Browsing by Subject "Neurodegeneration"

Hypoxia is a profound stressor of the central nervous system implicated in numerous neurodegenerative diseases. While it is increasingly evident that the early effects of hypoxia cause impairment at the level of the axon, the precise mechanisms through which hypoxia compromises axonal structure and function remain unclear. However, links between hypoxia-induced axonopathic disease and the amyloid cascade, as well as the upregulation of amyloid precursor protein (APP) and amyloid beta (Aβ) by hypoxic stress, give rise to the hypothesis that proteolytic cleavage of APP into Aβ may be specifically responsible for axonopathy under conditions of hypoxia.

The goal of this dissertation was thus to understand dependence of hypoxia-induced axonal morphological and functional impairment on APP cleavage and the production of Aβ. I have developed a model of hypoxia-induced axonopathy in retinal explants. Using this model, I have experimentally addressed the core hypothesis that APP cleavage, and in particular the formation of Aβ, is necessary and sufficient to mediate morphological and functional axonopathy caused by hypoxia. I have found that there is a dissociation between the mechanisms responsible for hypoxia-induced morphological and functional impairment of the axon in the explanted retina, with the former being dependent on APP-to-Aβ processing and the latter likely being dependent on cleavage of a non-APP substrate by the enzyme BACE1. These findings shed light on mechanisms of hypoxia-induced axonopathy.

Germinal center kinases (GCKs) belong to the mammalian Ste20-like family of serine/threonine kinases and participate in various signaling pathways needed to regulate a wide range of cellular activities. GCK-like kinase (GLK), also known as MAP4K3, belongs to the MAP kinase kinase kinase kinase (MAP4K) family of proteins and has recently been established as a key node in the amino acid response pathway and putative nutrient sensing regulator in cells, as it is required for the amino acid-dependent activation of the mechanistic target of rapamycin complex 1 (mTORC1)—a central regulator of cell growth and metabolism. The precise mechanism(s) by which MAP4K3 activates mTORC1 under conditions of amino acid satiety, however, are undefined. Recent studies in the La Spada lab suggest MAP4K3 activates mTORC1 by phosphorylating the NAD-dependent deacetylase sirtuin 1 (Sirt1) and subsequently, inhibiting the LKB1-AMPK pathway—a pathway that suppresses mTORC1 activation during starvation. MAP4K3 has additionally been linked to the regulation of cellular stress responses, autophagy, growth, survival, and organismal lifespan through largely unknown pathways. My working hypothesis is that MAP4K3 serves as an amino acid sensor and activates mTORC1 through phosphorylation of Sirt1 and subsequent inhibition of the mTORC1-suppressing Sirt1-LKB1-AMPK pathway under conditions of amino acid satiety and engages different biological pathways by virtue of its protein interacting partners to control critical cellular processes involved in cell growth, survival, and lifespan. In study 1, I used amino acid depletion/restimulation experiments and phospho mass spectrometry to establish a direct link between MAP4K3 and the Sirt1-LKB1-AMPK pathway and determines that Sirt1 is phosphorylated at Threonine 344 (T344) in a MAP4K3- and amino acid-dependent manner. Furthermore, I showed that phosphorylation of T344 inhibits Sirt1 and is sufficient to restore amino acid-dependent mTORC1 activation in cells lacking MAP4K3. To elucidate additional pathways regulated by MAP4K3, in study 2, I sought to discover novel MAP4K3 interacting partners by integrating proteomics interactome data and phosphoproteomics data followed by validation studies in cells. Experiments from these studies indicate a novel role for MAP4K3 in regulating DNA double-strand break (DSB) sensing and repair in the nucleus, mTOR localization to the lysosome through the GATOR2 complex, and endocytosis. Recent discoveries regarding the important role for MAP4K3 in nutrient sensing through mTORC1 activation and other cellular activities, including cell growth, autophagy, and survival are significant because deregulation of these cellular processes has been implicated in aging, as well as a wide array of human diseases including cancer, immunological disorders, and neurodegeneration. This dissertation, thus, sheds light on the molecular mechanisms by which MAP4K3 regulates these processes and provides significant insight into the modulation of these pathways in health and disease states.

Neurons, with their enormous membrane contents, depend heavily on regulated membrane trafficking processes to maintain their morphology and function. The phosphatidylinositol 3-kinase class III, or PIK3C3, plays a critical role in various membrane trafficking processes including both the endocytic and autophagic pathways. The functions of PIK3C3 in the nervous system in vivo are un-characterized. We reasoned that studying PIK3C3 in neurons would provide us an entry point into understanding the regulations and functions of the neuronal membrane trafficking processes and their roles in neuronal morphogenesis and homeostasis.

We generated a conditional allele of Pik3c3 and first deleted it specifically in the peripheral sensory neurons. Mutant large-diameter myelinated sensory neurons accumulated numerous enlarged vacuoles and ubiquitin-positive aggregates and underwent rapid degeneration. By contrast, Pik3c3-deficient small-diameter unmyelinated neurons accumulated excessive numbers of lysosome-like organelles and degenerated slower than large-diameter neurons. These differential degenerative phenotypes are unlikely caused by a disruption of the autophagy pathway, because inhibiting autophagy alone by conditional deletion of Atg7 results in a completely distinct subcellular phenotypes and very slow degenerations of all sensory neurons. More surprisingly, a noncanonical PIK3C3-independent LC3-positive autophagosome formation pathway was activated in Pik3c3-deficient small-diameter neurons. This work uncovered unexpected differences of the endo-lysosomal systems in different types of neurons and discovered a novel autophagy initiation pathway in vivo in neurons.

To examine the role of PIK3C3 in the central nervous system (CNS), we next deleted Pik3c3 in CNS neural progenitor cells using the Nestin-Cre transgenic line. The resulting conditional knockout mice displayed a severe cortical lamination abnormality caused by defective cortical neuron migration. This finding uncovered a previously under-appreciated role of endocytic trafficking in neural migration, which was further confirmed by electron microscopic analyses of the developing cortex. Moreover, overexpressing the dominant negative forms of Dynamin2 or Rab5, two regulators of endocytosis, caused similar migration defects as Pik3c3-deletion. Mechanistically, Pik3c3-deficient cortical neurons drastically reduced surface Reelin binding sites, and showed significantly decreased levels of Dab1 phosphorylation, despite expressing normal total amount of Reelin receptor ApoER2. This work suggests endocytosis and recycling of Reelin receptors are likely to play an important role in cortical migration regulated by the Reelin signaling pathway.

These studies represent the first in vivo characterization of PIK3C3 functions in mammals, and provide insight into the complexity and functional importance of neuronal endo-lysosomal and autophagic pathways.

The loss of nigrostriatal dopaminergic neurons is the fundamental hallmark of Parkinson’s disease (PD). In early PD stages, this is ameliorated by dopamine (DA) supplementation; however, as the disease progresses, the complete loss of this key dopaminergic pathway forces the central nervous system to find alternative routes to regain motor control. It has previously been shown that serotonergic routes must take on the role of the failed dopaminergic system throughout the progression of the disease. Previously studied 5-HT1A anxiolytic and anti-depressive therapeutics have yet to be successfully repurposed for Parkinson’s disease patients. Herein is described the current efforts towards the employment non-dopaminergic agonists in the investigation of motor control in Parkinson’s disease. This research outlines the development of non-dopaminergic therapeutics inspired by the core structure of the clinically approved 5-HT1A agonist Befiradol. This motif has been infused with a trans-2-arylcyclopropylamine moiety which has been independently shown to reduce motor symptoms in Parkinson’s disease via a prior collaboration from the McCafferty lab. While it was originally hypothesized that these therapeutics would act as bifunctional agonists at the 5-HT1A and M4 GPCRs, affinity assays reveal dualistic agonism at the 5-HT1A and 1 receptors, offering a new class of potential bifunctional therapeutics.

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Advances in modern medicine have helped to prolong human life. These advancements coupled with an ever-increasing population means that diseases associated with aging will become more prevalent in the coming years. As such, it is critical to understand the pathogenesis of disease where aging is the main risk factor. While not widely known, age is in fact a large risk factor in development of obesity and metabolic syndrome. More widely known and discussed are the neurodegenerative diseases that occur late in life. While age as a risk factor is a common point between these types of pathology, there are other similarities, such as the interaction between lipid metabolism and mitochondrial health.

To study the overlap between obesity and neurodegeneration, we investigated two pathways that regulate both. First, we find that loss of cytoplasmic deacetylase HDAC6 leads to aberrant accumulation of lipid in vitro and in vivo. HDAC6 knock-out (KO) mice gain more weight than WT counterparts after a high-fat diet regimen. Additionally, the intermediary metabolism of cells lacking HDAC6 is disrupted as they increase glucose uptake while downregulating fatty acid oxidation. HDAC6 not only plays a role in lipid metabolism, but regulates mitochondrial dynamics. Upon glucose-withdrawal, HDAC6 KO cells fail to elongate their mitochondria and display increased levels of mitochondrial toxic by-products. Therefore, HDAC6 has critical roles in lipid homeostasis and mitochondrial health.

The other pathway we investigated is critical in neurodegenerative disease, Parkinson's disease. Parkin, an E3 ubiquitin ligase, flags damaged mitochondria for destruction so they do not poison the other functional organelles. We found that Parkin promotes lipid remodeling at the surface of the mitochondria. Phosphatidic acid (PA) accumulates shortly after mitochondrial damage while diacylglycerol (DAG) appears several hours later. This lipid accumulation is dependent upon Parkin's translocation and E3 ligase activity. Additionally, we found that lipin-1, a PA phosphatase, and endophilin B1 (EndoB1) are critical for DAG accumulation and effective mitochondrial clearance.

Through this work, we show that two proteins critical in quality control mechanisms also play significant roles in energy homeostasis. We aim to highlight this overlap and posit that common diseases of aging, though presenting differently, might have disruptions in the same basic process.

An aging global population and accompanying increases in the prevalence of age-related disorders are leading to greater financial, social, and health burdens. Alzheimer’s disease and related dementias are one such category of age-related disorders that are associated with progressive loss of physical and cognitive ability. One proposed preventative measure against risk for such dementias is improving cardiovascular fitness, which may help reverse or buffer age-related brain atrophy associated with worse aging-related outcomes and cognitive decline. However, research into this potential of cardiovascular fitness has suffered from extreme heterogeneity in study design methodology leading to a lack of cohesion in the field and undermining any potential causal evidence that may exist. In addition, while direct measures of cardiovascular fitness (e.g., VO2Max) and healthy lifestyle behaviors typically associated with better cardiovascular fitness (e.g., exercise, diet, smoking status) are not necessarily highly correlated, they are often conflated in existing research. This has contributed to a lack of clarity in generalizing and comparing results.

This dissertation presents results from four original research projects addressing open empirical questions about possible links between cardiovascular fitness, healthy lifestyle behaviors, and structural brain integrity in midlife, which has become a target of intervention research seeking to stave off cognitive decline and risk for dementia before irreversible damage has accrued. Each section of the dissertation builds on increasingly complex aspects of these links with the goal of providing supporting evidence that may aid future biomarker research and clinical trials design. All studies involved were conducted using behavioral data, physiological data, and structural MRI data from the Dunedin Study, which has followed a population-representative birth cohort to midlife.

Across four empirical sections, results collectively suggest that there is a modest association between cardiovascular fitness and both grey and white matter structural integrity as well as between healthy lifestyle behaviors and grey matter structural integrity. Further, results indicate that these associations may extend beyond cross-sectional data and have relevance for measures capturing the extent of age-related atrophy in the brain. In addition, the results reinforce prior findings that cardiovascular fitness and healthy lifestyle behaviors are independent constructs and suggest that the differentially mapping of these constructs onto specific features of brain integrity in midlife may be useful in directing the search for risk biomarkers and designing clinical trials.

Mitochondrial dysfunction appears to be a hallmark of many neurodegenerative diseases. The link between mitochondrial and neuronal health is particularly strong in Parkinson’s disease (PD). Genes that have been shown to cause PD when mutated are known to interact with mitochondria to varying degrees; particularly, two of the genes associated with mitochondria-specific autophagy (mitophagy), PINK1 and PARK2, have been shown to cause autosomal recessive PD. Deletions and mutations in mitochondrial DNA (mtDNA) have been detected in the substantia nigra of PD patients. Furthermore, toxicants used to model PD in the laboratory are known to exert their toxicity by disrupting mitochondrial function. Despite all the advances in PD research, the precise mechanisms through which this disease arises are still not well understood.

The central goal of this dissertation was to investigate the role that mtDNA damage and mitochondrial dysfunction play in causing dopaminergic neurodegeneration in the context of PD. This work was performed with a particular focus on mitochondrial toxicants: their mechanism of action and effect on neuronal health, and how these toxic exposures might exacerbate genetic susceptibilities. Another goal was to identify windows of susceptibility to mtDNA damage, with early life and old age being of particular interest. To explore these ideas, the nematode Caenorhabditis elegans was utilized as a model organism.

To characterize age-related sensitivity to mtDNA damage, worms at different life stages were exposed to ultraviolet C (UVC) to accumulate mtDNA damage and the effect of this damage on mitochondrial genome copy number and dopaminergic neuron health was assessed. To evaluate the role of autophagy in maintaining mitochondrial and neuronal health after toxicant exposure, mitophagy gene knockouts (KOs) and wild type worms were exposed to 6-hydroxydopamine (6-OHDA) and UVC; to study non-selective macroautophagy, starvation was used to induce it, and nematodes were subsequently subjected to a toxic exposure. Dopaminergic neuron morphology, targeted gene expression, and mitochondrial morphology were evaluated. Lastly, the mechanism of toxicity of rotenone in C. elegans was investigated; specifically, mitochondrial respiration (with the Seahorse Bioanalyzer), targeted metabolomics, ATP levels, and targeted gene expression were evaluated.

Due to technical complications with our UVC exposure model and PCR-based evaluation of mtDNA damage in C. elegans eggs, the windows of susceptibility to mtDNA damage hypothesis could not be properly tested. However, our current results do not support this idea. Our studies on the role of autophagy in the context of toxicant-induced dopaminergic neurodegeneration suggest that both non-selective macroautophagy and mitophagy have a protective effect. We saw protection from 6-OHDA-induced neurodegeneration after starvation treatment, suggesting that macroautophagy could be playing a protective role. In the context of mitophagy mutations, we saw different responses to UVC and 6-OHDA. Both pink-1 and pdr-1 mutants were more sensitive to UVC-induced dopaminergic neurodegeneration than the control strain; however, only the pdr-1 strain was more sensitive than the control strain to 6-OHDA. The pink-1 mutant was actually protected from neurodegeneration caused by 6-OHDA. We proceeded to evaluate antioxidant responses in these strains (since 6-OHDA exerts its toxicity mainly via oxidative stress) but saw no differences across strains. We are currently testing the hypothesis that changes in the mitochondrial network in the pink-1 mutant after 6-OHDA exposure allow it to better withstand its toxic effect. Our studies into the mechanism of action of rotenone in C. elegans gave us surprising results: we saw no major alterations in oxygen consumption or ATP levels. However, various metabolites were altered, signaling a metabolic restructuring similar to that observed in C. elegans complex I mutants. Also similar to complex I mutants, it appears that the glyoxylate pathway is upregulated after rotenone exposure; this alternate pathway parallel to the citric acid cycle might be allowing the nematodes to acclimate to rotenone exposure, explaining our respiration and ATP results.

Overall, this dissertation work supports the link between mitochondrial dysfunction and dopaminergic neurodegeneration, and provides new hypotheses for research in this field.