Mutations disrupting the nuclear localization of the RNA-binding protein FUS characterize a subset of amyotrophic lateral sclerosis patients (ALS-FUS). FUS regulates nuclear RNAs, but its role at the ...synapse is poorly understood. Using super-resolution imaging we determined that the localization of FUS within synapses occurs predominantly near the vesicle reserve pool of presynaptic sites. Using CLIP-seq on synaptoneurosomes, we identified synaptic FUS RNA targets, encoding proteins associated with synapse organization and plasticity. Significant increase of synaptic FUS during early disease in a mouse model of ALS was accompanied by alterations in density and size of GABAergic synapses. mRNAs abnormally accumulated at the synapses of 6-month-old ALS-FUS mice were enriched for FUS targets and correlated with those depicting increased short-term mRNA stability via binding primarily on multiple exonic sites. Our study indicates that synaptic FUS accumulation in early disease leads to synaptic impairment, potentially representing an initial trigger of neurodegeneration.
In Vietnam, intensive shrimp farms heavily rely on a wide variety of antibiotics (ABs) to treat animals or prevent disease outbreak. Potential for the emergence of multi-resistant bacteria is high, ...with the concomitant contamination of adjacent natural aquatic habitats used for irrigation and drinking water, impairing in turn human health system. In the present study, quantification of AB multi-resistant bacteria was carried out in water and sediment samples from effluent channels connecting a shrimp farming area to the Vam Co River (Long An Province, Vietnam). Bacterial strains, e.g. Klebsiella pneumoniae and Aeromonas hydrophila, showing multi-resistance traits were isolated. Molecular biology analysis showed that these strains possessed from four to seven different AB resistance genes (ARGs) (e.g. sul1, sul2, qnrA, ermB, tetA, aac(6)lb, dfrA1, dfr12, dfrA5), conferring multidrug resistance capacity. Sequencing of plasmids present within these multi-resistant strains led to the identification of a total of forty-one resistance genes, targeting nine AB groups. qPCR analysis on the sul2 gene revealed the presence of high copy numbers in the effluent channel connecting to the Vam Co River. The results of the present study clearly indicated that multi-resistant bacteria present in intensive shrimp cultures may disseminate in the natural environment. This study offered a first insight in the impact of plasmid-born ARGs and the related pathogenic bacteria that could emerged due to inappropriate antibiotic utilization in South Vietnam.
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•Multi-resistant pathogenic bacteria were isolated from shrimp farming effluents.•Isolated strains possessed four to seven resistance genes on plasmids.•Forty-one plasmid-borne resistance genes were detected.•High numbers of sul2 gene copies were measured in the adjacent natural water course.•Antibiotic promotes the dispersion of multi-resistant bacteria in the environment.
Aggregation of the RNA‐binding protein TAR DNA‐binding protein 43 (TDP‐43) is the key neuropathological feature of neurodegenerative diseases, including amyotrophic lateral sclerosis (ALS) and ...frontotemporal lobar degeneration (FTLD). In physiological conditions, TDP‐43 is predominantly nuclear, forms oligomers, and is contained in biomolecular condensates assembled by liquid–liquid phase separation (LLPS). In disease, TDP‐43 forms cytoplasmic or intranuclear inclusions. How TDP‐43 transitions from physiological to pathological states remains poorly understood. Using a variety of cellular systems to express structure‐based TDP‐43 variants, including human neurons and cell lines with near‐physiological expression levels, we show that oligomerization and RNA binding govern TDP‐43 stability, splicing functionality, LLPS, and subcellular localization. Importantly, our data reveal that TDP‐43 oligomerization is modulated by RNA binding. By mimicking the impaired proteasomal activity observed in ALS/FTLD patients, we found that monomeric TDP‐43 forms inclusions in the cytoplasm, whereas its RNA binding‐deficient counterpart aggregated in the nucleus. These differentially localized aggregates emerged via distinct pathways: LLPS‐driven aggregation in the nucleus and aggresome‐dependent inclusion formation in the cytoplasm. Therefore, our work unravels the origins of heterogeneous pathological species reminiscent of those occurring in TDP‐43 proteinopathy patients.
Synopsis
Loss of TDP‐43 oligomerization and RNA binding promotes diverse TDP‐43 pathologies providing insight into the origins of heterogeneous pathological species occurring in human disease.
TDP‐43 oligomerization and RNA binding govern its stability, splicing regulation, LLPS, and subcellular localization.
TDP‐43 oligomerization is modulated by RNA binding.
Monomeric TDP‐43 forms inclusions in the cytoplasm, whereas its RNA binding‐deficient counterpart aggregates in the nucleus.
TDP‐43 aggregates emerge via distinct pathways: LLPS‐driven aggregation in the nucleus and aggresome‐dependent inclusion formation in the cytoplasm.
Our work unravels the origins of heterogeneous TDP‐43 inclusions reminiscent of those occurring in TDP‐43 proteinopathy patients.
Loss of TDP‐43 oligomerization and RNA binding promotes diverse TDP‐43 pathologies providing insights into the origins of heterogeneous pathological species occurring in human disease.
Autoimmune synaptic encephalitides are recently described human brain diseases leading to psychiatric and neurological syndromes through inappropriate brain-autoantibody interactions. The most ...frequent synaptic autoimmune encephalitis is associated with autoantibodies against extracellular domains of the glutamatergic N-methyl-d-aspartate receptor, with patients developing psychotic and neurological symptoms in an autoantibody titre-dependent manner. Although N-methyl-d-aspartate receptors are the primary target of these antibodies, the cellular and molecular pathway(s) that rapidly lead to N-methyl-d-aspartate receptor dysfunction remain poorly understood. In this report, we used a unique combination of high-resolution nanoparticle and bulk live imaging approaches to demonstrate that anti-N-methyl-d-aspartate receptor autoantibodies from patients with encephalitis strongly alter, in a time-dependent manner, the surface content and trafficking of GluN2-NMDA receptor subtypes. Autoantibodies laterally displaced surface GluN2A-NMDA receptors out of synapses and completely blocked synaptic plasticity. This loss of extrasynaptic and synaptic N-methyl-d-aspartate receptor is prevented both in vitro and in vivo, by the activation of EPHB2 receptors. Indeed, the anti-N-methyl-d-aspartate receptor autoantibodies weaken the interaction between the extracellular domains of the N-methyl-d-aspartate and Ephrin-B2 receptors. Together, we demonstrate that the anti-N-methyl-d-aspartate receptor autoantibodies from patients with encephalitis alter the dynamic retention of synaptic N-methyl-d-aspartate receptor through extracellular domain-dependent mechanism(s), shedding new light on the pathology of the neurological and psychiatric disorders observed in these patients and opening possible new therapeutic strategies.
Hexanucleotide G
C
repeat expansions in the
gene are the most common genetic cause of amyotrophic lateral sclerosis and frontotemporal dementia. Dipeptide repeat proteins (DPRs) generated by ...translation of repeat-containing RNAs show toxic effects in vivo as well as in vitro and are key targets for therapeutic intervention. We generated human antibodies that bind DPRs with high affinity and specificity. Anti-GA antibodies engaged extra- and intra-cellular poly-GA and reduced aggregate formation in a poly-GA overexpressing human cell line. However, antibody treatment in human neuronal cultures synthesizing exogenous poly-GA resulted in the formation of large extracellular immune complexes and did not affect accumulation of intracellular poly-GA aggregates. Treatment with antibodies was also shown to directly alter the morphological and biochemical properties of poly-GA and to shift poly-GA/antibody complexes to more rapidly sedimenting ones. These alterations were not observed with poly-GP and have important implications for accurate measurement of poly-GA levels including the need to evaluate all centrifugation fractions and disrupt the interaction between treatment antibodies and poly-GA by denaturation. Targeting poly-GA and poly-GP in two mouse models expressing G
C
repeats by systemic antibody delivery for up to 16 mo was well-tolerated and led to measurable brain penetration of antibodies. Long-term treatment with anti-GA antibodies produced improvement in an open-field movement test in aged
mice. However, chronic administration of anti-GA antibodies in AAV-(G
C
)
mice was associated with increased levels of poly-GA detected by immunoassay and did not significantly reduce poly-GA aggregates or alleviate disease progression in this model.
This paper presents an extension of the validation domain of a previously validated three-dimensional probabilistic semi-explicit cracking numerical model, which was initially validated for a ...specific concrete mix design. This model is implemented in a finite element code. The primary objective of this study is to propose a function that enables the estimation of the critical fracture energy parameter utilized in the model and validate its effectiveness for various concrete mix designs. The model focuses on macrocrack propagation and introduces significant aspects such as employing volume elements for simulating macrocrack propagation and incorporating two key factors in governing its behavior. Firstly, macrocrack initiation is linked to the uniaxial tensile strength (ft). Secondly, macrocrack propagation is influenced by a post-cracking dissipation energy in tension. This energy is taken equal to the mode I critical fracture energy (GIC) based on the linear elastic fracture mechanics theory. Importantly, both ft and GIC are probabilistic properties influenced by the volume of concrete under consideration. Consequently, in the numerical model, they are dependent on the volume of the finite elements employed. To achieve this objective, numerical simulations of fracture mechanical tests are conducted on a large double cantilever beam specimen. Through these simulations, we validate the proposed function, which is a crucial step towards expanding the model’s applicability to all concrete mix designs.
This paper introduces a three-dimensional (3D) semi-explicit probabilistic numerical model for simulating crack propagation within the framework of the finite element method. The model specifically ...addresses macrocrack propagation using linear volume elements. The criteria governing the macrocrack propagation is based on the softening behavior observed in concrete under uniaxial tension. This softening behavior corresponds to a dissipated cracking energy that is equal to the mode I critical fracture energy (GIC) used in the Linear Elastic Fracture Mechanics theory (LEFM). The probabilistic nature of this model revolves around the random distribution of two mechanical properties: tensile strength (ft) and fracture energy, which varies based on the volume of finite elements. The scattering of the fracture energy increases as the volume of finite elements decreases in order to consider the strong heterogeneity of the material. This work primarily aims to estimate the relationship between the standard deviation of GIC and the volume of finite elements through the development of the numerical model. For this purpose, an inverse analysis is conducted based on a fracture mechanical test simulation. This test involves macrocrack propagation in a large Double Cantilever Beam (DCB) specimen with a crack length exceeding two meters. The proposed inverse analysis procedure yields highly significant results, indicating that the numerical model effectively evaluates both crack length and crack opening during propagation.
While the initial pathology of Parkinson’s disease and other α‐synucleinopathies is often confined to circumscribed brain regions, it can spread and progressively affect adjacent and distant brain ...locales. This process may be controlled by cellular receptors of α‐synuclein fibrils, one of which was proposed to be the LAG3 immune checkpoint molecule. Here, we analysed the expression pattern of LAG3 in human and mouse brains. Using a variety of methods and model systems, we found no evidence for LAG3 expression by neurons. While we confirmed that LAG3 interacts with α‐synuclein fibrils, the specificity of this interaction appears limited. Moreover, overexpression of LAG3 in cultured human neural cells did not cause any worsening of α‐synuclein pathology ex vivo. The overall survival of A53T α‐synuclein transgenic mice was unaffected by LAG3 depletion, and the seeded induction of α‐synuclein lesions in hippocampal slice cultures was unaffected by LAG3 knockout. These data suggest that the proposed role of LAG3 in the spreading of α‐synucleinopathies is not universally valid.
SYNOPSIS
This study re‐evaluated the role of neuronal lymphocyte‐activation gene 3 (LAG3) in modulating the spreading of α‐synucleinopathies.
The expression of LAG3 in neurons could not be validated, using genomic and proteomic approaches.
The binding of α‐synuclein fibrils to LAG3 appeared to be limited regarding its specificity and affinity.
Overexpression of LAG3 in human neurons did not lead to an increased deposition of α‐synuclein aggregates ex vivo.
The genetic ablation of LAG3 did not lead to a prolonged survival of mice expressing human A53T α‐synuclein.
The absence of LAG3 did not influence the deposition of α‐synuclein inclusions in organotypic slice cultures.
This study re‐evaluated the role of neuronal lymphocyte‐activation gene 3 (LAG3) in modulating the spreading of α‐synucleinopathies.
BIN1, a member of the BAR adaptor protein family, is a significant late-onset Alzheimer disease risk factor. Here, we investigate BIN1 function in the brain using conditional knockout (cKO) models. ...Loss of neuronal Bin1 expression results in the select impairment of spatial learning and memory. Examination of hippocampal CA1 excitatory synapses reveals a deficit in presynaptic release probability and slower depletion of neurotransmitters during repetitive stimulation, suggesting altered vesicle dynamics in Bin1 cKO mice. Super-resolution and immunoelectron microscopy localizes BIN1 to presynaptic sites in excitatory synapses. Bin1 cKO significantly reduces synapse density and alters presynaptic active zone protein cluster formation. Finally, 3D electron microscopy reconstruction analysis uncovers a significant increase in docked and reserve pools of synaptic vesicles at hippocampal synapses in Bin1 cKO mice. Our results demonstrate a non-redundant role for BIN1 in presynaptic regulation, thus providing significant insights into the fundamental function of BIN1 in synaptic physiology relevant to Alzheimer disease.
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•The loss of BIN1 in neurons leads to impaired spatial memory consolidation•Neuronal Bin1 cKO mice have deficits in excitatory synaptic transmission•BIN1 regulates presynaptic vesicular release in hippocampal excitatory synapses•The results highlight a non-redundant role for BIN1 in presynaptic regulation
BIN1 is a significant risk factor for late-onset Alzheimer disease. BIN1 has a general role in endocytosis and membrane dynamics in non-neuronal cells. De Rossi et al. show that BIN1 localizes to presynaptic terminals and plays an indispensable role in excitatory synaptic transmission by regulating neurotransmitter vesicle dynamics.
Protein aggregation is the main hallmark of neurodegenerative diseases. Many proteins found in pathological inclusions are known to undergo liquid-liquid phase separation, a reversible process of ...molecular self-assembly. Emerging evidence supports the hypothesis that aberrant phase separation behavior may serve as a trigger of protein aggregation in neurodegeneration, and efforts to understand and control the underlying mechanisms are underway. Here, we review similarities and differences among four main proteins, α-synuclein, FUS, tau, and TDP-43, which are found aggregated in different diseases and were independently shown to phase separate. We discuss future directions in the field that will help shed light on the molecular mechanisms of aggregation and neurodegeneration.
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