| 1. |
- Klingstedt, Therése, et al.
(författare)
-
Desmin L345P transgenic mice exhibit morphological and biochemical features of amyloidosis of two distinct types
- 2010
-
Ingår i: European Heart Journal. - Oxford, UK : Oxford University Press. - 0195-668X .- 1522-9645. ; 31:Suppl. 1, s. 924-925
-
Tidskriftsartikel (övrigt vetenskapligt/konstnärligt)abstract
- Background: Being a chief intermediate filament of the muscle tissue, desmin isimplicated in the sarcomeric organization, organelle positioning and mitochondrialfunction. Various desmin mutations have been reported as a possible cause forcardiomyopathies. Several reports on transgenic mice expressing mutant desminshowed deleterious effects of mutant desmin incorporated into filaments on cardiomyocyte function, but most importantly that accumulation of unfolded proteinaggregates plays an important pathogenic role in development of desminassociatedcardiomyopathies. Thus, in desmin transgenic mice with the L345Pmutation, which interferes in a dominant-negative manner with desmin polymerization,the accumulation of intracellular and extracellular amyloidogenic proteinaggregates was shown to be the key feature along with alteration of mitochondrialstructure and function. Therefore, the aim of this study was to characterizethe nature of amyloidogenic protein aggregates in L345P desmin transgenic miceon molecular and protein level.Material and methods: L345P desmin transgenic mice (DM) and WT mice 40weeks old were analyzed. Myocardial cryostat 10 micron sections were stainedwith conventional techniques (hematoxilin-eosin, Congo Red). A detailed amyloidcharacterization was carried out using novel optical probes called luminescentconjugated oligothiophenes and polythiophenes (LCOs and LCPs) that specificallystain various protein aggregates and give rise to conformation dependentemission spectra.Results: The most prominent feature of DM mice myocardium was misfoldedprotein depositions in perivascular space and between muscle fibers. Analysisof samples from DM mouse stained with LCO or LCP revealed the presence ofaggregates emitting light with two different emission spectra. Since the spectralproperties of the LCOs or LCPs are dependent on their conformation, the appearanceof two dissimilar emission spectra indicates that the probes might bindto two different types of amyloid aggregates within the tissue. Interestingly, aggregateswith emission spectra similar to one of the two types found in the DMmouse could also be found in WT mice, but in a much lower extent, suggesting asporadic cardiac amyloid pathology in C57 Bl/6 mice at 40 weeks, probably, as anative aging attribute.Conclusions: The L345P desmin mutation causes focal amyloid protein depositionin heart muscle of two distinct types. White first one can be a natural attributeof C57 Bl/6 mice detected with age, another one can be specifically responsiblefor the development of desmin-related cardiomyopathy.
|
|
| 2. |
- Klingstedt, Therése, 1978-
(författare)
-
Fluorescent thiophene-based ligands for detection and characterization of disease-associated protein aggregates
- 2013
-
Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- In this thesis the unique optical properties of fluorescent ligands termed luminescent conjugated oligothiophenes (LCOs) have been used to study a variety of protein aggregates associated with human protein misfolding disease. This heterogeneous group of diseases contains well known and fatal members such as Alzheimer´s and Huntington´s disease and the development of sensitive tools for the detection and characterization of protein aggregates is crucial for unravelling the complexity of these pathologies. Conventionally, the molecular dyes Congo red and thioflavin T (ThT) have been the primary choices for detecting and monitoring protein misfolding events. However, the rigid scaffold of both Congo red and ThT only offers an on or off mode and limits their ability to make fine distinctions at the molecular level. In contrast, LCOs have a flexible conjugated backbone and in addition to detect a broader subset of misfolded proteins, LCO can be used to visualize the heterogeneity of protein aggregates.The work presented in this thesis has given novel insights regarding the close connection between LCO design and optical performance. By altering the backbone length and the arrangement of substituents as well as replacing thiophene units with moieties affecting conjugation length and conformational freedom, the structural requirements of an optimal LCO for a certain application have been revealed. LCOs having a pentameric thiophene backbone with carboxyl end-groups were able to i) cross the blood-brain barrier and selectively stain cerebral amyloid β (Aβ) plaques, ii) detect non-thioflavinophilic Aβ aggregates and non-congophilic prion aggregates, iii) spectrally discriminate Aβ from tau aggregates and iiii) strongly label protein inclusion bodies. However, in some applications this design was outdone by others and in general, the conjugation length and the level of conformational freedom of the backbone were important determinants of the performance of the LCO.Overall, the findings in this thesis illustrate how small alterations in the LCO molecular scaffold may have large impact on the ligand properties. The results highlight the importance of having a toolbox of diverse ligands in order to increase our knowledge regarding the complex nature of protein aggregates.
|
|
| 3. |
- Klingstedt, Therése, et al.
(författare)
-
Synthesis of a library of oligothiophenes and their utilization as fluorescent ligands for spectral assignment of protein aggregates
- 2011
-
Ingår i: Organic and biomolecular chemistry. - : Royal Society of Chemistry. - 1477-0520 .- 1477-0539. ; 9:24, s. 8356-8370
-
Tidskriftsartikel (refereegranskat)abstract
- Molecular probes for selective identification of protein aggregates are important to advance our understanding of the molecular pathogenesis underlying protein aggregation diseases. Here we report the chemical design of a library of anionic luminescent conjugated oligothiophenes (LCOs), which can be utilized as ligands for detection of protein aggregates. Certain molecular requirements were shown to be necessary for detecting (i) early non-thioflavinophilic protein assemblies of A beta 1-42 and insulin preceding the formation of amyloid fibrils and (ii) for obtaining distinct spectral signatures of the two main pathological hallmarks observed in human Alzheimers diease brain tissue (A beta plaques and neurofibrillary tangles). Our findings suggest that a superior anionic LCO-based ligand should have a backbone consisting of five to seven thiophene units and carboxyl groups extending the conjugated thiophene backbone. Such LCOs will be highly useful for studying the underlying molecular events of protein aggregation diseases and could also be utilized for the development of novel diagnostic tools for these diseases.
|
|
| 4. |
- Lord, Anna, 1979-, et al.
(författare)
-
Arctic Aβ selectively increases diffuse deposition of wild type Aβ in APP transgenic mice with the Swedish mutation
-
Annan publikation (övrigt vetenskapligt/konstnärligt)abstract
- Studies of familial Alzheimer´s disease (AD) suggest that misfolding and aggregation of amyloid-β (Aβ) peptides initiate the pathogenesis, which causes dementia. The Arctic amyloid precursor protein (APP) mutation results in AD, and Arctic Aβ is more prone to form Aβ protofibrils. Here we show that the number of diffuse Aβ deposits, but not amyloid plaques, is increased if tg-ArcSwe mice synthesizing a low level of Arctic Aβ are crossed with plaque-depositing tg-Swe mice. The diffuse deposits in bitransgenic mice, which contain mainly wild type Aβ42, accumulate in regions both with and without transgene expression. The selective increase of a single type of parenchymal Aβ deposit suggest that different pathways of Aβ aggregation lead to the formation of diffuse and compact Aβ deposits in the brain. The raise in diffuse deposits is most likely due to direct physical interactions between Arctic and wild type Aβ42, and not to altered APP processing in young bitransgenic mice. A mixture of Arctic and wild type Aβ42 facilitates the formation of prefibrillar and fibrillar Aβ assemblies, but inhibits the further elongation of Aβ fibrils in vitro. Our findings might have implications to the pathogenesis of patients who are heterozygous for the Arctic mutation. It also further illustrates how Aβ neuropathology can be manipulated in vivo in a manner reminiscent to prion disorders.
|
|
| 5. |
- Lord, Anna, et al.
(författare)
-
Observations in APP Bitransgenic Mice Suggest thatDiffuse and Compact Plaques Form via IndependentProcesses in Alzheimer’s Disease
- 2011
-
Ingår i: American Journal of Pathology. - : Elsevier. - 0002-9440 .- 1525-2191. ; 178:5, s. 2286-2298
-
Tidskriftsartikel (refereegranskat)abstract
- Studies of familial Alzheimer's disease suggest that misfolding and aggregation of amyloid-β (Aβ) peptides initiate the pathogenesis. The Arctic mutation of Aβ precursor protein (APP) results in AD, and Arctic Aβ is more prone to form Aβ protofibrils and extracellular deposits. Herein is demonstrated that the burden of diffuse Aβ deposits but not compact plaques is increased when tg-Swe mice are crossed with tg-ArcSwe mice synthesizing low levels of Arctic Aβ. The diffuse deposits in bitransgenic mice, which contain primarily wild-type Aβ42, accumulate in regions both with and without transgene expression. However, APP processing, when compared with tg-Swe, remains unchanged in young bitransgenic mice, whereas wild-type Aβ42 aggregation is accelerated and fibril architecture is altered in vitro and in vivo when a low level of Arctic Aβ42 is introduced. Thus, the increased number of diffuse deposits is likely due to physical interactions between Arctic Aβ and wild-type Aβ42. The selective increase of a single type of parenchymal Aβ deposit suggests that different pathways lead to formation of diffuse and compact plaques. These findings could have general implications for Alzheimer's disease pathogenesis and particular relevance to patients heterozygous for the Arctic APP mutation. Moreover, it further illustrates how Aβ neuropathologic features can be manipulated in vivo by mechanisms similar to those originally conceptualized in prion research.
|
|
| 6. |
- Sörgjerd, Karin, 1977-, et al.
(författare)
-
BiP can function as a molecular shepherd that alleviates oligomer toxicity and amass amyloid
-
Annan publikation (övrigt vetenskapligt/konstnärligt)abstract
- A wide range of diseases are linked to protein misfolding and aggregation inside and outside the cell. It is of utmost interest to understand how the molecular chaperone machinery of the endoplasmic reticulum (ER) handles the expression of highly amyloidogenic proteins. We explored the hypothesis that the ER located Hsp70 molecular chaperone BiP plays a crucial role in amyloid diseases and influence the misfolding process and disease progression. We used the transthyretin mutant TTR D18G associated with an unusual central nervous system amyloid disease as the model substrate because it represents the most destabilized and degraded TTR variant known. Over-expression of TTR D18G in concert with BiP showed that BiP selectively recognize the amyloidogenic mutant protein as compared to wild type in human cells and collects the mutant in stable intermediate size oligomers within the ER. Furthermore, whereas TTR D18G was found to be highly cytotoxic to neuroblastoma cells, TTR D18G preincubated with BiP was non-toxic indicating that BiP protects the cell from cytotoxicity. BiP was also found present in cerebellar amyloid deposits and co-localized with TTR in a TTR D18G patient suggesting that the complex can be found in the extracellular space. We promote a fundamental role of BiP in misfolding diseases and describe a molecular shepharding function of BiP in sequestrating amyloidogenic protein molecules in benign oligomeric states.
|
|
| 7. |
- Sörgjerd, Karin, 1977-, et al.
(författare)
-
Prefibrillar Amyloid Aggregates and Cold Shocked Tetrameric Wild Type Transthyretin are Cytotoxic
-
Annan publikation (övrigt vetenskapligt/konstnärligt)abstract
- Recent studies suggest that soluble, oligomeric species, which are intermediates in the fibril formation process in amyloid disease, might be the key species in amyloid pathogenesis. Soluble oligomers of TTR were produced by kinetic sampling from a TTR fibrillation reaction (A-state TTR, pH 2, 100 mM NaCl). The reaction was terminated at different time points, and different states in the aggregation process were captured and analyzed to elucidate the oligomer properties followed by sampling for cytotoxicity using exposure towards human SH-SYY5 neuroblastoma cells. Employing ThT fluorescence, time-resolved fluorescence anisotropy of pyrenelabeled TTR, chemical cross-linking and electron microscopy we demonstrated that early formed oligomers from A-state TTR were soluble and comprised on the average 20-30 TTR monomers. Early oligomers were highly cytotoxic and induced apoptosis as indicated by the MTT assay and caspase-3 activation, whereas mature fibrils were non-toxic. We also indicate an activated unfolded protein response in cells exposed to oligomers as evidenced by an increased expression of the endoplasmic reticulum located molecular chaperone BiP. Following exposure, BiP appeared relocalized to the cytoplasm. Surprisingly, we also found that native tetrameric TTR purified and stored under cold conditions (4 °C) was highly cytotoxic. The effect could be partially restored by increasing the temperature of the protein. The molecular basis for this pathogenicity is rather unclear but likely stems from previously reported increased sensitivity towards dissociation and denaturation of TTR at low temperatures and opens the possibility that rearranged tetrameric TTR is cytotoxic towards neuroblastoma cells.
|
|
| 8. |
- Sörgjerd, Karin, et al.
(författare)
-
Prefibrillar transthyretin oligomers and cold stored native tetrameric transthyretin are cytotoxic in cell culture
- 2008
-
Ingår i: Biochemical and Biophysical Research Communications - BBRC. - : Elsevier BV. - 0006-291X .- 1090-2104. ; 377:4, s. 1072-1078
-
Tidskriftsartikel (refereegranskat)abstract
- Recent studies Suggest that Soluble, oligomeric species, which are intermediates in the fibril formation process in amyloid disease, might be the key species in amyloid pathogenesis. Soluble oligomers of human wild type transthyretin (TTR) were produced to elucidate oligomer properties. Employing ThT fluorescence, time-resolved fluorescence anisotropy of pyrene-labeled TTR, chemical cross-linking, and electron microscopy we demonstrated that early formed soluble oligomers (within minutes) from A-state TTR comprised on the average 20-30 TTR monomers. When administered to neuroblastoma cells these early oligomers proved highly cytotoxic and induced apoptosis after 48 h of incubation. More Mature fibrils (> 24 h of fibrillation) were non-toxic. Surprisingly, we also found that native tetrameric TTR, when purified and stored under cold conditions (4 degrees C) was highly cytotoxic. The effect Could be partially restored by increasing the temperature of the protein. The cytotoxic effects of native tetrameric TTR likely stems from a hitherto unexplored low temperature induced rearrangement of the tetramer conformation that possibly is related to the conformation of misfolded TTR in amyloigogenic oligomers.
|
|
| 9. |
- Wegenast-Braun, Bettina M., et al.
(författare)
-
Spectral Discrimination of Cerebral Amyloid Lesions after Peripheral Application of Luminescent Conjugated Oligothiophenes
- 2012
-
Ingår i: American Journal of Pathology. - : Elsevier. - 0002-9440 .- 1525-2191. ; 181:6, s. 1953-1960
-
Tidskriftsartikel (refereegranskat)abstract
- In vivo imaging of pathological protein aggregates provides essential knowledge of the kinetics and implications of these lesions in the progression of proteopathies, such as Alzheimer disease. Luminescent conjugated oligothiophenes are amyloid-specific ligands that bind and spectrally distinguish different types of amyloid aggregates. Herein, we report that heptamer formyl thiophene acetic acid (hFTAA) passes the blood-brain barrier after systemic administration and specifically binds to extracellular beta-amyloid deposits in the brain parenchyma (A beta plaques) and in the vasculature (cerebral beta-amyloid angiopathy) of beta-amyloid precursor protein transgenic APP23 mice. Moreover, peripheral application of hFIAA also stained intracellular lesions of hyperphosphorylated Tau protein in P301S Tau transgenic mice. Spectral profiling of all three amyloid types was acquired ex vivo using two-photon excitation. hFTAA revealed a distinct shift in its emission spectra when bound to A beta plaques versus Tau lesions. Furthermore, a spectral shift was observed for A beta plaques versus cerebral beta-amyloid angiopathy, indicating that different amyloid types and structural variances of a specific amyloid type can be distinguished. In conclusion, by adding spectral signatures to amyloid lesions, our results pave the way for a new area of in vivo amyloid imaging, allowing in vivo differentiation of amyloid (sub)types and monitoring changes of their structure/composition over time. (Am J Pathol 2012, 181: 1953-1960 http://dx.doi.org/10.1016/j.ajpath.2012.08.031)
|
|
| 10. |
- Åslund, Andreas, et al.
(författare)
-
Novel Pentameric Thiophene Derivatives for in Vitro and in Vivo Optical Imaging of a Plethora of Protein Aggregates in Cerebral Amyloidoses
- 2009
-
Ingår i: ACS CHEMICAL BIOLOGY. - : American Chemical Society (ACS). - 1554-8929 .- 1554-8937. ; 4:8, s. 673-684
-
Tidskriftsartikel (refereegranskat)abstract
- Molecular probes for selective Identification of protein aggregates are important to advance our understanding of the molecular pathogenesis underlying cerebral amyloidoses. Here we report the chemical design of pentameric thiophene derivatives, denoted luminescent conjugated oligothiophenes (LCOs), which could be used for real-time visualization of cerebral protein aggregates in transgenic mouse models of neurodegenerative diseases by multiphoton microscopy. One of the LCOs, p-FTAA, could be utilized for ex vivo spectral assignment of distinct prion deposits from two, mouse-adapted prion strains. p-FTAA also revealed a transient soluble pre-fibrillar non-thioflavinophilic A beta-assemblies during in vitro fibrillation of A beta peptides. In brain tissue samples, A beta deposits and neurofibrillary tangles (NFTs) were readily identified by a strong fluorescence from p-FTAA and the LCO staining showed complete co-localliation with conventional antibodies (6E10 and AT8). In addition, a patchy islet-like staining of individual A beta plaque was unveiled by the anti-oligomer A11 antibody during co-staining with p-FTAA. The major hallmarks of Alzheimers disease, namely, A beta aggregates versus NFTs, could also be distinguished because of distinct emission spectra from p-FTAA. Overall, we demonstrate that LCOs can be utilized as powerful practical research tools for studying protein aggregation diseases and facilitate the study of amyloid origin, evolution and maturation, A beta-tau interactions, and pathogenesis both ex vivo and in vivo.
|
|
