Lithium chloride alters Tau phosphorylation, kinase activity, and Rho GTPase signaling in cell models.

Cytoplasmic aggregation of transactive response DNA-binding protein 43 (TDP-43) represents pathological hallmarks of TDP-43 proteinopathies. Accumulating evidence indicates that oxidative stress plays a pivotal role in these disorders by promoting TDP-43 aggregation and subsequent neurotoxicity. Glutaredoxin-1 (Grx1) is a key antioxidant enzyme that maintains cellular redox homeostasis. In this study, we investigated the role of Grx1 in TDP-43 proteinopathy. We examined the effects of Grx1 in neuro-2a cells expressing human wild-type TDP-43 (N2a-hTDP-43), a cellular model of TDP-43 proteinopathy characterized by increased oxidative stress, TDP-43 aggregation, and neurotoxicity. In N2a-hTDP-43 cells, Grx1 expression was increased in parallel with elevated oxidative stress. Increasing Grx1 significantly suppresses intracellular oxidative stress and cytoplasmic TDP-43 aggregation in N2a-hTDP-43 cells. Notably, increasing Grx1 significantly reduces cleaved caspase-3 levels in N2a-hTDP-43 cells, indicating reduced neurotoxicity. Collectively, our findings demonstrate that Grx1 attenuates neurotoxicity by suppressing oxidative stress and TDP-43 aggregation, highlighting its potential as a therapeutic target for TDP-43 proteinopathies.

Tauopathies are neurodegenerative diseases characterized by pathological tau accumulation, leading to motor and neuropsychiatric symptoms. Effective tau-targeting therapies remain a major challenge, in part because tau lacks well-defined druggable sites and accumulates as heterogeneous intracellular aggregates that are difficult to access and clear. Here, we present 1D9-LIRΔTP53INP2, a single-domain antibody (sdAb)-based protein degrader that facilitates tau clearance through the autophagy-lysosomal pathway. This engineered molecule combines the anti-tau sdAb 1D9 with an LC3-interacting region (LIRΔTP53INP2) to promote autophagosomal recruitment, mimicking autophagy receptors by simultaneously binding tau and LC3. In neurons derived from patients with frontotemporal dementia (FTD) and JNPL3 tauopathy mice, both harboring the P301L tau mutation, 1D9-LIRΔTP53INP2 promoted autophagy-lysosome-mediated tau degradation. It readily crossed the blood-brain barrier and improved motor function in JNPL3 tauopathy mice. These findings underscore the therapeutic potential of sdAb-based protein degraders for tauopathies. Given the challenges of brain delivery for conventional antibodies, sdAbs with enhanced brain penetration and efficacy offer a promising strategy for treatment of neurodegenerative diseases.

Parkinson's disease (PD) is characterised by progressive neurodegeneration and is marked by the formation of Lewy bodies, which are intracellular aggregates primarily composed of α-synuclein. Mitochondrial dysfunction and impaired protein degradation pathways are thought to play critical roles in PD progression, contributing to the loss of dopaminergic neurons in the substantia nigra. Phosphorylation of α-synuclein has been shown to promote its aggregation, underscoring its potential role in disease progression. Parkin, an E3 ubiquitin ligase, is widely regarded as a pleiotropic neuroprotective protein that modulates the mitochondrial quality control, as well as metabolic turnover and the accumulation of α-synuclein. Death-associated protein kinase 1 (DAPK1), which is involved in the regulation of apoptosis and autophagy, has recently emerged as an important factor in neurodegeneration. While DAPK1 has been implicated in Alzheimer's disease through its role in tau aggregation and amyloid-β production, our findings suggest that DAPK1 may also influence PD-related pathways by phosphorylating parkin at Ser136 and Ser198. This phosphorylation promotes the mitochondrial transport of parkin, enhancing interaction with mitochondria-localised E3 ubiquitin ligase MITOL and consequently leading to the degradation of parkin. Given the neuroprotective role of parkin, its reduction increases the vulnerability of neurons to 6-hydroxydopamine-induced toxicity, potentially contributing to decreased neuronal survival. Together, these findings suggest that DAPK1 functions as a previously unrecognised modulator of parkin and could potentially influence PD-related neurodegenerative processes. This pathway may provide a mechanistic link between mitochondrial dysfunction, α-synuclein pathology and neuronal cell death.

Autosomal dominant tubulointerstitial kidney disease due to pathogenic UMOD variants (ADTKD-UMOD) is a toxic proteinopathy caused by intracellular accumulation of mutant uromodulin and endoplasmic reticulum (ER) stress. Lipocalin-2 (LCN2) is an acute-phase protein induced by ER stress with context-dependent roles in kidney injury. To examine the role of LCN2 in ADTKD-UMOD, we used Umod knock-in mouse models (C171Y, R186S, C125R), urine samples from affected patients, and mIMCD3 cells expressing wild-type or mutant uromodulin. LCN2 expression was assessed by immunoblotting, immunostaining, and ELISA. Autophagy was stimulated with Torin1 to evaluate effects on LCN2 induction. UmodR186S/+ mice were crossed with Lcn2-/- mice to determine the impact of LCN2 deficiency on disease progression. Robust LCN2 induction was observed in kidneys and urine of UmodR186S/+, UmodC125R/+, and UmodC171Y/+ mice, correlating with uromodulin aggregates and ER stress severity in thick ascending limb cells. In patients, specific UMOD variants were associated with elevated urinary LCN2. In mIMCD3 cells expressing mutant uromodulin (C170Y, R185S), treatment with Torin1 reduced aggregates and attenuated LCN2 induction. Genetic deletion of Lcn2 in UmodR186S/+ mice decreased interstitial iron deposition but did not alter uromodulin accumulation, interstitial inflammation, or fibrosis. LCN2 was induced by intracellular uromodulin aggregates and ER stress in various models of ADTKD-UMOD. Although it influenced iron handling, LCN2 did not drive fibrosis or inflammation, supporting a role as a biomarker of toxic proteinopathy rather than a therapeutic target.

Physiological amyloidogenesis drives the formation of functional amyloids involved in various biochemical pathways. We recently showed that the RNA tailing and decay machinery controls the maturation of intracellular amyloid-like aggregates. This raises the question of whether enzymes that participate in the maturation of physiological amyloids are involved in pathological amyloidogenesis implicated in human proteopathies. Using Caenorhabditis elegans and mouse models of pathological amyloids, we show that manipulating the RNA tailing-decay axis alters the toxicity of β-amyloid and α-synuclein involved in Alzheimer's and Parkinson's diseases, respectively. The RNA tailing enzymes TENT4b and TENT2 protect against β-amyloid- and α-synuclein-induced toxicity by facilitating the formation of nontoxic amyloidogenic assemblies. In contrast, the RNA exonuclease Exosc10 potentiates pathological amyloid toxicity. Remarkably, Exosc10 depletion prevents cognitive decline and restores memory in two different mouse models of β-amyloid neurotoxicity. Taken together, these results suggest that pathways of physiological amyloidogenesis participate in pathological amyloid etiology.

The neuromuscular junction (NMJ) is the peripheral synapse controlling muscle contraction. Although aging and neurodegeneration result in NMJ denervation and synaptic dismantling, early indicators of this process remain elusive. Here, we analyzed the organization and dynamics of postsynaptic nicotinic acetylcholine receptors (nAChR) following muscle denervation. Using fluorescent conjugates of α-bungarotoxin (BTX), we found that loss of nAChR stability preceded morphological disintegration. Early after denervation, the combined use of receptor labeling and lectin staining revealed a rearrangement of long-lasting or newly inserted receptors that resulted in a novel compartmentalized postsynaptic pattern in which stable, pre-existing nAChRs concentrated centrally, while newly inserted, dynamic receptors localized peripherally. Small ectopic, highly dynamic nAChR clusters emerged since early denervation. Additionally, intracellular ring-like nAChR aggregates emerged since early denervation stages and were distributed in perinuclear regions, co-localizing with the lysosomal marker LAMP1, consistent with a degradative fate. Altogether, specific combinations of nAChR dynamics and morphologies serve as early markers of NMJ dismantling. These novel criteria to assess NMJ integrity may help define therapeutic windows to promote reinnervation in degenerative neuromuscular conditions.

The transcriptional repressor Fli1 inhibits proteostasis during nutrient stress to limit NK cell persistence in solid tumors.

Charcot-Marie-Tooth disease (CMT) is one of the most prevalent inherited peripheral neuropathies. CMT type X1 (CMTX1), caused by mutations in the GJB1 gene, represents the most common X-linked subtype with central nervous system (CNS) involvement. Here, we report the identification and functional characterization of a novel GJB1 variant (c.554C>T, p.Thr185Ile) in a CMTX1-affected family and its pathogenic impact using patient-derived induced pluripotent stem cells (iPSCs) and three-dimensional (3D) neural organoid models. The GJB1 gene encodes connexin 32 (Cx32), a gap junction protein. Immunofluorescent analysis revealed aberrant intracellular reduction and aggregation of the mutant Cx32 protein, suggesting impaired gap junction function. iPSC-derived neural organoids carrying the GJB1 mutation exhibited significant delay in neural differentiation and disrupted neural rosette organization. These findings underscore the critical role of Cx32 in neural development and provide a physiologically relevant platform for underlying CMTX1 pathological mechanisms on central nervous system. The established GJB1-variant organoid model holds promise for investigating genotype-phenotype correlations and facilitating the development of targeted therapeutic strategies for CMTX1.

Macromolecular crowding and protein aggregation: Friend, foe or contextual force?

Functional amyloids are a class of amyloids that serve important biological functions. One such bacterial functional amyloid is curli, assembled on the cell surface by Escherichia coli during biofilm biogenesis. Curli precursor proteins, CsgA and CsgB, synthesized in the cytoplasm, are highly amyloidogenic. It is imperative to keep the proteins in a soluble, non-aggregated form to prevent intracellular aggregation and cellular toxicity. Chaperones and chaperone-like proteins aid in solubility and proper translocation of curli subunits. Here, we have investigated a new functionality of a cytoplasmic protein, YedX, an E. coli transthyretin-related protein known to function as a hydrolase enzyme in purine metabolism. We established structure-influenced functional parallelism between YedX and CsgC, a chaperone-like protein that prevents immature aggregation of CsgA and CsgB in the periplasmic space. Our biophysical and biochemical studies suggest that YedX alleviates the in vitro amyloid assembly of CsgA, maintaining its native, soluble, and non-toxic state. Our findings unravel a novel function of YedX in keeping aggregation-prone proteins in the soluble state to aid protein homeostasis within the cell.

Microbes naturally grow exponentially, but this trait might not always be desirable for applications with genetically modified microorganisms. Especially in microorganisms engineered for therapeutic applications, uncurbed exponential proliferation might cause unpredictable liabilities in their behavior that in turn compromise their dosing and biocontainment. In an effort to fundamentally reprogram population growth dynamics, we constructed a bacterial chassis that adheres to linear proliferation for a finite number of generations. More specifically, growth of the chassis is directed by an intracellular protein aggregate that is engineered to reconstitute a split enzyme producing cAMP as a conditionally essential metabolite. Due to the asymmetric segregation and gradual disaggregation of this aggregate, it autonomously keeps growth restricted to the aggregate inheriting cell and to a limited number of divisions. By imposing such a transient and linear growth potential without the need for external intervention, this chassis offers a unique venue for the controlled application of engineered microorganisms.

Screening of small-molecule drugs to suppress both protein aggregation and reactive oxygen species (ROS) generation is critical for developing therapies for neurodegenerative diseases (NDs). However, existing methods are limited to characterizing only a single pathological feature (either protein aggregation or ROS generation) in a single measurement. Herein, taking α-synuclein (α-Syn) as the template protein, we developed a dual-mode electrochemical sensing platform for concurrently monitoring protein aggregation and ROS generation characteristics. A gold electrode functionalized with α-Syn via self-assembled monolayers (SAMs) was constructed as the sensing platform, realizing both ordered α-Syn immobilization and monitoring of metal ion (e.g., Cu(ii))-driven aggregation. This was accomplished by synchronously recording the electrochemiluminescence (ECL) and cyclic voltammetry (CV) dual responses of the tris(2,2'-bipyridine) ruthenium(ii) (Ru(bpy)3 2+) reporter in a single integrated assay. The catalysis of DNA oxidation by Ru(bpy)3 2+ enables the amplification of ECL and CV dual-mode signals, which increased the detection sensitivity for both aggregation and ROS generation accompanied by the α-Syn - Cu(ii) complex. Machine learning algorithms were then utilized to analyze ECL and CV responses of small molecules with known drug effects. This analysis culminated in the development of a linear discriminant analysis (LDA) screening model, which enabled the assessment of drug efficacy against the two pathological features. The predictive capability of the screening model was verified through transmission electron microscopy (TEM), cell viability and intracellular aggregation studies. This model was further successfully applied to assess two previously unexplored small molecules: diethylenetriaminepentaacetic dianhydride (DTPA) and deferiprone. Collectively, this dual-mode sensing platform, integrating DNA-amplified monitoring of protein aggregation and ROS generation, enables the robust establishment of a machine learning-assisted small-molecule drug screening model, offering a novel approach for the in vitro characterization of protein-related pathological features.

N-terminal KTKEGV motif lysine residues of α-Synuclein are critical for TLR2 interaction and activation.

The Escherichia coli bacterial expression system was the first platform developed for recombinant protein production and remains the fastest, simplest, and most cost-effective system for achieving high protein yields for fundamental research, as well as biotechnological and pharmaceutical applications. Bacterial surface display systems and secretion of target proteins have become widely used approaches. These strategies help prevent intracellular aggregation and proteolytic degradation of recombinant proteins, enabling the recovery of soluble, properly folded, and stable protein products. In the case of toxic proteins, secretion mitigates their inhibitory effects on essential host cell processes. Furthermore, secretion of target proteins and peptides significantly simplifies their purification. The review summarizes the data on E.coli secretion systems with a special focus on protein export and display strategies, and discusses their applications in scientific research, industrial biotechnology, and medicine.

Tauopathies represent a major class of neurodegenerative disorders associated with intracellular aggregates of the microtubule-associated protein Tau. To identify molecular modulators of Tau toxicity, we used a genetic screen to identify protein chaperones whose RNAi-mediated knockdown could modulate hTauV337M-induced eye-ommatidial degeneration in Drosophila. This screen identified the Prefoldins Pfdn5 and Pfdn6 as strong modifiers of hTauV337M cytotoxicity. Consistent with the known function of Pfdn as a cotranslational chaperone for tubulin, Pfdn5 mutants showed substantially reduced levels of tubulin monomer. However, additional microtubule-related functions were indicated by the robust unexpected association of Pfdn5 with axonal microtubules in vivo, as well as binding with stabilized microtubules in biochemical assays. Loss of Pfdn5 resulted in neuromuscular junctions (NMJ) defects similar to those previously described in hTau-expressing flies: namely, increased supernumerary boutons and fewer microtubule loops within mature presynaptic boutons. Significantly, synaptic phenotypes caused by hTauV337M overexpression were also strongly enhanced in a Pfdn5 mutant background. Consistent with a role in modulating Tau toxicity, not only did loss of Pfdn5 result in increased accumulations of Tau aggregates in hTauV337M-expressing neurons, but also neuronal overexpression of Prefoldin strikingly ameliorated age-dependent neurodegeneration and memory deficits induced by pathological hTau. Together, these and other observations described herein: (a) provide new insight into Prefoldin-microtubule interactions; (b) point to essential post-translational roles for Pfdn5 in controlling Tau toxicity in vivo; and (c) demonstrate that Pfdn5 overexpression is sufficient to restrict Tau-induced neurodegeneration.

The trimeric spike (S) protein on the envelope of the SARS-CoV-2 virus is the primary target structure for currently approved corona vaccines. For this reason, the two mRNA-based corona vaccines Comirnaty (BNT162b2, Pfizer/BioNTech) and Spikevax (mRNA-1273, Moderna) first induce the production of a spike monomer in body cells. After enzymatic cleavage by the endoprotease furin, two S subunits are formed, which are supposed to trigger the desired immune response following secretion. Based on this concept, a preventive measure against symptomatic SARS-CoV-2 infections became available within one year of the pandemic's onset. mRNA-based vaccines have proven highly effective in reducing severe disease and mortality. However, both the virus itself and mRNA vaccines have been associated with cardiac symptoms, which are commonly classified as myocarditis, pericarditis, or a combination thereof based on clinical presentation. Although vaccine-induced myocarditis remains a rare adverse event, recent longitudinal studies have raised questions regarding its long-term impact. To better understand the molecular mechanisms potentially involved in vaccine-associated cardiac side effects, we investigated the translation and proteolytic processing of the encoded spike monomers in human AC16 cardiomyocytes, as well as (for comparative purposes) in HEK-293 and HeLa cells. In all three cell types, both BNT162b2 and mRNA-1273 produced two divergently sized monomer translation products from which one S1 subunit was formed after enzymatic cleavage. However, the number of identified S2 subunits varied between two and four depending on the cell line and mRNA used. Within a few hours, covalently bonded high-molecular complexes formed from both the spike monomers and their subunits. The arrangement of these complexes always adhered to a consistent pattern in each cell type. Particularly in AC16 cardiomyocytes, the various spike protein derivatives impaired not only cell proliferation, but also induced a pro-inflammatory response and oxidative stress. Only the secreted S1 subunit was detected as an immunogen in the supernatant of all three cell lines. Our findings may help to improve the safety and specificity of future mRNA platform technologies by emphasizing the importance of evaluating intracellular protein processing and the potential cellular effects of translated immunogens already during preclinical development.

Non-cell autonomous autophagy in amyotrophic lateral sclerosis: A new promising target?

Self‐aggregation processes are ubiquitous in living systems and play vital roles in maintaining normal physiological functions. Inspired by these natural phenomena, constructing artificial aggregates within cells is of great significance for understanding the complex mechanisms of life activities and precisely regulating cellular processes. Owing to its excellent programmability and stimulus responsiveness, DNA nanotechnology offers unprecedented opportunities for building intelligent nanodevices inside living cells, far more dynamically regulable than traditional materials. This review systematically summarizes recent strategies for achieving intelligent intracellular aggregation of DNA nanostructures in response to various stimuli, including protons, metal ions, enzymes, nucleic acids, and sequential stimuli. Furthermore, we discuss the specific applications of these DNA‐based smart aggregates, including in situ biosensing and cellular function regulation. Finally, we provide an outlook on key challenges and prospects in this emerging field.

Interacting partners of Tau protein in Alzheimer's disease.