N-methyl-D-aspartate receptors (NMDAR) are ionotropic glutamate receptors which can be blocked by Mg2+ in a voltage-dependent manner and are highly permeable to Ca2+, hence they represent a medically relevant target for neurodegenerative disorders caused by excitotoxicity. The two main objectives of this study were, (i) to determine the impact of Q/R/N, +1 and -8 sites modification in the M2 pore region of GluN2A NMDAR subunit on Mg2+ block and other open channel blockers; and (ii) to evaluate novel multi-target-directed ligands (MTDL) for Alzheimer’s disease therapy. The Xenopus laevis oocyte expression system was employed where NMDAR subunit cRNAs were injected into the oocytes and responses to NMDA/glycine and channel blockers were recorded using two-electrode voltage clamp (TEVC) electrophysiology. Pore region mutations to investigate the impact of Q/R/N and adjacent sites were characterized using Mg2+, memantine, MK-801, philanthotoxin analogues and an MTDL compound, CR18. NN at the Q/R/N and +1 sites in GluN2A subunits were mutated to GR and RR, while W at the -8 position (in relation to the Q/R/N site), was mutated to N. Wild type and mutated GluN2A were co-expressed with GluN1-1a in Xenopus oocytes and antagonistic responses by channel blockers were recorded with TEVC. At -75 mV, the RR mutation significantly increased IC50s of Mg2+, memantine and MK-801 by 27-, 42- and 325-fold respectively, compared to wild-type. As for the GR mutation, IC50s were also significantly increased for memantine and MK-801 by 5- and 132-fold respectively, compared to wild type. W to N mutation at the -8 position did not significantly affect blocking potencies for all channel blockers. Blocking potency for PhTX-343 was not significantly altered by any mutations. This study provided evidence that the presence of G and R at the Q/R/N and +1 sites are likely responsible for the changes in blocking sensitivity and play important roles in ion permeability. The fact that PhTX-343 remained potent despite the mutations suggest that this compound might have a different mode of action or different binding site other than the M2 region and should be further characterized. In the MTDL study, twenty one novel compounds were tested on GluN1-1a/GluN2A NMDAR subunits. Thirteen were memantine-derivatives (MAB) incorporated with antioxidant moieties, three were spermine-derived polyamines also incorporated with antioxidants, and five were combinatorial forms of donepezil and carvedilol. The antagonistic properties of the compounds were tested electrophysiologically at -60 mV and compared with Mg2+ and memantine. The MAB series were found to be weak NMDAR channel blockers suggesting the loss of memantine functionality due to attachment of the antioxidant structure to its amine group. Subsequently, modification of the linker point to memantine moieties to free its amine group eventually resulted in weaker NMDAR channel blockers with IC50s of more than 100 µM. The spermine-derived polyamines (CR compounds) were potent NMDAR blockers with IC50s (0.69 to 2.35 µM) comparable to memantine (2.28 µM) and significantly lower than Mg2+ (10.1 µM) and also exhibited voltage-dependence block. Our mutation study revealed that CR18, the most potent MTDL compound was less sensitive in NMDAR containing GR or RR mutation in GluN2A subunits. This is a favourable property of an NMDAR blocker for potential Alzheimer’s disease treatment since GluN3 subunits containing GR or RR at the Q/R/N and +1 sites are less permeable to Ca2+ influx and has been shown to exert neuroprotective effects.

Previous work has demonstrated that rhythmic motor outputs, such as locomotion and scratch induce a hyperpolarization of the voltage threshold (Vth) for action potential initiation in spinal motoneurons, enhancing their excitability. Descending monoamines were implicated in mediating this effect; however, the recent observation that changes in Vth persist during fictive scratch in cats following acute cervical transection revealed that intraspinal systems, of unknown neuromodulatory identity, also have the ability to regulate motoneuron excitability during motor behaviour. This thesis addresses: 1) whether acetylcholine (ACh) is able to modulate spinal motoneuron Vth, and 2) whether endogenous ACh modulates motoneuron excitability during motor activity without intact descending modulation. Our first study investigates whether ACh from exogenous and/or endogenous sources alters motoneuron Vth. We made intracellular recordings of lumbar motoneurons from neonatal rats to pharmacologically manipulate muscarinic and nicotinic receptor activity. Results show that ACh induces either Vth hyperpolarization, Vth depolarization or no change in Vth depending on the activity state of the network, the ACh concentration, and influences from other systems. Our second study investigates whether intraspinal cholinergic inputs induce Vth hyperpolarization during rhythmic motor output when descending projections are disrupted. For this we developed an in vitro neonatal rat spinal cord preparation to elicit rhythmic activity independently of brainstem or lumbar cord stimulation. Intracellular recordings from motoneurons allowed comparison of the Vth prior to and during rhythmic output, both in the absence and presence of cholinergic antagonists in the lumbar cord. Results show that intraspinal cholinergic mechanisms are active and importantly contribute to modulation of motoneuron Vth during motor output. We suggest that in addition to descending modulation, the spinal cholinergic system regulates motoneuron Vth to either facilitate or inhibit recruitment according to the motor network state. Motoneuron excitability regulation by modification of distinct membrane properties resulting from separate modulatory systems activation during diverse motor behaviours is discussed. This work is the first to demonstrate the role of cholinergic mechanisms in regulating motoneuron excitability through modulation of Vth in an activity based context, and therefore outlines a spinal modulatory system that would contribute to motor control in both normal and pathological states. / May 2016

Thesis (M.S.)--Massachusetts Institute of Technology, Dept. of Biology, 1975. / Bibliography: leaves 42-44. / by Michael Binder. / M.S.

Alzheimer's disease (AD) is the most common form of dementia, now the leading cause of death in the UK, which affects more than 35 million people worldwide. Genome-wide association studies identified 20 genetic loci associated with disease susceptibility, however these only exerted small effects on risk. Next-generation sequencing is now being employed to identify more of the missing heritability. This project utilised whole-exome sequencing to explore genetic variation using the Brains for Dementia Research (BDR) resource, a well-characterised cohort of neuropathologically confirmed samples. Exome-wide and candidate gene approaches were employed to assess coding variants for association with AD, using single-variant and burden tests. Coding variants in other neurodegenerative disease genes were also analysed as potential susceptibility factors for AD. Furthermore, polygenic risk scores (PRS) were generated to explore the ability to classify case and control individuals based on their genetic profiles. A synonymous variant in PILRA (rs2405442) was nominally associated with 3-fold increased risk of AD, also contributing strongly to PILRA burden. It was previously linked to AD through risk gene ZCWPW1; however, it has not been directly associated until now. Additional variants in GWAS gene ABCA7 (rs3764645, rs3752234, rs3752237, rs4147915) and rare variants in CLU were also implicated, further supporting their roles in AD susceptibility. A variant in PD gene LRRK2 (rs35303786) inferred protection against AD, implicating potential pleiotropy across the two diseases. PRS could distinguish AD cases from controls with 85.3% accuracy and also identified controls with high PRS but no cognitive impairments. This could be useful for identifying individuals at risk of developing AD in the future. We have uncovered tentative associations both in established and newly identified loci; highlighting several interesting candidates for further investigation. Although there remains a large amount of missing heritability, we hope that as the BDR resource grows, we will achieve increased power to detect significant associations with AD.

Myotonic dystrophy type 1 (DM1) is a dominant human neuromuscular disorder caused by a CTG repeat expansion at the 3' end of the DMPK gene. Pathogenesis of DM1 is linked to a toxic gain of function due to mutant RNA and is manifested by nuclear retention of expanded CUG repeats and aberrant splicing. The development of an inducible model for DM1 with uninterrupted CTG repeats could help us to better understand early pathogenic changes in DM1 due to CUG repeat expansion. In the first part of this thesis, I report an inducible C2C12 mouse myoblast cell line in which the pTetOne inducible system was used to express 174 CTG repeats. This resulted in the production of RNA foci in 26% of cells. Efforts to make larger un-interrupted CTG repeats were unsuccessful due to their instability in the E.coli plasmid. In the second part of the thesis, I used the CRISPR/Cas9-induced genome-editing technique to knock-in an inducible promoter into the endogenous DMPK gene in a DM1 fibroblast cell line. For this, I employed two different CRISPR/Cas9 based strategies which exploit homology-directed repair (HDR) and non-homologous end joining (NHEJ). Our results suggest that CRISPR/Cas9 induced knock-in is enabled by non-homologous end joining more efficiently as compared to homology-directed repair. In the last part of the thesis, TruSeq RNA Sequencing was used to quantify the number of mutant DMPK transcripts and other molecular markers of DM1 pathogenesis that could be a valuable tool for the evaluation of the efficacy of therapeutic compounds. The sequencing results reveal the significant low abundance of mutant DMPK transcripts in the cytoplasmic fraction of DM1 lines and confirm the previously reported nuclear retention of mutant DMPK transcripts. We identify six potential genes which are dysregulated in DM1 fibroblasts and the absolute quantification of mutant DMPK transcripts along with these six reported dysregulated genes can be suitable biomarkers for disease severity and therapeutic response in DM1 fibroblasts. Additionally, these assays could be further refined to provide valuable tools to assess therapeutic compounds.

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Our sensory systems provide us with our ability to perceive and ultimately act upon the world around us. One of the challenges constantly placed on the nervous system is the task of detecting important stimuli among ongoing sensory inputs and then correctly discriminating between possible sources of the stimulus. This dissertation is focused on the study of tactile processing in the rodent vibrissa system to better understand how the nervous system moves between these two operating regimes. The work used a combination of behavioral tasks in trained rats and electrophysiological measurement of neural activity in both anesthetized and awake animals. Results demonstrate that persistent exposure to a sensory stimulus - sensory adaptation - improves spatial discrimination performance, but at the expense of the ability to detect weak stimuli. Parallel experiments involved cortical imaging of anesthetized animals demonstrated important processing changes that could explain these behavioral effects. In a final set of experiments, recordings were obtained from the thalamus, the input stage to the cortex, in awake, behaving animals during performance of a detection task including sensory adaptation. Important changes in the information processing characteristics of the thalamus helped to explain the subsequent cortical and behavioral effects of sensory adaptation. Taken together, results from this set of studies suggest a general principle of adaptive shaping of feature selectivity in sensory processing, resulting from both self-generated sensor motion and the properties of the ongoing stimulus.

Thesis (Ph. D.)--University of Texas at Austin, 2008. / Vita. Includes bibliographical references.