Designed Trpzip‑3 β‑Hairpin Inhibits Amyloid Formation in Two Different Amyloid Systems

The trpzip peptides are small, monomeric, and extremely stable β-hairpins that have become valuable tools for studying protein folding. Here, we show that trpzip-3 inhibits aggregation in two very different amyloid systems: transthyretin and Aβ(1–42). Interestingly, Trp → Leu mutations renders the peptide ineffective against transthyretin, but Aβ inhibition remains. Computational docking was used to predict the interactions between trpzip-3 and transthyretin, suggesting that inhibition occurs via binding to the outer region of the thyroxine-binding site, which is supported by dye displacement experiments

Changes in neuronal markers demonstrate specific neuronal populations targeted to certain brain regions.

<p>(a) Tyrosine hydroxylase staining of midbrain neurons. No visible change in staining pattern could be observed in any GSS/101LL animal tested at 240 dpi (<i>n</i> = 6) compared to age- and region-matched NBH/101LL controls (<i>n</i> = 6). A marked loss of staining pattern is observed in the midbrain neurons upon clinical onset (291.1 ± 5.3 dpi), indicative of a loss of tyrosine hydroxylase neurons upon clinical onset of disease. Scale bar = 200 μm. (b) MAP2 staining in brain stem has marked loss of MAP2 cell–associated staining compared to NBH/101LL brain stem age-matched control. Overall, levels of MAP2 staining are visibly lost in the ventral-medial parts of the thalamus compared to region- and age-matched NBH/101LL controls. No change could be observed in the staining pattern of MAP2 in any part of the cortex or cerebellum compared to region- and age-matched NBH/101LL controls. These findings are observed consistently across all animals tested; GSS/101LL (<i>n</i> = 9), NBH/101LL (<i>n</i> = 4). Scale bars = 100 μm. (c) Higher magnification examples of MAP2 neurons lost in the gigantoreticular nuclei of the brain stem but no loss of neurons evidenced in the cortex. (d) Parvalbumin staining of Purkinje cells of the cerebellum at clinical stages of disease in GSS/101LL animals (291.1 ± 5.3 dpi; <i>n</i> = 3) compared to age-matched NBH/101LL controls (<i>n</i> = 3). Scale bars = 100 μm. (e) Neuronal cell counts of substantia nigra (SN) neurons of the midbrain, gigantocellular reticular nuclei (Gi) of the brain stem, and retrosplenial granular region (RSGc) of the cortex from three representative animals. Cells counted based upon the number of cells showing positive staining for either tyrosine hydroxylase (TyHy+) in the SN or MAP2 in the Gi and RSGc. (f) Quantification of MAP2+ staining intensity from three representative animals showing a loss of MAP2 staining in brain stem and the thalamus but no change in the cortex or cerebellum. Quantification of staining was performed using colour deconvolution plug-in of Image-J software.</p

Case note: Pres. Rb Utrecht, (No. 111)

End-point dilutions by αSyn RT-QuIC of synucleinopathy CSF samples listed in Fig. 4. Each sample trace represents the average ThT signal of quadruplicate wells. Traces represent 15 (Blue), 7.5 (Red), 3.75 (Green), 1.89 (Purple), 0.94 (Orange), 0.47 (Black), 0.2 (Brown) and 0.1 (Dark Blue) μL of DLBD CSF diluted into normal pooled CSF, when needed, to give overall CSF sample volumes of 15 µL. (TIFF 1383 kb

Morphological glial cell responses are restricted to specific brain regions.

<p>(a) Severe astrogliosis is observed in the brain stem and thalamus of GSS/101LL but is not detected in NBH/101LL age-matched controls or in the cortex or cerebellum of GSS/101LL mice. (b) High magnification image demonstrating the change in astrocyte expression of GFAP in GSS/101LL mice compared to equivalent NBH/101LL brain regions. (c) A distinct change in cell morphology to that of a hypertrophied cell body and short thick processes could be observed in Iba1+ cells, indicative of activated microglia, in GSS/101LL brain stem and thalamus. No change in cell morphology was observed in either NBH/101LL age- and region-matched control samples or in GSS/101LL cortex and cerebellum samples. (d) High magnification image to highlight the shortening and thickening of microglial processes, a characteristic common to morphologically activated microglia. These findings are observed consistently across all animals tested; GSS/101LL (<i>n</i> = 9), NBH/101LL (<i>n</i> = 4). Scale bars = 100 μm.</p

RT-QuIC analysis of CWD-positive clinically ill white-tailed deer.

<p>Both 1:10 diluted and PTA precipitated saliva show positive results by RT-QuIC. Each line represents an individual experiment with the average of four replicates. Blue and light blue lines represent 1:10 diluted saliva experiments and red and dark red lines represent PTA precipitated counterpart of the same saliva samples. The left axis (blue) denotes ThT fluorescence for 1:10 diluted saliva samples and the right axis (red) denotes ThT fluorescence for PTA saliva samples. Dashed lines represent threshold for a positive sample. Blue for 1:10 diluted saliva experiments and red for PTA saliva experiments. Positive RT-QuIC results were observed for saliva from clinically ill white-tailed deer in 5 of 9 1:10 diluted saliva samples and 8 of 9 PTA precipitated saliva samples. Each saliva sample was from an individual deer.</p

All GSS/101LL brain regions show disease-associated gene expression changes.

<p>(A) Biolayout <i>Express</i>^3D graph showing a spatial representation of genes orientated according to their correlation to one another. Three major and separate components of highly correlated genes were formed using this software, which we term components A–C. This structure was used by the Markov clustering algorithm to divide the graph into clusters of co-expressed transcripts, which are shown in the graph as different colours. Representative clusters, shown as numbers 1–6, are shown on the graph, which highlights the expression differences found between each of the three major components identified. These can be viewed as average gene expression as bar graphs (B). (C) Filtered gene lists from the GSS/101LL cerebellum, thalamus, and brain stem are overlaid onto the original graph. Shown here are the filtered genes highlighted in yellow (cerebellum), purple (thalamus), or turquoise (brain stem) as part of component B of the main graph shown in (A). The highlighted genes in each brain region were observed predominantly within the same part of the graph rather than segregating into distinct groups, demonstrating that these differentially expressed genes between brain regions were highly correlated.</p

SHrPrP substrate can detect CWD-positive brain over a range of dilutions from 10^-4 to 10^-7.

<p>CWD positive and negative brain was serially diluted and analyzed by RT-QuIC assay. Each line for positive CWD samples represents the average of 4 individual wells from 4 independent experiments.</p

Kaplan-Meier survival curve for mice inoculated with CWD+ saliva.

<p>The same saliva samples from deer assayed in Fig. 1 (#133 and 144) were inoculated into cervidized mice (Tg[CerPrP] 5037) as well as saliva from a sham inoculated deer. 50% of mice were euthanized due to clinical disease after ~500 days.</p

Detection of misfolded PrP using IHC at different time-points in different brain regions.

<p>(a) At 150 dpi, small quantities of fine-punctate misfolded PrP deposits can be detected in the midbrain. This positive staining could be observed in five of twelve GSS/101LL mice tested, but no staining was observed in any NBH/101LL animal in any brain region (<i>n</i> = 12). At 220 dpi, fine-punctate misfolded PrP deposits were detectable in both the midbrain and brain stem, which was observed in four of six GSS/101LL mice tested, but no staining was observed in any NBH/101LL animal in any brain region (<i>n</i> = 6). At clinical onset of disease (291.1 ± 5.3 dpi), misfolded PrP staining could be observed in midbrain, brain stem, and the thalamus but not in cortex or cerebellum in GSS/101LL mice. This staining pattern was observed in all mice tested at this stage (<i>n</i> = 9), whereas no staining was observed in any NBH/101LL animal in any brain region tested (<i>n</i> = 4). Scale bars: midbrain = 100 μm, brain stem, thalamus, cortex, and cerebellum = 200 μm. (b) Quantification of PrP+ staining intensity. The levels of PrP+ staining are originally high in the midbrain, but at later time-points in other brain regions, such as brain stem and thalamus, the levels of PrP+ staining increase to comparable levels to that of the midbrain. In cortex and cerebellum, no change in PrP+ staining was observed. Quantitation was performed using colour deconvolution plug-in to Image-J software.</p

Disease-associated gene expression changes can be predominantly attributed to microglia in all GSS/101LL brain regions tested.

<p>(a) Spider graph representation of component B genes after filtering. Up-regulation of genes in all GSS/101LL brain regions, but particularly increased in GSS/101LL brain stem and thalamus compared to GSS/101LL cerebellum and cortex. N = number of genes present after data are filtered that constitute the average intensity value. The number of genes represented is highest in GSS/101LL brain stem but lowest in GSS/101LL cortex. (b) Gene expression can be attributed to specific cell types when overlaid onto previous microarray datasets. These data show a simplified version to demonstrate how different genes that are known to have selective expression in specific cell types in vivo can be attributed to their expected cell type. For example, Cd11b is a gene generally regarded as a pan-macrophage marker, and hence we show the increased expression of this gene in macrophage/microglial cell populations compared to other cell types. Colony-stimulating factor 1 (Csf1) is a gene that is up-regulated during immune cell activation, shown here by its increased expression in lipopolysaccharide-activated macrophages. Gfap is a gene expressed highly in astrocytes, and, indeed, we show the high and specific expression of Gfap in astrocytes in this dataset. Finally, synapsin I is a synaptic-specific protein and therefore will most commonly be expressed in neurons, as is shown here. (c) Attribution of genes that are identified in component b to their respective cell type shows that a majority of genes that are identified in component b can be attributed to macrophage/microglia. (d) Representation of the macrophage/microglia gene list overlap of different brain regions tested.</p