Comparison of the Effects of Saporin-IgG Injections into the Nucleus Basalis Magnocellularis and Medial Septal Area of Male Rat as Assessed by the Morris Water Maze Task

Alzheimer\u27s disease currently afflicts approximately 4 million people in the United States, with 100,000 new cases being reported each year. As post mortem examination of AD patientsI brains has revealed a significant decrease in the number of cholinergic neurons, one approach we have taken is to look at the correlation between the depletion of certain cholinergic markers in animals and the resulting behavioral deficits. Two regions of specific interest are the medial septal area (MSA) and the nucleus basalis magnocellularis (NBM). These regions are important because they are the major source of cholinergic neurons in the brain, they are selectively targeted during aging and AD, and there have been many reports of their importance in learning and memory tasks. Therefore in this study we examined the effects on spatial learning, as assessed by the Morris water maze (MWM), in the male rat following intracerebral injections of the selective cholinergic neurotoxin, saporin-IgG. The results of this study indicate that saporin injections into the NBM impaired the performance in the MWM when compared to controls and injections of saporin into the MSA. This was revealed by significantly longer latencies to find a submerged platform and longer latencies during the spatial discrimination test

Protein misfolding in neurodegenerative diseases : implications and strategies

A hallmark of neurodegenerative proteinopathies is the formation of misfolded protein aggregates that cause cellular toxicity and contribute to cellular proteostatic collapse. Therapeutic options are currently being explored that target different steps in the production and processing of proteins implicated in neurodegenerative disease, including synthesis, chaperone-assisted folding and trafficking, and degradation via the proteasome and autophagy pathways. Other therapies, like mTOR inhibitors and activators of the heat shock response, can rebalance the entire proteostatic network. However, there are major challenges that impact the development of novel therapies, including incomplete knowledge of druggable disease targets and their mechanism of action as well as a lack of biomarkers to monitor disease progression and therapeutic response. A notable development is the creation of collaborative ecosystems that include patients, clinicians, basic and translational researchers, foundations and regulatory agencies to promote scientific rigor and clinical data to accelerate the development of therapies that prevent, reverse or delay the progression of neurodegenerative proteinopathies.Peer reviewe

Amyloid-Mediated Sequestration of Essential Proteins Contributes to Mutant Huntingtin Toxicity in Yeast

BACKGROUND: Polyglutamine expansion is responsible for several neurodegenerative disorders, among which Huntington disease is the most well-known. Studies in the yeast model demonstrated that both aggregation and toxicity of a huntingtin (htt) protein with an expanded polyglutamine region strictly depend on the presence of the prion form of Rnq1 protein ([PIN+]), which has a glutamine/asparagine-rich domain. PRINCIPAL FINDINGS: Here, we showed that aggregation and toxicity of mutant htt depended on [PIN+] only quantitatively: the presence of [PIN+] elevated the toxicity and the levels of htt detergent-insoluble polymers. In cells lacking [PIN+], toxicity of mutant htt was due to the polymerization and inactivation of the essential glutamine/asparagine-rich Sup35 protein and related inactivation of another essential protein, Sup45, most probably via its sequestration into Sup35 aggregates. However, inhibition of growth of [PIN+] cells depended on Sup35/Sup45 depletion only partially, suggesting that there are other sources of mutant htt toxicity in yeast. CONCLUSIONS: The obtained data suggest that induced polymerization of essential glutamine/asparagine-rich proteins and related sequestration of other proteins which interact with these polymers represent an essential source of htt toxicity

Transcriptional co -activator dysfunction in polyglutamine disease

Spinal and bulbar muscular atrophy (SBMA) is caused by a CAG repeat expansion in exon 1 of the androgen receptor (AR). This CAG repeat expansion is translated as a polyglutamine repeat, making SBMA one of the eight known polyglutamine expansion diseases. The polyglutamine expansion confers a novel toxic function to the host protein. A central issue in the field is identification of this novel function and its molecular consequences. This thesis addresses changes in transcriptional regulation caused by nuclear expression of polyglutamine, with emphasis on the sequestration of CREB-binding protein (CBP). CBP distribution is altered in the presence of nuclear accumulation of mutant polyglutamine in cell culture, mouse models, and in patient tissue. When nuclear inclusions of polyglutamine form, CBP redistributes from a diffuse nuclear pattern to the inclusions. Coincident with the redistribution are a decrease in soluble CBP and an increase in CBP mRNA. The ability of CBP function to drive transcription from a reported plasmid is impaired in cells expressing mutant polyglutamine. These data prompted the central hypothesis of this thesis, that CBP sequestration by polyglutamines is a cause of cell dysfunction and death in polyglutamine disease. A prediction of the hypothesis is that over-expression of exogenous CBP should reduce the amount of cell death. The hypothesis was tested by developing a system with transient transfection of mutant polyglutamine targeted to the nucleus. CBP over-expression reduced polyglutamine-induced cell loss, as did expression of fragments containing the amino terminus alone. Since CBP is one of several transcriptional coactivators with histone acetyltransferase activity found in polyglutamine inclusions, the acetylation state of histones was assessed. Nuclear expression of mutant polyglutamine causes decreased histone acetylation. Reversal of this deacetylation, either by CBP over-expression or by addition of deacetylase inhibitors reduces cell death. The findings presented in this thesis support a model for polyglutamine disease in which critical coactivators are sequestered, altering acetylation states in the cells, and triggering cell death. Furthermore, deacetylase inhibitors are identified as potential therapeutic agents

Transcriptional co -activator dysfunction in polyglutamine disease

Spinal and bulbar muscular atrophy (SBMA) is caused by a CAG repeat expansion in exon 1 of the androgen receptor (AR). This CAG repeat expansion is translated as a polyglutamine repeat, making SBMA one of the eight known polyglutamine expansion diseases. The polyglutamine expansion confers a novel toxic function to the host protein. A central issue in the field is identification of this novel function and its molecular consequences. This thesis addresses changes in transcriptional regulation caused by nuclear expression of polyglutamine, with emphasis on the sequestration of CREB-binding protein (CBP). CBP distribution is altered in the presence of nuclear accumulation of mutant polyglutamine in cell culture, mouse models, and in patient tissue. When nuclear inclusions of polyglutamine form, CBP redistributes from a diffuse nuclear pattern to the inclusions. Coincident with the redistribution are a decrease in soluble CBP and an increase in CBP mRNA. The ability of CBP function to drive transcription from a reported plasmid is impaired in cells expressing mutant polyglutamine. These data prompted the central hypothesis of this thesis, that CBP sequestration by polyglutamines is a cause of cell dysfunction and death in polyglutamine disease. A prediction of the hypothesis is that over-expression of exogenous CBP should reduce the amount of cell death. The hypothesis was tested by developing a system with transient transfection of mutant polyglutamine targeted to the nucleus. CBP over-expression reduced polyglutamine-induced cell loss, as did expression of fragments containing the amino terminus alone. Since CBP is one of several transcriptional coactivators with histone acetyltransferase activity found in polyglutamine inclusions, the acetylation state of histones was assessed. Nuclear expression of mutant polyglutamine causes decreased histone acetylation. Reversal of this deacetylation, either by CBP over-expression or by addition of deacetylase inhibitors reduces cell death. The findings presented in this thesis support a model for polyglutamine disease in which critical coactivators are sequestered, altering acetylation states in the cells, and triggering cell death. Furthermore, deacetylase inhibitors are identified as potential therapeutic agents

Regulation of Protein Quality Control by UBE4B and LSD1 through p53-Mediated Transcription

<div><p>Protein quality control is essential for clearing misfolded and aggregated proteins from the cell, and its failure is associated with many neurodegenerative disorders. Here, we identify two genes, <i>ufd-2</i> and <i>spr-5</i>, that when inactivated, synergistically and robustly suppress neurotoxicity associated with misfolded proteins in <i>Caenorhabditis elegans</i>. Loss of human orthologs ubiquitination factor E4 B (UBE4B) and lysine-specific demethylase 1 (LSD1), respectively encoding a ubiquitin ligase and a lysine-specific demethylase, promotes the clearance of misfolded proteins in mammalian cells by activating both proteasomal and autophagic degradation machineries. An unbiased search in this pathway reveals a downstream effector as the transcription factor p53, a shared substrate of UBE4B and LSD1 that functions as a key regulator of protein quality control to protect against proteotoxicity. These studies identify a new protein quality control pathway via regulation of transcription factors and point to the augmentation of protein quality control as a wide-spectrum antiproteotoxicity strategy.</p></div

p53 promotes the clearance of misfolded SOD1 mutant proteins.

<p>(<b>A</b>) p53 small molecule activators Tenovin-1 and CP-31398 reduce the levels of misfolded SOD1 proteins, as determined by the SOD1^G85R solubility assay in HEK293 cells. Increasing concentrations of the p53 activators significantly decrease the levels of SOD1^G85R but not the endogenous WT SOD1 proteins in western blots of both supernatant and pellet fractions. (<b>B</b>) A decrease in p53 as the result of shRNA knockdown increases the levels of SOD1^G85R but not WT SOD1 proteins in the SOD1^G85R aggregation assay, as shown by western blots of both supernatant (S) (<i>n</i> = 2) and pellet (P) (<i>n</i> = 3) fractions. (<b>C</b>) A complete absence of p53 increases the accumulation of SOD1^G85R mutant proteins in p53–/– HCT116 cells when compared with controls. Representative western blots (left panels) and quantification of SOD1^G85R levels in the supernatant lysates are shown. The middle graph indicates the ratio of G85R to WT SOD1 proteins in the presence or absence of p53 with varying amounts of transfected mutant SOD1. The right graph panel shows the same data as shown in the middle panel, but normalized to the average SOD1^G85R level for each amount of the transfected plasmid. Data represent means ± SEM. The numerical data used to make this figure can be found in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1002114#pbio.1002114.s001" target="_blank">S1 Data</a>.</p

UBE4B and LSD1 double-knockdown accelerates SOD1^G85R protein degradation.

<p>(<b>A</b>) Western blots of cell lysates derived from mock (CTRL), single UBE4B or LSD1, or double UBE4B and LSD1 knockdowns. Supernatant (S) and pellet (P) fractions were probed with indicated antibodies. While the LSD1 or UBE4B single-knockdown reduces the SOD1^G85R aggregates in both supernatant and pellet fractions, the combined knockdown produces the strongest reduction in the aggregates. (<b>B</b>) Quantification of SOD1^G85R protein levels by western blotting (A). <i>n</i> = 3 (supernatant); <i>n</i> = 8 (pellet). (<b>C</b>) Western blots of a representative cycloheximide chase experiment to determine SOD1 protein half-lives in the double UBE4B and LSD1 knockdown cells versus controls. (<b>D</b>) Quantification of SOD1^G85R clearance, as analyzed by western blotting in (C). The graph indicates the relative band intensity of SOD1^G85R at each chase time point. <i>n</i> = 5; Overall <i>p</i> = 0.02 (paired <i>t</i> test, CTRL versus UBE4B and LSD1 double knockdown). Individual <i>p</i> = 0.03 (3 h), <i>p</i> = 0.003 (6 h), <i>p</i> = 0.06 (9 h), and <i>p</i> = 0.004 (12–21 h). (<b>E</b>) The half-life of SOD1^G85R is reduced from 8.5 h to 5 h upon knockdown of UBE4B and LSD1. Data represent means ± SEM. The numerical data used to make this figure can be found in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1002114#pbio.1002114.s001" target="_blank">S1 Data</a>.</p