Browsing by Subject "Neurodegenerative Diseases"
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Item Development of P7C3-Class of Neuroprotective Molecules(2015-07-29) De Jesús-Cortés, Héctor José; Huber, Kimberly M.; Pieper, Andrew A.; Kavalali, Ege T.; Johnson, Jane E.Neurodegenerative diseases and disorders are physically, emotionally and financially devastating for patients and their families, and are also associated with great costs to caregivers and society. All of these neuropsychiatric conditions have in common neuronal cell death, for which there is currently no effective pharmacologic treatment. Here, I describe the development of the P7C3-series of neuroprotective compounds, which were originally discovered through an unbiased in-vivo screen for new chemicals with proneurogenic and/or neuroprotective properties. My dissertation work focused on evaluating the efficacy of these compounds in multiple models of neurodegeneration with respect to potency and efficacy of blocking neuronal cell death, as well as associated behavioral outcomes. I used multiple animal and cellular models of neuronal cell death, including Parkinson's disease, traumatic brain injury, and mice lacking expression of L-type calcium channels in the brain. I showed that the P7C3-series of compounds protect mature neurons outside of the hippocampus from otherwise overwhelming toxic insult, and that this protection is associated with preservation of normal neurological function. In these efforts, I also discovered a new function of the P7C3-series of compounds: preservation of normal axonal and mitochondrial integrity after injury. Together, these findings provide starting points for the development of new treatments for neurodegenerative disease, as well as tools to study the biology underlying these disorders.Item Distinct Tau Strains: Exploring Variability in Cell Uptake and Seeding(2019-03-15) Prueitt, William Lloyd; Diamond, Marc; Stopschinski, Barbara; Joachimiak, LukaszBACKGROUND: Tauopathies are neurodegenerative diseases characterized by the pathological aggregation of the microtubule-associated protein tau in neurons and glia. These conditions are incurable, progressive, and deadly. Alzheimer's Disease, the most common tauopathy, affects more than 30 million people worldwide and will afflict more than 120 million by 2050. Evidence suggests that tau aggregates spread pathology as do prions, infectious proteins that transmit a pathologic conformation to native proteins via disease-specific conformers (strains). Various tau strains have been identified which propagate stably in cultured cells over many generations. Additionally, evidence shows that tau aggregates enter cells through heparan sulfate proteoglycan (HSPG) mediated macropinocytosis. However, it is unknown if: 1) different tau strains bind HSPGs uniquely or generically to trigger uptake; 2) which HSPG size and sulfation patters are important for cellular uptake of tau. OBJECTIVE: Test for differential inhibition of cellular uptake using heparin, heparinoids, and HSPG modifications; and test effects of HSPG size and sulfation patterns on binding to tau. METHODS: A "biosensor" cell line responsive to tau aggregates was used to measure intracellular tau aggregation based on fluorescence resonance energy transfer (FRET). The biosensors were HEK-293T cells which overexpress the tau repeat domain (RD) with the disease-associated P301S mutation and were tagged with cyan or yellow fluorescent proteins (RD-CFP/YFP). Cell lysate from various strains of tau was used as source material for pathologic tau seeds to induce aggregation of native tau protein within the biosensors. Lysate was incubated with heparin or heparinoids (heparin-derived molecules of varying length and sulfation patterns) for 24-hours and then added to biosensor cells in culture. When incubated in this way, heparin and heparinoids block cellular uptake of tau by preventing its binding to HSPGs. In a separate assay, lysate was added to cultured biosensor cells with CRISPR/Cas9 knockouts of important genes in the HSPG synthesis pathway. In both assays, cells were harvested 48 hours after lysate/lysate-heparinoid addition and seeding was quantified using FRET flow cytometry. RESULTS: All tau strains tested (DS 5, 6, 8, 9, 10, 13, 14, 15, 16, 17) were highly sensitive to heparin inhibition of seeding and most maintained a highly similar dose response (IC50 of ~100 nM). Some strains, however, showed subtle differences. At maximal heparin concentrations (200 ug/mL), noticeably higher seeding vs baseline was observed in DS 5 and 6 (17%, 9%) as compared to the other strains (<5%). When using heparinoids of 4, 8, 12, and 16 disaccharide units to inhibit tau uptake, similar patterns were seen in DS 9 and 10 (seeding reduction: dp4 = 21% vs 19%; dp8 = 27% vs 33%; dp12 = 70% vs 64%; dp16 = 63% vs 46%). Heparinoids that were desulfated at the 2-O, 6-O, and N positions also showed similar patterns of tau uptake inhibition in DS 9 and 10 (De-2-O = 65% vs 53%; De-6-O = 52% vs 25%; De-N = 35% vs 13%). Finally, seeding in HSPG genetic knockout cells was reduced substantially across strains tested in two knockout cell lines (for genes EXT1 and NDST1). Interestingly, DS 5, DS 6, and DS 15 showed less reduction than the other strains in the knockout cell lines (-38%, -50%, and -51% respectively vs roughly -65% for other strains). Finally, seeding in the third knockout cell line (HS6ST2) increased across all strains tested ranging from +13% to +58%. CONCLUSIONS: Cellular uptake of many tau strains is similarly inhibited by heparin, hinting that the same heparinoid (or small molecule analog) could be used to treat diverse tauopathies. However, the unique behavior of some strains suggests that a one-size-fits-all treatment approach may not always be sufficient. Additionally, certain heparin size and sulfation patterns have specific importance for tau binding. Larger heparinoids better inhibited tau seeding (dp16 & dp12 > dp8 & dp4). Regarding sulfation patterns, the relative importance for tau binding of the sulfate moieties tested is: N-sulfation > 6-O-sulfation > 2-O-sulfation. This pattern remains consistent in recombinant tau, DS 9, DS 10, and in the genetic knockout data gathered in this project (using strains) and by others in the laboratory (using recombinant tau). Overall, this data shows many similarities and some differences in cellular uptake between strains of tau. Additional research to further characterize these differences could have important implications for understanding the diversity of tauopathies and finding unique approaches to diagnosis and treatment.Item Herp Reduces ER Calcium Content by Proteasomal Degradation of SERCA(2007-08-08) Mao, Yuntao Steve; Kodadek, Thomas J.Herp, an endoplasmic reticulum (ER) stress inducible protein, reduces ER Ca2+ content in neurons and prevents their apoptosis. An understanding of the mechanism by which Herp decreases ER Ca2+ content requires studies of Herp interacting proteins, which could be SERCA and the proteasome. Herp may recruit the proteasome from the cytosol to the ER membrane, thereby facilitating the ER associated degradation (ERAD) of SERCA. The proteasome recruitment and the subsequent degradation of SERCA reduce ER lumenal Ca2+ concentration and the Ca2+ release during ER stress which counteracts the activation of apoptosis. This proposal describes how to determine the mechanism through which Herp reduces ER Ca2+ content, how to test the proteasomal degradation of SERCA, how to illustrate the proteasome recruitment to the ER membrane, and how to demonstrate the interaction between Herp and SERCA. The work will provide a new regulatory link between ER stress and Ca2+ homeostasis. In addition, studies of the proteasomal degradation of SERCA will broaden our present understanding of the regulation of SERCA. Since dysregulation of Ca2+ homeostasis has been implicated in the pathophysiology of several neurodegenerative diseases like Alzheimer's and Huntington's, research focused on Herp may lead to insights regarding therapies for those.Item An Insight into Alpha-Synulcien's Biological Function and Its Pathogenesis in Neurodegenerative Disease(2007-08-08) Gallardo, Gilbert; Südhof, Thomas C.The discovery of two missense mutations (A53T and A30P) in alpha -synuclein that are genetically linked to Parkinson's disease, together with alpha -synuclein being the major component in Lewy bodies, has generated extensive interest in alpha -synuclein as a key component in neurodegenerative diseases. In recent years modeling this disease in transgenic mice and flies has lead to new understandings of alpha -synuclein function and pathogenesis in neurodegeneration. In the current study we analyzed transgenic mice overexpressing human alpha -synuclein and human alpha -synuclein mutations (A53T&A30P) to; First establish these transgenic mice as a model for degenerative diseases; second to identify potential contributing factors in neurodegeneration; third to decipher a potential function of alpha -synuclein. We first established that transgenic mice expressing human variants of alpha -synuclein developed an age dependant motor dysfunction with symptom logy characteristic of Parkinson's disease. Immunohistological studies revealed the presence of alpha -synuclein inclusions and a loss of motor neurons. Biochemical analysis identified a 4-5 fold increase in ubiquitin with altered expression of proteasomal subunits, characteristic of proteasomal impairment. In addition, we identified a significant increase in amyloid beta -peptides. Protein quantification of apolipoprotein E (ApoE) a protein that has been associated with the development of Alzheimer's disease, demonstrated a 5-15 fold increase in symptomatic transgenic mice. Ablation of ApoE in alpha -synuclein transgenic mice by genetic crosses revealed a delayed onset for motor dysfunction and an overall increase in survival. ApoE deficient transgenic mice displayed a decrease in ubiquitin and amyloid beta -peptides. This study illustrates ApoE, ubiquitin and Abeta - peptides contribute to the onset and progression of the neurodegeneration in transgenic alpha -synuclein mice. Genetic crosses of transgenic alpha -synuclein with a csystine string protein-alpha (CSPalpha ) knockout mouse revealed a potential function for alpha -synuclein. CSPalpha deficient mice develop an early age neurodegenerative disease that is lethal at 3-4 months. Transgenic expression of human alpha -synuclein prevented the deleterious effects of CSPalpha deficiency. Immunofluorescence studies illustrated alpha -synuclein function in a cell autonomous manner. Biochemical analysis demonstrated CSPalpha deficient mice have impaired SNARE complexes that are partially reverted by transgenic alpha -synuclein. This study illustrates a protective function of alpha -synuclein in preventing neurodegeneration.Item Mechanisms of Protein Mislocalization in Neurodegenerative Disease(2017-09-06) Pinarbasi, Emile S.; Yu, Gang; Thomas, Philip J.; Chook, Yuh Min; Bezprozvanny, IlyaFronto Temporal Lobar Degeneration (FTLD) and Amyotrophic Lateral Sclerosis (ALS) are two fatal and rapidly progressing neurodegenerative diseases. A unifying characteristic of these diseases is the mislocalization of an RNA-binding protein, TDP-43. In unstressed cells, TDP-43 is predominantly nuclear and constantly shuttling to the cytosol; in ALS/FTLD, TDP-43 is aggregated in the cytosol. Two lines of evidence suggest this shift is a cause, rather than an effect, of disease. First, point mutations in the C-terminus of TDP-43, which enhance its aggregation, are a rare cause of familial ALS. Second, animal models which replicate the disease-linked redistribution of TDP-43 in motor neurons demonstrate the progressive muscle weakness and loss of spinal cord mass seen in patients. However, little is known about the cellular insults that promote TDP-43 mislocalization. My graduate work makes two contributions to this understanding. First, I elucidated a major determinant of normal TDP-43 trafficking. TDP-43 localization is governed by the balance between nuclear import and nuclear export. While a model for TDP-43 nuclear export had been proposed, there was no direct experimental evidence supporting it. I have shown that the proposed model of TDP-43 nuclear export is incorrect; the putative nuclear export signal (NES) does not mediate nuclear export, and TDP-43 nuclear export is XPO1 independent. Additionally, my data suggest no discrete trafficking signal within TDP-43. Rather, I propose that TDP-43 nuclear export is primarily driven by diffusion through the nuclear pore. Second, I focused on an upstream event known to affect TDP-43 localization: progranulin secretion. One genetic cause of FTLD is a single loss-of-function mutation in GRN, which causes progranulin haploinsufficiency. For reasons that are still unclear, a lifetime of progranulin haploinsufficiency in a patient causes FTLD with TDP-43 mislocalization. I focused on how a subset of GRN mutations- the signal sequence mutations- prevent progranulin secretion. I found that the W7R and A9D mutations disrupt co-translational recruitment of the targeting factor SRP (Signal Recognition Particle). This triggers a quality control pathway called RAPP (Regulation of Aberrant Protein Production), which results in degradation of both mutant protein and mutant mRNA. Thus, RAPP mediates progranulin haploinsufficiency in these patients.Item Polyglutamines & neurodegenerative diseases /(2004-04-29) Cox, Rody P.Item The role of inflammation and infection in neurodegeneration(2024-01-12) Beckham, J. DavidItem [Southwestern News](2004-11-11) Donihoo, Rachel