domains, a phosphotyrosine-binding domain (PTB) and a proline-rich region (PRR). Recent studies showed that the PTB domain of Numb interacts with the cytoplasmic tails of APP and Notch but the functional relevance of these interactions with respect to AD pathogenesis is not clear. In the current studies, we proposed to investigate the biological consequences of the interaction of the Numb proteins with APP and Notch in neural cells stably overexpressing each of the four human Numb proteins. In the first part of our studies, we found that expression of the Numb isoforms lacking the insert in the PTB (SPTB-Numb) caused the abnormal accumulation of cellular APP in the early endosomes, and increased the levels of C-terminal APP fragments and A-beta. By contrast, expression of the Numb isoforms with the insert in PTB (LPTB-Numb) leads to the depletion of cellular APP and coincides with significantly lower production of APP derivatives and A-beta. The contrasting effects of the Numb isoforms on APP metabolism were not attributed to differences in the expression of APP nor the activities of the various APP-processing secretases. In the second part of our studies, we found that expression of SPTB-Numb protein enhances neuronal vulnerability to serum deprivation-induced cell death by a mechanism involving the dysregulation of cellular calcium homeostasis. Neural cells expressing SPTB-Numb exhibited enhanced Notch activity, which markedly upregulated the expression of transient receptor potential canonical 6 (TRPC6) channels enhancing calcium entry in response to store depletion. We also found that serum deprivation increased TRPC6 expression, mediating the serum deprivation-induced death in neural cells. Interestingly, expression of LPTB-Numb protein suppressed serum deprivation-induced activation of Notch and the subsequent upregulation of TRPC6 and cell death. Finally, we showed that the Numb proteins differentially impact Notch activation by altering the endocytic trafficking and processing of Notch. Taken together, these studies demonstrate that aberrant expression of the Numb proteins may influence APP metabolism and Notch-mediated cellular responses to injury by altering their endocytic trafficking and processing.