Karin Sörgjerd

Prefibrillar transthyretin oligomers and cold stored native tetrameric transthyretin are cytotoxic in cell culture

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.

Prefoldin Protects Neuronal Cells from Polyglutamine Toxicity by Preventing Aggregation Formation

Background: Prefoldin, a molecular chaperone composed of six subunits, prevents misfolding of newly synthesized nascent polypeptides. Results: Prefoldin inhibited aggregation of pathogenic Huntingtin and subsequent cell death. Conclusion: Prefoldin suppressed Huntingtin aggregation at the small oligomer stage. Significance: Prefoldin plays a role in preventing protein aggregation in Huntington disease. Huntington disease is caused by cell death after the expansion of polyglutamine (polyQ) tracts longer than ∼40 repeats encoded by exon 1 of the huntingtin (HTT) gene. Prefoldin is a molecular chaperone composed of six subunits, PFD1–6, and prevents misfolding of newly synthesized nascent polypeptides. In this study, we found that knockdown of PFD2 and PFD5 disrupted prefoldin formation in HTT-expressing cells, resulting in accumulation of aggregates of a pathogenic form of HTT and in induction of cell death. Dead cells, however, did not contain inclusions of HTT, and analysis by a fluorescence correlation spectroscopy indicated that knockdown of PFD2 and PFD5 also increased the size of soluble oligomers of pathogenic HTT in cells. In vitro single molecule observation demonstrated that prefoldin suppressed HTT aggregation at the small oligomer (dimer to tetramer) stage. These results indicate that prefoldin inhibits elongation of large oligomers of pathogenic Htt, thereby inhibiting subsequent inclusion formation, and suggest that soluble oligomers of polyQ-expanded HTT are more toxic than are inclusion to cells.

Human Prefoldin Inhibits Amyloid-β (Aβ) Fibrillation and Contributes to Formation of Nontoxic Aβ Aggregates

Amyloid-β (Aβ) peptides represent key players in the pathogenesis of Alzheimers disease (AD), and mounting evidence indicates that soluble Aβ oligomers mediate the toxicity. Prefoldin (PFD) is a molecular chaperone that prevents aggregation of misfolded proteins. Here we investigated the role of PFD in Aβ aggregation. First, we demonstrated that PFD is expressed in mouse brain by Western blotting and immunohistochemistry and found that PFD is upregulated in AD model APP23 transgenic mice. Then we investigated the effect of recombinant human PFD (hPFD) on Aβ(1-42) aggregation in vitro and found that hPFD inhibited Aβ fibrillation and induced formation of soluble Aβ oligomers. Interestingly, cell viability measurements using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay showed that Aβ oligomers formed by hPFD were 30-40% less toxic to cultured rat pheochromocytoma (PC12) cells or primary cortical neurons from embryonic C57BL/6CrSlc mice than previously reported Aβ oligomers (formed by archaeal PFD) and Aβ fibrils (p < 0.001). Thioflavin T measurements and immunoblotting indicated different structural properties for the different Aβ oligomers. Our findings show a relation between cytotoxicity of Aβ oligomers and structure and suggest a possible protective role of PFD in AD.

A Structure-Toxicity Study of Aß42 Reveals a New Anti-Parallel Aggregation Pathway

Amyloid beta (Aβ) peptides produced by APP cleavage are central to the pathology of Alzheimer’s disease. Despite widespread interest in this issue, the relationship between the auto-assembly and toxicity of these peptides remains controversial. One intriguing feature stems from their capacity to form anti-parallel ß-sheet oligomeric intermediates that can be converted into a parallel topology to allow the formation of protofibrillar and fibrillar Aβ. Here, we present a novel approach to determining the molecular aspects of Aß assembly that is responsible for its in vivo toxicity. We selected Aß mutants with varying intracellular toxicities. In vitro, only toxic Aß (including wild-type Aß42) formed urea-resistant oligomers. These oligomers were able to assemble into fibrils that are rich in anti-parallel ß-sheet structures. Our results support the existence of a new pathway that depends on the folding capacity of Aß .

Degeneration of amyloid-ß fibrils caused by exposure to low-temperature atmospheric-pressure plasma in aqueous solution

Low-temperature atmospheric-pressure plasma was applied to degenerate amyloid-s (As) fibrils, which are a major component of neuritic plaque associated with Alzheimers disease (AD). We showed that an As fibril exposed to a low-frequency (LF) plasma jet in aqueous solution retained its morphology, molecular weight, and cytotoxicity, but, intriguingly, decreased in protease resistance and s-sheet content. These results suggested that an LF plasma jet could be utilized for the treatment of AD to eliminate neuritic plaque by accelerating the proteolysis of As fibrils.

Cell interaction study of amyloid by using luminescent conjugated polythiophene: implication that amyloid cytotoxicity is correlated with prolonged cellular binding.

Needles and noodles: Studying amyloid toxicity is important for understanding protein misfolding diseases. Using a luminescent conjugated polythiophene, we found that cell binding of nontoxic filamentous amyloids of insulin and β2-microglobulin was less efficient than that of toxic fibrillar amyloids; this suggests a correlation between amyloid toxicity and cell binding.

Adsorption and separation of amyloid beta aggregates using ferromagnetic nanoparticles coated with charged polymer brushes

Amyloid beta (Aβ) protein aggregates, which include fibrils and oligomers, are neurotoxic and are considered to cause Alzheimers disease. Thus, separation of these Aβ aggregates from biological samples is important. Herein, we report the use of strongly ferromagnetic few-layer graphene-coated magnetic nanoparticles (C/Co), which were functionalized with a cationic polymer, poly[3-(methacryloyl amino)propyl]trimethylammonium chloride (polyMAPTAC), C/Co@polyMAPTAC, for the adsorption and magnetic separation of Aβ aggregates. Fast adsorption (∼1 min) of Aβ fibrils and oligomers onto the particles was observed. Interestingly, the Aβ monomer was not captured by the particles, suggesting that binding to Aβ molecules is toxic species-selective. Selective adsorption was also observed in the presence of serum albumin protein. We also showed that C/Co@polyMAPTAC could reduce the cytotoxicity of the Aβ aggregate solutions. This study should be useful for further elucidation of the application of nanoparticle adsorption in mediating Aβ toxicity.

Fluorescence molecular probes for sensitive point detection of amyloid fibrils and protofibrils

Protein based infections such as prion diseases have lately attracted a large amount of interest, primarily due to the Mad Cow Epidemic in Great Britain, and the increase of Alzheimers disease and related diseases in the ageing Western society. Infective proteins are very stable and almost untraceable prior to infection making them ideal as biological weapons. Particularly if the used agent is of human origin, the immunoresponse can be avoided, leaving no trace of the infectious agent. The transient nature of infectious oligomeric intermediates of misfolded proteins or peptide fragments that later matures into fibrillar aggregates makes them hard to study, and methods to detect and study these species are sparse. There exist a number of fluorescent probes that bind specifically to protein amyloidic structures. Thioflavins (ThT, ThS), Congo and Nile red, 4-(dicyanovinyl)-julolidine (DCVJ), as well as derivatives amino-8-naphtalene sulphonate (ANS, Bis-ANS) which are known to bind to the fibrillar or pre-fibrillar states with dissociation constants of typically 1 - 20 μM. Here, transthyretin (TTR), insulin and lysozyme were used as model proteins to detect different amyloid precursor states for diseases such as senile systemic amyloidosis, familial amyloidotic polyneuropathy (FAP) and iatrogenic amyloidosis. Specifically, the probes were employed in static assays to characterize protofibrillar and mature amyloid fibrillar states using steady state and time-resolved fluorescence techniques. Particularly, we investigate and report on the possibility to detect protofibrillar states at low concentration levels using modern fluorescence array detector systems in conjunction with lasers operating in the blue or ultraviolett wavelengths as excitation source. Results of ANS, ThT and a ThT analogue (abbreviated ThC) are discussed.