Neuroprotective Function of Non-Proteolytic Amyloid-β Chaperones in Alzheimer’s Disease

This chapter attempts to explore protective role of chaperone proteins in the neurodegenerative diseases caused by amyloidosis. These chaperones prevent amyloid pathology either directly, through chemical interactions with amyloidogenic species to mediate their refolding, solubilization and degradation, or indirectly, by scavenging reactive oxygen species produced as by-products of amyloid aggregation. Here we focus on structural and morphological changes during aggregation of amyloids which have been identified using Nuclear magnetic resonance spectroscopy, X-ray crystallography, Electron microscopy, Atomic force microscopy and other biophysical techniques as well as interactions between chaperone proteins and amyloid moieties. Non-proteolytic chaperones mediate amyloid clearance and metabolism through conformational changes due to proximity binding. In this chapter, we delineate these interactions as well as the molecular mechanism of chaperones used to sequester ROS products of amyloidosis with focus on amyloid-β peptides associated with the Alzheimer’s disease

Developing amyloid-β chaperone lipocalin-type prostaglandin D synthase protein as a magnetic resonance active early diagnostic tool

Lipocalin-type prostaglandin D synthase (L-PGDS) is an endogenous brain protein, shown previously as a significant chaperone for amyloid β (Aβ) peptide. It binds to monomeric Aβ as well as mature fibrils and is capable of inhibiting their aggregation. We recently showed that L-PGDS could successfully breakdown mature Aβ fibrils (manuscript submitted), which opens avenues for its use as a therapeutic agent in cases of Alzheimer's disease (AD). This thesis is aimed at utilizing the theranostic (therapeutic + diagnostic) potential of L-PGDS in AD by early detection of Aβ using MRI. For this purpose, we covalently conjugated the recombinant L-PGDS protein with iron oxide-based nanoparticles with different outer coatings. Four different nanoparticles were compared for their T2 contrast enhancement. The functionality of the conjugated protein, its inhibition activity towards Aβ, the effect on cellular viability and the tendency to form aggregates were compared to choose the most efficient composition. For this purpose, we used a multi-disciplinary approach using chemical and biophysical assays, spectrophotometric techniques, structural and morphological studies, cellular assays and tissue histology, and pre-clinical mouse imaging to develop an L-PGDS-based theranostic molecular probe. Based on the results of in vitro assay, the biological probes designed by covalent conjugation of magnetic resonance active ferritin protein nanocages conjugated with L-PGDS were used for studies in AD mice. Injections in diseased mice showed hypointensity in mouse brain areas correlated with the presence of amyloid-rich structures compared to age-matched healthy mice. All MRI data were acquired using Bruker spectrometers, and Paravision 6.0 software was used for processing and analysis. T2 relaxation curves were prepared for nanoparticles to compare relaxivity differences in the biological buffer. Differences in T2-star weighted image intensities were identified in the brains of healthy mice after non-invasive intranasal administration of conjugated L-PGDS probes. L-PGDS conjugated with ferritin nanocages injected in the ventricular chambers were seen to disperse towards the amyloid-rich brain regions in AD mice. To this end, we have shown here that L-PGDS protein has great outlook as a diagnostic agent for early identification of AD hallmarks in disease-prone populations, and potential as a therapeutic intervention.Doctor of Philosoph

Magnetic Nanoparticles as In Vivo Tracers for Alzheimer’s Disease

Drug formulations and suitable methods for their detection play a very crucial role in the development of therapeutics towards degenerative neurological diseases. For diseases such as Alzheimer’s disease, magnetic resonance imaging (MRI) is a non-invasive clinical technique suitable for early diagnosis. In this review, we will discuss the different experimental conditions which can push MRI as the technique of choice and the gold standard for early diagnosis of Alzheimer’s disease. Here, we describe and compare various techniques for administration of nanoparticles targeted to the brain and suitable formulations of nanoparticles for use as magnetically active therapeutic probes in drug delivery targeting the brain. We explore different physiological pathways involved in the transport of such nanoparticles for successful entry in the brain. In our lab, we have used different formulations of iron oxide nanoparticles (IONPs) and protein nanocages as contrast agents in anatomical MRI of an Alzheimer’s disease (AD) brain. We compare these coatings and their benefits to provide the best contrast in addition to biocompatibility properties to be used as sustainable drug-release systems. In the later sections, the contrast enhancement techniques in MRI studies are discussed. Examples of contrast-enhanced imaging using advanced pulse sequences are discussed with the main focus on important studies in the field of neurological diseases. In addition, T1 contrast agents such as gadolinium chelates are compared with the T2 contrast agents mainly made of superparamagnetic inorganic metal nanoparticles

Magnetic nanoparticles as in vivo tracers for Alzheimer’s disease

Drug formulations and suitable methods for their detection play a very crucial role in the development of therapeutics towards degenerative neurological diseases. For diseases such as Alzheimer’s disease, magnetic resonance imaging (MRI) is a non-invasive clinical technique suitable for early diagnosis. In this review, we will discuss the different experimental conditions which can push MRI as the technique of choice and the gold standard for early diagnosis of Alzheimer’s disease. Here, we describe and compare various techniques for administration of nanoparticles targeted to the brain and suitable formulations of nanoparticles for use as magnetically active therapeutic probes in drug delivery targeting the brain. We explore different physiological pathways involved in the transport of such nanoparticles for successful entry in the brain. In our lab, we have used different formulations of iron oxide nanoparticles (IONPs) and protein nanocages as contrast agents in anatomical MRI of an Alzheimer’s disease (AD) brain. We compare these coatings and their benefits to provide the best contrast in addition to biocompatibility properties to be used as sustainable drug-release systems. In the later sections, the contrast enhancement techniques in MRI studies are discussed. Examples of contrast-enhanced imaging using advanced pulse sequences are discussed with the main focus on important studies in the field of neurological diseases. In addition, T1 contrast agents such as gadolinium chelates are compared with the T2 contrast agents mainly made of superparamagnetic inorganic metal nanoparticles.Ministry of Education (MOE)Published versionThis work was funded by Grant from Ministry of Education of Singapore to Konstantin Pervushin, grant number M4012175

PREreview of "The circadian clock is a pacemaker of the axonal regenerative ability"

<p><strong>This Zenodo record is a permanently preserved version of a PREreview. You can view the complete PREreview at <a href="https://prereview.org/reviews/10079230">https://prereview.org/reviews/10079230</a>.</strong></p> <p><i>This review reflects comments and contributions by Bhargy Sharma, Ryan Cubero & Anna Oliveras. Review synthesized by Ryan Cubero.</i></p><p>In this preprint, De Virgiliis and colleagues use a murine model of sciatic injury to provide functional evidence that peripheral nerve regeneration is affected by circadian rhythm. The authors show that regenerative capacity of mice DRG neurons is time-of-day dependent and that the disruption of the intrinsic neuronal circadian rhythm via knockdown or deletion of Bmal1, a non-redundant core clock gene, decreases regeneration capability after injury. They found that injuries performed at ZT20 induce a transcriptional program that enhances regeneration and targets long-lasting re-innervation. Also, lithium, a chono-active compound, is  pinpointed as a new therapeutic avenue for nerve repair. Although the role of the circadian clock has already been shown for other wound healing processes, these novel findings describe for the first time the regenerative potential in the PNS is controlled by neuronal intrinsic circadian clock and is time-of-day dependent. </p><p>Positive aspects of the study:</p><ul><li><p>The preprint starts with a meta-analysis of previously published transcriptomes, which allowed them to establish "circadian rhythms" as a potential common pathway. Studies like this that make use of the vast published transcriptomes should be highly encouraged and commended.</p></li><li><p>Overall, the experiments are well designed and well explained with images. The supplementary material provides extensive control experiments that help to draw and strengthen the conclusions.</p></li><li><p>The conclusions suggest a careful re-evaluation of previous data interpretation taking in account the time dependency of data acquisition. This might help to solve previous controversial data. As well, the conclusions highlight  important considerations for future experimental designs. We believe that the findings provided by the authors are remarkably relevant for the scientific community, specially those in the field of axonal regeneration research.</p></li><li><p>The implications of the study for future therapeutic strategies:</p><ul><li><p>Focusing current neurorehabilitation therapies to time-of-day effects</p></li><li><p>Using chrono-active compounds for axon regeneration therapies</p></li><li><p>Potential gene therapy targeting circadian clock molecular machinery</p></li></ul></li></ul><p>Major aspects to be addressed:</p><ul><li><p>Although authors found transcriptional differences in ZT20 vs ZT8 after injury, we wonder if there is already a background regeneration machinery that is highly active at ZT20 in unchallenged DRGs that allows for increased regenerative capacity.</p></li><li><p>Although authors clearly show that the regeneration ability depends on Baml1, they miss proving that Baml1 is directly binding regulatory gene sequences and actually orchestrating the transcriptional response. If not Baml1, who is the main orchestrator?</p></li><li><p>While the authors denote lithium as a chrono-active drug, the way it affects circadian rhythm-dependent enhancement of regenerative ability is unclear. We suggest a thorough characterization of the effect of lithium treatment across all ZT during 24h.</p></li><li><p>The authors should discuss the results of the recent publication <a href="https://www.nature.com/articles/s41467-023-40816-7">https://www.nature.com/articles/s41467-023-40816-7</a> were they found that Baml1 controls axon regeneration via Tet3 epigenetics</p></li><li><p>The authors show that the role of inflammation triggered by non-neuronal cells in nerve regeneration is not clock-dependent. Nevertheless, neutrophin, BDNF and NFG levels were measured in naive DRG neurons. The authors should specify if "naive" means uninjured neurons. In this case, we recommend comparing these data with post-injury levels.</p></li></ul><p>Minor comments:</p><ul><li><p>In the introduction, we would suggest to include more background information (more details on the different mechanisms of PNS regeneration or evolutionary perspective on why regeneration might be controlled by circadian rhythms) and limit the description of the results to a very short summary.</p></li><li><p>We consider that Figure 1 can gain some clarity by reworking the following aspects:</p><ul><li><p>The heatmap shows biological processes instead of genes. Caption of the figure legend should be corrected accordingly.</p></li><li><p>Mention abbreviation of "DE genes" in figure legend</p></li><li><p>In the comparative GO analysis, we wonder how much overlap there is in the differentially expressed circadian rhythm genes across the regenerative models?</p></li><li><p>Mention abbreviation of SGC10 in the main text. A reference for this marker would be helpful as well.</p></li><li><p>We suggest having the fluorescence intensity distribution plotted beneath the representative image so readers can get a better sense of the regeneration index.</p></li><li><p>Legend for 1F is incorrect. Should be modified for G and H accordingly.</p></li></ul></li><li><p>We would like to encourage the authors to give some reason why they chose a sciatic nerve crush over sciatic nerve axotomy</p></li><li><p>Supplementary figure 1 shows representative images ZT0, 4, 8, 12, 16 and 20 instead of only ZT8 and 20. Caption should be revised. In the same figure legend, authors explain that "Fluorescence intensity was measured in one series of tissue sections for each nerve". We suggest providing more details on the average size of each tissue section, and how many sections in one series.</p></li><li><p>Edit y-axis label of supplementary figure 2B to 'CTB+ DRG neurons' </p></li><li><p>In supplementary figures 3 and 4, describing abbreviations for LY6G and CD68 will help the reader. As well, we suggest to mark on the images the site of the injury and double-check consistency between the number of replicates shown in the plots and the figure legend.</p></li><li><p>How many hours post-injury refers to the data shown in supplementary figure 5?</p></li><li><p>The text of supplementary figure 7 legend should be checked for clarity,  "mRNA levels of Bmal1 mRNA levels" is confusing and also the references to other figures.</p></li><li><p>Also, why in supplementary figure 7B, 5 replicates are chosen instead of 6 that were included in figure 1D?</p></li><li><p>Figure 2B and supplementary Figure 8A should be consistently labeled using the same labels (ZT8_1, ZT8_2, and so on) in the heatmap and the PCA analysis of the clustered differentially expressed genes.</p></li><li><p>Regarding the data presented in Supplementary Figure 8B, we wonder if there could be any reason why the ZT20 transcriptome is more likely to resemble IF than EE, if both non-injury models promote regeneration? Also, when comparing their data with previously published dataset, we find important to comment at which ZT the samples were collected, if available</p></li><li><p>Clarify in Figure 2C how to interpret dot-plot data regarding size and color code of the dots</p></li><li><p>In Figure 2D, we wonder whether there could be RAGs that are already upregulated in ZT20 even without the SNC? On that figure the y-label states -log12FC, while the figure legend states -log2 Fold Change.</p></li><li><p>In Table 2, we recommend to add indications for green colored fold changes and specify whether  are these log2(FC) or log10(FC)</p></li><li><p>In Figure 3B the x-labels don't match between the histogram and the legend. The so</p></li></ul><p>Comments on reporting:</p><ul><li><p>As the number of biologically independent groups varies from 3 to 6 for different experiments for similar samples throughout this study, an explanation for these differences in discussion would be good.</p></li></ul> <h2>Competing interests</h2> <p> The author declares that they have no competing interests. </p&gt

Proton conductivity of the protein-based velvet worm slime

Summary: The properties of complex bodily fluids are linked to their biological functions through natural selection. Velvet worms capture their prey by ensnaring them with a proteinaceous fluid (slime). We examined the electrical conductivity of slime and found that dry slime is an insulator. However, its conductivity can increase by up to 106 times in its hydrated state, which can be further increased by an order in magnitude under acidic hydration (pH ≈ 2.3). The transient current measured using ion-blocking electrodes showed a continuous decay for up to 7 h, revealing slime’s nature as a proton conducting material. Slime undergoes a spontaneous fibrilization process producing high aspect ratio ≈ 105 fibers that exhibit an average conductivity ≈2.4 ± 1.1 mS cm−1. These findings enhance our understanding of slime as a natural biopolymer and provide molecular-level guidelines to rationally design biomaterials that may be employed as hygroscopic conductors

Utf1 contributes to intergenerational epigenetic inheritance of pluripotency

Undifferentiated embryonic cell transcription factor 1 (Utf1) is expressed in pluripotent embryonic stem cells (ESCs) and primordial germ cells (PGCs). Utf1 expression is directly controlled by pluripotency factors Oct4 and Sox2, which form a ternary complex with the Utf1 enhancer. The Utf1 protein plays a role in chromatin organization and epigenetic control of bivalent gene expression in ESCs in vitro, where it promotes effective cell differentiation during exit from pluripotency. The function of Utf1 in PGCs in vivo, however, is not known. Here, we report that proper development of Utf1 null embryos almost entirely depends on the presence of functional Utf1 alleles in the parental germline. This indicates that Utf1’s proposed epigenetic role in ESC pluripotency in vitro may be linked to intergenerational epigenetic inheritance in vivo. One component - or at least facilitator - of the relevant epigenetic mark appears to be Utf1 itself, since Utf1-driven tomato reporter and Utf1 are detected in mature germ cells. We also provide initial evidence for a reduced adult testis size in Utf1 null mice. Our findings thus point at unexpected functional links between the core ESC pluripotency factor network and epigenetic inheritance of pluripotency.MOE (Min. of Education, S’pore)NMRC (Natl Medical Research Council, S’pore)Published versio

Magnetoferritin enhances T₂ contrast in magnetic resonance imaging of macrophages

Imaging of immune cells has wide implications in understanding disease progression and staging. While optical imaging is limited in penetration depth due to light properties, magnetic resonance (MR) imaging provides a more powerful tool for the imaging of deep tissues where immune cells reside. Due to poor MR signal to noise ratio, tracking of such cells typically requires contrast agents. This report presents an in-depth physical characterization and application of archaeal magnetoferritin for MR imaging of macrophages - an important component of the innate immune system that is the first line of defense and first responder in acute inflammation. Magnetoferritin is synthesized by loading iron in apoferritin in anaerobic condition at 65 °C. The loading method results in one order of magnitude enhancement of r1 and r2 relaxivities compared to standard ferritin synthesized by aerobic loading of iron at room temperature. Detailed characterizations of the magnetoferritin revealed a crystalline core structure that is distinct from previously reported ones indicating magnetite form. The magnetite core is more stable in the presence of reducing agents and has higher peroxidase-like activities compared to the core in standard loading. Co-incubation of macrophage cells with magnetoferritin in-vitro shows significantly higher enhancement in T2-MRI contrast of the immune cells compared to standard ferritin.Nanyang Technological UniversityThis work was supported by NTU-Northwestern Institute for Nano-medicine located at the International Institute for Nanotechnology, Northwestern University, USA and the Nanyang Technological Univer-sity, Singapore (Grant No. M4081504.F40.706022)

Complete sequences of the velvet worm slime proteins reveal that slime formation is enabled by disulfide bonds and intrinsically disordered regions

The slime of velvet worms (Onychophora) is a strong and fully biodegradable protein material, which upon ejection undergoes a fast liquid-to-solid transition to ensnare prey. However, the molecular mechanisms of slime self-assembly are still not well understood, notably because the primary structures of slime proteins are yet unknown. Combining transcriptomic and proteomic studies, the authors have obtained the complete primary sequences of slime proteins and identified key features for slime self-assembly. The high molecular weight slime proteins contain cysteine residues at the N- and C-termini that mediate the formation of multi-protein complexes via disulfide bonding. Low complexity domains in the N-termini are also identified and their propensity for liquid-liquid phase separation is established, which may play a central role in slime biofabrication. Using solid-state nuclear magnetic resonance, rigid and flexible domains of the slime proteins are mapped to specific peptide domains. The complete sequencing of major slime proteins is an important step toward sustainable fabrication of polymers inspired by the velvet worm slime.Agency for Science, Technology and Research (A*STAR)Ministry of Education (MOE)National Research Foundation (NRF)Published versionThis research was funded by ExxonMobil through the Singapore Energy Research Center (SgEC). The authors also acknowledge financial support from the Singapore Ministry of Education (MOE) through an Academic Research Fund (AcRF) Tier 3 grant (grant no. MOE 2019-T3-1-012). Y.T.L. and R.M.S. thank the support of A*STAR Core funding and the Singapore National Research Foundation under its NRF-SIS “SingMass” scheme (RMS)

Abundant neuroprotective chaperone Lipocalin-type prostaglandin D synthase (L-PGDS) disassembles the Amyloid-β fbrils

Misfolding of Amyloid β (Aβ) peptides leads to the formation of extracellular amyloid plaques. Molecular chaperones can facilitate the refolding or degradation of such misfolded proteins. Here, for the first time, we report the unique ability of Lipocalin-type Prostaglandin D synthase (L-PGDS) protein to act as a disaggregase on the pre-formed fibrils of Aβ(1–40), abbreviated as Aβ40, and Aβ(25–35) peptides, in addition to inhibiting the aggregation of Aβ monomers. Furthermore, our proteomics results indicate that L-PGDS can facilitate extraction of several other proteins from the insoluble aggregates extracted from the brain of an Alzheimer’s disease patient. In this study, we have established the mode of binding of L-PGDS with monomeric and fibrillar Aβ using Nuclear Magnetic Resonance (NMR) Spectroscopy, Small Angle X-ray Scattering (SAXS), and Transmission Electron Microscopy (TEM). Our results confirm a direct interaction between L-PGDS and monomeric Aβ40 and Aβ(25–35), thereby inhibiting their spontaneous aggregation. The monomeric unstructured Aβ40 binds to L-PGDS via its C-terminus, while the N-terminus remains free which is observed as a new domain in the L-PGDS-Aβ40 complex model.MOE (Min. of Education, S’pore)Published versio