Neuromodulatory systems, such as the locus coeruleus (LC) - norepinephrine (NE) system, are integral in the modulation of behavioral state, which in turn exerts a heavy influence on sensory processing, perception, and behavior. LC neurons project diffusely through the forebrain as the sole source of NE. LC tonic firing rate has been shown to correlate with arousal level and behavioral performance. As the LC-NE system innervates sensory pathways and NE has been shown to affect neuronal response, the LC-NE system could potentially allow for state-dependent modulation of sensory processing. However, the precise link between LC activation and sensory processing in the various stages of the sensory pathway that underly perception remained elusive. It is well established that thalamic relay nuclei play an essential role in gating the flow of sensory information to the neocortex, serving to establish cortical representation of sensory environment. Thalamocortical information transmission has been proposed to be strongly modulated by the dynamic interplay between the thalamic relay nuclei and the thalamic reticular nucleus (TRN). Neurons in the early stages of sensory pathways selectively respond to specific features of sensory stimuli. In the rodent vibrissa pathway, thalamocortical neurons in the ventral posteromedial nucleus (VPm) encode kinetic features of whisker movement, allowing stimuli to be encoded by distinctive, temporally precise firing patterns. Therefore, understanding feature selectivity is crucial to understanding sensory processing and perception. However, whether LC activation modulates this feature selectivity, and if it does, the mechanisms through which this modulation occurs, remained largely unknown. This work investigates LC modulation of thalamic feature selectivity through reverse correlation analysis of single-unit recordings from different stages of the rat vibrissa pathway. LC activation increased feature selectivity, drastically improving thalamic information transmission. This improvement was dependent on both local activation of α-adrenergic receptors and modulation of T-type calcium channels in the thalamus and was not due to LC modulation of trigeminothalamic feedforward or corticothalamic feedback inputs. LC activation reduced thalamic bursting, but this change in thalamic firing mode was not the primary cause of the improved information transmission as tonic spikes with LC stimulation carried three-times the information than tonic spikes without LC stimulation. Modelling confirmed NE regulation of intrathalamic circuit dynamics led to the improved information transmission as LC-NE modulation of either relay or reticular nucleus alone cannot account for the improvement. These results suggest a new sub-dimension within the tonic mode in which brain state can optimize thalamic sensory processing through modulation of intrathalamic circuit dynamics. Subsequent computational work was then performed to determine exactly how the encoding of sensory information by thalamic relay neurons was altered to allow for an increase in both information transmission efficiency and rate. The results show that LC-NE induced improvements in feature selectivity are not simply due to an increased signal-to-noise ratio, a shift from bursting to tonic firing, or improvements in reliability or precision. Rather, LC-NE-induced modulation of intrathalamic dynamics changed the temporal response structure thalamic neurons used to encode the same stimuli to a new structure that increased the information carried by both tonic and burst spikes. The shift in events times favors optimal encoding, as more events occur at ideal positions, i.e. when the stimulus most closely matches the neuron’s feature selectivity. Further, this work analyzed the ability to reconstruct the original stimulus using the evoked spike trains of multiple neurons and their recovered feature selectivity from an ideal observer point-of-view. The results showed that LC-activation improved the accuracy of this reconstruction, indicating it may improve the accuracy of perception of whisker stimuli. Finally, to make this work translatable, the use of vagus nerve stimulation (VNS) was investigated as a potential method for minimally invasive enhancement of thalamic sensory processing. The vagus nerve, which runs through the side of the neck, has long been known to have profound effects on brain-state and VNS has been shown to evoke LC firing. This work elucidates the previously uninvestigated short-term effects of VNS on thalamic sensory processing. Similar to direct LC stimulation, VNS enhanced the feature selectivity of thalamic neurons, resulting in a significant increase in the efficiency and rate of stimulus-related information conveyed by thalamic spikes. VNS-induced improvement in thalamic sensory processing also coincided with a decrease in thalamic burst firing, suggesting the same underlying mechanism as the improvements induced with direct LC stimulation.

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The generation of amyloid-β (Aβ) peptides through proteolytic processing of the amyloid precursor protein (APP) is a key pathogenic event in Alzheimer’s disease (AD). Aβ generation begins with APP endocytosis, which is mediated by the endocytic YENPTY sequence located in the cytoplasmic tail of APP. Mints, a family of cytosolic adaptor proteins, directly bind to the YENPTY motif of APP and facilitate APP endocytosis and amyloidogenic processing. In addition, loss of any one of the three Mint proteins decreases Aβ production in aging mouse models of AD, supporting the hypothesis that the APP-Mint interaction may provide a novel therapeutic target to selectively reduce Aβ production in AD. Characterizing the biochemical and cellular dynamics of the APP-Mint interaction is critical for understanding Aβ generation. Thus, we generated Mint1 mutants that bind with high affinity (Mint1Y633A) or low affinity (Mint1Y549A/F610A) to APP. These Mint1 mutants exhibited profound alterations in cellular localization, APP endocytosis, and Aβ production. Therapeutically, we generated a novel cell-permeable APP mimetic peptide (APPMP) that interferes with the APP-Mint interaction. This APPMP was designed to outcompete endogenous APP binding, with a 46-fold improved affinity to Mint. Treatment of primary neurons from an AD mouse model with several cell permeable APPMP variants reduced Aβ production with minimal cellular toxicity, supporting Mints as a promising novel therapeutic target for AD. The PTB domain of Mint1 that mediates APP binding is autoinhibited by an adjacent C-terminal α-helix. However, the molecular mechanisms underlying the relief of Mint1 autoinhibition are unclear. Since post-translational modification is one mechanism for alleviating protein autoinhibition, and Mint1 is highly regulated by phosphorylation, we performed mass spectrometry and identified several Mint1 phosphosites. In addition, we found constitutively-active Src kinase, a kinase implicated in Mint phosphorylation, enhanced APP-Mint1 binding. These results suggest that Src kinase-mediated phosphorylation of Mint1 may relieve Mint1 autoinhibition and promote APP-Mint1 interaction. Overall, this work biochemically characterized the Mint-APP interaction and how it affects amyloidogenic processing, provided a proof of concept for targeting the APP-Mint1 interaction as an AD therapeutic target, and suggested a novel mechanism for the relief of Mint1 autoinhibition.

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abstract: LKB1/STK11 is a serine/threonine kinase first identified in C.elegans as a gene important for cell polarity and proliferation. Mutations in LKB1 are the primary cause of Peutz-Jegher’s cancer syndrome, an autosomal dominantly inherited disease, in which patients are predisposed to benign and malignant tumors. Past studies have focused on defining LKB1 functions in various tissue types, for example LKB1 regulates axonal polarization and dendritic arborization by activating downstream substrates in excitatory neurons of the developing neocortex. However, the implications of LKB1, specifically in the developing cortical inhibitory GABAergic interneurons is unknown. LKB1 deletion was achieved by using Cre-lox technology to induce LKB1 loss in cells localized in the medial ganglionic eminence (MGE) that express Nkx2.1 and generate cortical GABAergic neurons. In this research study it is suggested that LKB1 plays a role in GABAergic interneuron specification by specifically regulating the expression of parvalbumin during the development of fast-spiking interneurons. Preliminary evidence suggest LKB1 also modulates the number of Nkx2.1-derived oligodendrocytes in the cortex. By utilizing a GABAergic neuron specific LKB1 deletion mutant, we confirmed that the loss of parvalbumin expression was due to a GABAergic neuron autonomous function for LKB1. These data provide new insight into the cell specific functions of LKB1 in the developing brain. / Dissertation/Thesis / Masters Thesis Biology 2019

The major goals of my research were to characterize the hypothalamic neural progenitors and to understand how Hh/Gli signaling plays a role in regulating cell proliferation in the hypothalamic neurogenic zone. In contrast to mammals, the zebrafish brain has a life-long potential to grow continuously. Thus, for comparative neurogenesis studies, zebrafish become an indispensible model organism to understand adult neurogenesis and regulatory signaling pathways. Identification of the regulatory mechanisms underlying the controlled cell proliferation in adult zebrafish brain will pave the way to manipulate the healing potential of the mammalian brain. Using immunohistochemistry and in situ hybridization techniques to label known markers for neural stem/ and progenitor cells I have identified three different populations of cells with radial glia (RG) like morphology in the adult zebrafish hypothalamic ventricular zone. In adult zebrafish, cells with RG-like morphology in the ventricular regions are thought to be the neurogenic population. The first population of cells I identified was positive for the neural stem cell marker NESTIN and showed additional characteristics of neural stem cells. Using a label retention assay we showed that Nestin(+) cells are slow cycling. The second population of RG-like cells was Hh responsive, and expressed markers of neural progenitor/transit amplifier cells. Double labeling experiments reveal that the Hh responsive cells were distinct from the Nestin(+) cells These cells were proliferative and cycled faster compared to nestin(+) neural stem cells. The third population of cells with RG morphology in the hypothalamic ventricular zone expressed shh ligand, indicating a regulatory role for Hh signaling in the hypothalamic ventricular zone. Down-regulation of Hh signaling at larval and adult stages reduced proliferation in the hypothalamic ventricle, indicating that Hh acts as a positive regulator of proliferation, as in the dorsal brain. According to our working model, nestin(+) cells are slow cycling, and/or quiescent neural stem cell population in the hypothalamic ventricular zone, whereas Hh responsive cells are the fast cycling transit amplifier cells which proliferate and give rise to new neurons and glia in the adult. My comprehensive analysis of the neural stem/progenitors in the adult zebrafish hypothalamic ventricular zone provides a starting point for the continued study of the mammalian hypothalamic ventricular zone. This study also demonstrates Hh signaling functions as a positive regulator of cell proliferation in the post-embryonic zebrafish hypothalamus consistent with its role in the dorsal brain. (Abstract shortened by UMI.)