Accumulation Of Fibrils Research Articles

Cathepsin L prevents the accumulation of alpha-synuclein fibrils in the cell.

The deposition of α-synuclein (α-Syn) fibrils in neuronal cells has been implicated as a causative factor in Parkinson's disease (PD) and dementia with Lewy Bodies (DLB). α-Syn can be degraded by autophagy, proteasome, and chaperone-mediated autophagy, and previous studies have suggested the potency of certain cathepsins, lysosomal proteases, for α-Syn degradation. However, no studies have comprehensively evaluated all cathepsins. Here, we evaluated the efficacy of all 15 cathepsins using a cell model of α-Syn fibril propagation and found that overexpression of cathepsin L (CTSL) was the most effective in preventing the accumulation of α-Syn aggregates. CTSL-mediated degradation of α-Syn aggregates was dependent on the autophagy machinery, and CTSL itself promoted autophagy flux. Interestingly, CTSL was effective in autophagic degradation of wild-type (WT) α-Syn, but not in the case of A53T and E46K missense mutations, which are causative for familial PD. These results suggest that CTSL is a potential therapeutic strategy for sporadic PD pathology in WT α-Syn.

TC14012 enhances the anti-fibrosis effects of UC-MSCs on the liver by reducing collagen accumulation and ameliorating inflammation

BackgroundMesenchymal stem cells (MSCs) are attracting attention as a promising cell-based therapy for the treatment of liver fibrosis or cirrhosis. However, the strategies and potential mechanisms of MSCs therapy need further investigation. The CXCL12/CXCR4/CXCR7 chemokine axis is well known to regulate cell migration and is involved in the regulation of liver fibrosis. This study aims to treat MSCs with a CXCR7-specific agonist to evaluate its therapeutic effects on hepatic fibrosis and potential mechanisms.MethodsTC14012, a potent agonist of CXCR7, has been used to pretreat human umbilical cord-derived MSCs (UC-MSCs) and assess its effect on proliferation, apoptosis, migration, immunoregulation, and gene regulatory network. Then, CCl4-induced liver fibrosis mice models were used to evaluate the therapeutic effect and mechanism of TC14012-treated UC-MSCs for treating hepatic fibrosis.ResultsTC14012 increased CXCR7 expression in UC-MSCs. Notably, co-culture of liver sinusoidal endothelial cells (LSEC) with TC14012-pretreated UC-MSCs increased CXCR7 expression in LSEC. Additionally, TC14012 promoted cell migration and mediated the immunoregulation of UC-MSCs. Compared to UC-MSCs without TC14012 pretreatment, UC-MSCs treated with TC14012 ameliorated live fibrosis by restoring CXCR7 expression, reducing collagen fibril accumulation, inhibiting hepatic stellate cells activation, and attenuating the inflammatory response.ConclusionThis study suggests that TC14012 pretreatment can enhance the therapeutic effects of UC-MSCs on liver fibrosis, mainly by promoting the migration and immunoregulation of MSCs.

Disaggregation of amyloid-beta fibrils via natural metabolites using long timescale replica exchange molecular dynamics simulation studies.

Amyloid fibrils are self-assembled fibrous protein aggregates that are associated with several presently incurable diseases such as Alzheimer's. disease that is characterized by the accumulation of amyloid fibrils in the brain, which leads to the formation of plaques and the death of brain cells. Disaggregation of amyloid fibrils is considered a promising approach to cure Alzheimer's disease. The mechanism of amyloid fibril formation is complex and not fully understood, making it difficult to develop drugs that can target the process. Diacetonamine and cystathionine are potential lead compounds to induce disaggregation of amyloid fibrils. In the current research, we have used long timescale molecular simulation studies and replica exchange molecular dynamics (REMD) for 1000 ns (1 μs) to examine the mechanisms by which natural metabolites can disaggregate amyloid-beta fibrils. Molecular docking was carried out using Glide and with prior protein minimization and ligand preparation. We focused on a screening a database of natural metabolites, as potential candidates for disaggregating amyloid fibrils. We used Desmond with OPLS 3e as a force field. MM-GBSA calculations were performed. Blood-brain barrier permeability, SASA, and radius of gyration parameters were calculated.

Spatial Neurolipidomics at the Single Amyloid-β Plaque Level in Postmortem Human Alzheimer's Disease Brain.

Lipid dysregulations have been critically implicated in Alzheimer's disease (AD) pathology. Chemical analysis of amyloid-β (Aβ) plaque pathology in transgenic AD mouse models has demonstrated alterations in the microenvironment in the direct proximity of Aβ plaque pathology. In mouse studies, differences in lipid patterns linked to structural polymorphism among Aβ pathology, such as diffuse, immature, and mature fibrillary aggregates, have also been reported. To date, no comprehensive analysis of neuronal lipid microenvironment changes in human AD tissue has been performed. Here, for the first time, we leverage matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI-MSI) through a high-speed and spatial resolution commercial time-of-light instrument, as well as a high-mass-resolution in-house-developed orbitrap system to characterize the lipid microenvironment in postmortem human brain tissue from AD patients carrying Presenilin 1 mutations (PSEN1) that lead to familial forms of AD (fAD). Interrogation of the spatially resolved MSI data on a single Aβ plaque allowed us to verify nearly 40 sphingolipid and phospholipid species from diverse subclasses being enriched and depleted, in relation to the Aβ deposits. This included monosialo-gangliosides (GM), ceramide monohexosides (HexCer), ceramide-1-phosphates (CerP), ceramide phosphoethanolamine conjugates (PE-Cer), sulfatides (ST), as well as phosphatidylinositols (PI), phosphatidylethanolamines (PE), and phosphatidic acid (PA) species (including Lyso-forms). Indeed, many of the sphingolipid species overlap with the species previously seen in transgenic AD mouse models. Interestingly, in comparison to the animal studies, we observed an increased level of localization of PE and PI species containing arachidonic acid (AA). These findings are highly relevant, demonstrating for the first time Aβ plaque pathology-related alteration in the lipid microenvironment in humans. They provide a basis for the development of potential lipid biomarkers for AD characterization and insight into human-specific molecular pathway alterations.

Potential Novel Treatments for Parkinson’s Disease

This review aims to evaluate novel developments in treatments for Parkinson’s disease that focus on slowing, stopping, and/or reversing the neurodegeneration associated with the disease. Parkinson’s disease (PD) is a neurodegenerative disease that affects dopaminergic neurons in the substantia nigra, located in the basal ganglia of the brain. This region also contains high levels of alpha synuclein, a protein found abundantly in the brain due to its important role in neurotransmitter release. α-synuclein has been found to be misfolded and overexpressed in people with PD, which leads to fibril formation and aggregation, which directly interfere with basic cellular processes and results in neurodegeneration. In PD patients, dopaminergic neurons are most affected; therefore, an artificial supply of dopamine must be provided to them via dopamine precursors that can cross the blood-brain barrier. However, these medications fail to slow, stop, or reverse the progression of PD and merely pose as treatments for physical symptoms, such as tremors. Researchers are now focusing on 2 major avenues for new PD treatments: treatments that target α-synuclein aggregates/Lewy body assemblies and treatments that focus on replacement of lost dopaminergic neurons in the brain. The use of antibodies (immunotherapy) to target and clear αsynuclein aggregates or the upregulation of genes that encode autophagic mechanisms to destroy dysfunctional proteins were shown to reduce α-synuclein aggregation and behavioral deficits in mice. Results from clinical trials have shown a 96.5% reduction in the concentration of extracellular α-synuclein aggregates, and doses were well tolerated with no serious side effects. In order to rebuild the neurons that were lost to PD neurodegeneration, a renewable source of dopamine-producing cells that can integrate into the host brain and survive for years is required. More specifically, the reprogramming of astrocytes can be used to develop functional dopaminergic neurons. To reprogram the astrocytes and fibroblasts, the RNA binding protein PTB was suppressed; PTB suppresses numerous neuronal genes required for neuronal maturation, thus, downregulation of PTB generates functional, mature neurons. When in vivo astrocyte reprogramming was performed in a mouse model of PD, researchers found new, successful dopaminergic neurons in the substantia nigra and increased dopamine levels. Although both avenues for treatment require further clinical trials and testing in human subjects, they both hold significant promise in the future.

Identification and Characterization of Neuroprotective Properties of Thaumatin-like Protein 1a from Annurca Apple Flesh Polyphenol Extract.

Alzheimer's disease (AD) and Parkinson's disease (PD) are multifactorial neurodegenerative disorders that are mostly treated with drugs inhibiting key enzymes of cholinergic and aminergic neurotransmission, such as acetyl and butyryl cholinesterase (AChE, BuChE) or monoamine oxidases (MAO)-A/B, and of Aβ1-40 aggregation. Diet plant components with multitarget functions are promising compounds in the prevention of AD and PD. Our aim was to identify neuroprotective compounds from Annurca apple polyphenol extract (AFPE). AFPE was fractionated by gel filtration, and the eluted peaks were subjected to chemical analyses (i.e., RP-HPLC and mass spectrometry), determination of inhibitory enzyme activity and cell effects by MTT, and morphology assays. In AFPE, we identified thaumatin-like protein 1a, belonging to the pathogenesis-related protein (PR) family. This protein showed the best inhibitory activity on AChE, MAO-A (IC50 = 5.53 µM and 1.71 µM, respectively), and Aβ1-40 fibril aggregation (IC50 = 9.16 µM), compared to AFPE and other polyphenol-containing fractions. Among the latter, Peak 4 reverted Aβ fibril formation (IC50 = 104.87 µM). Moreover, thaumatin-like protein 1a protected AGS and MKN-28 cells from serum-deprivation-induced stress conditions. We showed that AFPE exerted neuroprotective functions not only through its polyphenols but also through thaumatin-like protein 1a, which acted like a multitarget molecule.

Therapeutic potential of Coumarin-polyphenolic acid hybrids in PD: Inhibition of α-Syn aggregation and disaggregation of preformed fibrils, leading to reduced neuronal inclusion formation

This study focuses on the discovery of new potential drugs for treating PD by targeting the aggregation of α-Syn. A series of hybrids combining Coumarin and phenolic acid were designed and synthesized. Four particularly promising compounds were identified, showing strong inhibitory effects with IC50 values ranging from low micromolar to submicromolar concentrations, as low as 0.63 μM. These compounds exhibited a higher binding affinity to α-Syn residues and effectively hindered the entire aggregation process, maintaining the proteostasis conformation of α-Syn and preventing the formation of β-sheet aggregates. This approach holds significant promise for PD prevention. Additionally, these candidate compounds demonstrated the ability to break down preformed α-Syn oligomers and fibrils, resulting in the formation of smaller aggregates and monomers. Moreover, the candidate compounds showed impressive effectiveness in inhibiting α-Syn aggregation within nerve cells, thereby reducing the likelihood of α-Syn inclusion formation resembling Lewy bodies, which highlights their potential for treating PD.

Modeling transthyretin (TTR) amyloid diseases, from monomer to amyloid fibrils.

ATTR amyloidosis is caused by deposition of large, insoluble aggregates (amyloid fibrils) of cross-β-sheet TTR protein molecules on the intercellular surfaces of tissues. The process of amyloid formation from monomeric TTR protein molecules to amyloid deposits has not been fully characterized and is therefore modeled in this paper. Two models are considered: 1) TTR monomers in the blood spontaneously fold into a β-sheet conformation, aggregate into short proto-fibrils that then circulate in the blood until they find a complementary tissue where the proto-fibrils accumulate to form the large, insoluble amyloid fibrils found in affected tissues. 2) TTR monomers in the native or β-sheet conformation circulate in the blood until they find a tissue binding site and deposit in the tissue or tissues forming amyloid deposits in situ. These models only differ on where the selection for β-sheet complementarity occurs, in the blood where wt-wt, wt-v, and v-v interactions determine selectivity, or on the tissue surface where tissue-wt and tissure-v interactions also determine selectivity. Statistical modeling in both cases thus involves selectivity in fibril aggregation and tissue binding. Because binding of protein molecules into fibrils and binding of fibrils to tissues occurs through multiple weak non-covalent bonds, strong complementarity between β-sheet molecules and between fibrils and tissues is required to explain the insolubility and tissue selectivity of ATTR amyloidosis. Observation of differing tissue selectivity and thence disease phenotypes from either pure wildtype TTR protein or a mix of wildtype and variant molecules in amyloid fibrils evidences the requirement for fibril-tissue complementarity. Understanding the process that forms fibrils and binds fibrils to tissues may lead to new possibilities for interrupting the process and preventing or curing ATTR amyloidosis.

A closer look at amyloid ligands, and what they tell us about protein aggregates.

The accumulation of amyloid fibrils is characteristic of neurodegenerative diseases such as Alzheimer's disease (AD) and Parkinson's disease. Detecting these fibrils with fluorescent or radiolabelled ligands is one strategy for diagnosing and better understanding these diseases. A vast number of amyloid-binding ligands have been reported in the literature as a result. To obtain a better understanding of how amyloid ligands bind, we have compiled a database of 3457 experimental dissociation constants for 2076 unique amyloid-binding ligands. These ligands target Aβ, tau, or αSyn fibrils, as well as relevant biological samples including AD brain homogenates. From this database significant variation in the reported dissociation constants of ligands was found, possibly due to differences in the morphology of the fibrils being studied. Ligands were also found to bind to Aβ(1-40) and Aβ(1-42) fibrils with similar affinities, whereas a greater difference was found for binding to Aβ and tau or αSyn fibrils. Next, the binding of ligands to fibrils was shown to be largely limited by the hydrophobic effect. Some Aβ ligands do not fit into this hydrophobicity-limited model, suggesting that polar interactions can play an important role when binding to this target. Finally several binding site models were outlined for amyloid fibrils that describe what ligands target what binding sites. These models provide a foundation for interpreting and designing site-specific binding assays.

Evolution of Disease-modifying Therapy for Transthyretin Cardiac Amyloidosis.

Transthyretin cardiac amyloidosis (ATTR-CA) represents an inexorably progressive and fatal cardiomyopathy. Increased understanding of the underlying pathogenesis responsible for the misfolding of transthyretin and the subsequent accumulation of amyloid fibrils within the myocardium has led to the development of several disease-modifying therapies that act on different stages of the disease pathway. Tafamidis is the first, and to date remains the only, therapy approved for the treatment of ATTR-CA, which, alongside acoramidis, stabilizes the transthyretin tetramer, preventing disaggregation, misfolding and formation of amyloid fibrils. Gene-silencing agents, such as patisiran, vutrisian and eplontersen, and novel gene-editing therapies, such as NTLA-2001, act to reduce the hepatic synthesis of transthyretin. Anti-amyloid therapies represent another strategy in the treatment of ATTR-CA and are designed to bind amyloid fibril epitopes and stimulate macrophage-mediated removal of amyloid fibrils from the myocardium. Many of these treatments are at an early investigational stage but represent an important area of unmet clinical need and could potentially reverse disease and restore cardiac functions even in patients with advanced disease.

Accelerating protein aggregation and amyloid fibrillation for rapid inhibitor screening.

The accumulation and deposition of amyloid fibrils, also known as amyloidosis, in tissues and organs of patients has been found to be linked to numerous devastating neurodegenerative diseases. The aggregation of proteins to form amyloid fibrils, however, is a slow pathogenic process, and is a major issue for the evaluation of the effectiveness of inhibitors in new drug discovery and screening. Here, we used microdroplet reaction technology to accelerate the amyloid fibrillation process, monitored the process to shed light on the fundamental mechanism of amyloid self-assembly, and demonstrated the value of the technology in the rapid screening of potential inhibitor drugs. Proteins in microdroplets accelerated to form fibrils in milliseconds, enabling an entire cycle of inhibitor screening for Aβ40 within 3 minutes. The technology would be of broad interest to drug discovery and therapeutic design to develop treatments for diseases associated with protein aggregation and fibrillation.

Physicochemical mechanisms of aggregation and fibril formation of α-synuclein and apolipoprotein A-I.

Deposition and accumulation of amyloid fibrils is a hallmark of a group of diseases called amyloidosis and neurodegenerative disorders. Although polypeptides potentially have a fibril-forming propensity, native proteins have evolved into proper functional conformations to avoid aggregation and fibril formation. Understanding the mechanism for regulation of fibril formation of native proteins provides clues for the rational design of molecules for inhibiting fibril formation. Although fibril formation is a complex multistep reaction, experimentally obtained fibril formation curves can be fitted with the Finke-Watzky (F-W) two-step model for homogeneous nucleation followed by autocatalytic fibril growth. The resultant F-W rate constants for nucleation and fibril formation provide information on the chemical kinetics of fibril formation. Using the F-W two-step model analysis, we investigated the physicochemical mechanisms of fibril formation of a Parkinson's disease protein α-synuclein (αS) and a systemic amyloidosis protein apolipoprotein A-I (apoA-I). The results indicate that the C-terminal region of αS enthalpically and entropically suppresses nucleation through the intramolecular interaction with the N-terminal region and the intermolecular interaction with existing fibrils. In contrast, the nucleation of the N-terminal fragment of apoA-I is entropically driven likely due to dehydration of large hydrophobic segments in the molecule. Based on our recent findings, we discuss the similarity and difference of the fibril formation mechanisms of αS and the N-terminal fragment of apoA-I from the physicochemical viewpoints.

Therapeutic approaches in proteinopathies.

A family of maladies known as amyloid disorders, proteinopathy, or amyloidosis, are characterized by the accumulation of abnormal protein aggregates containing cross-β-sheet amyloid fibrils in many organs and tissues. Often, proteins that have been improperly formed or folded make up these fibrils. Nowadays, most treatments for amyloid illness focus on managing symptoms rather than curing or preventing the underlying disease process. However, recent advances in our understanding of the biology of amyloid diseases have led to the development of innovative therapies that target the emergence and accumulation of amyloid fibrils. Examples of these treatments include the use of small compounds, monoclonal antibodies, gene therapy, and others. In the end, even if the majority of therapies for amyloid diseases are symptomatic, greater research into the biology behind these disorders is identifying new targets for potential therapy and paving the way for the development of more effective treatments in the future.

White Light Stimulation at Gamma Frequency to Modify the Aβ42 and tau Proteins in SH-SY5Y Cells

Background Non-invasive 40 Hz gamma frequency stimulation has shown improved cognition and memory recall in Alzheimer’s disease (AD). Purpose The present study investigates the effects of white light stimulation at gamma frequency on SH-SY5Y cells using Delta M + BrainCare Light without causing discomfort. Methods SH-SY5Y cells were exposed to The Delta M + BrainCare Light for 15, 30, 45, and 60 minutes. The secretion of amyloid-β42 (Aβ42), p-AKT (Ser473), AKT, p-mTOR (Ser2448), mTOR, p-4E-BP1 (Thr37/46), 4E-BP1, p-tau (Thr181), and tau were analyzed. Aβ42 fibril aggregation and phagocytosis of FITC-Aβ42 in BV-2 cells were also observed. Results Delta M + BrainCare Light reduced tau, AKT, and p-mTOR and increased p-4E-BP1 in SH-SY5Y cells. Further, it inhibited secretion and aggregation of Aβ42, alongside increased phagocytosis in microglial cells. Conclusion These findings underscore the potential of the Delta M + BrainCare Light in mitigating the associated proteins and pathways implicated in AD.

A smart adaptable metal sequestering peptide conjugate to modulate Aβ fibrillar aggregation.

The aggregation of amyloid β peptide (Aβ) in the presence of elevated levels of transition-metal ions, e.g., Fe3+, Cu2+, Zn2+, is accountable for enhanced cellular toxicity in Alzheimer's disease. Many strategies are reported to inhibit either Cu2+, Zn2+, or Fe3+-induced Aβ fibrillation, focused on one metal. Herein, a taurine-containing adaptable metal sequestering peptide (AMSP) has been developed as the modulator of any of the cited metal-induced Aβ-aggregation in vitro. We designed the peptide conjugate comprising VFFA as a recognition motif and a taurine moiety coupled with a pendant chain of glutamic acid such that the -SO3H groups of taurine lie nearby 13His and 14His of Aβ, and sequester such metal ions that construct the salt bridge preponderantly via13His-metal-14His composition as well as bridges with 6His of Aβ. We checked the modulation of fibrillar aggregates of Aβ in the presence of metal ions by monitoring the fibril accumulation using several biophysical methods. The results established that non-aggregating AMSP sequesters Zn2+ preferably, along with Fe3+ and Cu2+ ions from the metal-Aβ complex at the physiological condition, efficiently inhibiting Aβ aggregation. While such adaptable metal binders that can chelate various metals are new, AMSP inhibits aggregation through selective recognition and metal scavenging.

Unifying the Puzzle: Cardiac Amyloidosis in the Spectrum of Generalized Edematous Syndromes

Introduction: Addressing the diagnostic challenges posed by nephrotic syndrome and congestive heart failure, this article underscores the pivotal role of comprehensive investigations in unraveling underlying etiologies. Amid the diverse spectrum of potential causes, the focus is directed towards amyloidosis—a distinct category characterized by abnormal amyloid fibril accumulation in organs. Despite its infrequency, the gravity and potential for targeted interventions accentuate the importance of considering amyloidosis in the differential diagnosis. Case Presentation: A detailed case study of a 68-year-old female with generalized edematous syndrome revealed a complex clinical scenario involving cardiac and renal manifestations. AL amyloidosis was confirmed through thorough diagnostic assessments, guiding a targeted therapeutic approach. Discussion: The discussion delves into the pathophysiology of AL amyloidosis, diagnostic considerations, and the significance of ECG and echocardiographic findings. It highlights the importance of laboratory assessments, especially immunologic tests, and emphasizes the role of biopsy in confirming the diagnosis. Conclusion: The complexity of AL amyloidosis is underscored, emphasizing the interplay of cardiac, renal, and hematologic aspects. Early diagnosis and a tailored multidisciplinary therapeutic approach yielded positive outcomes, contributing valuable insights to the evolving understanding of amyloidosis management.

Changes in the macro and nano-structure of paper during moisture cycling

Hygroexpansion, CP/MAS 13C-NMR, WAXS and SAXS measurements were carried out on sheets made from four different commercial pulps of varying lignin content. Non-directional laboratory sheets were made at different press levels from the pulps following different degrees of beating. The sheets were dried both freely and with restraints. Measurements were made on sheets before and after moisture cycling to determine hygroexpansion coefficients, changes in cellulose average lateral fibril dimensions and average cellulose crystallite sizes, with the aim of understanding macroscale and nanoscale changes as the result of moisture cycling. Within the sheets consistent and statistically significant structural changes were observed on both macro and nanoscale. On the macroscale, moisture cycling consistently induced irreversible shrinkage in sheets dried with restraints, but less so in the case of sheets dried freely. The hygroexpansion coefficients were typically higher for freely dried sheets compared with sheets dried with constraints. On the nanoscale, moisture cycling consistently caused an increase in the average crystallite sizes (WAXS) and the average lateral fibril dimensions (CP/MAS 13C-NMR), though the latter occurred with poor statistical significance. These changes were interpreted as an increase in the degree of order in the cellulose fibril interior/cellulose crystallite. There were no profound differences in the nanoscale changes observed for sheets dried with restraints and for sheets dried freely. Changes in the fibre wall nanostructure were of similar magnitudes when comparing results from freely dried low grammage sheets (less abundant inter-fibre joints) with freely dried sheets of higher grammage (more abundant inter-fibre joints). No obvious correlations were found between the macroscale and nanoscale measurements. The proposed explanation for this was that the nanoscale structural changes occurred similarly throughout the entirety of the fibre wall, independent of the proximity to an inter-fibre joint, and that the nanoscale structural changes were mainly the result of water penetrating into the interior of cellulose fibril aggregates. By using the same fibril model for evaluation of CP/MAS 13C-NMR and WAXS data, good-to-reasonable agreement were found for estimates of the degree of cellulose crystallinity.

Gut mucosal cells transfer α-synuclein to the vagus nerve.

Epidemiological and histopathological findings have raised the possibility that misfolded α-synuclein protein might spread from the gut to the brain and increase the risk of Parkinson's disease. Although past experimental studies in mouse models have relied on gut injections of exogenous recombinant α-synuclein fibrils to study gut-to-brain α-synuclein transfer, the possible origins of misfolded α-synuclein within the gut have remained elusive. We recently demonstrated that sensory cells of intestinal mucosa express α-synuclein. Here, we employed mouse intestinal organoids expressing human α-synuclein to observe the transfer of α-synuclein protein from epithelial cells in organoids to cocultured nodose neurons devoid of α-synuclein. In mice expressing human α-synuclein, but no mouse α-synuclein, α-synuclein fibril-templating activity emerged in α-synuclein-seeded fibril aggregation assays in intestine, vagus nerve, and dorsal motor nucleus. In newly engineered transgenic mice that restrict pathological human α-synuclein expression to intestinal epithelial cells, α-synuclein fibril-templating activity transfered to the vagus nerve and dorsal motor nucleus. Subdiaphragmatic vagotomy prior to induction of α-synuclein expression in intestinal epithelial cells effectively protected the hindbrain from emergence of α-synuclein fibril-templating activity. Overall, these findings highlight a potential non-neuronal source of fibrillar α-synuclein protein that might arise in gut mucosal cells.

Characterizing cannabis-prevalent terpenes for neuroprotection reveal a role for α and β-pinenes in mitigating amyloid β-evoked neurotoxicity and aggregation in vitro

BackgroundCannabis sativa L. (C. sativa) can efficiently synthesize of over 200 terpenes, including monoterpenes, sesquiterpenes and triterpenes that may contribute to the known biological activities of phytocannabinoids of relevance for the burgeoning access to medicinal cannabis formulations globally; however, to date have been uncharacterized. We assessed twelve predominant terpenes in C. sativa for neuroprotective and anti-aggregative properties in semi-differentiated PC12 neuronal cell line that is robust and validated as a cell model responsive to amyloid β (Aβ1–42) protein exposure and oxidative stress. MethodsCell viability was assessed biochemically using the MTT assay in the presence of myrcene, β-caryophyllene, terpinolene, limonene, linalool, humulene, α-pinene, nerolidol, β-pinene, terpineol, citronellol and friedelin (1–200 μM) for 24 hr. Sub-toxic threshold test concentrations of each terpene were then applied to cells, alone or with concomitant incubation with the lipid peroxidant tert-butyl hyrdroperoxide (t-BHP; 0–250 μM) or amyloid β (Aβ1–42; 0–1 μM) to assess neuroprotective effects. Direct effects of each terpene on Aβ fibril formation and aggregation were also evaluated using the Thioflavin T (ThT) fluorometric kinetic assay and transmission electron microscopy (TEM) to visualize fibril and aggregate morphology. ResultsTerpenes were intrinsically benign to PC12 cells up to 50 μM, with higher concentrations of β-caryophyllene, humulene and nerolidol inducing some loss of PC12 cell viability. No significant protective effects of terpenes were observed following t-BHP (0–200 µM) administration, with some enhanced toxicity instead demonstrated from both β-caryophyllene and humulene treatment (each at 50 µM). α-pinene and β-pinene demonstrated a significant neuroprotective effect against amyloid β exposure. α-pinene, β-pinene, terpineol, terpinolene and friedelin were associated with a variable inhibition of Aβ1–42 fibril and aggregate density. ConclusionsThe outcomes of this study underline a neuroprotective role of α-pinene and β-pinene against Aβ-mediated neurotoxicity associated with an inhibition of Aβ1–42 fibrilization and density. This demonstrates the bioactive potential of selected terpenes for consideration in the development of medicinal cannabis formulations targeting neurodegenerative diseases.

Disease-Associated Mutations in Tau Encode for Changes in Aggregate Structure Conformation.

The accumulation of tau fibrils is associated with neurodegenerative diseases, which are collectively termed tauopathies. Cryo-EM studies have shown that the packed fibril core of tau adopts distinct structures in different tauopathies, such as Alzheimer's disease, corticobasal degeneration, and progressive supranuclear palsy. A subset of tauopathies are linked to missense mutations in the tau protein, but it is not clear whether these mutations impact the structure of tau fibrils. To answer this question, we developed a high-throughput protein purification platform and purified a panel of 37 tau variants using the full-length 0N4R splice isoform. Each of these variants was used to create fibrils in vitro, and their relative structures were studied using a high-throughput protease sensitivity platform. We find that a subset of the disease-associated mutations form fibrils that resemble wild-type tau, while others are strikingly different. The impact of mutations on tau structure was not clearly associated with either the location of the mutation or the relative kinetics of fibril assembly, suggesting that tau mutations alter the packed core structures through a complex molecular mechanism. Together, these studies show that single-point mutations can impact the assembly of tau into fibrils, providing insight into its association with pathology and disease.