Focused ultrasound (FUS) can temporarily increase blood-brain barrier (BBB) permeability and locally deliver therapeutic agents to the brain. To date, applications of FUS for treatment of Alzheimer’s disease (AD) have not been explored. Here, I propose that FUS can facilitate a rapid reduction in amyloid-β peptide (Aβ) pathology in a mouse model of AD. Firstly, FUS was used to enhance delivery of an antibody directed against Aβ, which aggregates and forms extracellular plaques. FUS mediated the delivery of antibodies to the targeted right cortex by 4 hours post-treatment and antibodies remained bound to Aβ plaques for 4 days. At 4 days post-treatment, stereological quantification of plaque burden demonstrated a significant reduction of 23%. Secondly, FUS treatment alone resulted in a significant reduction in plaque load (13%). I then investigated effects of FUS that may contribute to Aβ plaque reduction, specifically the delivery of endogenous antibodies to the brain and, activation of microglia and astrocytes. Endogenous immunoglobulin was found bound to plaques within the treated cortex at 4 days post-FUS. Western blot analysis confirmed that immunoglobulin levels were increased significantly. Further, FUS led to a time-dependent increase in glial response. The expression of ionized calcium-binding adaptor molecule 1, a marker of phagocytic microglia, was increased at 4 hours and 4 days, and it was resolved by 15 days. Astrocytes had a slightly delayed response, with an increase in the expression of glial fibrillary acidic protein at 4 days, which declined by 15 days. After 4 days, microglia and astrocytes had significantly greater volumes and surface areas, signifying enhanced activation in the FUS-treated cortex, without an apparent increase in cell count. Co-localization of Aβ within activated glia revealed a significant increase in Aβ internalization following FUS. In conclusion, it was demonstrated that the delivery of exogenous antibodies by FUS, and FUS alone can lead to plaque reduction. Mechanisms by which FUS alone reduces plaque load may include entry of endogenous antibodies to the brain and the induction of a transient glial response. This work details acute effects of FUS that highlight the promise of this delivery method for AD treatment.

Submitted by MARCIA ROVADOSCHI (marciar@unifra.br) on 2018-08-16T14:24:58Z No. of bitstreams: 2 license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) Dissertacao_FrancineRodriguesIaniski.pdf: 3871772 bytes, checksum: ea06b31c8ed51f3bf9a88cbfb0d4956c (MD5) / Made available in DSpace on 2018-08-16T14:24:58Z (GMT). No. of bitstreams: 2 license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) Dissertacao_FrancineRodriguesIaniski.pdf: 3871772 bytes, checksum: ea06b31c8ed51f3bf9a88cbfb0d4956c (MD5) Previous issue date: 2011-12-02 / Alzheimer's disease (AD) is a chronic neurodegenerative pathologic process associated with aging. This disease causes cognition deterioration and memory loss. The formation of senile plaques containing amyloid-β peptide (aβ) is the main characteristic of this disease. Also, AD related with the inflammation and oxidative stress. The lack of drugs used in the prevention and treatment of AD has stimulated the search for new agents that may represent a novel therapeutic alternative. In the present study, we investigated the beneficial effect of meloxicam-loaded nanocapsules in a model of AD induced by intracerebroventricular (i.c.v.) injection of aβ peptide (fragment 25-35) in mice, comparing the effect with free meloxicam. Mice were divided into six groups: (I) control, (II) aβ, (III) Nano, (IV) Free, (V) Nano + aβ and (VI) Free + aβ. Mice were treated with meloxicam-loaded nanocapsules (5 mg/kg, by gavage), free-meloxicam (5 mg/kg, by gavage) or blank nanocapsules. Thirty minutes after treatments, aβ (3 nmol) or filtered water were i.c.v. injected (day 1). Learning and memory were assessed with the Morris water-maze and step-down-type passive-avoidance tasks at the days 4–7 and 7–8 after the aβ injection, respectively. At the end of the experimental protocol, animals were died and brains were removed for determination of reactive species (RS) and non-protein thiols (NPSH) levels, and superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx), glutathione reductase (GR), glutathione S-transferase (GST) activities. The results demonstrated that aβ injection caused learning and memory deficits in mice, which were verified using the Morris water-maze and step-down-type passiveavoidance tasks. Furthermore, this study showed that oxidative stress was increased in mice that received aβ. The most important findings of the present study was that meloxicam-loaded nanocapsules protected the learning and memory impairments induced by aβ. Moreover, meloxicam-loaded nanocapsules also protected against the increase of oxidative stress. However, free-meloxicam did not have protective effect. All these findings support the beneficial role of meloxicam-loaded nanocapsules in a model of AD induced by aβ. We can suggest that nanocapsules favor the passage of meloxicam through the blood-brain barrier and entry of the drug in the central nervous system. / A doença de Alzheimer (DA) é um processo patológico neurodegenerativo crônico associado ao envelhecimento. Essa patologia ocasiona deterioração da cognição e perda da memória. A formação de placas senis contendo o peptídeo β-amiloide (βa) é a principal característica dessa doença, que também está associada à inflamação e ao estresse oxidativo. A falta de fármacos empregados na prevenção e no tratamento da DA tem estimulado a pesquisa por novos agentes que possam representar uma inovadora alternativa terapêutica. No presente estudo, investigamos o efeito benéfico das nanocápsulas contendo meloxicam sobre o déficit de aprendizagem e de memória em um modelo da DA induzido pela injeção intracerebroventricular (i.c.v.) do peptídeo βa (fragmento 25-35) nos camundongos, comparando o efeito com o fármaco na forma livre. Os camundongos foram divididos em seis grupos: (I) Controle, (II) βa, (III) Nano, (IV) Livre, (V) Nano + βa, (VI) Livre + βa. Os camundongos foram pré-tratados com as nanocápsulas contendo meloxicam (5 mg/kg, por gavagem), ou com o fármaco na forma livre (5 mg/kg, por gavagem), ou com as nanocápsulas brancas. Trinta minutos após os tratamentos, foram injetados i.c.v. o peptídeo βa (3 nmol) ou água filtrada (dia 1). A aprendizagem e a memória foram avaliadas através dos testes do labirinto aquático de Morris e da esquiva passiva, nos dias 4-7 e 7-8 após a injeção do peptídeo βa, respectivamente. No final dos testes comportamentais, os animais foram mortos e os cérebros removidos para a determinação dos níveis de espécies reativas (ER) e tióis nãoproteicos (SHNP), e a atividade das enzimas superóxido dismutase (SOD), catalase (CAT), glutationa peroxidase (GPx), glutationa redutase (GR) e glutationa S-transferase (GST). Os resultados demonstraram, através dos testes do labirinto aquático de Morris e da esquiva passiva, que a injeção i.c.v. do peptídeo βa causou um déficit na aprendizagem e na memória dos camundongos. Além disso, esse estudo demonstrou que o estresse oxidativo foi aumentado nos camundongos que receberam a injeção i.c.v. do peptídeo βa. Os achados mais importantes desse estudo foram que as nanocápsulas contendo meloxicam protegeram o déficit de aprendizado e de memória induzidas pela injeção i.c.v. do peptídeo βa, assim como foram capazes de proteger contra o aumento do estresse oxidativo. No entanto, o meloxicam na forma livre não apresentou esse efeito protetor. Todos esses achados reforçam o papel benéfico do meloxicam nanoencapsulado em um modelo da DA induzido pela injeção i.c.v. do peptídeo βa, sugerindo que as nanocápsulas favorecem a passagem do meloxicam através da barreira hematoencefálica (BHE) e a entrada do fármaco no sistema nervoso central (SNC)

Alzheimer’s disease (AD) is a neurodegenerative disorder characterized by two major pathological hallmarks, amyloid plaques and neurofibrillary tangles. The accumulation of amyloid-β protein (Aβ) is an early event associated with synaptic and mitochondrial damage in AD. Therefore, molecular pathways underlying the neurotoxicity and generation of Aβ represent promising therapeutic targets for AD. Recent studies have shown that actin severing protein, Cofilin plays an important role in synaptic remodeling, mitochondrial dysfunction, and AD pathogenesis. However, whether Cofilin is an essential component of AD pathogenesis and how Aβ induced neurotoxicity impinges its signals to Cofilin are unclear. In my dissertation studies, we found Aβ oligomers bind with intermediate activation conformers of β1-integrin to induce the loss of surface β1-integrin and activation of Cofilin via Slingshot homology-1 (SSH1) activation. Specifically, conditional loss of β1-integrin prevented Aβ induced Cofilin activation, and allosteric modulation or activation of β1-integrin significantly reduced Aβ binding to neurons and mitigated Aβ42-induced reactive oxygen species (ROS) generation, mitochondrial dysfunction, synaptic proteins depletion, and apoptosis. Furthermore, we found that SSH1 reduction, which mitigated Cofilin activation, prevented Aβ-induced mitochondrial Cofilin translocation and apoptosis, while AD brain mitochondria contained significantly increased activated/oxidized Cofilin. In mechanistic support in vivo, we demonstrated that APP transgenic mice brains contain decreased SSH/Cofilin and SSH1/14-3-3 complexes which indicates that SSH-Cofilin activation occurred by releasing of SSH from 14-3-3. We also showed that genetic reduction in Cofilin rescues APP/Aβ-induced synaptic protein loss and gliosis, as well as impairments in synaptic plasticity and contextual memory in vivo. Our lab previously found that overexpression of the scaffolding protein RanBP9 increases Aβ production in cell lines and in transgenic mice, while promoting Cofilin activation and mitochondrial dysfunction. However, how endogenous RanBP9 activates cofilin and whether endogenous RanBP9 accelerates Aβ-induced deficits in synaptic plasticity, cofilin-dependent pathology, and cognitive impairments were unknown. In my dissertation studies, we found that endogenous RanBP9 positively regulates SSH1 levels and mediates A-induced translocation of Cofilin to mitochondria. Moreover, we demonstrated that endogenous RanBP9 mediates A-induced formation of Cofilin-actin rods in primary neurons. Endogenous level of RanBP9 was also required for Aβ-induced collapse of growth cones in immature neurons and depletion of synaptic proteins in mature neurons. In vivo, we also found APP transgenic mice exhibit significantly increased endogenous RanBP9 levels and that genetic reduction in RanBP9 rescued APP/Aβ-induced synaptic protein loss, gliosis, synaptic plasticity impairments, and contextual memory deficits. These findings indicated that endogenous RanBP9 not only promotes Aβ production but also meditate Aβ induced neurotoxicity via positively regulating SSH1. Taken together, these novel findings implicate essential involvement of β1-integrin–SSH1/RanBP9–Cofilin pathway in mitochondrial and synaptic dysfunction in AD pathogenesis.

京都大学 / 0048 / 新制・課程博士 / 博士(農学) / 甲第22505号 / 農博第2409号 / 新制||農||1077(附属図書館) / 学位論文||R2||N5285(農学部図書室) / 京都大学大学院農学研究科食品生物科学専攻 / (主査)教授 保川 清, 教授 宮川 恒, 教授 入江 一浩 / 学位規則第4条第1項該当 / Doctor of Agricultural Science / Kyoto University / DGAM

京都大学 / 0048 / 新制・課程博士 / 博士(人間健康科学) / 甲第21459号 / 人健博第66号 / 新制||人健||5(附属図書館) / 京都大学大学院医学研究科人間健康科学系専攻 / (主査)教授 青山 朋樹, 教授 岡 昌吾, 教授 髙橋 良輔 / 学位規則第4条第1項該当 / Doctor of Human Health Sciences / Kyoto University / DFAM

This work investigates the influence of mutations at selected positions on the structure formation of the Alzheimer’s disease peptide amyloid β. Amyloid β is a member of the class of intrinsically disordered proteins that can aggregate into fibrils, which are characterized by a highly stable secondary structure, called cross-β structure. A central contact during fibrillation is the hydrophobic F19-L34 contact, which is located within the core of the cross-β structure. Modifications of this contact are known to influence the local molecular structure whereas the fibril morphology and the cross-β structure remain stable. In contrast, toxicity of amyloid β was completely lost for all previously investigated mutants of F19 and L34. This work characterizes the properties of this contact and answers the question what the minimally tolerated modifications are. To characterize the structure, structure formation process and biological activity of the Aβ variants a set of experiments was carried out. The local structure and dynamics were investigated using NMR experiments focusing on 13C-chemical shift changes and 1H-13C dipolar couplings, respectively. The fibril morphology and cross- β structure was verified by electron microscopy, circular dichroism spectroscopy and X-ray diffraction. Toxicity and biological activity was investigated using complementary cell culture experiments. The work was divided in three parts. First, L34 was substituted with three highly similar amino acids: the isomer isoleucine, valine that is one methylene group shorter but also a branched chain amino acid and the stereoisomer D-leucine. The L34 position proved to be important for the initiation of the structure formation, oligomer stability, fibril growth and the biological activity of amyloid β. These characteristics and properties were highly sensitive also to minor modifications but the different mutants showed no specific but qualitatively similar effects. The second part complemented previous mutation studies of the F19 position. Four new mutants were designed testing mild modification of the F19-L34 contact: phenylglycine and the homophenylalanine (S)-2-amino-4-phenyl-butyric acid change the length of the side chain, cyclohexyl-alanine eliminates the π-aromaticity of the ring system and increases the 3D steric demand, and (1-naphtyl)-alanine increases the 2D steric demand while maintaining the aromaticity. Mutations at the F19 position caused qualitatively similar effects as L34 modifications but proved to have quantitatively greater impact. Furthermore, they showed some specificity as steric constraints caused larger changes than modifications of the ring system. The third part investigates the influence of β-methylamino-L-alanine (BMAA) substitutions at positions F19, S8, and S26. The serine to BMAA substitutions were included because of their potential medical relevance. A F19BMAA substitution caused similar effects like other modifications at this position. Replacement of serine lead to a structural reorientation of the Aβ N-terminus and turn region. Furthermore, the pathways of the cell response changed from mitochondrial activity and plasma membrane integrity to apoptosis and neuronal stress reaction. Summarizing, it could be shown that, although the formation and structure of amyloid β fibrils is robust against different modifications the fibrillation kinetics, local structure and especially biological activity is highly sensitive and to some extend specific to even minor modifications.

A hallmark of Alzheimer's disease (AD) is the accumulation of insoluble senile plaques in the brain.[1] The major component of the insoluble plaques is the amyloid-β peptide (Aβ) that is produced through cleavage of the amyloid-β precursor protein (APP).[2] It is well understood that once the monomeric Aβ is generated, it has the potential to aggregate into soluble oligomers and further into insoluble fibrils. Recently it has been proposed that early oligomers are the main toxic species in the aggregation cascade.[3] However, it has been shown that the formation of toxic early oligomers is inhibited by several endogenous plasma proteins, including albumin and transferrin (Tf). In this investigation we are focusing on the mechanism of inhibition of the Aβ early oligomerization by Tf. Specifically, we have targeted the early stages of Aβ aggregation using a deletion mutant of the Aβ peptide, i.e. the Aβ12-28 fragment, which selectively stabilizes the early Aβ oligomers. Self-association of this peptide was controlled by adding-NaCl to filtered monomeric Aβ samples and the effect of Tf inhibition on these aggregates was probed by 1H relaxation NMR experiments.[4-7] Our data shows that Tf directly targets intermediary Aβ oligomers via a coating mechanism. 1. Kirkitadze, M.D., Condron, M.M. and Teplow, D.B, JMB 2001 312;1103-1119. 2. Stefan F. Lichtenthaler and Christian Haass, JCI 2004 113(10);1384-1387. 3. Necula M., Kayed R., Milton, S. and Glabe C.G, JBC 2007 282(14);10311-10324. 4. Klement K., Wieligmann K., Meinhardt J., Hortschansky P., Richter W., and Fändrich M., JMB 2007 373;1321-1333. 5. Huang H, Milojevic J, Melacini G. J Phys Chem B. 2008 112(18):5795-802. 6. Milojevic J, Esposito V, Das R, Melacini G. JACS. 2007 129(14):4282-90. 7. Milojevic J, Esposito V, Das R, Melacini G. J Phys Chem B. 2006 110(41):20664-70. / Thesis / Master of Science (MSc)

The morphology, structure, and dynamics of mature amyloid β (Aβ) fibrils formed by the Aβ variant, which is truncated at residue 11 and chemically modified by enzymatic pyroglutamate formation (pGlu11-Aβ(11–40)), was studied along with the investigation of the toxicity of these Aβ variants to neurons and astrocytes. The fibrils of pGlu11-Aβ (11–40) were more toxic than wildtype Aβ (1–40) and the longer pGlu3-Aβ (3–40) especially at higher concentration, whereas the overall morphology was quite similar. The secondary structure of pGlu11-Aβ (11–40) fibrils shows the typical two β-strands connected by a short turn as known for mature fibrils of Aβ (1–40) and also pGlu3-Aβ (3–40). Further insights into tertiary contacts exhibit some similarities of pGlu11-Aβ (11–40) fibrils with wildtype Aβ (1–40), but also a so far not described contact between Gly25 and Ile31. This highlights the biological importance of chemical modifications on the molecular structure of Aβ.

The formation of amyloid-β (Aβ) peptides is causally involved in the development of Alzheimer’s disease (AD). A significant proportion of deposited Aβ is N-terminally truncated and modified at the N-terminus by a pGlu-residue (pGlu-Aβ). These forms show enhanced neurotoxicity compared to full-length Aβ. Although the truncation may occur by aminopeptidases after formation of Aβ, recently discovered processing pathways of amyloid-β protein precursor (AβPP) by proteases such as meprin β may also be involved. Here, we assessed a role of meprin β in forming Aβ3 -40/42 , which is the precursor of pGlu-Aβ3 -40/42 generated by glutaminyl cyclase (QC). Similar to QC, meprin β mRNA is significantly upregulated in postmortem brain from AD patients. A histochemical analysis supports the presence of meprin β in neurons and astrocytes in the vicinity of pGlu-Aβ containing deposits. Cleavage of AβPP-derived peptides by meprin β in vitro results in peptides Aβ1 -x , Aβ2 -x , and Aβ3 -x . The formation of N-truncated Aβ by meprin β was also corroborated in cell culture. A subset of the generated peptides was converted into pGlu-Aβ3 -40 by an addition of glutaminyl cyclase, supporting the preceding formation of Aβ3 -40 . Further analysis of the meprin β cleavage revealed a yet unknown dipeptidyl-peptidase–like activity specific for the N-terminus of Aβ1 -x . Thus, our data suggest that meprin β contributes to the formation of N-truncated Aβ by endopeptidase and exopeptidase activity to generate the substrate for QC-catalyzed pGlu-Aβ formation.

Fibril formation of amyloid β (Aβ) peptides is one of the key molecular events connected to Alzheimer's disease. The pathway of formation and mechanism of action of Aβ aggregates in biological systems is still object of very active research. To this end, systematic modifications of the Phe19-Leu34 hydrophobic contact, which has been reported in almost all structural studies of Aβ40 fibrils, helps understanding Aβ folding pathways and the underlying free energy landscape of the amyloid formation process. In our approach, a series of Aβ40 peptide variants with two types of backbone modifications, namely incorporation of (i) a methylene or an ethylene spacer group and (ii) a N-methylation at the amide functional group, of the amino acids at positions 19 or 34 was applied. These mutations are expected to challenge the inter-β-strand side chain contacts as well as intermolecular backbone β-sheet hydrogen bridges. Using a multitude of biophysical methods, it is shown that these backbone modifications lead, in most of the cases, to alterations in the fibril formation kinetics, a higher local structural heterogeneity, and a somewhat modified fibril morphology without generally impairing the fibril formation capacity of the peptides. The toxicological profile found for the variants depend on the type and extent of the modification.