The stability and aggregation properties of the GTPase domain from human SEPT4

The septins are a family of conserved proteins involved in cytokinesis and cortical organization. An increasing amount of data implicates different septins in diverse pathological conditions including neurodegenerative disorders, neoplasia and infections. Human SEPT4 is a member of this family and its tissue-specific ectopic expression profile in colorectal and urologic cancer makes it a useful diagnostic biomarker. Thermal unfolding of the GTPase domain of SEPT4 (SEPT4-G) revealed an unfolding intermediate which rapidly aggregates into amyloid-like fibers under physiological conditions. In this study, we examined the effects of protein concentration, pH and metals ions on the aggregation process of recombinant SEPT4-G using a series of biophysical techniques, which were also employed to study chemical unfolding and stability. Divalent metal ions caused significant acceleration to the rate of SEPT4-G aggregation. Urea induced unfolding was shown to proceed via the formation of a partially unfolded intermediate state which unfolds further at higher urea concentrations. The intermediate is a compact dimer which is unable to bind GTR At 1 M urea concentration, the intermediate state was plagued by irreversible aggregation at temperatures above 30 degrees C. However, higher urea concentration resulted in a marked decay of the aggregation, indicating that the partially folded structures may be necessary for the formation of these aggregates. The results presented here are consistent with the recently determined crystal structure of human septins and shed light on the aggregation properties of SEPT4 pertinent to its involvement in neurodegenerative disease. (C) 2008 Elsevier B.V. All rights reserved.

El tratamiento fisioterapéutico ¿es efectivo en el mantenimiento físico y en la reducción del declive cognitivo de las demencias?

Introduction. Dementia is a chronic and neurodegenerative disease responsible for a progressive decline of cognition and physical capacity. Objective: To verify the effectiviness of three months of physiotherapy intervention on cognitive functions and balance of institutionilized elderly people with dementia. Patients and methods. This study has comprised thirty subjects, divided in two groups: EG (experimental group) and CG (control group). EG was composed by 15 participants with mean age of 71,6 ± 8,5 years and scholarity of 4,7 ± 4,2 years. CG was formed by 15 subjects with mean age of 78,5 ± 7,6 years and scholarity of 2,2 ± 1,8 years. The sample was evaluated by means of the following instruments: Mini-Mental State Examination, Semantic Verbal Fluency test, Clock Drawing test, Berg Balance Scale and Timed Get Up and Go test. The statistical procedures were done by means of Multivariate Analisis of Variance (MANOVA), with significance of 5 % (p < 0,05). Results. The results has shown a heterogeneity of both groups related to age (F = 0,2, p < 0,05) and scholarity (F = 2,6, p < 0,05). MANOVA pointed beneficts of physiotherapy on balance (F = 7,3, p < 0,01) but not on cognitive functions (F = 0,3, p > 0,05). Conclusions. Three months of physiotherapy were effective to improuve physical capacity of institucionilized elderly people with dementia. Neverthless, it was not able to minimize cognitive decline commonly seen on dementia. The question if a prolonged intervention could propitiate cognitive beneficts remains, specially in a more homogeneous groups.

Análise citogenética como bioindicador para pacientes com diagnóstico sugestivo de Alzheimer

A doença de Alzheimer (DA) é uma doença neurodegenerativa que provoca morte neuronal e consequente perda progressiva das funções cognitivas, reduzindo as capacidades de trabalho, interferindo na relação social e no comportamento do paciente. Entre as doenças causadoras de demência, a DA é a mais incidente que as de cunho vascular, numa proporção de 4:1, respectivamente. Além das terapias farmacológicas, os métodos diagnósticos auxiliam na identificação precoce da doença auxiliando o tratamento prévio, assim diminuído a progressão da doença. Atualmente estudos citogenéticos vêm demonstrando alterações cromossômicas em portadoras da DA e podem auxiliar no diagnósticos da doença. O objetivo desse trabalho foi verificar o potencial da análise cariotípica de linfócitos do sangue periférico como bioindicador diagnostico da doença de Alzheimer. Para a realização deste trabalho, utilizamos dois grupos de mulheres com 65 anos ou mais, sendo um grupo com (10) portadoras de DA e outro grupo (10) normais. Cada indivíduo foi submetida ao questionário socioeconômico, teste de rastreio cognitivo (MEEM) e à coleta de sangue venoso para cultura de linfócitos e análise cromossômica. Nossos resultados demonstram que o grupo de mulheres portadoras da DA apresentaram elevada taxa de monossomia e trissomia em relação às mulheres normais. Através de estudo de anamnese via questionário, verificamos o estilo de vida de ambos os grupos. Quando comparado a relação das alterações cromossômicas com o nível cognitivo do grupo DA, nós evidenciamos uma tendência inversamente proporcional entre o número de monossomia/trissomia e o desempenho cognitivo. Outro aspecto de nossas análises foi o papel de cada cromossomo ligado à DA. Os cromossomos 1, 14 e 21 não apresentaram trissomia e na verificação da frequência de monossomia, cada cromossomo possui frequência abaixo de 3 % de aneuploidia, ou seja, os cromossomos estudados não possuem uma grande representatividade nas alterações cromossômicas encontradas no estudo.

Sintomas não motores na doença de Parkinson: modelo de lesão intraestriatal por 6-OHDA em camundongos

A doença de Parkinson (DP) é a segunda doença neurodegenerativa mais comum em idosos, caracterizada pela neurodegeneração de neurônios dopaminérgicos da substância negra (SN), com etiologia não claramente estabelecida, entretanto as causas podem estar associadas a exposição de toxinas ambientais e fatores genéticos. Os processos patológicos envolvidos na DP são disfunção mitocondrial, estresse oxidativo, inflamação e excitotoxicidade. A sintomatologia da DP são alterações motoras, cognitivas e autonômicas. Contudo, poucos estudos analisam os sintomas não-motores da DP, principalmente em modelos animais. Nesse contexto o objetivo deste trabalho foi avaliar sintomas não-motores da DP em modelo animal com lesão provocada pela neurotoxina 6-hidroxidopamina com duas doses diferentes, injetadas bilateralmente no estriado. Para alcançar nossos objetivos realizamos os testes de campo aberto, apomorfina, labirinto aquático de Morris e testes de discriminação olfativa, além de análises histológicas. Nossos resultados mostraram alterações motoras, déficits de memória e aprendizado, associadas a diminuição de células dopaminérgicas na SN, neurônios estriatais e neurônios da região hipocampal CA1. Dessa forma, esse modelo para os sintomas não-motores da DP pode ser utilizado para a compreensão dos mecanismos que envolvem a doença, assim como para avaliar medidas terapêuticas que possam retardar ou interromper a progressão da DP.

Efeitos do exercício físico multimodal nas concentrações plasmáticas de biomarcadores, funções cognitivas e funcionalidade em pacientes com doença de Alzheimer

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Atividade física para promoção da saúde de idosos com doença de Alzheimer e seus cuidadores

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Vascular dementia

The epidemic growth of dementia causes great concern for the society. It is customary to consider Alzheimer's disease (AD) as the most common cause of dementia, followed by vascular dementia (VaD). This dichotomous view of a neurodegenerative disease as opposed to brain damage caused by extrinsic factors led to separate lines of research in these two entities. Indeed, accumulated data suggest that the two disorders have additive effects and probably interact; however it is still unknown to what degree. Furthermore, epidemiological studies have shown "vascular" risk factors to be associated with AD. Therefore, a clear distinction between AD and VaD cannot be made in most cases, and is furthermore unhelpful. In the absence of efficacious treatment for the neurodegenerative process, special attention must be given to the vascular component, even in patients with presumed mixed pathology. Symptomatic treatment of VaD and AD is similar, although the former is less effective. For prevention of dementia it is important to treat all factors aggressively, even in stroke survivors who do not show evidence of cognitive decline. In this review, we will give a clinical and pathological picture of the processes leading to VaD and discuss its interaction with AD. (c) 2012 Elsevier B.V. All rights reserved.

Genotyping of polymorphisms in the prnp gene in Santa Ines sheep in the State of Sao Paulo, Brazil

Santos C.R., Mori E., Leao D.A. & Maiorka P.C. 2012. [Genotyping of polymorphisms in the prnp gene in Santa Ines sheep in the State of Sao Paulo, Brazil.] Genotipagem de polimorfismos no gene prnp em ovinos Santa Ines no Estado de Sao Paulo. Pesquisa Veterinaria Brasileira 32(3):221-226. Laboratorio de Neuropatologia Experimental e Comparada, Departamento de Patologia, Faculdade de Medicina Veterinaria e Zootecnia, Universidade de Sao Paulo, Av. Prof. Dr. Orlando Marques de Paiva 87, Cidade Universitaria, Sao Paulo, SP 05508-270, Brazil. E-mail: caio-patologia@usp.br Enzootic paraplexia or scrapie is a fatal neurodegenerative disease affecting mainly sheep and rarely goats. The disease is influenced by polymorphisms at codons 136, 154 and 171 of prnp gene that encodes the prion protein. The animals may be susceptible or resistant to the development of the disease according to the allelic sequences observed in these codons. In Brazil there were only cases of scrapie in imported animals, therefore the country is considered free of the disease. This study performed the genotyping of different polymorphisms associated to the development of scrapie. Then, based on these findings the animals were categorized in resistant and susceptible. A total of 118 samples were sequenced from the Santa Ines sheep raised on properties located in the State of Sao Paulo. From these samples, 6 alleles and 11 genotypes were identified (ARQ / ARQ, ARR / ARQ, ARQ / AHQ, ARQ / VRQ, AHQ / AHQ, ARR / ARR, ARR / AHQ, VRQ / VRQ, ARQ / TRQ, TRR / TRR, TRQ / TRQ), the genotype ARQ / ARQ presented a frequency of 56.7%. It was also detected the presence of tyrosine at codon 136, which may be considered a rare observation, since there is no report regarding Santa Ines breeding presenting this polymorphism. These results showed the great genetic variability in Santa Ines in Sao Paulo and only 1,69% of the genotypes observed are extremely resistant to scrapie. These data demonstrate that the Santa Ines sheep can be considered potentially susceptible to scrapie.

IL-12p40 Deficiency Leads to Uncontrolled Trypanosoma cruzi Dissemination in the Spinal Cord Resulting in Neuronal Death and Motor Dysfunction

Chagas' disease is a protozoosis caused by Trypanosoma cruzi that frequently shows severe chronic clinical complications of the heart or digestive system. Neurological disorders due to T. cruzi infection are also described in children and immunosuppressed hosts. We have previously reported that IL-12p40 knockout (KO) mice infected with the T. cruzi strain Sylvio X10/4 develop spinal cord neurodegenerative disease. Here, we further characterized neuropathology, parasite burden and inflammatory component associated to the fatal neurological disorder occurring in this mouse model. Forelimb paralysis in infected IL-12p40KO mice was associated with 60% (p<0.05) decrease in spinal cord neuronal density, glutamate accumulation (153%, p<0.05) and strong demyelization in lesion areas, mostly in those showing heavy protein nitrosylation, all denoting a neurotoxic degenerative profile. Quantification of T. cruzi 18S rRNA showed that parasite burden was controlled in the spinal cord of WT mice, decreasing from the fifth week after infection, but progressive parasite dissemination was observed in IL-12p40KO cords concurrent with significant accumulation of the astrocytic marker GFAP (317.0%, p<0.01) and 8-fold increase in macrophages/microglia (p<0.01), 36.3% (p<0.01) of which were infected. Similarly, mRNA levels for CD3, TNF-alpha, IFN-gamma, iNOS, IL-10 and arginase I declined in WT spinal cords about the fourth or fifth week after infection, but kept increasing in IL-12p40KO mice. Interestingly, compared to WT tissue, lower mRNA levels for IFN-gamma were observed in the IL-12p40KO spinal cords up to the fourth week of infection. Together the data suggest that impairments of parasite clearance mechanisms in IL-12p40KO mice elicit prolonged spinal cord inflammation that in turn leads to irreversible neurodegenerative lesions.

Genetic and environmental factors associated with the risk of cognitive decline and dementia

AD is the most common age related neurodegenerative disease in the industrialized world. Clinically AD is defined as a progressing decline of cognitive functions. Neuropathologically, AD is characterized by the aggregation of b-amyloid (Ab) peptide in the form of extracellular senile plaques, and hyperphosphorlylated tau protein in the form of intracellular neurofibrillary tangles. These neuropathological hallmarks are often accompanied by abundant microvascular damage and pronounced inflammation of the affected brain regions. In this thesis we investigated several aspects of AD focusing on the genetic aspect. We confirmed that Alpha 1 antichymotrypsin (ACT), an acute phase protein, was associated to AD subjects, being plasma levels higher in AD cases than controls. In addition, in a GWA study we demonstrated that two different gene, Clusterin and CR1 were strongly associated to AD. A single gene association not explain such a complex disease like AD. The goal should be to created a network of genetic, phenotypic and clinical data associated to AD. We used a new algorithm, the ANNs, aimed to map variables and search for connectivity among variables. We found specific variables associated to AD like cholesterol levels, the presence of variation in HMGCR enzyme and the age. Other factors such as the BMI, the amount of HDL and blood folate levels were also associated with AD. Pathogen infections, above all viral infections, have been previously associated to AD. The hypothesis suggests that virus and in particular herpes virus could enter the brain when an individual becomes older, perhaps because of a decline in the immune system. Our new hypothesis is that the presence of SNPs in our GWA gene study results in a genetic signature that might affect individual brain susceptibility to infection by herpes virus family during aging.

Der Einfluss der Zellalterung auf die pathobiochemischen Prozesse von Morbus Alzheimer und weiteren neurodegenerativen Erkrankungen

Die am häufigsten auftretende altersassoziierte neurodegenerative Krankheit ist die Alzheimer Demenz. Ein mit entscheidender Schritt bei der Entstehung der Alzheimer Erkrankung ist wahrscheinlich die Produktion des Aβ-Peptids durch proteolytische Spaltung das Amyloid-Vorläuferproteins APP. In der vorliegenden Arbeit wurde die altersabhängige Prozessierung des Amyloid-Vorläuferproteins (APP) in Fibroblasten von Hautbiopsien von Familiärer Alzheimer-, Trisomie21 und Niemann-Pick Typ C-Krankheit untersucht. Die in dieser Arbeit verwendeten Fibroblasten wurden bis zum Erreichen des zellulären Wachstumsstopps (replikative Seneszenz) seriell passagiert und die Untersuchungen erfolgten an Zellen aufsteigender PDL. Dabei zeigte sich, dass, unabhängig von dem durch die Krankheit vorliegenden genetischen biochemischen Hintergrund, die APP-Prozessierung im Laufe der Zellalterung progressiv verringert wird. Die altersabhängig ansteigenden Cholesterinspiegel führten zu einer Reduktion der APP-Reifung und infolge dessen nahmen sowohl die intrazellulären APP-Spaltfragmente (C99, C83 und AICD) als auch die extrazellulären APP-Fragmente (sAPPα, sAPP) ab. Ebenso konnte gezeigt werden, dass die γ-Sekretase-Aktivität abnimmt. Dies war verbunden mit einem Rückgang der Proteinspiegel von Nicastrin und Presenilin, beides Komponenten des γ-Sekretase-Komplexes. Obwohl die Proteinexpression der α-Sekretase ADAM10 altersassoziiert konstant blieb, nahm die α-Sekretase-Aktivität mit steigendem Lebensalter ab. Erste Untersuchungen zeigten, dass die NAD+-abhängige Histon-Deacetylase SIRT1 eine wichtige Rolle im Bezug auf die α-Sekretase-Aktivität spielen könnte. Im Gegensatz zu den Abnahmen der α- und γ-Sekretase-Aktivitäten konnte eine erhöhte Aktivität der β-Sekretase in seneszenten Zellen beobachtet werden. Die mRNA-Menge und Proteinspiegel der ß-Sekretase BACE1 blieben dabei unverändert. Des Weiteren zeigte sich eine Zunahme der β-Sekretase-Aktivität bei Behandlung von jungen Zellen mit konditioniertem Medium seneszenter Zellen. Da sensezente Zellen einem Proliferationsstopp in der G1-Phase unterliegen, wurde der Einfluss des Zellzyklus-Inhibitors Aphidicolin auf die β-Sekretase untersucht. Hier wurde sowohl in IMR90 Fibroblasten als auch in Neuroblastoma-Zellen N2a eine Zunahme der β-Sekretase-Aktivität nach Zugabe der Inhibitoren beobachtet. Auch kommt es im Zuge der Alterung zu einer verstärkten Expression inflammatorischer Zytokine, die mit der Entstehung von Aβ-Peptiden in Verbindung gebracht werden. Deshalb wurde der Einfluss von Zytokinen auf die β-Sekretase-Aktivität untersucht. Die Zugabe von Interferon-γ und Interleukin 6 führte bei jungen IMR90-Zellen zu einem Anstieg der β-Sekretase-Aktivität, während bei alten Zellen keine Änderung zu verzeichnen war.

Longitudinal evolution of cognitive functions in patients with multiple system atrophy: a prospective study

Introduction and Background: Multiple system atrophy (MSA) is a sporadic, adult-onset, progressive neurodegenerative disease characterized clinically by parkinsonism, cerebellar ataxia, and autonomic failure. We investigated cognitive functions longitudinally in a group of probable MSA patients, matching data with sleep parameters. Patients and Methods: 10 patients (7m/3f) underwent a detailed interview, a general and neurological examination, laboratory exams, MRI scans, a cardiovascular reflexes study, a battery of neuropsychological tests, and video-polysomnographic recording (VPSG). Patients were revaluated (T1) a mean of 16±5 (range: 12-28) months after the initial evaluation (T0). At T1, the neuropsychological assessment and VPSG were repeated. Results: The mean patient age was 57.8±6.4 years (range: 47-64) with a mean age at disease onset of 53.2±7.1 years (range: 43-61) and symptoms duration at T0 of 60±48 months (range: 12-144). At T0, 7 patients showed no cognitive deficits while 3 patients showed isolated cognitive deficits. At T1, 1 patient worsened developing multiple cognitive deficits from a normal condition. At T0 and T1, sleep efficiency was reduced, REM latency increased, NREM sleep stages 1-2 slightly increased. Comparisons between T1 and T0 showed a significant worsening in two tests of attention and no significant differences of VPSG parameters. No correlation was found between neuropsychological results and VPSG findings or RBD duration. Discussion and Conclusions: The majority of our patients do not show any cognitive deficits at T0 and T1, while isolated cognitive deficits are present in the remaining patients. Attention is the cognitive function which significantly worsened. Our data confirm the previous findings concerning the prevalence, type and the evolution of cognitive deficits in MSA. Regarding the developing of a condition of dementia, our data did not show a clear-cut diagnosis of dementia. We confirm a mild alteration of sleep structure. RBD duration does not correlate with neuropsychological findings.

The characterization of the Atxn2-CAG42-knock-in mouse as a model for Spinocerebellar Ataxia Type 2

Die Ursache der neurodegenerativen Erkrankung Spinozerebelläre Ataxie Typ 2 (SCA2) ist eine expandierte Polyglutamin-Domäne im humanen ATXN2-Gen von normalerweise 22 auf über 31 CAGs. Von der Degeneration sind vorwiegend die zerebellären Purkinje Neuronen betroffen, in denen zunehmend zytoplasmatische Aggregate sichtbar werden. Auch wenn die genaue Funktion von ATXN2 und die zugrunde liegenden molekularen Mechanismen noch immer ungeklärt sind, werden ein toxischer Funktionsgewinn sowie der Verlust der normalen Proteinfunktion als mögliche Ursachen diskutiert.rnUm ein wirklichkeitsgetreues Tiermodell für die SCA2 zu haben, wurde eine knock-in Maus generiert, deren einzelnes CAG im Atxn2-Gen durch 42 CAGs ersetzt wurde. Dieses Mausmodell ist durch eine stabile Vererbung der Expansion charakterisiert. Weiterhin zeigt sie ein verringertes Körpergewicht sowie eine spät beginnende motorische Inkoordination, was dem Krankheitsbild von SCA2 entspricht. rnIm Weiteren konnte gezeigt werden, dass, obwohl die Atxn2 mRNA-Spiegel in Großhirn und Kleinhirn erhöht waren, die Menge an löslichem ATXN2 im Laufe der Zeit abnahm und dies mit einem Auftreten an unlöslichem ATXN2 korrelierte. Dieser im Kleinhirn progressive Prozess resultierte schließlich in zytoplasmatischen Aggregaten innerhalb der Purkinje Neuronen alter Mäuse. Der Verlust an löslichem ATXN2 könnte Effekte erklären, die auf einen partiellen Funktionsverlust von ATXN2 zurückzuführen sind, wobei die Aggregatbildung einen toxischen Funktionsgewinn wiederspiegeln könnte. Neben ATXN2 wurde auch sein Interaktor PABPC1 zunehmend unlöslich. Während dies im Großhirn eine Erhöhung der PABPC1 mRNA- und löslichen Proteinspiegel zur Folge hatte, konnte keine kompensatorische Veränderung seiner mRNA und zudem eine Verminderung an löslichem PABPC1 im Kleinhirn beobachtet werden. Auch PABPC1 wurde in Aggregate sequestriert. Diese Unterschiede zwischen Großhirn und Kleinhirn könnten zu der spezifischen Vulnerabilität des Kleinhirns beitragen.rnUm die Folgen auf mRNA-Prozessierung zu untersuchen, wurde ein Transkriptomprofil im mittleren sowie fortgeschrittenen Alter der Mäuse erstellt. Hierbei war eine erhöhte Expression von Fbxw8 im Kleinhirn alter Mäuse auffällig. Als Komponente eines Ubiquitin-E3-Ligase-Komplexes, hilft FBXW8 in der Degradierung von Zielproteinen und könnte somit die Toxizität des expandieren ATXN2 verringern. rnZur näheren Beschreibung der physiologischen Funktion von ATXN2, konnte in ATXN2-knock-out Mäusen gezeigt werden, dass das Fehlen von ATXN2 zu einer reduzierten globalen Proteinsyntheserate führte und somit eine Rolle als Translationsaktivator möglich erscheint. Kompensatorisch wurde eine erhöhte S6-Phosphorylierung gemessen.

Der Einfluss einer wildtypischen SOD1-Untereinheit auf Konformation und Stabilität mutanter SOD1-Heterodimere

Die Erkrankung Amyotrophe Lateralsklerose (ALS) ist gekennzeichnet durch eine progressive Degeneration der Motoneurone. Die hierdurch im Patienten hervorgerufene fortschreitende Paralyse kann von wenigen Wochen über Monate bis zu mehreren Jahren variieren. Im Durchschnitt beträgt die Krankheitsdauer 3 - 5 Jahre. Häufig führt respiratorische Insuffizienz letztendlich zum Tod des Patienten. ALS ist bis heute unheilbar. Etwa 10 % aller ALS Fälle zeigen einen familiären Hintergrund. Hiervon werden ~20 % durch Mutationen im Gen des antioxidativen Enzyms CuZnSuperoxiddismutase (SOD1) verursacht. Mehr als 150 Mutationen im Gen der SOD1 wurden bisher als Auslöser der ALS beschrieben. Durch die Mutation erlangen SOD1 Proteine zusätzliche, bisher jedoch unbekannte toxische Eigenschaften. Ein dismutaseaktives SOD1 Enzym setzt sich aus zwei SOD1 Untereinheiten zusammen. Aufgrund der autosomal dominanten Vererbung der Krankheit kann ein SOD1 Dimer im Patienten als wildtypisches Homodimer (SOD1WT‑WT), als mutantes Homodimer (SOD1mut‑mut) oder als Heterodimer (SOD1mut-WT) vorliegen. In dieser Arbeit wurden SOD1 Dimere untersucht, deren Untereinheiten kovalent miteinander verbunden waren. Es konnte gezeigt werden, dass sich die biochemischen und biophysikalischen Eigenschaften mutanter SOD1 Heterodimere von mutanten SOD1 Homodimeren mit der gleichen Mutation unterschieden. Mutante SOD1 Heterodimere wiesen eine höhere Resistenz gegen einen Abbau durch Proteinase K auf als ihre korrespondierenden Homodimere. Des Weiteren verminderte eine wildtypische Untereinheit die Interaktion der Heterodimere mit Antikörpern gegen fehlgefaltete SOD1. Die Sekundärstruktur der mutanten SOD1 Heterodimere unterschied sich hierbei nicht auffällig von der Sekundärstruktur ihrer zugehörigen Homodimere. Eine wildtypische Untereinheit verändert somit möglicherweise die Tertiärstruktur seiner kovalent gebundenen mutanten SOD1 Untereinheit und/oder die Konformation des gesamten Dimerproteins. Durch die Mutation bedingte Missfaltungen werden hierdurch reduziert, die Stabilität des Dimers gegenüber proteolytischem Abbau erhöht. Nach der Aufreinigung der Dimerproteine wies das mutanten SOD1 Heterodimer diese Eigenschaften nicht mehr auf. Ein potentieller Interaktionspartner, der eine verminderte Fehlfaltung des Heterodimers oder eine verstärkte Missfaltung des Homodimers fördert, könnte hierbei während der Aufreinigungsprozedur verlorengegangen sein. Die hier nachgewiesene Konformationsänderung könnte über einen Prionen-ähnlichen Effekt übertragen werden und die erhöhte Stabilität das mutante, toxische Protein vor Degradation schützen. Dies korreliert mit der Beobachtung früherer Studien, in denen nachgewiesen wurde, dass mutante SOD1 Heterodimere potentiell toxischer sind als ihre korrespondierenden Homodimere.

DEFINITION OF THE LANDSCAPE OF CHROMATIN STRUCTURE AT THE FRATAXIN GENE IN FRIEDREICH’S ATAXIA

Friedreich’s ataxia (FRDA) is caused by the transcriptional silencing of the frataxin (FXN) gene. FRDA patients have expansion of GAA repeats in intron 1 of the FXN gene in both alleles. A number of studies demonstrated that specific histone deacetylase inhibitors (HDACi) affect either histone modifications at the FXN gene or FXN expression in FRDA cells, indicating that the hyperexpanded GAA repeat may facilitate heterochromatin formation. However, the correlation between chromatin structure and transcription at the FXN gene is currently limited due to a lack of more detailed analysis. Therefore, I analyzed the effects of the hyperexpanded GAA repeats on transcription status and chromatin structure using lymphoid cell lines derived from FRDA patients. Using chromatin immunoprecipitation and quantitative PCR, I observed significant changes in the landscape of histone modifications in the vicinity of the GAA tract in FRDA cells relative to control cells. Similar epigenetic changes were observed in GFP reporter construct containing 560 GAA repeats. Further, I detected similar levels of FXN pre-mRNA at a region upstream of hyperexpanded GAA repeats in FRDA and control cells, indicating similar efficiency of transcription initiation in FRDA cells. I also showed that histone modifications associated with hyperexpanded GAA repeats are independent of transcription progression using the GFP reporter system. My data strongly support evidence that FXN deficiency in FRDA patients is consequence of defective transition from initiation to elongation of FXN transcription due to heterochromatin-like structures formed in the proximity of the hyperexpanded GAAs.