There has been much interest in the use of therapeutics based on ribonucleic acid interference(RNAi) to inhibit synthesis of mutant proteins ever since Elbashir et al. (Elbashir, S. M., Harborth, J., Lendeckel, W., Yalcin, A., Weber, K. and Tuschl, T., 2001. Duplexes of 21-nucleotide RNAs mediate RNA interference in cultured mammalian cells. Nature. 411, 494-498.) found that synthetic double stranded small interfering ribonucleic acids (siRNAs) can initiate this evolutionarily conserved process in mammalian cells. Since RNAi is able to target single genes and therefore mitigate the underlying molecular pathology of diseases, RNAi-based therapeutics will most likely benefit monogenic neurodegenerative diseases such as Huntington’s disease. It is however particularly difficult to deliver exogenous materials such as siRNAs into neurons in vivo as the blood-brain barrier (BBB) isolates the brain from the vascular system and prevents permeation of most materials. Neurons also do not take up exogenous materials readily. Therefore, effective delivery of siRNAs into the brain remains one of the biggest challenges impeding their use as a potential neurotherapeutic. Layered double hydroxide (LDH) nanoparticles are a class of anionic clay materials that have demonstrated great potential as a DNA (deoxyribonucleic acid) delivery system for a variety of mammalian cell lines due to their unique physiochemical properties. This thesis examined the feasibility of LDH as a siRNA delivery system for cultured neurons and demonstrated that the delivered siRNAs are able to effectively down-regulate synthesis of a target protein with minimal toxicity. Experiments were conducted using double stranded DNAs (dsDNAs) initially, and siRNAs were then used to verify these results. It was shown that nucleic acids(dsDNAs and siRNAs) could successfully intercalate into pristine LDHs to form nucleic acid-LDH complexes that had properties suitable for use as a delivery system in mammalian cells. These studies established that LDHs and nucleic acid-LDH complexes were biocompatible with neurons isolated from embryonic day 17.5 mouse cerebral cortex, suggesting that LDH can be used for nucleic acid delivery into cultured neurons. LDHs were also shown to successfully deliver nucleic acids into a non-neural mammalian cell line (NIH 3T3 cells). Finally, this thesis demonstrated for the first time that LDHs were able to deliver siRNAs into neurons, providing encouraging preliminary evidence that sequence specific gene silencing of the Mus Musculus Deleted in Colorectal Cancer (DCC) gene had occurred. However, down-regulation of the DCC protein did not occur consistently, suggesting that further optimisation is needed to improve the efficacy of siRNA-LDH complexes to inhibit expression of target protein in neurons. In future, LDHs should be further developed as an efficient siRNA delivery system for therapeutic gene silencing in the central nervous system using a neurodegenerative disease model such as the Huntington’s disease mouse model, which closely phenocopies the human disease. This model will allow the in vivo efficacy of these nanoparticles to be tested and subsequently improved in order to deliver siRNAs locally and systematically into the brain.

As head injuries and their sequelae have become an increasingly salient matter of public health, experts in the field have made great progress elucidating the biological processes occurring within the brain at the moment of injury and throughout the recovery thereafter. Given the extraordinary rate at which our collective knowledge of neurotrauma has grown, new insights may be revealed by examining the existing literature across disciplines with a new perspective. This article will aim to expand the scope of this rapidly evolving field of research beyond the confines of the central nervous system (CNS). Specifically, we will examine the extent to which the bidirectional influence of the gut-brain axis modulates the complex biological processes occurring at the time of traumatic brain injury (TBI) and over the days, months, and years that follow. In addition to local enteric signals originating in the gut, it is well accepted that gastrointestinal (GI) physiology is highly regulated by innervation from the CNS. Conversely, emerging data suggests that the function and health of the CNS is modulated by the interaction between 1) neurotransmitters, immune signaling, hormones, and neuropeptides produced in the gut, 2) the composition of the gut microbiota, and 3) integrity of the intestinal wall serving as a barrier to the external environment. Specific to TBI, existing pre-clinical data indicates that head injuries can cause structural and functional damage to the GI tract, but research directly investigating the neuronal consequences of this intestinal damage is lacking. Despite this void, the proposed mechanisms emanating from a damaged gut are closely implicated in the inflammatory processes known to promote neuropathology in the brain following TBI, which suggests the gut-brain axis may be a therapeutic target to reduce the risk of Chronic Traumatic Encephalopathy and other neurodegenerative diseases following TBI. To better appreciate how various peripheral influences are implicated in the health of the CNS following TBI, this paper will also review the secondary biological injury mechanisms and the dynamic pathophysiological response to neurotrauma. Together, this review article will attempt to connect the dots to reveal novel insights into the bidirectional influence of the gut-brain axis and propose a conceptual model relevant to the recovery from TBI and subsequent risk for future neurological conditions.

Spinal Muscular Atrophy (SMA) is a neuronal degenerative disease caused by the mutation or loss of the Survival Motor Neuron (SMN) gene. The cause for the specific motor neuron susceptibility in SMA has not been identified. The high axonal transport/localization demand on motor neurons may be one potentially disrupted function, more specific to these cells. We therefore used a large-scale immunoprecipitation (IP) experiment, to identify potential interactors of SMN involved in neuronal transport and localization of mRNA targets. We identified KH-type splicing regulatory protein (KSRP), a multifunctional RNA-binding protein that has been implicated in transcriptional regulation, neuro-specific alternative splicing, and mRNA decay. KSRP is closely related to chick zipcode-binding protein 2 and rat MARTA1, proteins involved in neuronal transport/localization of beta-actin and microtubule-associated protein 2 mRNAs, respectively. We demonstrated that KSRP is arginine methylated, a novel SMN interactor (specifically with the SMN Tudor domain; and not with SMA causing mutants). We also found this protein to be misregulated in the absence of SMN, resulting in increased mRNA stability of KSRP mRNA target, p21cip/waf1. A role for SMN as an axonal chaperone of methylated RBPs could thus be key in SMA pathophysiology.

Traumatic brain injury is a topic that in recent years has received increased scrutiny by the media and is viewed as a cause for public health concern in athletes that are participating in contact sports. There has been an apparent rise in the reported number of traumatic brain injuries over the last decade possibly due to a number of factors such as an increase in enrollment of sports and suspected better understanding of brain injury in the sports world. Direct or indirect impact forces applied involving acceleration/deceleration and linear/angular forces primarily cause trauma to the brain. This insult results in evident diffuse axonal and focal injuries to varying degrees in brain tissue. The spectrum of pathophysiology in traumatic brain injury involves structural, neurochemical, metabolic, vascular, inflammatory, immunologic, and ultimately cell death, which plays a hand directly in the nonspecific presentation of symptoms reported by athletes as well as the progression of recovery. Traumatic brain injury is typically associated with short- and long-term sequelae, however, inducing repetitive episodes of trauma over a career, as may happen in sports, may lead to a progressive neurodegenerative disease known as chronic traumatic encephalopathy. Chronic traumatic encephalopathy has been known to affect boxers previously, but in recent years the attention has shifted and found this disease in athletes from other sports. The spectrum of disease in chronic traumatic encephalopathy involves a progressive tauopathy that spreads across different regions of the brain in a classified four staged grading system. Several risk factors have been identified in placing athletes at risk for traumatic brain episodes, however no risk factors have been directly linked to chronic traumatic encephalopathy. Much information is lacking in a complete understanding of traumatic brain injury and chronic traumatic encephalopathy, therefore emphasizing the importance of further research and consistently improving modifications in the protocols for assessment, recognition, management, and return to play criteria for injured athletes. Furthermore, despite the gaps in knowledge, preventative measures should serve a particular role in reducing the incidence of detected traumatic brain injuries, which should include policy changes, sport rule changes, and especially changes to the accepted sports culture through mandatory education.

Although the central nervous system was long perceived as the ivory tower without immune entities, there is growing evidence that the immune and nervous systems are intimated connected. These two systems have been shown to communicate both cellularly and molecularly under physiological and pathological conditions. Despite our increasing understanding of the interplay between these two systems, there are still numerous open questions. In this thesis, I address such unanswered questions related to: the role of microglia and their mechanism in contributing to pathologies in Rett syndrome; the beneficial effects of T-cell secreted cytokines in supporting social brain function; the evolutionary link of the interactions between the nervous and immune systems; the transcription regulation of a subset of microglia population in common neurodegenerative diseases. Collectively, the current thesis is focused on the joint frontier of bioinformatics and experimental work in neuroimmunology. A multifaceted approach, that includes transcriptomics, genomics and other biomolecular modules, was implemented to unearth signaling pathways and mechanisms underlying the presenting biological phenomena. The findings of this thesis can be summarized as follows: 1) MeCP2 acts as a master regulator in the transcriptional repression of inflammatory stimuli in macrophages; 2) T-cell secreted IFN-γ supports social brain function through an evolutionally conserved interaction between the immune and nervous systems; 3) The APOE-TREM2 pathway regulates the microglia phenotype switch in neurodegenerative diseases. Provided that recent technologies allow for readily manipulating the immune system, the findings presented herein may create new vistas for therapeutic interventions in various neurological disorders.

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One of the pathological hallmarks of Alzheimer's disease (AD) is the amyloid plaque deposition in aging brains by aggregation of amyloid-β (Aβ) peptides. In this study, the effect of chloroplast derived myelin basic protein (MBP) fused with cholera toxin subunit B (CTB) was investigated in advanced diseased stage of human and mouse AD brains. The CTB-fusion protein in chloroplasts facilitates transmucosal delivery in the gut by the natural binding ability of CTB pentameric form with GM1 receptors on the intestinal epithelium. Further, bioencapsulation of the MBP within plant cells confers protection from enzymes and acids in the digestive system. Here, 12-14 months old triple transgenic AD mice were fed with CTB-MBP bioencapsulated in the plant cells for 3 months. A reduction of 67.3% and 33.3% amyloid levels in hippocampal and cortical regions, respectively were observed by immunostaining of brain sections with anti- Aβ antibody. Similarly, 70% decrease in plaque number and 40% reduction of plaque intensity was observed through thioflavin S (ThS) staining that specifically stains amyloid in the AD brain. Furthermore, ex vivo 3xTg AD mice brain sections showed up to 45% reduction of ThS stained amyloid levels when incubated with enriched CTB-MBP in a concentration dependent manner. Similarly, incubation of enriched CTB-MBP with ex vivo postmortem human brain tissue sections with advanced stage of AD resulted up to 47% decrease of ThS stained amyloid plaque intensity. Lastly, lyophilization of plant material facilitates dehydration and long term storage of capsules at room temperature, in addition to increasing CTB-MBP concentration by 17 fold. These observations offer a low cost solution for treatment of even advanced stages of the AD by facilitating delivery of therapeutic proteins to central nervous system to address other neurodegenerative disease.

No description available.