Thesis (Ph.D.)--University of Alabama at Birmingham, 2008. / Title from first page of PDF file (viewed on June 29, 2009). Includes bibliographical references.

Abstract Alzheimer's disease (AD) and frontotemporal lobar degeneration (FTLD) are the two most common neurodegenerative diseases leading to early onset dementia (< 65 years). Mutations in the amyloid precursor protein (APP), presenilin 1 (PSEN1) and presenilin 2 (PSEN2) genes cause a proportion of familial early-onset AD (eoAD), while the microtubule-associated protein tau (MAPT) and progranulin (PGRN) mutations have been identified in FTLD patients. Only a few PSEN1 and APP mutations have previously been found in Finnish AD patients, and one MAPT mutation in a FTLD family, while the role of PGRN in Finnish FTLD patients is unknown. Increasing evidence suggests that mitochondrial dysfunction and oxidative stress also play an important role in neurodegenerative diseases. The aim here was to investigate the genetics of eoAD and FTLD in the population of the province of Northern Ostrobothnia, Finland. Sequencing analysis of the APP, PSEN1 and PSEN2 genes was performed to determine whether mutations in these genes could be detected. The MAPT and PGRN genes were analysed in the FTLD patients by sequencing and MAPT haplotypes were determined. The contributions of mtDNA and its maintenance enzymes to eoAD and FTLD were studied by comparing the frequencies of mtDNA haplogroups and their clusters between the patient groups and controls and by screening for the five common POLG1 mutations (T251I, A467T, P587L, W748S, Y955C), two common mtDNA mutations (m.3243A>G, m.8344A>G) and mutations in the PEO1 and ANT1 genes. This is the first report of a significant association between the mtDNA haplogroup cluster IWX and FTLD. The H2 MAPT haplotype was also associated with FTLD in our cohort. No significant differences in the frequencies of the mtDNA haplogroups were observed between the eoAD patients and controls, nor were there any pathogenic mutations detected in the genes analysed. The findings suggest that possession of the mtDNA haplogroup cluster IWX and the H2 MAPT haplotype may be possible risk factors for FTLD in our cohort. The absence of any pathogenic mutations in the MAPT, PGRN, APP or PSEN genes in our series, together with the previous reports of only a few mutations found in this region, supports a minor role for these genes in the aetiology of eoAD and FTLD in Northern Ostrobothnia and indicates that this population may have its own genetic features. There may be other, still unknown genetic factors to be discovered, that explain familial diseases in the region.

There is a paucity of studies, clinical and experimental, attesting to the benefit of cerebral perfusion pressure (CPP) directed pressor therapy following traumatic brain injury (TBI). The current study evaluates this therapy in a swine model of TBI and hypotension. Forty-five anesthetized and ventilated swine received TBI followed by a 45% blood volume bleed. After 1 h, all animals were resuscitated with 0.9% sodium chloride equal to three times the shed blood volume. The experimental group (PHE) received phenylephrine to maintain CPP > 80 mm Hg; the control group (SAL) did not. Outcomes in the first phase (n = 33) of the study were as follows: cerebro-venous oxygen saturation (S cvO2), cerebro-vascular carbon dioxide reactivity (δScvO2), and brain structural damage (β-amyloid precursor protein [βAPP] immunoreactivity). In the second phase (n = 12) of the study, extravascular blood free water (EVBFW) was measured in the brain and lung. After resuscitation, intracranial and mean arterial pressures were >15 and >80 mm Hg, respectively, in both groups. CPP declined to 64 ± 5 mm Hg in the SAL group, despite fluid supplements. CPP was maintained at >80 mm Hg with pressors in the PHE group. PHE animals maintained better ScvO2 (p < 0.05 at 180, 210, 240, 270, and 300 min post-TBI). At baseline, 5% CO2 evoked a 16 ± 4% increase in ScvO2, indicating cerebral vasodilatation and luxury perfusion. By 240 min, this response was absent in SAL animals and preserved in PHE animals (p < 0.05). Brain EVBFW was higher in SAL animals; however, lung EVBFW was higher in PHE animals. There was no difference in βAPP immunoreactivity between the SAL and PHE groups (p > 0.05). In this swine model of TBI and hypotension, CPP directed pressor therapy improved brain oxygenation and maintained cerebro-vascular CO2 reactivity. Brain edema was lower, but lung edema was greater, suggesting a higher propensity for pulmonary complications.

Neurological disorders are the biggest concern globally and ageing contributes in worsening the disease scenarios. In AD or AD like diseases, there is abnormal accumulation of extracellular amyloid beta produced due to abnormal processing of the transmembrane amyloid precursor protein, by β and γ-secretases. It spreads in the cortical and limbic regions of the brain leading to neuronal toxicity, impairment in memory and neurological functions. Aβ deposition in the CNS is common in aging HIV patients. Neurotoxic protein Tat, results in increased Aβ in combination with drugs of abuse cocaine. We examined the role of Withaferin A, against Aβ induced neurotoxicity. Our in-vitro dose optimization study demonstrates that lower concentrations (0.5–2 μM) of WA significantly reduce the Aβ40, without inducing cytotoxicity in the APP plasmid transfected SH-SY5Y cells (SHAPP). We demonstrate that Aβ secretion is increased in the presence of Tat (50 ng/ml) and coc (0.1 μM), WA reduces the Tat and coc induced increase in Aβ40. Additionally, we studied the role of WA against NF-kB mediated neuroinflammation, and observed that WA inhibits the expression of NFkB2 and RELA transcription factors, which play a major role in the expression of inflammatory chemokines. Further, to address the issue of minimal drug bioavailability in the CNS, we developed the WA loaded liposomal nanoformulation (WA-LNF) and characterized its size (499+/-50nm), toxicity and drug binding efficacy (28%). Our in-vitro 3D BBB transmigration of WA-LNF demonstrated ~40% transmigration efficiency. Furthermore, it was imperative for us to understand the mechanism of action of WA, therefore we studied the molecular mechanism of interaction of WA with Aβ protein by in-silico molecular dynamics simulations. We demonstrated that WA binds to the middle region of Aβ protein and the amino acid motif involved were FAEDVGS highlighting the mid-region Aβ capture by WA. 3 Hydrogen bonds were formed between WA and the amino acids, ASN17, GLY15 and SER16. This study reports WA as a potent neuroprotectant against amyloid induced neurotoxicity. Our study may have an immense therapeutic potential to target Aβ in the CNS, in the ageing patients and/or PLWH and/or ageing drug abusers.

A doença de Alzheimer (DA) é uma doença neurodegenerativa caracterizada pelo declínio progressivo da memória e de outras funções cognitivas, acometendo, sobretudo, indivíduos idosos. A anormalidade do metabolismo da proteína precursora do amilóide (APP) e a hiperfosforilação da proteína TAU são processos celulares característicos desta doença. A enzima glicogênio sintase quinase 3B (GSK3B) é altamente expressa no sistema nervoso central e apresenta grande importância na regulação da plasticidade neuronal e também nos mecanismos de sobrevivência celular. Estudos têm associado a GSK3B aos mecanismos que levam à formação das placas senis e dos emaranhados neurofibrilares na DA. Diante da dificuldade para se estabelecer com precisão o diagnóstico clínico da DA, sobretudo nas fases iniciais da doença, a identificação de biomarcadores se torna particularmente importante, principalmente em tecidos periféricos. Este trabalho avaliou, por meio de dois preparos distintos, dois possíveis candidatos a marcadores bioquímicos para a DA. Em plaquetas de pacientes com DA, comprometimento cognitivo leve (CCL) e idosos saudáveis, determinamos: (1) a razão de APP (APPr), que consiste na proporção entre fragmentos de 130kDa e 110kDa da APP; e (2) a expressão das formas fosforilada (fosfo-GSK3B) e total (total-GSK3B) da enzima GSK3B, possibilitando o cálculo da razão de GSK3B (fosfo-GSK3B/total-GSK3B). Ambas as razões foram avaliadas por Western Blot utilizando anticorpos específicos. Não observamos diferença estatisticamente significante nos valores de APPr entre os três grupos estudados (p=0,847). Para o cálculo da razão de GSK3B, foram necessárias adaptações do protocolo de preparo e análise de plaquetas, prevenindo a ativação plaquetária durante o procedimento, bem como a degradação do substrato pela ação de proteases e fosfatases presentes nesta matriz biológica; deste modo foram encontradas diferenças estatisticamente significantes entre os grupos nas médias de GSK3B total e da razão de GSK3B (p=0,05 e p=0,06 respectivamente). Não foi encontrada correlação entre a razão de APP e a razão de GSK3B. Contudo, a razão de GSK3B mostrou correlação com o desempenho em testes de memória, segundo o escore na bateria cognitiva CAMCOG. Estes resultados sugerem que a razão de GSK3B em plaquetas sinaliza algumas alterações biológicas que ocorrem na progressão do CCL para a demência na DA / Alzheimer\'s disease (AD) is a neurodegenerative disease characterized by progressive decline of memory and other cognitive functions, affecting mainly elderly. The abnormal metabolism of amyloid precursor protein (APP) and protein TAU hyperphosphorylation are cellular hallmarks of this disease. Glycogen synthase kinase 3B (GSK3B) is an enzyme highly expressed in the central nervous system and of great importance in the regulation of neuronal plasticity and also the mechanisms of cell survival. Studies have associated GSK3B with the mechanisms that lead to the formation of senile plaques and neurofibrillary tangles in AD. Given the difficulty to establish the precise clinical diagnosis of AD, especially in the early stages of the disease, identification of biomarkers is particularly important, especially in peripheral tissues. This work evaluated two candidate biochemical markers for AD, using two different preparations. We investigated the ratio between 130kDa and 110kDa APP fragments (APPr) and between phosphorylated and total GSK3B (phospho-GSK3B / total-GSK3B) in platelets of patients with AD and mild cognitive impairment (MCI), comparing their results with those from healthy older adults (controls). The expression of APP fragments and GSK3B was assessed by Western blot using specific antibodies. No statistically significant differences in APPr were found between the study groups (p=0.847). We found statistically significant differences in mean total GSK3B and GSK3B ratio across disgnostic groups (p=0.05 and p=0.06, respectively). APPr and GSK3B ratio were not correlated, but the latter parameter did correlate with the performance on memory tests, as shown by the CAMCOG sub-score. The present data indicate that platelet GSK3B ratio may indicate biological changes that occur in the MCI-AD continuum

This thesis work describes the role of Rab proteins in amyloid processing and clearance in different cell pathways. It also describes an experimental approach used to analyze the expression effects of Rab10 in amyloid beta production. Since the main theory behind neurodegeneration in Alzheimer's disease claims that high levels of amyloid beta 42 (Aβ42) molecules trigger widespread neuronal death, control of Aβ42 has been a main target in Alzheimer's disease research. In addition, several studies show increased levels of particular Rab proteins in Alzheimer's pathogenesis. However, no review consolidates current findings in neurodegeneration of Alzheimer's with Rab protein dysfunction. The first chapter of this thesis aims to address this need by providing a current review of Rab proteins associated with APP and neurodegeneration. The second chapter constitutes an experimental approach used to characterize the effects of Rab10 and Sar1A GTPases in APP and amyloid processing. We found that Rab10 expression does not affect APP production but significantly changes Aβ generation, particularly the toxic Aβ42 and Aβ42:40 ratio. On the other hand, we found no significant effect of Sar1A expression on either APP or amyloid beta generation. These findings partially confirm the work done by Kauwe et al (2015) and provide preliminary evidence for two potential targets for protective effects in neurodegeneration.

Die Bildung von Amyloidablagerungen ist ein Kennzeichen der Alzheimerschen Erkrankung. Hauptbestandteil dieser senilen Plaques sind sogenannte A beta Peptide, die durch proteolytische Prozessierung aus einem Vorläufermolekül (APP) gebildet werden. Die vorliegende Arbeit beschreibt die Klonierung des Meerschweinchen - APP. Diese cDNA-Sequenz zeigt auf DNA-Ebene eine Homologie zum Human-APP von ca. 90%, auf Proteinebene beträgt die Identität ca. 97 %. Damit wird ein weiterer experimenteller Beweis für die evolutionäre Konservierung des Amyloidvorläuferproteins in Säugetieren erbracht. APP mRNA wird in Meerschweinchen-Geweben ubiquitär exprimiert. Durch alternatives Spleißen wird ein zum Human-APP im wesentlichen ähnliches Isoformenmuster gebildet: Isoformen, welche eine Proteaseinhibitordomäne enthalten, werden dominierend in peripheren Organen exprimiert, dagegen ist im Zentralnervensystem das APP 695 mit über 60 % der Gesamttranskripte die bevorzugt exprimierte Isoform. Die klonierte cDNA des Meerschweinchen-APP wurde in prokaryontischen wie auch eukaryontischen Zellsystemen exprimiert. Dabei wurde die Eignung einer Anzahl von gegen Human-APP gewonnenen Antikörpern zur Detektion des Meerschweinchen-APP und seiner Prozessierungsprodukte gezeigt. Die Expression der neuronal dominierend exprimierten Isoform APP 695 des Meerschweinchen-APP in humanen Neuroblastom-Zellen zeigte keine Unterschiede hinsichtlich der APP-Prozessierung und A beta-Bildung im direkten Vergleich zu Human-APP 695. Die proteolytische Prozessierung des Proteins wurde durch Detektion der typischen Spaltprodukte in vivo (im Liquor) als auch in einem neu etablierten in vitro-Modell primär kultivierter neuronaler Zellen untersucht. Diese Zellkulturen wurden zunächst immunhistochemisch und biochemisch charakterisiert und als "mixed brain"-Typ mit einem hohen neuronalen Anteil beschrieben. Die Prozessierung des endogenen Meerschweinchen-APP in kultivierten Zellen führt dabei zur Bildung und Akkumulation aggregationsfähiger A beta - Peptide. Zur Detektion dieser Peptide wurde ein sensitiver Nachweis durch Western-Blot etabliert. Es wird damit ein Modellsystem für in vitro-Untersuchungen vorgeschlagen, welches ein Studium der Expression und Prozessierung des Amyloidvorläuferproteins unter angenähert physiologischen Bedingungen ermöglicht. / A beta peptides, the major component of neuritic plaques found in the brains of patients with Alzheimer’s disease, are derived by proteolytic processing from a larger precursor molecule (amyloid precursor protein - APP). A combination of PCR methods was used to clone and sequence APP cDNA from guinea pig (Cavia porcellus). Guinea pig APP exhibits extensive similarities to human APP in terms of primary structure, mRNA expression of differentially spliced isoforms as shown by Northern blot and RT-PCR analysis as well as proteolytic processing to amyloidogenic A beta peptides. In contrast to rat and mouse APP, guinea pig APP - recombinantly expressed in human neuroblastoma-cells - was processed indistinguishable from human APP thus excluding intrinsic sequence-specific factors influencing processing. Further studies were performed using newly established primary cell cultures of guinea pig neurons. Refined methods have been used to detect and characterize major proteolytic processing products of APP in vitro and in vivo. In conclusion, guinea pigs provide a model to study expression and processing of APP that closely resembles the physiological situation in humans and should, therefore, be important in elucidating potential strategies to prevent amyloid formation in Alzheimers Disease.

Thesis (Ph. D.)--University of Oklahoma. / Bibliography: leaves 122-136.

A doença de Alzheimer (DA) é uma doença neurodegenerativa caracterizada pelo declínio progressivo da memória e de outras funções cognitivas, acometendo, sobretudo, indivíduos idosos. A anormalidade do metabolismo da proteína precursora do amilóide (APP) e a hiperfosforilação da proteína TAU são processos celulares característicos desta doença. A enzima glicogênio sintase quinase 3B (GSK3B) é altamente expressa no sistema nervoso central e apresenta grande importância na regulação da plasticidade neuronal e também nos mecanismos de sobrevivência celular. Estudos têm associado a GSK3B aos mecanismos que levam à formação das placas senis e dos emaranhados neurofibrilares na DA. Diante da dificuldade para se estabelecer com precisão o diagnóstico clínico da DA, sobretudo nas fases iniciais da doença, a identificação de biomarcadores se torna particularmente importante, principalmente em tecidos periféricos. Este trabalho avaliou, por meio de dois preparos distintos, dois possíveis candidatos a marcadores bioquímicos para a DA. Em plaquetas de pacientes com DA, comprometimento cognitivo leve (CCL) e idosos saudáveis, determinamos: (1) a razão de APP (APPr), que consiste na proporção entre fragmentos de 130kDa e 110kDa da APP; e (2) a expressão das formas fosforilada (fosfo-GSK3B) e total (total-GSK3B) da enzima GSK3B, possibilitando o cálculo da razão de GSK3B (fosfo-GSK3B/total-GSK3B). Ambas as razões foram avaliadas por Western Blot utilizando anticorpos específicos. Não observamos diferença estatisticamente significante nos valores de APPr entre os três grupos estudados (p=0,847). Para o cálculo da razão de GSK3B, foram necessárias adaptações do protocolo de preparo e análise de plaquetas, prevenindo a ativação plaquetária durante o procedimento, bem como a degradação do substrato pela ação de proteases e fosfatases presentes nesta matriz biológica; deste modo foram encontradas diferenças estatisticamente significantes entre os grupos nas médias de GSK3B total e da razão de GSK3B (p=0,05 e p=0,06 respectivamente). Não foi encontrada correlação entre a razão de APP e a razão de GSK3B. Contudo, a razão de GSK3B mostrou correlação com o desempenho em testes de memória, segundo o escore na bateria cognitiva CAMCOG. Estes resultados sugerem que a razão de GSK3B em plaquetas sinaliza algumas alterações biológicas que ocorrem na progressão do CCL para a demência na DA / Alzheimer\'s disease (AD) is a neurodegenerative disease characterized by progressive decline of memory and other cognitive functions, affecting mainly elderly. The abnormal metabolism of amyloid precursor protein (APP) and protein TAU hyperphosphorylation are cellular hallmarks of this disease. Glycogen synthase kinase 3B (GSK3B) is an enzyme highly expressed in the central nervous system and of great importance in the regulation of neuronal plasticity and also the mechanisms of cell survival. Studies have associated GSK3B with the mechanisms that lead to the formation of senile plaques and neurofibrillary tangles in AD. Given the difficulty to establish the precise clinical diagnosis of AD, especially in the early stages of the disease, identification of biomarkers is particularly important, especially in peripheral tissues. This work evaluated two candidate biochemical markers for AD, using two different preparations. We investigated the ratio between 130kDa and 110kDa APP fragments (APPr) and between phosphorylated and total GSK3B (phospho-GSK3B / total-GSK3B) in platelets of patients with AD and mild cognitive impairment (MCI), comparing their results with those from healthy older adults (controls). The expression of APP fragments and GSK3B was assessed by Western blot using specific antibodies. No statistically significant differences in APPr were found between the study groups (p=0.847). We found statistically significant differences in mean total GSK3B and GSK3B ratio across disgnostic groups (p=0.05 and p=0.06, respectively). APPr and GSK3B ratio were not correlated, but the latter parameter did correlate with the performance on memory tests, as shown by the CAMCOG sub-score. The present data indicate that platelet GSK3B ratio may indicate biological changes that occur in the MCI-AD continuum

Mutations of three genes encoding amyloid precursor protein (APP), presenilin-1 (PSEN1), and presenilin-2 (PSEN2) have been shown to reliably result in familial early-onset Alzheimer's disease (FAD); a rare, but catastrophic, subtype of Alzheimer's disease (AD) marked by symptom emergence before age 65 as well as accelerated cognitive deterioration. The current study represents the first known meta-analysis on the association of APP, PSEN1 or PSEN2 on neurocognitive variables. A total of 278 FAD mutation-carriers (FAD-MC) and 284 cognitively healthy non-mutation-carriers (NC) across 10 independent investigations meeting inclusion criteria were chosen for the current meta-analysis (random effects design). Findings revealed an overarching trend of poorer performance by FAD-MC individuals compared to NC individuals across the majority of cognitive domains identified. Significant differences in effect sizes suggested FAD-MC individuals exhibited worse performance on measures of attention, explicit memory, fluency, primary memory, verbal, and visuospatial functioning. Findings indicative of differential sensitivity to cognitive domain impairments across FAD-MC and NC groups inform neuropsychological descriptions of individuals in preclinical phases of FAD.