bims-proned 2024-03-24 papers

bims-proned

Biomed News

on Proteostasis in neurodegeneration

Issue of 2024‒03‒24
24 papers selected by
Verena Kohler, Umeå University

HTRA1 disaggregates α-synuclein amyloid fibrils and converts them into non-toxic and seeding incompetent species.
Discovery of a Gut Bacterial Metabolic Pathway that Drives α-Synuclein Aggregation.
Understanding the Modulation of α-Synuclein Fibrillation by N-Acetyl Aspartate: A Brain Metabolite.
LETC inhibits α-Syn aggregation and ameliorates motor deficiencies in the L62 mouse model of synucleinopathy.
Single-Molecule Two-Color Coincidence Detection of Unlabeled alpha-Synuclein Aggregates.
Rapid FRET Assay for the Early Detection of Alpha-Synuclein Aggregation in Parkinson's Disease.
α-synuclein regulates Cyclin D1 to promote abnormal initiation of the cell cycle and induce apoptosis in dopamine neurons.
Liquid-liquid phase separation of α-synuclein is highly sensitive to sequence complexity.
Decreased Water Mobility Contributes To Increased α-Synuclein Aggregation.
Amyloid-Induced Neurodegeneration: A Comprehensive Review through Aggregomics Perception of Proteins in Health and Pathology.
N-acetylation of α-synuclein enhances synaptic vesicle clustering mediated by α-synuclein and lysophosphatidylcholine.
Photo-Excited Dyes: Emerging Technique Against Tau Protein Aggregation.
Effect of Natural Osmolytes on Recombinant Tau Monomer: Propensity of Oligomerization and Aggregation.
Preparation of Tau Condensates by Liquid-Liquid Phase Separation to Study Tau Amyloid Aggregation.
A partial Drp1 knockout improves autophagy flux independent of mitochondrial function.
Assays for the Screening and Characterization of Tau Aggregation Inhibitors.
Biochemical and Biophysical Characterization of Tau and α-Linolenic Acid Vesicles In Vitro.
Copper ion incorporation in α-synuclein amyloids.
Seeding Activity of Skin Misfolded Tau as a Biomarker for Tauopathies.
Endoplasmic reticulum stress and therapeutic strategies in metabolic, neurodegenerative diseases and cancer.
Sedimentation and Laser Light Scattering Methods for Quantifying Synthetic Tau Aggregation Propensity.
Quercetin-3-O-glc-1-3-rham-1-6-glucoside decreases Aβ production, inhibits Aβ aggregation and neurotoxicity, and prohibits the production of inflammatory cytokines.
Are age-related neurodegenerative diseases caused by a lack of the diet-derived compound ergothioneine?
Kinetic Ensemble of Tau Protein through the Markov State Model and Deep Learning Analysis.

Nat Commun. 2024 Mar 18. 15(1): 2436

HTRA1 disaggregates α-synuclein amyloid fibrils and converts them into non-toxic and seeding incompetent species.

Sheng Chen, Anuradhika Puri, Braxton Bell, Joseph Fritsche, Hector H Palacios, Maurie Balch, Macy L Sprunger, Matthew K Howard, Jeremy J Ryan, Jessica N Haines, Gary J Patti, Albert A Davis, Meredith E Jackrel.

Parkinson's disease (PD) is closely linked to α-synuclein (α-syn) misfolding and accumulation in Lewy bodies. The PDZ serine protease HTRA1 degrades fibrillar tau, which is associated with Alzheimer's disease, and inactivating mutations to mitochondrial HTRA2 are implicated in PD. Here, we report that HTRA1 inhibits aggregation of α-syn as well as FUS and TDP-43, which are implicated in amyotrophic lateral sclerosis (ALS) and frontotemporal dementia. The protease domain of HTRA1 is necessary and sufficient for inhibiting aggregation, yet this activity is proteolytically-independent. Further, HTRA1 disaggregates preformed α-syn fibrils, rendering them incapable of seeding aggregation of endogenous α-syn, while reducing HTRA1 expression promotes α-syn seeding. HTRA1 remodels α-syn fibrils by targeting the NAC domain, the key domain catalyzing α-syn amyloidogenesis. Finally, HTRA1 detoxifies α-syn fibrils and prevents formation of hyperphosphorylated α-syn accumulations in primary neurons. Our findings suggest that HTRA1 may be a therapeutic target for a range of neurodegenerative disorders.

DOI: https://doi.org/10.1038/s41467-024-46538-8
ACS Chem Biol. 2024 Mar 22.

Discovery of a Gut Bacterial Metabolic Pathway that Drives α-Synuclein Aggregation.

Lizett Ortiz de Ora, Julia M Balsamo, Kylie S Uyeda, Elizabeth N Bess.

Parkinson's disease (PD) etiology is associated with aggregation and accumulation of α-synuclein (α-syn) proteins in midbrain dopaminergic neurons. Emerging evidence suggests that in certain subtypes of PD, α-syn aggregates originate in the gut and subsequently spread to the brain. However, mechanisms that instigate α-syn aggregation in the gut have remained elusive. In the brain, the aggregation of α-syn is induced by oxidized dopamine. Such a mechanism has not been explored in the context of the gastrointestinal tract, a niche harboring 46% of the body's dopamine reservoirs. Here, we report that Enterobacteriaceae, a bacterial family prevalent in human gut microbiotas, induce α-syn aggregation. More specifically, our in vitro data indicate that respiration of nitrate by Escherichia coli K-12, which results in production of nitrite that mediates oxidation of Fe2+ to Fe3+, creates an oxidizing redox potential. These oxidizing conditions enabled the formation of dopamine-derived quinones and α-syn aggregates. Exposing nitrite, but not nitrate, to enteroendocrine STC-1 cells induced aggregation of α-syn that is natively expressed in these cells, which line the intestinal tract. Taken together, our findings indicate that bacterial nitrate reduction may be critical for initiating intestinal α-syn aggregation.

DOI: https://doi.org/10.1021/acschembio.4c00095
ACS Omega. 2024 Mar 12. 9(10): 12262-12271

Understanding the Modulation of α-Synuclein Fibrillation by N-Acetyl Aspartate: A Brain Metabolite.

Tanzeel Khan, Rashid Waseem, Mohammad Shahid, Jaoud Ansari, Md Imtaiyaz Hassan, Anas Shamsi, Asimul Islam.

α-Synuclein (α-Syn) fibrillation is a prominent contributor to neuronal deterioration and plays a significant role in the advancement of Parkinson's Disease (PD). Considering this, the exploration of novel compounds that can inhibit or modulate the aggregation of α-Syn is a topic of significant research. This study, for the first time, elucidated the effect of N-acetyl aspartate (NAA), a brain osmolyte, on α-Syn aggregation using spectroscopic and microscopic approaches. Thioflavin T (ThT) assay revealed that a lower concentration of NAA inhibits α-Syn aggregation, whereas higher concentrations of NAA accelerate the aggregation. Further, this paradoxical effect of NAA was complemented by ANS, RLS, and the turbidity assay. The secondary structure transition was more pronounced at higher concentrations of NAA by circular dichroism, corroborating the fluorescence spectroscopic observations. Confocal microscopy also confirmed the paradoxical effect of NAA on α-Syn aggregation. Interaction studies including fluorescence quenching and molecular docking were employed to determine the binding affinity and critical residues involved in the α-Syn-NAA interaction. The explanation for this paradoxical nature of NAA could be a solvophobic effect. The results offer a profound understanding of the modulatory mechanism of α-Syn aggregation by NAA, thereby suggesting the potential role of NAA at lower concentrations in therapeutics against α-Syn aggregation-related disorders.

DOI: https://doi.org/10.1021/acsomega.4c00595
Eur J Pharmacol. 2024 Mar 17. pii: S0014-2999(24)00193-6. [Epub ahead of print] 176505

LETC inhibits α-Syn aggregation and ameliorates motor deficiencies in the L62 mouse model of synucleinopathy.

Karima Schwab, Silke Frahm, Mandy Magbagbeolu, David Horsley, Elizabeth A Goatman, Valeria Melis, Franz Theuring, Ahtsham Ishaq, John M D Storey, Charles R Harrington, Claude M Wischik, Gernot Riedel.

  Alpha-Synuclein (α-Syn) aggregation is a pathological feature of synucleinopathies, neurodegenerative disorders that include Parkinson's disease (PD). Here, we explored the efficacy of N,N,N',N'-tetraethyl-10H-phenothiazine-3,7-diamine dihydrochloride (LETC), a protein aggregation inhibitor, on α-Syn aggregation. In both cellular models and transgenic mice, α-Syn aggregation was achieved by the overexpression of full-length human α-Syn fused with a signal sequence peptide. α-Syn accumulated in transfected DH60.21 neuroblastoma cells and α-Syn aggregation was inhibited by LETC with an EC50 of 0.066 ± 0.047 μM. Full-length human α-Syn overexpressing Line 62 (L62) mice accumulated neuronal α-Syn that was associated with a decreased motor performance in the open field and automated home cage. LETC, administered orally for 6 weeks at 10 mg/kg significantly decreased α-Syn-positive neurons in multiple brain regions and this resulted in a rescue of movement deficits in the open field in these mice. LETC however, did not improve activity deficits of L62 mice in the home cage environment. The results suggest that LETC may provide a potential disease modification therapy in synucleinopathies through the inhibition of α-Syn aggregation.

Keywords:  Aggregation inhibitor; Alpha-synuclein; Mouse model; Parkinson's disease

DOI:  https://doi.org/10.1016/j.ejphar.2024.176505
Angew Chem Weinheim Bergstr Ger. 2023 Apr 03. 135(15): e202216771

Single-Molecule Two-Color Coincidence Detection of Unlabeled alpha-Synuclein Aggregates.

Alexandre Chappard, Craig Leighton, Rebecca S Saleeb, Kiani Jeacock, Sarah R Ball, Katie Morris, Owen Kantelberg, Ji-Eun Lee, Elsa Zacco, Annalisa Pastore, Margaret Sunde, David J Clarke, Patrick Downey, Tilo Kunath, Mathew H Horrocks.

  Protein misfolding and aggregation into oligomeric and fibrillar structures is a common feature of many neurogenerative disorders. Single-molecule techniques have enabled characterization of these lowly abundant, highly heterogeneous protein aggregates, previously inaccessible using ensemble averaging techniques. However, they usually rely on the use of recombinantly-expressed labeled protein, or on the addition of amyloid stains that are not protein-specific. To circumvent these challenges, we have made use of a high affinity antibody labeled with orthogonal fluorophores combined with fast-flow microfluidics and single-molecule confocal microscopy to specifically detect α-synuclein, the protein associated with Parkinson's disease. We used this approach to determine the number and size of α-synuclein aggregates down to picomolar concentrations in biologically relevant samples.

Keywords:  Aggregation or Oligomerization; Fluorescence; Microscopy; Proteins; Single-Molecule

DOI:  https://doi.org/10.1002/ange.202216771
ACS Chem Neurosci. 2024 Mar 20.

Rapid FRET Assay for the Early Detection of Alpha-Synuclein Aggregation in Parkinson's Disease.

Hang Yu, Rui Feng, Fenglin Chen, Zuodong Wu, Dehai Li, Xue Qiu.

  Alpha-synuclein (α-Syn) is a key protein of Parkinson's disease (PD). Oligomers formed by misfolding and aggregation of α-Syn can cause many pathological phenomena and aggravate the development of PD. Therefore, sensitive and accurate detection of oligomers is essential to understanding the pathology of PD and beneficial to screening and developing new drugs against PD. Here, we demonstrated a simple and sensitive method to detect the early aggregation of α-Syn via Förster resonance energy transfer (FRET) technology. We performed systematic investigations of the FRET sensitizations, efficiencies, and donor-to-acceptor distances during α-Syn aggregation, which was proved to be more sensitive to reflect small distance changes in the early stage of α-Syn aggregation, especially for α-Syn oligomers. The FRET assays were also applied to study the influence of Ser129 phosphorylation (pS129) on the aggregation rate of α-Syn. Our results showed that pS129 modification promotes α-Syn aggregation and enhances the ability of preformed fibrils to induce monomer aggregation. pS129 also increased the cytotoxicity of α-Syn. These results are of great significance for a better understanding of the pathological mechanisms of PD and future PD drug development.

Keywords:  FRET; PTM; Parkinson’s disease; aggregation; alpha-synuclein

DOI:  https://doi.org/10.1021/acschemneuro.3c00617
Biomed Pharmacother. 2024 Mar 18. pii: S0753-3322(24)00328-7. [Epub ahead of print]173 116444

α-synuclein regulates Cyclin D1 to promote abnormal initiation of the cell cycle and induce apoptosis in dopamine neurons.

Xiaokang Jia, Qiliang Chen, Ciyu Yao, Tetsuya Asakawa, Yuanyuan Zhang.

  The etiology of Parkinson's disease (PD) is characterized by the death of dopamine neurons in the substantia nigra pars compacta, while misfolding and abnormal aggregation of α-synuclein (α-syn) are core pathological features. Previous studies have suggested that damage to dopamine neurons may be related to cell cycle dysregulation, but the specific mechanisms remain unclear. In this study, a PD mouse model was induced by stereotactic injection of α-syn into the nucleus, and treated with the cell cycle inhibitor, roscovitine (Rosc). The results demonstrated that Rosc improved behavioral disorders caused by α-syn, increased TH protein expression, inhibited α-syn and p-α-syn protein expression, and reduced the expression levels of G1/S phase cell cycle genes Cyclin D1, Cyclin E, CDK2, CDK4, E2F and pRB. Additionally, Rosc decreased Bax and Caspase-3 expression caused by α-syn, while increasing Bcl-2 protein expression. Meanwhile, we observed that α-syn can influence neuronal cell autophagy by decreasing the expression level of Beclin 1 and increasing the expression level of P62. However, Rosc can improve this phenomenon. In a cell model induced by α-syn in dopamine neuron injury cells, knockdown of Cyclin D1 led to similar results as those observed in animal experiments: Knocking down Cyclin D1 improved the abnormal initiation of the cell cycle caused by α-syn and regulated cellular autophagy, resulting in a reduction of apoptosis in dopamine neurons. In summary, exogenous α-syn can lead to the accumulation of α-syn and phosphorylated α-syn in dopamine neurons, increase key factors of the G1/S phase cell cycle such as Cyclin D1, and regulate downstream related indicators, causing the cell cycle to restart and leading to apoptosis of dopamine neurons. This exacerbates PD symptoms. However, knockdown of Cyclin D1 inhibits the progression of the cell cycle and can reverse this situation. These findings suggest that a Cyclin D inhibitor may be a novel therapeutic target for treating PD.

Keywords:  Cell cycle; Dopamine neurons; Parkinson's disease; α-synuclein

DOI:  https://doi.org/10.1016/j.biopha.2024.116444
Protein Sci. 2024 Apr;33(4): e4951

Liquid-liquid phase separation of α-synuclein is highly sensitive to sequence complexity.

Anindita Mahapatra, Robert W Newberry.

  The Parkinson's-associated protein α-synuclein (α-syn) can undergo liquid-liquid phase separation (LLPS), which typically leads to the formation of amyloid fibrils. The coincidence of LLPS and amyloid formation has complicated the identification of the molecular determinants unique to LLPS of α-syn. Moreover, the lack of strategies to selectively perturb LLPS makes it difficult to dissect the biological roles specific to α-syn LLPS, independent of fibrillation. Herein, using a combination of subtle missense mutations, we show that LLPS of α-syn is highly sensitive to its sequence complexity. In fact, we find that even a highly conservative mutation (V16I) that increases sequence complexity without perturbing physicochemical and structural properties, is sufficient to reduce LLPS by 75%; this effect can be reversed by an adjacent V-to-I mutation (V15I) that restores the original sequence complexity. A18T, a complexity-enhancing PD-associated mutation, was likewise found to reduce LLPS, implicating sequence complexity in α-syn pathogenicity. Furthermore, leveraging the differences in LLPS propensities among different α-syn variants, we demonstrate that fibrillation of α-syn does not necessarily correlate with its LLPS. In fact, we identify mutations that selectively perturb LLPS or fibrillation of α-syn, unlike previously studied mutations. The variants and design principles reported herein should therefore empower future studies to disentangle these two phenomena and distinguish their (patho)biological roles.

Keywords:  aggregation; alpha‐synuclein; phase separation; sequence complexity

DOI:  https://doi.org/10.1002/pro.4951
Angew Chem Weinheim Bergstr Ger. 2023 Feb 06. 135(7): e202212063

Decreased Water Mobility Contributes To Increased α-Synuclein Aggregation.

Amberley D Stephens, Johanna Kölbel, Rani Moons, Chyi Wei Chung, Michael T Ruggiero, Najet Mahmoudi, Talia A Shmool, Thomas M McCoy, Daniel Nietlispach, Alexander F Routh, Frank Sobott, J Axel Zeitler, Gabriele S Kaminski Schierle.

  The solvation shell is essential for the folding and function of proteins, but how it contributes to protein misfolding and aggregation has still to be elucidated. We show that the mobility of solvation shell H2O molecules influences the aggregation rate of the amyloid protein α-synuclein (αSyn), a protein associated with Parkinson's disease. When the mobility of H2O within the solvation shell is reduced by the presence of NaCl, αSyn aggregation rate increases. Conversely, in the presence CsI the mobility of the solvation shell is increased and αSyn aggregation is reduced. Changing the solvent from H2O to D2O leads to increased aggregation rates, indicating a solvent driven effect. We show the increased aggregation rate is not directly due to a change in the structural conformations of αSyn, it is also influenced by a reduction in both the H2O mobility and αSyn mobility. We propose that reduced mobility of αSyn contributes to increased aggregation by promoting intermolecular interactions.

Keywords:  Amyloid; Hydration Shell; Hydrogen Bond; Solvation Shell; Solvent

DOI:  https://doi.org/10.1002/ange.202212063
Ageing Res Rev. 2024 Mar 16. pii: S1568-1637(24)00094-1. [Epub ahead of print] 102276

Amyloid-Induced Neurodegeneration: A Comprehensive Review through Aggregomics Perception of Proteins in Health and Pathology.

Sania Bashir, Ayesha Aiman, Mohammad Shahid, Anis Ahmad Chaudhary, Neha Sami, Seemi Farhat Basir, Imtaiyaz Hassan, Asimul Islam.

  Amyloidosis of protein caused by fibrillation and aggregation are some of the most exciting new edges not only in protein sciences but also in molecular medicines. The present review discusses recent advancements in the field of neurodegenerative diseases and therapeutic applications with ongoing clinical trials, featuring new areas of protein misfolding resulting in aggregation. The endogenous accretion of protein fibrils having fibrillar morphology symbolizes the beginning of neuro-disorders. Prognostic amyloidosis is prominent in numerous degenerative infections such as Alzheimer's and Parkinson's disease, Amyotrophic lateral sclerosis (ALS), etc. However, the molecular basis determining the intracellular or extracellular evidence of aggregates, playing a significant role as a causative factor in neurodegeneration is still unclear. Structural conversions and protein self-assembly resulting in the formation of amyloid oligomers and fibrils are important events in the pathophysiology of the disease. This comprehensive review sheds light on the evolving landscape of potential treatment modalities, highlighting the ongoing clinical trials and the potential socio-economic impact of novel therapeutic interventions in the realm of neurodegenerative diseases. Furthermore, many drugs are undergoing different levels of clinical trials that would certainly help in treating these disorders and will surely improve the socio-impact of human life.

Keywords:  Neurodegenerative diseases; aggregation; protein misfolding

DOI:  https://doi.org/10.1016/j.arr.2024.102276
bioRxiv. 2024 Mar 06. pii: 2024.03.04.583437. [Epub ahead of print]

N-acetylation of α-synuclein enhances synaptic vesicle clustering mediated by α-synuclein and lysophosphatidylcholine.

Chuchu Wang, Chunyu Zhao, Xiao Hu, Jiali Qiang, Zhenying Liu, Jinge Gu, Shengnan Zhang, Dan Li, Yaoyang Zhang, Jacqueline Burré, Jiajie Diao, Cong Liu.

Post-translational modifications (PTMs) of α-synuclein (α-syn) such as acetylation and phosphorylation play important yet distinct roles in regulating α-syn conformation, membrane binding, and amyloid aggregation. However, how PTMs regulate α-syn function in presynaptic terminals remains unclear. Previously, we reported that α-syn clusters synaptic vesicles (SV) 1 , and neutral phospholipid lysophosphatidylcholine (LPC) can mediate this clustering 2 . Here, based on our previous findings, we further demonstrate that N-terminal acetylation, which occurs under physiological condition and is irreversible in mammalian cells, significantly enhances the functional activity of α-syn in clustering SVs. Mechanistic studies reveal that this enhancement is caused by the N-acetylation-promoted insertion of α-syn's N-terminus and increased intermolecular interactions on the LPC-containing membrane. Our work demonstrates that N-acetylation fine-tunes α-syn-LPC interaction for mediating α-syn's function in SV clustering.

DOI: https://doi.org/10.1101/2024.03.04.583437
Methods Mol Biol. 2024 ;2754 105-116

Photo-Excited Dyes: Emerging Technique Against Tau Protein Aggregation.

Subashchandrabose Chinnathambi, Tushar Dubey.

  Tau aggregates are considered a pathological hallmark of Alzheimer's disease. The screening of molecules against Tau aggregation is a novel strategy for Alzheimer's disease. The photo-excited molecules have proven to be effective as a therapeutic agent in several diseases. In recent studies, the photo-excited dyes showed an inhibitory effect on Alzheimer's disease-related Tau protein aggregation and toxicity. The present chapter deals with the effect of rose bengal on the aggregation of Tau. The in vitro studies carried out with the help of electron microscopy, ThS fluorescence, and circular dichroism suggested that RB attenuated the Tau aggregation under in vitro conditions, whereas PE-RB disaggregated the mature Tau fibrils. Photo-excited rose bengal and the classical rose bengal induced a low degree of toxicity in cells. Thus, for the treatment of Alzheimer's disease, the rose bengal could be considered a potential molecule.

Keywords:  Alzheimer’s disease; Photodynamic therapy; Photosensitizer; Rose bengal; Tau aggregates

DOI:  https://doi.org/10.1007/978-1-0716-3629-9_6
ACS Chem Neurosci. 2024 Mar 19.

Effect of Natural Osmolytes on Recombinant Tau Monomer: Propensity of Oligomerization and Aggregation.

Sharif Arar, Md Anzarul Haque, Nemil Bhatt, Yingxin Zhao, Rakez Kayed.

  The pathological misfolding and aggregation of the microtubule associated protein tau (MAPT), a full length Tau2N4R with 441aa, is considered the principal disease relevant constituent in tauopathies including Alzheimer's disease (AD) with an imbalanced ratio in 3R/4R isoforms. The exact cellular fluid composition, properties, and changes that coincide with tau misfolding, seed formation, and propagation events remain obscure. The proteostasis network, along with the associated osmolytes, is responsible for maintaining the presence of tau in its native structure or dealing with misfolding. In this study, for the first time, the roles of natural brain osmolytes are being investigated for their potential effects on regulating the conformational stability of the tau monomer (tauM) and its propensity to aggregate or disaggregate. Herein, the effects of physiological osmolytes myo-inositol, taurine, trimethyl amine oxide (TMAO), betaine, sorbitol, glycerophosphocholine (GPC), and citrulline on tau's aggregation state were investigated. The overall results indicate the ability of sorbitol and GPC to maintain the monomeric form and prevent aggregation of tau, whereas myo-inositol, taurine, TMAO, betaine, and citrulline promote tau aggregation to different degrees, as revealed by protein morphology in atomic force microscopy images. Biochemical and biophysical methods also revealed that tau proteins adopt different conformations under the influence of these osmolytes. TauM in the presence of all osmolytes expressed no toxicity when tested by a lactate dehydrogenase assay. Investigating the conformational stability of tau in the presence of osmolytes may provide a better understanding of the complex nature of tau aggregation in AD and the protective and/or chaotropic nature of osmolytes.

Keywords:  Alzheimer’s disease; aggregation; cytotoxicity; osmolytes; proteinase K digestion mass spectrometry

DOI:  https://doi.org/10.1021/acschemneuro.3c00614
Methods Mol Biol. 2024 ;2754 185-192

Preparation of Tau Condensates by Liquid-Liquid Phase Separation to Study Tau Amyloid Aggregation.

Yanxian Lin.

  Protein liquid-liquid phase separation (LLPS) has been associated with protein amyloid aggregation. Amyloid aggregation of tau is a hallmark of Alzheimer's disease and other neurodegenerative diseases. This protocol provides steps to prepare tau condensates via LLPS, so that researchers can further study its driving forces and its relationship with tau amyloid aggregation.

Keywords:  Amyloid aggregation; Complex coacervation; Liquid–liquid phase separation; Tau proteinTauprotein

DOI:  https://doi.org/10.1007/978-1-0716-3629-9_10
Mol Neurodegener. 2024 Mar 19. 19(1): 26

A partial Drp1 knockout improves autophagy flux independent of mitochondrial function.

Rebecca Z Fan, Carolina Sportelli, Yanhao Lai, Said S Salehe, Jennifer R Pinnell, Harry J Brown, Jason R Richardson, Shouqing Luo, Kim Tieu.

  BACKGROUND: Dynamin-related protein 1 (Drp1) plays a critical role in mitochondrial dynamics. Partial inhibition of this protein is protective in experimental models of neurological disorders such as Parkinson's disease and Alzheimer's disease. The protective mechanism has been attributed primarily to improved mitochondrial function. However, the observations that Drp1 inhibition reduces protein aggregation in such neurological disorders suggest the involvement of autophagy. To investigate this potential novel protective mechanism of Drp1 inhibition, a model with impaired autophagy without mitochondrial involvement is needed.METHODS: We characterized the effects of manganese (Mn), which causes parkinsonian-like symptoms in humans, on autophagy and mitochondria by performing dose-response studies in two cell culture models (stable autophagy HeLa reporter cells and N27 rat immortalized dopamine neuronal cells). Mitochondrial function was assessed using the Seahorse Flux Analyzer. Autophagy flux was monitored by quantifying the number of autophagosomes and autolysosomes, as well as the levels of other autophagy proteins. To strengthen the in vitro data, multiple mouse models (autophagy reporter mice and mutant Drp1+/- mice and their wild-type littermates) were orally treated with a low chronic Mn regimen that was previously reported to increase α-synuclein aggregation and transmission via exosomes. RNAseq, laser captured microdissection, immunofluorescence, immunoblotting, stereological cell counting, and behavioural studies were used. RESULTS IN VITRO: data demonstrate that at low non-toxic concentrations, Mn impaired autophagy flux but not mitochondrial function and morphology. In the mouse midbrain, RNAseq data further confirmed autophagy pathways were dysregulated but not mitochondrial related genes. Additionally, Mn selectively impaired autophagy in the nigral dopamine neurons but not the nearby nigral GABA neurons. In cells with a partial Drp1-knockdown and Drp1+/- mice, Mn induced autophagic impairment was significantly prevented. Consistent with these observations, Mn increased the levels of proteinase-K resistant α-synuclein and Drp1-knockdown protected against this pathology.
CONCLUSIONS: This study demonstrates that improved autophagy flux is a separate mechanism conferred by Drp1 inhibition independent of its role in mitochondrial fission. Given that impaired autophagy and mitochondrial dysfunction are two prominent features of neurodegenerative diseases, the combined protective mechanisms targeting these two pathways conferred by Drp1 inhibition make this protein an attractive therapeutic target.

Keywords:  Autophagy; Dynamin related protein 1; Manganese; Mitochondrial dynamics; Mitochondrial dysfunction; Parkinson’s disease; Protein aggregation; α-synuclein

DOI:  https://doi.org/10.1186/s13024-024-00708-w
Methods Mol Biol. 2024 ;2754 93-104

Assays for the Screening and Characterization of Tau Aggregation Inhibitors.

David Horsley, Janet E Rickard, Thomas Vorley, Matilda F Leeper, Claude M Wischik, Charles R Harrington.

  Aggregation of tau protein is a pathological hallmark of Alzheimer's disease and other neurodegenerative tauopathies. Inhibition of tau aggregation may provide a method for treatment of these disorders. Methods to identify tau aggregation inhibitors (TAIs) in vitro are useful and here we describe assays for TAIs using purified recombinant tau protein fragments in a cell-free immunoassay format and in a stably transfected cell model to create a more physiological environment.

Keywords:  Alzheimer’s disease; ELISA; Immunoassay; Tau aggregation inhibitor; Tau protein

DOI:  https://doi.org/10.1007/978-1-0716-3629-9_5
Methods Mol Biol. 2024 ;2754 193-203

Biochemical and Biophysical Characterization of Tau and α-Linolenic Acid Vesicles In Vitro.

Smita Eknath Desale, Hariharakrishnan Chidambaram, Subashchandrabose Chinnathambi.

  Alzheimer's disease (AD) is characterized by the abnormal accumulation of disordered protein, that is, extracellular senile plaques of amyloid-β (Aβ) and intracellular neurofibrillary tangles of Tau. Tau protein has gained the attention in recent years owing to the ability to propagate in a "prion-like" nature. The disordered protein Tau possesses a high positive charge, which allows its binding to anionic proteins and factors. The native disorder of proteins attends the β-sheet structure from its random-coiled conformation upon charge compensation by various polyanionic agents such as heparin, RNA, etc. Anionic lipids such as arachidonic acid (AA) and oleic acid (OA) are also one of the factors which can induce aggregation of Tau in physiological conditions. The free units of Tau protein can bind to lipid membranes through its repeat domain (RD), the anionic side chains of the membrane lipids induce aggregation of Tau by reducing the activation barrier. In this study, we investigated the role of α-linolenic acid (ALA) as an inducing agent for Tau aggregation in vitro conditions. Omega-3 fatty acids bear a capacity to reduce the pathology of Tau by downregulating the Tau phosphorylation pathway. We have studied by using various biochemical or biophysical methods the potency of ALA as an aggregating agent for Tau. We have implemented different techniques such as SDS-PAGE, transmission electron microscopy, CD spectroscopy to evaluated higher-order aggregates of Tau upon induction by ALA.

Keywords:  Alzheimer’s disease; Tau aggregation; VesiclesVesicles; α-Linolenic acid

DOI:  https://doi.org/10.1007/978-1-0716-3629-9_11
Protein Sci. 2024 Apr;33(4): e4956

Copper ion incorporation in α-synuclein amyloids.

Gulshan Walke, Ranjeet Kumar, Pernilla Wittung-Stafshede.

  Copper ion dys-homeostasis is linked to neurodegenerative diseases involving amyloid formation. Even if many amyloidogenic proteins can bind copper ions as monomers, little is known about copper interactions with the resulting amyloid fibers. Here, we investigate copper interactions with α-synuclein, the amyloid-forming protein in Parkinson's disease. Copper (Cu(II)) binds tightly to monomeric α-synuclein in vitro involving the N-terminal amine and the side chain of His50. Using purified protein and biophysical methods in vitro, we reveal that copper ions are readily incorporated into the formed amyloid fibers when present at the start of aggregation reactions, and the metal ions also bind if added to pre-formed amyloids. Efficient incorporation is observed for α-synuclein variants with perturbation of either one of the high-affinity monomer copper-binding residues (i.e., N-terminus or His50) whereas a variant with both N-terminal acetylation and His50 substituted with Ala does not incorporate any copper into the amyloids. Both the morphology of the resulting α-synuclein amyloids (amyloid fiber pitch, secondary structure, proteinase sensitivity) and the copper chemical properties (redox activity, chemical potential) are altered when copper is incorporated into amyloids. We speculate that copper chelation by α-synuclein amyloids contributes to the observed copper dys-homeostasis (e.g., reduced bioavailable levels) in Parkinson's disease patients. At the same time, amyloid-copper interactions may be protective to neuronal cells as they will shield aberrantly free copper ions from promotion of toxic reactive oxygen species.

Keywords:  aggregation; alpha‐synuclein; amyloid; copper; reactive oxygen species

DOI:  https://doi.org/10.1002/pro.4956
Res Sq. 2024 Mar 04. pii: rs.3.rs-3968879. [Epub ahead of print]

Seeding Activity of Skin Misfolded Tau as a Biomarker for Tauopathies.

Zerui Wang, Ling Wu, Maria Gerasimenko, Tricia Gilliland, Steven A Gunzler, Vincenzo Donadio, Rocco Liguori, Bin Xu, Wen-Quan Zou.

Background Tauopathies are a group of age-related neurodegenerative diseases characterized by the accumulation of pathologically phosphorylated tau protein in the brain, leading to prion-like propagation and aggregation. They include Alzheimer's disease (AD), progressive supranuclear palsy (PSP), corticobasal degeneration (CBD), and Pick's disease (PiD). Currently, reliable diagnostic biomarkers that directly reflect the capability of propagation and spreading of misfolded tau aggregates in peripheral tissues and body fluids are lacking. Methods We utilized the seed-amplification assay (SAA) employing ultrasensitive real-time quaking-induced conversion (RT-QuIC) to assess the prion-like seeding activity of pathological tau in the skin of cadavers with neuropathologically confirmed tauopathies, including AD, PSP, CBD, and PiD, compared to normal controls. Results We found that the skin prion-SAA demonstrated a significantly higher sensitivity (75-80%) and specificity (95-100%) for detecting tauopathy, depending on the tau substrates used. Moreover, increased tau-seeding activity was also observed in biopsy skin samples from living AD and PSP patients examined. Analysis of the end products of skin-tau SAA confirmed that the increased seeding activity was accompanied by the formation of tau aggregates with different physicochemical properties related to two different tau substrates used. Conclusions Overall, our study provides proof-of-concept that the skin tau-SAA can differentiate tauopathies from normal controls, suggesting that the seeding activity of misfolded tau in the skin could serve as a diagnostic biomarker for tauopathies.

DOI: https://doi.org/10.21203/rs.3.rs-3968879/v1
Mol Med. 2024 Mar 20. 30(1): 40

Endoplasmic reticulum stress and therapeutic strategies in metabolic, neurodegenerative diseases and cancer.

Siqi Yuan, Dan She, Shangming Jiang, Nan Deng, Jiayi Peng, Ling Ma.

  The accumulation of unfolded or misfolded proteins within the endoplasmic reticulum (ER), due to genetic determinants and extrinsic environmental factors, leads to endoplasmic reticulum stress (ER stress). As ER stress ensues, the unfolded protein response (UPR), comprising three signaling pathways-inositol-requiring enzyme 1, protein kinase R-like endoplasmic reticulum kinase, and activating transcription factor 6 promptly activates to enhance the ER's protein-folding capacity and restore ER homeostasis. However, prolonged ER stress levels propels the UPR towards cellular demise and the subsequent inflammatory cascade, contributing to the development of human diseases, including cancer, neurodegenerative disorders, and diabetes. Notably, increased expression of all three UPR signaling pathways has been observed in these pathologies, and reduction in signaling molecule expression correlates with decreased proliferation of disease-associated target cells. Consequently, therapeutic strategies targeting ER stress-related interventions have attracted significant research interest. In this review, we elucidate the critical role of ER stress in cancer, metabolic, and neurodegenerative diseases, offering novel therapeutic approaches for these conditions.

Keywords:  Cancer; Endoplasmic reticulum stress; Metabolic; Neurodegenerative diseases; Signaling pathway; Therapeutic strategies

DOI:  https://doi.org/10.1186/s10020-024-00808-9
Methods Mol Biol. 2024 ;2754 117-129

Sedimentation and Laser Light Scattering Methods for Quantifying Synthetic Tau Aggregation Propensity.

Dmitry Malyshka, Daniela Jimenez-Harrison, Jeff Kuret.

  Tau aggregation assays detect and quantify the conversion of soluble tau monomers into species having filamentous or oligomeric structure. Assays for filamentous aggregates in cross-β-sheet conformation leverage optical, biochemical, or biophysical methods, each with their own advantages and throughput capacity. Here we provide protocols for two medium-throughput assays based on sedimentation and laser light scattering and compare their performance, their utility for characterizing tau aggregation dynamics, and their limitations relative to other approaches. Additionally, a protocol for transmission electron microscopy analysis is updated so as to be compatible with the truncated tau variants that have emerged as powerful tools for interrogating the structural basis of tau polymorphism. Together these methods contribute to a rich tool kit for interrogating tau aggregation kinetics and propensity over a wide range of experimental conditions.

Keywords:  Aggregation dynamics; Laser light scattering; Sedimentation; Tau protein; Transmission electron microscopy

DOI:  https://doi.org/10.1007/978-1-0716-3629-9_7
Eur J Pharmacol. 2024 Mar 19. pii: S0014-2999(24)00179-1. [Epub ahead of print]970 176491

Quercetin-3-O-glc-1-3-rham-1-6-glucoside decreases Aβ production, inhibits Aβ aggregation and neurotoxicity, and prohibits the production of inflammatory cytokines.

Shuo Tan, Linmei Wu, Jiayi Liu, Zhaoyuan Wu, Qiang Cheng, Qiuhao Qu, Lianghao Zhu, Yizhu Yan, Hao Wu, Tie-Jun Ling, Rui-Tian Liu, Shigao Yang.

  Alzheimer's disease (AD) is a progressive neurodegenerative disease with the hallmark of aggregation of beta-amyloid (Aβ) into extracellular fibrillar deposition. Accumulating evidence suggests that soluble toxic Aβ oligomers exert diverse roles in neuronal cell death, oxidative stress, neuroinflammation, and the eventual pathogenesis of AD. Aβ is derived from the sequential cleavage of amyloid-β precursor protein (APP) by β-secretase (BACE1) and γ-secretase. The current effect of single targeting is not ideal for the treatment of AD. Therefore, developing multipotent agents with multiple properties, including anti-Aβ generation and anti-Aβ aggregation, is attracting more attention for AD treatment. Previous studies indicated that Quercetin was able to attenuate the effects of several pathogenetic factors in AD. Here, we showed that naturally synthesized Quercetin-3-O-glc-1-3-rham-1-6-glucoside (YCC31) could inhibit Aβ production by reducing β-secretase activity. Further investigations indicated that YCC31 could suppress toxic Aβ oligomer formation by directly binding to Aβ. Moreover, YCC31 could attenuate Aβ-mediated neuronal death, ROS and NO production, and pro-inflammatory cytokines release. Taken together, YCC31 targeting multiple pathogenetic factors deserves further investigation for drug development of AD.

Keywords:  APP; Aggregation; Amyloid-beta; Neurotoxicity; β-secretase

DOI:  https://doi.org/10.1016/j.ejphar.2024.176491
Free Radic Biol Med. 2024 Mar 14. pii: S0891-5849(24)00135-7. [Epub ahead of print]

Are age-related neurodegenerative diseases caused by a lack of the diet-derived compound ergothioneine?

Barry Halliwell, Irwin Cheah.

  We propose that the diet-derived compound ergothioneine (ET) is an important nutrient in the human body, especially for maintenance of normal brain function, and that low body ET levels predispose humans to significantly increased risks of neurodegenerative (cognitive impairment, dementia, Parkinson's disease) and possibly other age-related diseases (including frailty, cardiovascular disease, and eye disease). Hence, restoring ET levels in the body could assist in mitigating these risks, which are rapidly increasing due to ageing populations globally. Prevention of neurodegeneration is especially important, since by the time dementia is usually diagnosed damage to the brain is extensive and likely irreversible. ET and vitamin E from the diet may act in parallel or even synergistically to protect different parts of the brain; both may be "neuroprotective vitamins". The present article reviews the substantial scientific basis supporting these proposals about the role of ET.

Keywords:  Ageing; Antioxidant; Cognitive impairment; Dementia; Ergothioneine; Neurodegenerative diseases

DOI:  https://doi.org/10.1016/j.freeradbiomed.2024.03.009
J Chem Theory Comput. 2024 Mar 19.

Kinetic Ensemble of Tau Protein through the Markov State Model and Deep Learning Analysis.

Yongna Yuan, Xuqi Mao, Xiaohang Pan, Ruisheng Zhang, Wei Su.

The ordered assembly of Tau protein into filaments characterizes Alzheimer's and other neurodegenerative diseases, and thus, stabilization of Tau protein is a promising avenue for tauopathies therapy. To dissect the underlying aggregation mechanisms on Tau, we employ a set of molecular simulations and the Markov state model to determine the kinetics of ensemble of K18. K18 is the microtubule-binding domain of Tau protein and plays a vital role in the microtubule assembly, recycling processes, and amyloid fibril formation. Here, we efficiently explore the conformation of K18 with about 150 μs lifetimes in silico. Our results observe that all four repeat regions (R1-R4) are very dynamic, featuring frequent conformational conversion and lacking stable conformations, and the R2 region is more flexible than the R1, R3, and R4 regions. Additionally, it is worth noting that residues 300-310 in R2-R3 and residues 319-336 in R3 tend to form sheet structures, indicating that K18 has a broader functional role than individual repeat monomers. Finally, the simulations combined with Markov state models and deep learning reveal 5 key conformational states along the transition pathway and provide the information on the microsecond time scale interstate transition rates. Overall, this study offers significant insights into the molecular mechanism of Tau pathological aggregation and develops novel strategies for both securing tauopathies and advancing drug discovery.

DOI: https://doi.org/10.1021/acs.jctc.3c01211