Cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL) represents the most common hereditary form of cerebral small vessel disease characterized by early-onset stroke and premature dementia. It is caused by mutations in the transmembrane receptor Notch3, which promote the aggregation and accumulation of the Notch3 extracellular domain (Notch3-ECD) within blood vessel walls. This process is believed to mediate the abnormal recruitment and dysregulation of additional factors including extracellular matrix (ECM) proteins resulting in brain vessel dysfunction. Based on recent evidence indicating a role for the transforming growth factor-β (TGF-β) pathway in sporadic and familial small vessel disease we studied fibronectin, fibrillin-1 and latent TGF-β binding protein 1 (LTBP-1), three ECM constituents involved in the regulation of TGF-β bioavailability, in post-mortem brain tissue from CADASIL patients and control subjects.
Fibronectin and fibrillin-1 were found to be enriched in CADASIL vessels without co-localizing with Notch3-ECD deposits, likely as a result of fibrotic processes secondary to aggregate formation. In contrast, LTBP-1 showed both an accumulation and a striking co-localization with Notch3-ECD deposits suggesting specific recruitment into aggregates. We also detected increased levels of the TGF-β prodomain (also known as latency-associated peptide, LAP) indicating dysregulation of the TGF-β pathway in CADASIL development. In vitro analyses revealed a direct interaction between LTBP-1 and Notch3-ECD and demonstrated a specific co-aggregation of LTBP-1 with mutant Notch3.
We propose LTBP-1 as a novel component of Notch3-ECD deposits and suggest its involvement in pathological processes triggered by Notch3-ECD aggregation.
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The online version of this article (doi:10.1186/s40478-014-0096-8) contains supplementary material, which is available to authorized users.
Latent TGF-β binding protein 1 (LTBP-1); Small vessel disease; CADASIL; TGF-β signaling; Granular osmiophilic material (GOM); Extracellular matrix (ECM)
In several neurodegenerative diseases, hyperphosphorylation at position Ser129 is found in fibrillar deposits of alpha-synuclein (asyn), implying a pathophysiological role of asyn phosphorylation in neurodegeneration. However, recent animal models applying asyn phosphorylation mimics demonstrated a protective effect of phosphorylation. Since metal-ion induced asyn oligomers were identified as a potential neurotoxic aggregate species with membrane pore-forming abilities, the current study was undertaken to determine effects of asyn phosphorylation on oligomer membrane binding.
We investigated the influence of S129 phosphorylation on interactions of metal-ion induced asyn oligomers with small unilamellar lipid vesicles (SUV) composed of POPC and DPPC applying the phosphorylation mimic asyn129E. Confocal single-particle fluorescence techniques were used to monitor membrane binding at the single-particle level.
Binding of asyn129E monomers to gel-state membranes (DPPC-SUV) is slightly reduced compared to wild-type asyn, while no interactions with membranes in the liquid-crystalline state (POPC-SUV) are seen for both asyn and asyn129E. Conversely, metal-ion induced oligomer formation is markedly increased in asyn129E. Surprisingly, membrane binding to POPC-SUV is nearly absent in Fe3+ induced asyn129E oligomers and markedly reduced in Al3+ induced oligomers.
The protective effect of pseudophosphorylation seen in animal models may be due to impeded oligomer membrane binding. Phosphorylation at Ser129 may thus have a protective effect against neurotoxic asyn oligomers by preventing oligomer membrane binding and disruption of the cellular electrophysiological equilibrium. Importantly, these findings put a new complexion on experimental pharmaceutical interventions against POLO-2 kinase.
Despite antibiotic therapy, acute and long-term complications are still frequent in pneumococcal meningitis. One important trigger of these complications is oxidative stress, and adjunctive antioxidant treatment with N-acetyl-l-cysteine was suggested to be protective in experimental pneumococcal meningitis. However, studies of effects on neurological long-term sequelae are limited. Here, we investigated the impact of adjunctive N-acetyl-l-cysteine on long-term neurological deficits in a mouse model of meningitis. C57BL/6 mice were intracisternally infected with Streptococcus pneumoniae. Eighteen hours after infection, mice were treated with a combination of ceftriaxone and placebo or ceftriaxone and N-acetyl-l-cysteine, respectively. Two weeks after infection, neurologic deficits were assessed using a clinical score, an open field test (explorative activity), a t-maze test (memory function), and auditory brain stem responses (hearing loss). Furthermore, cochlear histomorphological correlates of hearing loss were assessed. Adjunctive N-acetyl-l-cysteine reduced hearing loss after pneumococcal meningitis, but the effect was minor. There was no significant benefit of adjunctive N-acetyl-l-cysteine treatment in regard to other long-term complications of pneumococcal meningitis. Cochlear morphological correlates of meningitis-associated hearing loss were not reduced by adjunctive N-acetyl-l-cysteine. In conclusion, adjunctive therapy with N-acetyl-l-cysteine at a dosage of 300 mg/kg of body weight intraperitoneally for 4 days reduced hearing loss but not other neurologic deficits after pneumococcal meningitis in mice. These results make a clinical therapeutic benefit of N-acetyl-l-cysteine in the treatment of patients with pneumococcal meningitis questionable.
Disrupted intracellular calcium homeostasis is believed to occur early in the cascade of events leading to Alzheimer's disease (AD) pathology. Particularly familial AD mutations linked to Presenilins result in exaggerated agonist-evoked calcium release from endoplasmic reticulum (ER). Here we report the development of a fully automated high-throughput calcium imaging assay utilizing a genetically-encoded FRET-based calcium indicator at single cell resolution for compound screening. The established high-throughput screening assay offers several advantages over conventional high-throughput calcium imaging technologies. We employed this assay for drug discovery in AD by screening compound libraries consisting of over 20,000 small molecules followed by structure-activity-relationship analysis. This led to the identification of Bepridil, a calcium channel antagonist drug in addition to four further lead structures capable of normalizing the potentiated FAD-PS1-induced calcium release from ER. Interestingly, it has recently been reported that Bepridil can reduce Aβ production by lowering BACE1 activity. Indeed, we also detected lowered Aβ, increased sAPPα and decreased sAPPβ fragment levels upon Bepridil treatment. The latter findings suggest that Bepridil may provide a multifactorial therapeutic modality for AD by simultaneously addressing multiple aspects of the disease.
PrPSc, the only known constituent of prions, the infectious agents causing prion diseases, can be detected by real-time quaking-induced conversion (RT-QuIC). However, there is no efficient method to quantify the amount of PrPSc by RT-QuIC.
Here we introduce quantitative RT-QuIC (qRT-QuIC) to quantify with high accuracy minute amounts of PrPSc in the brain and various peripheral tissues at levels far below detection by in vivo transmission. PrPSc is relatively resistant to treatment with proteinase K (PK). However, as there can also be a fraction of pathological PrP that is digested by PK, we use the term PrP27-30 to denote to the amount of PrPSc that can be detected by immunoblot after PK treatment. qRT-QuIC is based upon the quantitative correlation between the seeded amount of PrP27-30 and the lag time to the start of the conversion reaction detected by RT-QuIC. By seeding known amounts of PrP27-30 quantified by immunoblot into qRT-QuIC a standard calibration curve can be obtained. Based on this calibration curve, seeded undetermined amounts of PrP27-30 can be directly calculated. qRT-QuIC allowed to quantify PrP27-30 concentrations at extremely low levels as low as 10-15.5 g PrP27-30, which corresponds to 0.001 LD50 units obtained by in vivo i.c. transmission studies. We find that PrP27-30 concentration increases steadily in the brain after inoculation and can be detected at various time points during the incubation period in peripheral organs (spleen, heart, muscle, liver, kidney) in two experimental scrapie strains (RML, ME7) in the mouse.
We suggest that an automatic quantitative system to measure disease progression as well as prion contamination of organs, blood and food product is feasible. Moreover, the concept of qRT-QuIC should be applicable to measure other disease-associated proteins rich in β-pleated structures (amyloid) that bind ThT and that show seeded aggregation.
Prion; PrPSc; PrP27-30; Quantitative RT-QuIC
The current classification of human sporadic prion diseases recognizes six major phenotypic subtypes with distinctive clinicopathological features, which largely correlate at the molecular level with the genotype at the polymorphic codon 129 (methionine, M, or valine, V) in the prion protein gene and with the size of the protease-resistant core of the abnormal prion protein, PrPSc (i.e. type 1 migrating at 21 kDa and type 2 at 19 kDa). We previously demonstrated that PrPSc typing by Western blotting is a reliable means of strain typing and disease classification. Limitations of this approach, however, particularly in the interlaboratory setting, are the association of PrPSc types 1 or 2 with more than one clinicopathological phenotype, which precludes definitive case classification if not supported by further analysis, and the difficulty of fully recognizing cases with mixed phenotypic features. In this study, we tested the inter-rater reliability of disease classification based only on histopathological criteria. Slides from 21 cases covering the whole phenotypic spectrum of human sporadic prion diseases, and also including two cases of variant Creutzfeldt–Jakob disease (CJD), were distributed blindly to 13 assessors for classification according to given instructions. The results showed good-to-excellent agreement between assessors in the classification of cases. In particular, there was full agreement (100 %) for the two most common sporadic CJD subtypes and variant CJD, and very high concordance in general for all pure phenotypes and the most common subtype with mixed phenotypic features. The present data fully support the basis for the current classification of sporadic human prion diseases and indicate that, besides molecular PrPSc typing, histopathological analysis permits reliable disease classification with high interlaboratory accuracy.
In neurodegenerative diseases such as Alzheimer’s disease (AD), Parkinson’s disease (PD) and prion diseases, deposits of aggregated disease-specific proteins are found. Oligomeric aggregates are presumed to be the key neurotoxic agent. Here we describe the novel oligomer modulator anle138b [3-(1,3-benzodioxol-5-yl)-5-(3-bromophenyl)-1H-pyrazole], an aggregation inhibitor we developed based on a systematic high-throughput screening campaign combined with medicinal chemistry optimization. In vitro, anle138b blocked the formation of pathological aggregates of prion protein (PrPSc) and of α-synuclein (α-syn), which is deposited in PD and other synucleinopathies such as dementia with Lewy bodies (DLB) and multiple system atrophy (MSA). Notably, anle138b strongly inhibited all prion strains tested including BSE-derived and human prions. Anle138b showed structure-dependent binding to pathological aggregates and strongly inhibited formation of pathological oligomers in vitro and in vivo both for prion protein and α-synuclein. Both in mouse models of prion disease and in three different PD mouse models, anle138b strongly inhibited oligomer accumulation, neuronal degeneration, and disease progression in vivo. Anle138b had no detectable toxicity at therapeutic doses and an excellent oral bioavailability and blood–brain-barrier penetration. Our findings indicate that oligomer modulators provide a new approach for disease-modifying therapy in these diseases, for which only symptomatic treatment is available so far. Moreover, our findings suggest that pathological oligomers in neurodegenerative diseases share structural features, although the main protein component is disease-specific, indicating that compounds such as anle138b that modulate oligomer formation by targeting structure-dependent epitopes can have a broad spectrum of activity in the treatment of different protein aggregation diseases.
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Synucleinopathies such as Parkinson's disease, multiple system atrophy and dementia with Lewy bodies are characterized by deposition of aggregated α-synuclein. Recent findings indicate that pathological oligomers rather than fibrillar aggregates may represent the main toxic protein species. It has been shown that α-synuclein oligomers can increase the conductance of lipid bilayers and, in cell-culture, lead to calcium dyshomeostasis and cell death. In this study, employing a setup for single-channel electrophysiology, we found that addition of iron-induced α-synuclein oligomers resulted in quantized and stepwise increases in bilayer conductance indicating insertion of distinct transmembrane pores. These pores switched between open and closed states depending on clamped voltage revealing a single-pore conductance comparable to that of bacterial porins. Pore conductance was dependent on transmembrane potential and the available cation. The pores stably inserted into the bilayer and could not be removed by buffer exchange. Pore formation could be inhibited by co-incubation with the aggregation inhibitor baicalein. Our findings indicate that iron-induced α-synuclein oligomers can form a uniform and distinct pore species with characteristic electrophysiological properties. Pore formation could be a critical event in the pathogenesis of synucleinopathies and provide a novel structural target for disease-modifying therapy.
Fibrillar amyloid-like deposits and co-deposits of tau and α-synuclein are found in several common neurodegenerative diseases. Recent evidence indicates that small oligomers are the most relevant toxic aggregate species. While tau fibril formation is well-characterized, factors influencing tau oligomerization and molecular interactions of tau and α-synuclein are not well understood.
We used a novel approach applying confocal single-particle fluorescence to investigate the influence of tau phosphorylation and metal ions on tau oligomer formation and its coaggregation with α-synuclein at the level of individual oligomers. We show that Al3+ at physiologically relevant concentrations and tau phosphorylation by GSK-3β exert synergistic effects on the formation of a distinct SDS-resistant tau oligomer species even at nanomolar protein concentration. Moreover, tau phosphorylation and Al3+ as well as Fe3+ enhanced both formation of mixed oligomers and recruitment of α-synuclein in pre-formed tau oligomers.
Our findings provide a new perspective on interactions of tau phosphorylation, metal ions, and the formation of potentially toxic oligomer species, and elucidate molecular crosstalks between different aggregation pathways involved in neurodegeneration.
α-Synuclein, Metal ion, Oligomer, Phosphorylation, Tau, Iron, Aluminium, GSK-3 beta, Alzheimer’s disease; Parkinson’s disease
Transmissible spongiform encephalopathies (TSEs) represent a group of fatal neurodegenerative disorders that can be transmitted by natural infection or inoculation. TSEs include scrapie in sheep, bovine spongiform encephalopathy (BSE) in cattle, and Creutzfeldt-Jakob disease (CJD) in humans. The emergence of a variant form of CJD (vCJD), which has been associated with BSE, produced strong pressure to search for effective treatments with new drugs. Up to now, however, TSEs have proved incurable, although many efforts have been made both in vitro and in vivo to search for potent therapeutic and prophylactic compounds. For this purpose, we analyzed a compound library consisting of 10,000 compounds with a cell-based high-throughput screening assay dealing with scrapie-infected scrapie mouse brain and ScN2A cells and identified a new class of inhibitors consisting of 3,5-diphenylpyrazole (DPP) derivatives. The most effective DPP derivative showed half-maximal inhibition of PrPSc formation at concentrations (IC50) of 0.6 and 1.2 μM, respectively. This compound was subsequently subjected to a number of animal experiments using scrapie-infected wild-type C57BL/6 and transgenic Tga20 mice. The DPP derivative induced a significant increase of incubation time both in therapeutic and prophylactic experiments. The onset of the prion disease was delayed by 37 days after intraperitoneal and 42 days after oral application, respectively. In summary, we demonstrate a high in vitro efficiency of DPP derivatives against prion infections that was substantiated in vivo for one of these compounds. These results indicate that the novel class of DPP compounds should comprise excellent candidates for future therapeutic studies.
Six clinico-pathological phenotypes of sporadic Creutzfeldt–Jakob disease have been characterized which correlate at the molecular level with the type (1 or 2) of the abnormal prion protein, PrPTSE, present in the brain and with the genotype of polymorphic (methionine or valine) codon 129 of the prion protein gene. However, to what extent these phenotypes with their corresponding molecular combinations (i.e. MM1, MM2, VV1 etc.) encipher distinct prion strains upon transmission remains uncertain. We studied the PrPTSE type and the prion protein gene in archival brain tissues from the National Institutes of Health series of transmitted Creutzfeldt–Jakob disease and kuru cases, and characterized the molecular and pathological phenotype in the affected non-human primates, including squirrel, spider, capuchin and African green monkeys. We found that the transmission properties of prions from the common sporadic Creutzfeldt–Jakob disease MM1 phenotype are homogeneous and significantly differ from those of sporadic Creutzfeldt–Jakob disease VV2 or MV2 prions. Animals injected with iatrogenic Creutzfeldt–Jakob disease MM1 and genetic Creutzfeldt–Jakob disease MM1 linked to the E200K mutation showed the same phenotypic features as those infected with sporadic Creutzfeldt–Jakob disease MM1 prions, whereas kuru most closely resembled the sporadic Creutzfeldt–Jakob disease VV2 or MV2 prion signature and neuropathology. The findings indicate that two distinct prion strains are linked to the three most common Creutzfeldt–Jakob disease clinico-pathological and molecular subtypes and kuru, and suggest that kuru may have originated from cannibalistic transmission of a sporadic Creutzfeldt–Jakob disease of the VV2 or MV2 subtype.
prion diseases; neuropathology; neurodegenerative disorders; phenotype; strain typing
Hearing loss is a frequent long-term complication of pneumococcal meningitis (PM). Its main pathological correlate is damage to the organ of Corti and loss of spiral ganglion neurons. The only current treatment option is cochlear implants which require surviving neurons. Here, we investigated the impact of systemically applied neurotrophin-3 (NT-3) on long-term hearing loss and the survival of neurons.
Eighteen hours after infection with S. pneumoniae, C57BL/6 mice were treated with a combination of ceftriaxone with NT-3 or dexamethasone or placebo. Hearing, cochlear damage, and brain damage were assessed by audiometry and histology.
The main findings from immunohistochemical visualization of neurotrophins (NT-3, BDNF) and their receptors (TrkB, TrkC, and p75) in the cochlea were (i) enhanced staining for the cell survival-promoting receptor TrkB and (ii) increased NT-3 staining in NT-3 treated mice, showing that systemically applied NT-3 reaches the cochlea. The major effects of adjunctive NT-3 treatment were (i) a reduction of meningitis-induced hearing impairment and (ii) a reduction of spiral ganglion neuronal loss. The efficacy of NT-3 therapy was comparable to that of dexamethasone.
Systemically applied NT-3 might be an interesting candidate to improve hearing outcome after pneumococcal meningitis.
Intracellular α-synuclein (α-syn) aggregates are the pathological hallmark in several neurodegenerative diseases including Parkinson's disease, dementia with Lewy bodies and multiple system atrophy. Recent evidence suggests that small oligomeric aggregates rather than large amyloid fibrils represent the main toxic particle species in these diseases. We recently characterized iron-dependent toxic α-syn oligomer species by confocal single molecule fluorescence techniques and used this aggregation model to identify several N′-benzylidene-benzohydrazide (NBB) derivatives inhibiting oligomer formation in vitro. In our current work, we used the bioluminescent protein-fragment complementation assay (BPCA) to directly analyze the formation of toxic α-syn oligomers in cell culture and to investigate the effect of iron and potential drug-like compounds in living cells. Similar to our previous findings in vitro, we found a converse modulation of toxic α-syn oligomers by NBB derivates and ferric iron, which was characterized by an increase in aggregate formation by iron and an inhibitory effect of certain NBB compounds. Inhibition of α-syn oligomer formation by the NBB compound 293G02 was paralleled by a reduction in cytotoxicity indicating that toxic α-syn oligomers are present in the BPCA cell culture model and that pharmacological inhibition of oligomer formation can reduce toxicity. Thus, this approach provides a suitable model system for the development of new disease-modifying drugs targeting toxic oligomer species. Moreover, NBB compounds such as 293G02 may provide useful tool compounds to dissect the functional role of toxic oligomer species in cell culture models and in vivo.
Parkinson's disease; Alpha-synuclein; Drug development; Oligomer; Amyloid; Protein-fragment complementation assay
Six subtypes of sporadic Creutzfeldt–Jakob disease with distinctive clinico-pathological features have been identified largely based on two types of the abnormal prion protein, PrPSc, and the methionine (M)/valine (V) polymorphic codon 129 of the prion protein. The existence of affected subjects showing mixed phenotypic features and concurrent PrPSc types has been reported but with inconsistencies among studies in both results and their interpretation. The issue currently complicates diagnosis and classification of cases and also has implications for disease pathogenesis. To explore the issue in depth, we carried out a systematic regional study in a large series of 225 cases. PrPSc types 1 and 2 concurrence was detected in 35% of cases and was higher in MM than in MV or VV subjects. The deposition of either type 1 or 2, when concurrent, was not random and always characterized by the coexistence of phenotypic features previously described in the pure subtypes. PrPSc type 1 accumulation and related pathology predominated in MM and MV cases, while the type 2 phenotype prevailed in VVs. Neuropathological examination best identified the mixed types 1 and 2 features in MMs and most MVs, and also uniquely revealed the co-occurrence of pathological variants sharing PrPSc type 2. In contrast, molecular typing best detected the concurrent PrPSc types in VV subjects and MV cases with kuru plaques. The present data provide an updated disease classification and are of importance for future epidemiologic and transmission studies aimed to identify etiology and extent of strain variation in sporadic Creutzfeldt–Jakob disease.
Prion protein; Brain mapping; Molecular typing; Neurodegeneration; Classification
Conformational changes and aggregation of specific proteins are hallmarks of a number of diseases, like Alzheimer's disease, Parkinson's disease, and prion diseases. In the case of prion diseases, the prion protein (PrP), a neuronal glycoprotein, undergoes a conformational change from the normal, mainly alpha-helical conformation to a disease-associated, mainly beta-sheeted scrapie isoform (PrPSc), which forms amyloid aggregates. This conversion, which is crucial for disease progression, depends on direct PrPC/PrPSc interaction. We developed a high-throughput assay based on scanning for intensely fluorescent targets (SIFT) for the identification of drugs which interfere with this interaction at the molecular level. Screening of a library of 10,000 drug-like compounds yielded 256 primary hits, 80 of which were confirmed by dose response curves with half-maximal inhibitory effects ranging from 0.3 to 60 μM. Among these, six compounds displayed an inhibitory effect on PrPSc propagation in scrapie-infected N2a cells. Four of these candidate drugs share an N′-benzylidene-benzohydrazide core structure. Thus, the combination of high-throughput in vitro assay with the established cell culture system provides a rapid and efficient method to identify new antiprion drugs, which corroborates that interaction of PrPC and PrPSc is a crucial molecular step in the propagation of prions. Moreover, SIFT-based screening may facilitate the search for drugs against other diseases linked to protein aggregation.
Objective: To analyse serum concentrations of brain specific S100 protein in patients with Creutzfeldt-Jakob disease and in controls.
Design: Prospective case-control study.
Setting: National Creutzfeldt-Jakob disease surveillance unit.
Subjects: 224 patients referred to the surveillance unit with suspected Creutzfeldt-Jakob disease and 35 control patients without dementia.
Main outcome measure: Serum concentration of S100 protein in patients with Creutzfeldt-Jakob disease, in patients with other diseases causing dementia, and in the control group.
Results: Of the 224 patients with suspected Creutzfeldt-Jakob disease, 65 were classed as definitely having the disease after neuropathological verification, an additional 6 were classed as definitely having the disease as a result of a genetic mutation, 43 as probably having the disease, 36 as possibly having the disease, and 74 patients were classed as having other disease. In the 108 patients classed as definitely or probably having Creutzfeldt-Jakob disease the median serum concentration of S100 was 395 pg/ml (SD 387 pg/ml). This was significantly higher than concentrations found in the 74 patients classed as having other diseases (median 109 pg/ml; SD 177 pg/ml; P=0.0001). At a cut off point of 213 pg/ml sensitivity for the diagnosis of the disease was 77.8% (95% confidence interval 68.8% to 85.2%) and specificity was 81.1% (70.3% to 89.3%). There was a significant difference in survival at different concentrations of S100 in Kaplan-Meier curves (P=0.023).
Conclusion: Measurement of serum concentrations of S100 is a valuable tool which can be used more easily than tests on cerebrospinal fluid in the differential diagnosis of Creutzfeldt-Jakob disease. More studies are needed to determine whether serial testing of serum S100 improves diagnostic accuracy.
Key messages Creutzfeldt-Jakob disease is a rare, fatal neurodegenerative disease. Diagnosis is made clinically and neuropathologically There is no serum test which allows the diagnosis to be made while the patient is alive In this study raised serum concentrations of S100 protein were found in patients with Creutzfeldt-Jakob disease Serum concentrations of S100 could be used with a sensitivity of 77.8% and a specificity of 81.1% to confirm Creutzfeldt-Jakob disease in the differential diagnosis of diseases that cause dementia Serial measurement of S100 concentrations will enhance diagnostic accuracy