Biomarkers for parkinsonian disorders in CNS-originating EVs: promise and challenges.

Extracellular vesicles (EVs), including exosomes, microvesicles, and oncosomes, are nano-sized particles enclosed by a lipid bilayer. EVs are released by virtually all eukaryotic cells and have been shown to contribute to intercellular communication by transporting proteins, lipids, and nucleic acids. In the context of neurodegenerative diseases, EVs may carry toxic, misfolded forms of amyloidogenic proteins and facilitate their spread to recipient cells in the central nervous system (CNS). CNS-originating EVs can cross the blood-brain barrier into the bloodstream and may be found in other body fluids, including saliva, tears, and urine. EVs originating in the CNS represent an attractive source of biomarkers for neurodegenerative diseases, because they contain cell- and cell state-specific biological materials. In recent years, multiple papers have reported the use of this strategy for identification and quantitation of biomarkers for neurodegenerative diseases, including Parkinson's disease and atypical parkinsonian disorders. However, certain technical issues have yet to be standardized, such as the best surface markers for isolation of cell type-specific EVs and validating the cellular origin of the EVs. Here, we review recent research using CNS-originating EVs for biomarker studies, primarily in parkinsonian disorders, highlight technical challenges, and propose strategies for overcoming them.

α-Synuclein in blood exosomes immunoprecipitated using neuronal and oligodendroglial markers distinguishes Parkinson's disease from multiple system atrophy.

The diagnosis of Parkinson's disease (PD) and atypical parkinsonian syndromes is difficult due to the lack of reliable, easily accessible biomarkers. Multiple system atrophy (MSA) is a synucleinopathy whose symptoms often overlap with PD. Exosomes isolated from blood by immunoprecipitation using CNS markers provide a window into the brain's biochemistry and may assist in distinguishing between PD and MSA. Thus, we asked whether α-synuclein (α-syn) in such exosomes could distinguish among healthy individuals, patients with PD, and patients with MSA. We isolated exosomes from the serum or plasma of these three groups by immunoprecipitation using neuronal and oligodendroglial markers in two independent cohorts and measured α-syn in these exosomes using an electrochemiluminescence ELISA. In both cohorts, α-syn concentrations were significantly lower in the control group and significantly higher in the MSA group compared to the PD group. The ratio between α-syn concentrations in putative oligodendroglial exosomes compared to putative neuronal exosomes was a particularly sensitive biomarker for distinguishing between PD and MSA. Combining this ratio with the α-syn concentration itself and the total exosome concentration, a multinomial logistic model trained on the discovery cohort separated PD from MSA with an AUC = 0.902, corresponding to 89.8% sensitivity and 86.0% specificity when applied to the independent validation cohort. The data demonstrate that a minimally invasive blood test measuring α-syn in blood exosomes immunoprecipitated using CNS markers can distinguish between patients with PD and patients with MSA with high sensitivity and specificity. Future optimization and validation of the data by other groups would allow this strategy to become a viable diagnostic test for synucleinopathies.

The genetics of niche-specific behavioral tendencies in an adaptive radiation of cichlid fishes.

Behavior is critical for animal survival and reproduction, and possibly for diversification and evolutionary radiation. However, the genetics behind adaptive variation in behavior are poorly understood. In this work, we examined a fundamental and widespread behavioral trait, exploratory behavior, in one of the largest adaptive radiations on Earth, the cichlid fishes of Lake Tanganyika. By integrating quantitative behavioral data from 57 cichlid species (702 wild-caught individuals) with high-resolution ecomorphological and genomic information, we show that exploratory behavior is linked to macrohabitat niche adaptations in Tanganyikan cichlids. Furthermore, we uncovered a correlation between the genotypes at a single-nucleotide polymorphism upstream of the AMPA glutamate-receptor regulatory gene cacng5b and variation in exploratory tendency. We validated this association using behavioral predictions with a neural network approach and CRISPR-Cas9 genome editing.

Homosexual behaviour increases male attractiveness to females.

Male homosexual behaviour-although found in most extant clades across the Animal Kingdom-remains a conundrum, as same-sex mating should decrease male reproductive fitness. In most species, however, males that engage in same-sex sexual behaviour also mate with females, and in theory, same-sex mating could even increase male reproductive fitness if males improve their chances of future heterosexual mating. Females regularly use social information to choose a mate; e.g. male attractiveness increases after a male has interacted sexually with a female (mate choice copying). Here, we demonstrate that males of the tropical freshwater fish Poecilia mexicana increase their attractiveness to females not only by opposite-sex, but likewise, through same-sex interactions. Hence, direct benefits for males of exhibiting homosexual behaviour may help explain its occurrence and persistence in species in which females rely on mate choice copying as one component of mate quality assessment.

Development of a Novel Electrochemiluminescence ELISA for Quantification of α-Synuclein Phosphorylated at Ser129 in Biological Samples.

Synucleinopathies are a group of neurodegenerative diseases including Parkinson's disease (PD), dementia with Lewy bodies (DLB), and multiple system atrophy (MSA). These diseases are characterized by the aggregation and deposition of α-synuclein (α-syn) in Lewy bodies (LBs) in PD and DLB or as glial cytoplasmic inclusions in MSA. In healthy brains, only ∼4% of α-syn is phosphorylated at Ser129 (pS129-α-syn), whereas >90% pS129-α-syn may be found in LBs, suggesting that pS129-α-syn could be a useful biomarker for synucleinopathies. However, a widely available, robust, sensitive, and reproducible method for measuring pS129-α-syn in biological fluids is currently missing. We used Meso Scale Discovery (MSD)'s electrochemiluminescence platform to create a new assay for sensitive detection of pS129-α-syn. We evaluated several combinations of capture and detection antibodies and used semisynthetic pS129-α-syn as a standard for the assay at a concentration range from 0.5 to 6.6 × 104 pg/mL. Using the antibody EP1536Y for capture and an anti-human α-syn antibody (MSD) for detection was the best combination in terms of assay sensitivity, specificity, and reproducibility. We tested the utility of the assay for the detection and quantification of pS129-α-syn in human cerebrospinal fluid, serum, plasma, saliva, and CNS-originating small extracellular vesicles, as well as in mouse brain lysates. Our data suggest that the assay can become a widely used method for detecting pS129-α-syn in biomedical studies including when only a limited volume of sample is available and high sensitivity is required, offering new opportunities for diagnostic biomarkers, monitoring disease progression, and quantifying outcome measures in clinical trials.

Designed peptides as nanomolar cross-amyloid inhibitors acting via supramolecular nanofiber co-assembly.

Amyloid self-assembly is linked to numerous devastating cell-degenerative diseases. However, designing inhibitors of this pathogenic process remains a major challenge. Cross-interactions between amyloid-ß peptide (Aß) and islet amyloid polypeptide (IAPP), key polypeptides of Alzheimer's disease (AD) and type 2 diabetes (T2D), have been suggested to link AD with T2D pathogenesis. Here, we show that constrained peptides designed to mimic the Aß amyloid core (ACMs) are nanomolar cross-amyloid inhibitors of both IAPP and Aß42 and effectively suppress reciprocal cross-seeding. Remarkably, ACMs act by co-assembling with IAPP or Aß42 into amyloid fibril-resembling but non-toxic nanofibers and their highly ordered superstructures. Co-assembled nanofibers exhibit various potentially beneficial features including thermolability, proteolytic degradability, and effective cellular clearance which are reminiscent of labile/reversible functional amyloids. ACMs are thus promising leads for potent anti-amyloid drugs in both T2D and AD while the supramolecular nanofiber co-assemblies should inform the design of novel functional (hetero-)amyloid-based nanomaterials for biomedical/biotechnological applications.

The molecular tweezer CLR01 improves behavioral deficits and reduces tau pathology in P301S-tau transgenic mice.

BACKGROUND: Molecular tweezers (MTs) are broad-spectrum inhibitors of abnormal protein aggregation. A lead MT, called CLR01, has been demonstrated to inhibit the aggregation and toxicity of multiple amyloidogenic proteins in vitro and in vivo. Previously, we evaluated the effect of CLR01 in the 3 × Tg mouse model of Alzheimer's disease, which overexpresses mutant human presenilin 1, amyloid ß-protein precursor, and tau and found that subcutaneous administration of the compound for 1 month led to a robust reduction of amyloid plaques, neurofibrillary tangles, and microgliosis. CLR01 also has been demonstrated to inhibit tau aggregation in vitro and tau seeding in cell culture, yet because in Alzheimer's disease (AD) and in the 3 × Tg model, tau hyperphosphorylation and aggregation are thought to be downstream of Aß insults, the study in this model left open the question whether CLR01 affected tau in vivo directly or indirectly. METHODS: To determine if CLR01 could ameliorate tau pathology directly in vivo, we tested the compound similarly using the P301S-tau (line PS19) mouse model. Mice were administered 0.3 or 1.0 mg/kg per day CLR01 and tested for muscle strength and behavioral deficits, including anxiety- and disinhibition-like behavior. Their brains then were analyzed by immunohistochemical and biochemical assays for pathological forms of tau, neurodegeneration, and glial pathology. RESULTS: CLR01 treatment ameliorated muscle-strength deterioration, anxiety-, and disinhibition-like behavior. Improved phenotype was associated with decreased levels of pathologic tau forms, suggesting that CLR01 exerts a direct effect on tau in vivo. Limitations of the study included a relatively short treatment period of the mice at an age in which full pathology is not yet developed. In addition, high variability in this model lowered the statistical significance of the findings of some outcome measures. CONCLUSIONS: The findings suggest that CLR01 is a particularly attractive candidate for the treatment of AD because it targets simultaneously the two major pathogenic proteins instigating and propagating the disease, amyloid ß-protein (Aß), and tau, respectively. In addition, our study suggests that CLR01 can be used for the treatment of other tauopathies in the absence of amyloid pathology.

CNS-Derived Blood Exosomes as a Promising Source of Biomarkers: Opportunities and Challenges.

Eukaryotic cells release different types of extracellular vesicles (EVs) including exosomes, ectosomes, and microvesicles. Exosomes are nanovesicles, 30-200 nm in diameter, that carry cell- and cell-state-specific cargo of proteins, lipids, and nucleic acids, including mRNA and miRNA. Recent studies have shown that central nervous system (CNS)-derived exosomes may carry amyloidogenic proteins and facilitate their cell-to-cell transfer, thus playing a critical role in the progression of neurodegenerative diseases, such as tauopathies and synucleinopathies. CNS-derived exosomes also have been shown to cross the blood-brain-barrier into the bloodstream and therefore have drawn substantial attention as a source of biomarkers for various neurodegenerative diseases as they can be isolated via a minimally invasive blood draw and report on the biochemical status of the CNS. However, although isolating specific brain-cell-derived exosomes from the blood is theoretically simple and the approach has great promise, practical details are of crucial importance and may compromise the reproducibility and utility of this approach, especially when different laboratories use different protocols. In this review we discuss the role of exosomes in neurodegenerative diseases, the usefulness of CNS-derived blood exosomes as a source of biomarkers for these diseases, and practical challenges associated with the methodology of CNS-derived blood exosomes and subsequent biomarker analysis.

A century later: Adaptive plasticity and rapid evolution contribute to geographic variation in invasive mosquitofish.

One century after their introduction to Europe, eastern mosquitofish (Gambusia holbrooki) represent a natural experiment to determine the relative contributions of adaptive plasticity and rapid evolutionary change in creating large-scale geographic variation in phenotypes. We evaluated the population-genetic structure and invasion history based on allele length polymorphisms of 15 nuclear microsatellites, which we quantified for N = 660 individuals from 23 populations sampled in 2013 across the invasive range of G. holbrooki in Europe. We analysed body-shape and life-history variation in N = 1331 individuals from 36 populations, sampled in 2013 and 2017, and tested heritability of phenotypic differences in a subset of four populations using a common-garden experiment. The genetic structure of wild-caught individuals suggested a single introduction for all European mosquitofish, which were genetically impoverished compared to their native counterparts. We found some convergent patterns of phenotypic divergence across native and invasive climatic gradients (e.g., increased body size in colder/more northern populations); however, several phenotypic responses were not consistent between sampling years, pointing towards plastic phenotypes. Our analysis of common-garden reared individuals uncovered moderate heritability estimates only for two measures of male body size (intraclass correlation coefficient, ICC = 0.628 and 0.556) and offspring fat content (ICC = 0.734), while suggesting high levels of plasticity in most other phenotypic traits (ICC ≤ 0.407). Our results highlight the importance of phenotypic plasticity in invasive species during range expansions and demonstrate that strong selective pressures-in this case towards increased body size in colder environments-simultaneously promote rapid evolutionary divergence.

An improved process for the production of highly purified recombinant thaumatin tagged-variants.

The sweetest tasting molecule known is the protein thaumatin, first isolated from the katemfe fruit, Thaumatococcus daniellii. Thaumatin is used in the food and beverage industry as a low-calorie sugar substitute. Thaumatin interacts with taste receptors in the oral cavity eliciting a persistent sweet taste and a bitter, liquorice flavor. Recombinant thaumatin was expressed in Pichia pastoris and through a co-expression strategy with a molecular chaperone, yields of one engineered thaumatin variant increased by greater than two-fold. A detailed purification strategy for thaumatin is reported resulting in a homogenous sample recovered at a yield of 42%. The recombinant thaumatins were extensively characterised using size exclusion chromatography for homogeneity, reversed-phase HPLC for purity (99%), peptide digest LC-MS/MS for sequence determination, and circular dichroism and tryptophan fluorescence spectroscopies for conformational characterisation. These new thaumatin variants are amenable for bioconjugation, providing chemical biology tools for thaumatin:taste receptor interaction studies.