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The central nervous system (CNS) is protected by the blood-brain barrier (BBB), which acts as a physical barrier to regulate and prevent the uptake of endogenous metabolites and xenobiotics. However, the BBB prevents most non-lipophilic drugs from reaching the CNS following systematic administration. Therefore, there is considerable interest in identifying drug carriers that can maintain the biostability of therapeutic molecules and target their transport across the BBB. In this regard, upconversion nanoparticles (UCNPs) have become popular as a nanoparticle-based solution to this problem, with the additional benefit that they display unique properties for in vivo visualization. The majority of studies to date have explored basic spherical UCNPs for drug delivery applications. However, the biophysical properties of UCNPs, cell uptake and BBB transport have not been thoroughly investigated. In this study, we described a one-pot seed-mediated approach to precisely control longitudinal growth to produce bright UCNPs with various aspect ratios. We have systematically evaluated the effects of the physical aspect ratios and PEGylation of UCNPs on cellular uptake in different cell lines and an in vivo zebrafish model. We found that PEGylated the original UCNPs can enhance their biostability and cell uptake capacity. We identify an optimal aspect ratio for UCNP uptake into several different types of cultured cells, finding that this is generally in the ratio of 2 (length/width). This data provides a crucial clue for further optimizing UCNPs as a drug carrier to deliver therapeutic agents into the CNS. STATEMENT OF SIGNIFICANCE: The central nervous system (CNS) is protected by the blood-brain barrier (BBB), which acts as a highly selective semipermeable barrier of endothelial cells to regulate and prevent the uptake of toxins and pathogens. However, the BBB prevents most non-lipophilic drugs from reaching the CNS following systematic administration. The proposed research is significant because identifying the aspect ratio of drug carriers that maintains the biostability of therapeutic molecules and targets their transport across the blood-brain barrier (BBB) is crucial for designing an efficient drug delivery system. Therefore, this research provides a vital clue for further optimizing UCNPs as drug carriers to deliver therapeutic molecules into the brain.

Amyotrophic lateral sclerosis (ALS)- and frontotemporal dementia (FTD)-linked mutations in CCNF have been shown to cause dysregulation to protein homeostasis. CCNF encodes for cyclin F, which is part of the cyclin F-E3 ligase complex SCFcyclinF known to ubiquitylate substrates for proteasomal degradation. In this study, we identified a function of cyclin F to regulate substrate solubility and show how cyclin F mechanistically underlies ALS and FTD disease pathogenesis. We demonstrated that ALS and FTD-associated protein sequestosome-1/p62 (p62) was a canonical substrate of cyclin F which was ubiquitylated by the SCFcyclinF complex. We found that SCFcyclin F ubiquitylated p62 at lysine(K)281, and that K281 regulated the propensity of p62 to aggregate. Further, cyclin F expression promoted the aggregation of p62 into the insoluble fraction, which corresponded to an increased number of p62 foci. Notably, ALS and FTD-linked mutant cyclin F p.S621G aberrantly ubiquitylated p62, dysregulated p62 solubility in neuronal-like cells, patient-derived fibroblasts and induced pluripotent stem cells and dysregulated p62 foci formation. Consistently, motor neurons from patient spinal cord tissue exhibited increased p62 ubiquitylation. We suggest that the p.S621G mutation impairs the functions of cyclin F to promote p62 foci formation and shift p62 into the insoluble fraction, which may be associated to aberrant mutant cyclin F-mediated ubiquitylation of p62. Given that p62 dysregulation is common across the ALS and FTD spectrum, our study provides insights into p62 regulation and demonstrates that ALS and FTD-linked cyclin F mutant p.S621G can drive p62 pathogenesis associated with ALS and FTD.

Glioblastoma (GBM) is among the most treatment-resistant solid tumors and often recurrs after resection. One of the mechanisms through which GBM escapes various treatment modalities is the overexpression of anti-apoptotic Bcl-2 family proteins (

SUMMARYTDP-43 is a nuclear RNA-binding protein that can undergo liquid-liquid phase separation (LLPS) and forms pathological insoluble aggregates in frontotemporal dementia and amyotrophic lateral sclerosis (ALS). Perturbations of TDP-43 function are linked to mislocalization and neurodegeneration. By studying TDP-43 in vivo, we confirmed for the first time that TDP-43 undergoes LLPS and forms biomolecular condensates in spinal motor neurons (MNs). Importantly, we discovered that interfering with the K136 SUMOylation site of TDP-43 altered its phase separation behavior, reducing cytoplasmic mislocalization and aggregation. Introduction of the ALS-linked mutation G294V did not alter these LLPS characteristics, indicating that posttranslational modifications such as lysine-specific alterations can modulate TDP-43 pathogenesis through regulating phase separation. Altogether, our in vivo characterization of TDP-43 confirms the formation of dynamic nuclear TDP-43 condensates in zebrafish spinal neurons and establishes a critical platform to validate the molecular grammar of phase separation that underpins TDP-43 aggregation in ALS and other proteinopathies.

Amyotrophic lateral sclerosis (ALS) is a form of motor neuron disease (MND) that is characterized by the progressive loss of motor neurons within the spinal cord, brainstem and motor cortex. Although ALS clinically manifests as a heterogeneous disease, with varying disease onset and survival, a unifying feature is the presence of ubiquitinated cytoplasmic protein inclusion aggregates containing TDP-43. However, the precise mechanisms linking protein inclusions and aggregation to neuronal loss are currently poorly understood.Bimolecular Fluorescence Complementation (BiFC) takes advantage the association of fluorophore fragments (non-fluorescent on their own) that are attached to an aggregation prone protein of interest. Interaction of the proteins of interest allows for the fluorescent reporter protein to fold into its native state and emit a fluorescent signal. Here, we combined the power of BiFC with the advantages of the zebrafish system to validate, optimize and visualize of the formation of ALS-linked aggregates in real time in a vertebrate model. We further provide in vivo validation of the selectivity of this technique and demonstrate reduced spontaneous self-assembly of the non-fluorescent fragments in vivo by introducing a fluorophore mutation. Additionally, we report preliminary findings on the dynamic aggregation of the ALS-linked hallmark proteins Fus and TDP-43 in their corresponding nuclear and cytoplasmic compartments using BiFC.Overall, our data demonstrates the suitability of this BiFC approach to study and characterize ALS-linked aggregate formation in vivo. Importantly, the same principle can be applied in the context of other neurodegenerative diseases and has therefore critical implications to advance our understanding of pathologies that underlie aberrant protein aggregation.

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease characterised by the loss of upper and lower motor neurons in the brain and spinal cord. ALS and frontotemporal dementia (FTD) are overlapping diseases with shared pathological features. Affected neurons of people with ALS and FTD typically contain ubiquitin-immunoreactive inclusions, of which TDP-43 (Tar DNA-binding protein of 43 kDa) is a major component. However, what triggers the formation of these abnormal TDP-43 inclusions is unclear. Previously, we identified CCNF mutations in cohorts of familial and sporadic cases of ALS and FTD. CCNF encodes cyclin F, the substrate-binding component of a multiprotein E3 ubiquitin ligase complex that ubiquitylates and subsequently directs a set of protein substrates for proteasomal degradation. Here, we explored the relationship between cyclin F and TDP-43.We used a series of complementary biochemical approaches including immunoprecipitations, in vitro ubiquitylation assays, immunofluorescence imaging and immunocytochemistry. Unpaired student t-tests were used to determine statistical significance of the results.In this study, we demonstrate that that the SCFIn summary, this study reports a direct ubiquitylation mechanism for TDP-43, revealing important insights into the regulation of cyclin F-mediated TDP-43 turnover and clues towards understanding the molecular origins of the ubiquitylated TDP-43 inclusions that are the hallmark pathological feature in ALS and FTD.

Amyotrophic lateral sclerosis (ALS) is the most common form of motor neuron disease and is characterized by the degeneration of upper and lower motor neurons of the brain and spinal cord. ALS is also linked clinically, genetically, and pathologically to a form of dementia known as frontotemporal dementia (FTD). Identifying gene mutations that cause ALS/FTD has provided valuable insight into the disease process. Several ALS/FTD-causing mutations occur within proteins with roles in protein clearance systems. This includes ALS/FTD mutations in CCNF, which encodes the protein cyclin F: a component of a multiprotein E3 ubiquitin ligase that mediates the ubiquitylation of substrates for their timely degradation. In this review, we provide an update on the link between ALS/FTD CCNF mutations and neurodegeneration.

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease commonly treated with riluzole, a small molecule that may act via modulation of glutamatergic neurotransmission. However, riluzole only modestly extends lifespan for people living with ALS, and its precise mechanisms of action remain unclear. Most ALS cases are characterised by accumulation of cytoplasmic TAR DNA binding protein of 43 kDa (TDP-43), and understanding the effects of riluzole in models that closely recapitulate TDP-43 pathology may provide insights for development of improved therapeutics. We therefore investigated the effects of riluzole in female transgenic mice that inducibly express nuclear localisation sequence (NLS)-deficient human TDP-43 in neurons (NEFH-tTA/tetO-hTDP-43ΔNLS, 'rNLS8', mice). Riluzole treatment from the first day of hTDP-43ΔNLS expression did not alter disease onset, weight loss or performance on multiple motor behavioural tasks. Riluzole treatment also did not alter TDP-43 protein levels, solubility or phosphorylation. Although we identified a significant decrease in GluA2 and GluA3 proteins in the cortex of rNLS8 mice, riluzole did not ameliorate this disease-associated molecular phenotype. Likewise, riluzole did not alter the disease-associated atrophy of hindlimb muscle in rNLS8 mice. Finally, riluzole treatment beginning after disease onset in rNLS8 mice similarly had no effect on progression of late-stage disease or animal survival. Together, we demonstrate specific glutamatergic receptor alterations and muscle fibre-type changes reminiscent of ALS in female rNLS8 mice, but riluzole had no effect on these or any other disease phenotypes. Future targeting of pathways related to accumulation of TDP-43 pathology may be needed to develop better treatments for ALS.

Ubiquitin ligases play an integral role in fine-tuning signaling cascades necessary for normal cell function. Aberrant regulation of ubiquitin ligases has been implicated in several neurodegenerative diseases, generally, due to mutations within the E3 ligase itself. Several proteomic-based methods have recently emerged to facilitate the rapid identification of ligase-substrate pairs-a previously challenging feat due to the transient nature of ligase-substrate interactions. These novel methods complement standard immunoprecipitations (IPs) and include proximity-dependent biotin identification (BioID), ubiquitin ligase-substrate trapping, tandem ubiquitin-binding entities (TUBEs), and a molecular trapping unit known as the NEDDylator. The implementation of these techniques is expected to facilitate the rapid identification of novel substrates of E3 ubiquitin ligases, a process that is likely to enhance our understanding of neurodegenerative diseases and highlight novel therapeutic targets for the treatment of neurodegenerative diseases.

The past decade has seen a rapid acceleration in the discovery of new genetic causes of ALS, with more than 20 putative ALS-causing genes now cited. These genes encode proteins that cover a diverse range of molecular functions, including free radical scavenging (e.g., SOD1), regulation of RNA homeostasis (e.g., TDP-43 and FUS), and protein degradation through the ubiquitin-proteasome system (e.g., ubiquilin-2 and cyclin F) and autophagy (TBK1 and sequestosome-1/p62). It is likely that the various initial triggers of disease (either genetic, environmental and/or gene-environment interaction) must converge upon a common set of molecular pathways that underlie ALS pathogenesis. Given the complexity, it is not surprising that a catalog of molecular pathways and proteostasis dysfunctions have been linked to ALS. One of the challenges in ALS research is determining, at the early stage of discovery, whether a new gene mutation is indeed disease-specific, and if it is linked to signaling pathways that trigger neuronal cell death. We have established a proof-of-concept proteogenomic workflow to assess new gene mutations, using CCNF (cyclin F) as an example, in cell culture models to screen whether potential gene candidates fit the criteria of activating apoptosis. This can provide an informative and time-efficient output that can be extended further for validation in a variety of in vitro and in vivo models and/or for mechanistic studies. As a proof-of-concept, we expressed cyclin F mutations (K97R, S195R, S509P, R574Q, S621G) in HEK293 cells for label-free quantitative proteomics that bioinformatically predicted activation of the neuronal cell death pathways, which was validated by immunoblot analysis. Proteomic analysis of induced pluripotent stem cells (iPSCs) derived from patient fibroblasts bearing the S621G mutation showed the same activation of these pathways providing compelling evidence for these candidate gene mutations to be strong candidates for further validation and mechanistic studies (such as E3 enzymatic activity assays, protein–protein and protein–substrate studies, and neuronal apoptosis and aberrant branching measurements in zebrafish). Our proteogenomics approach has great utility and provides a relatively high-throughput screening platform to explore candidate gene mutations for their propensity to cause neuronal cell death, which will guide a researcher for further experimental studies.

Aim Splicing factor proline and glutamine rich (SFPQ) is an RNA-DNA binding protein that is dysregulated in Alzheimer's disease and frontotemporal dementia. Dysregulation of SFPQ, specifically increased intron retention and nuclear depletion, has been linked to several genetic subtypes of amyotrophic lateral sclerosis (ALS), suggesting that SFPQ pathology may be a common feature of this heterogeneous disease. Our study aimed to investigate this hypothesis by providing the first comprehensive assessment of SFPQ pathology in large ALS case-control cohorts. Methods We examined SFPQ at the RNA, protein and DNA levels. SFPQ RNA expression and intron retention were examined using RNA-sequencing and quantitative PCR. SFPQ protein expression was assessed by immunoblotting and immunofluorescent staining. At the DNA level, SFPQ was examined for genetic variation novel to ALS patients. Results At the RNA level, retention of SFPQ intron nine was significantly increased in ALS patients' motor cortex. In addition, SFPQ RNA expression was significantly reduced in the central nervous system, but not blood, of patients. At the protein level, neither nuclear depletion nor reduced expression of SFPQ was found to be a consistent feature of spinal motor neurons. However, SFPQ-positive ubiquitinated protein aggregates were observed in patients' spinal motor neurons. At the DNA level, our genetic screen identified two novel and two rare SFPQ sequence variants not previously reported in the literature. Conclusions Our findings confirm dysregulation of SFPQ as a pathological feature of the central nervous system of ALS patients and indicate that investigation of the functional consequences of this pathology will provide insight into ALS biology.

BackgroundSplicing factor proline and glutamine rich (SFPQ, also known as polypyrimidine tract-binding protein-associated-splicing factor, PSF) is a RNA-DNA binding protein with roles in key cellular pathways such as DNA transcription and repair, RNA processing and paraspeckle formation. Dysregulation of SFPQ is emerging as a common pathological feature of multiple neurodegenerative diseases including amyotrophic lateral sclerosis (ALS). Increased retention of SFPQ intron nine and nuclear loss of the protein have been linked to multiple genetic subtypes of ALS. Consequently, SFPQ dysregulation has been hypothesised to be a common pathological feature of this highly heterogeneous disease.MethodsThis study provides a comprehensive assessment of SFPQ pathology in large ALS patient cohorts. SFPQ gene expression and intron nine retention were examined in multiple neuroanatomical regions and blood from ALS patients and control individuals using RNA sequencing (RNA-Seq) and quantitative PCR (RT-qPCR). SFPQ protein levels were assessed by immunoblotting of patient and control motor cortex and SFPQ expression pattern was examined by immunofluorescent staining of patient and control spinal cord sections. Finally, whole-genome sequencing data from a large cohort of sporadic ALS patients was analysed for genetic variation in SFPQ.ResultsSFPQ intron nine retention was significantly increased in ALS patient motor cortex. Total SFPQ mRNA expression was significantly downregulated in ALS patient motor cortex but not ALS patient blood, indicating tissue specific SFPQ dysregulation. At the protein level, nuclear expression of SFPQ in both control and patient spinal motor neurons was highly variable and nuclear depletion of SFPQ was not a consistent feature in our ALS cohort. However, we did observe SFPQ-positive cytoplasmic ubiquitinated protein aggregates in ALS spinal motor neurons. In addition, our genetic screen of ALS patients identified two novel, and two rare sequence variants in SFPQ not previously reported in ALS.ConclusionsThis study shows that dysregulation of SFPQ is a feature of ALS patient central nervous system tissue. These findings confirm SFPQ pathology as a feature of ALS and indicate that investigations into the functional consequences of this pathology will provide insight into the biology of ALS.

Intrinsically fluorescent poly(amidoamine) dendrimers (IF-PAMAM) are an emerging class of versatile nanoplatforms for in vitro tracking and bio-imaging. However, limited tissue penetration of their fluorescence and interference due to auto-fluorescence arising from biological tissues limit its application in vivo. Herein, a green IF-PAMAM (FGP) dendrimer is reported and its biocompatibility, circulation, biodistribution and potential role for traceable central nervous system (CNS)-targeted delivery in zebrafish is evaluated, exploring various routes of administration. Key features of FGP include visible light excitation (488 nm), high fluorescence signal intensity, superior photostability and low interference from tissue auto-fluorescence. After intravenous injection, FGP shows excellent imaging and tracking performance in zebrafish. Further conjugating FGP with transferrin (FGP-Tf) significantly increases its penetration through the blood-brain barrier (BBB) and prolongs its circulation in the blood stream. When administering through local intratissue microinjection, including intracranial and intrathecal injection in zebrafish, both FGP and FGP-Tf exhibit excellent tissue diffusion and effective cellular uptake in the brain and spinal cord, respectively. This makes FGP/FGP-Tf attractive for in vivo tracing when transporting to the CNS is desired. The work addresses some of the major shortcomings in IF-PAMAM and provides a promising application of these probes in the development of drug delivery in the CNS.

Previously, we identified missense mutations in CCNF that are causative of familial and sporadic amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). Hallmark features of these diseases include the build-up of insoluble protein aggregates as well as the mislocalization of proteins such as transactive response DNA binding protein 43 kDa (TDP-43). In recent years, the dysregulation of SFPQ (splicing factor proline and glutamine rich) has also emerged as a pathological hallmark of ALS/FTD. CCNF encodes for the protein cyclin F, a substrate recognition component of an E3 ubiquitin ligase. We have previously shown that ALS/FTD-linked mutations in CCNF cause disruptions to overall protein homeostasis that leads to a build-up of K48-linked ubiquitylated proteins as well as defects in autophagic machinery. To investigate further processes that may be affected by cyclin F, we used a protein-proximity ligation method, known as Biotin Identification (BioID), standard immunoprecipitations and mass spectrometry to identify novel interaction partners of cyclin F and infer further process that may be affected by the ALS/FTD-causing mutation. Results demonstrate that cyclin F closely associates with proteins involved with RNA metabolism as well as a number of RNA-binding proteins previously linked to ALS/FTD, including SFPQ. Notably, the overexpression of cyclin F(S621G) led to the aggregation and altered subcellular distribution of SFPQ in human embryonic kidney (HEK293) cells, while leading to altered degradation in primary neurons. Overall, our data links ALS/FTD-causing mutations in CCNF to converging pathological features of ALS/FTD and provides a link between defective protein degradation systems and the pathological accumulation of a protein involved in RNA processing and metabolism.

BackgroundPreviously, we identified missense mutations in CCNF that are causative of familial and sporadic amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). CCNF encodes for the protein cyclin F, a substrate recognition component of the E3-ubiquitin ligase, SCFcyclin F. We have previously shown that mutations in CCNF cause disruptions to overall protein homeostasis; causing a build-up of ubiquitylated proteins (1) as well as defects in autophagic machinery (2).MethodsHere, we have used an unbiased proteomic screening workflow using BioID, as well as standard immunoprecipitations to identify novel interaction partners of cyclin F, identifying the interaction between cyclin F and a series of paraspeckle proteins. The homeostasis of these new cyclin F interaction partners, RBM14, NONO and SFPQ were monitored in primary neurons using immunoblotting. In addition, the homeostasis of RBM14 was compared between control and ALS/FTD patient tissue using standard IHC studies.ResultsUsing BioID, we found over 100 putative interaction partners of cyclin F and demonstrated that cyclin F closely associates with a number of essential paraspeckle proteins, which are stress-responsive proteins that have recently been implicated in ALS pathogenesis. We further demonstrate that the turnover of these novel binding partners are defective when cyclin F carries an ALS/FTD-causing mutation. In addition the analysis of RBM14 levels in ALS patient post-mortem tissue revealed that RBM14 levels were significantly reduced in post-mortem ALS patient motor cortex and significantly reduced in the neurons of spinal cord tissue.ConclusionOverall, our data demonstrate that the dysregulation of paraspeckle components may be contributing factors to the molecular pathogenesis of ALS/FTD.HighlightsPreviously, we identified missense mutations in CCNF that are linked to Amyotrophic lateral sclerosis/Frontotemporal dementia (ALS/FTD) and have shown that a single mutation in cyclin F can cause defects to major protein degradation systems in dividing cells.Cyclin F has very few known interaction partners, many of which have roles in cell cycle progression. Accordingly, we used BioID and mass spectrometry to identify novel binding partners of cyclin F that may reveal insight into the role of cyclin F in neurodegeneration.Mass spectrometry and bioinformatic studies demonstrate that cyclin F interacts with several RNA binding proteins. This includes the essential paraspeckle proteins, RBM14. Notably, this interaction could be validated by standard immunoprecipitations and immunoblotting. Cyclin F could also be found to interact with a series of essential proteins which form the paraspeckle complex.We further evaluated the effect of cyclin F(S621G) on the homeostasis of these novel interaction partners in primary neurons in response to a known paraspeckle inducer, MG132. Notably, we demonstrate significant defects in the homeostasis of RBM14 and SFPQ, but not NONO, when cyclin F carries an S621G mutation.Unlike other paraspeckle proteins, RBM14 levels have not previously been reported in the post-mortem brain and spinal cord of ALS patient post-mortem tissue. Here, we note significant defects in the homeostasis of RBM14 in the post-mortem tissue of ALS patients.

Generation of reactive oxygen species (ROS) has been shown to be important for many physiological processes, ranging from cell differentiation to apoptosis. With the development of the genetically encoded photosensitiser KillerRed (KR) it is now possible to efficiently produce ROS dose-dependently in a specific cell type upon green light illumination. Zebrafish are the ideal vertebrate animal model for these optogenetic methods because of their transparency and efficient transgenesis. Here we describe a zebrafish model that expresses membrane-targeted KR selectively in motor neurons. We show that KR-activated neurons in the spinal cord undergo stress and cell death after induction of ROS. Using single-cell resolution and time-lapse confocal imaging, we selectively induced neurodegeneration in KR-expressing neurons leading to characteristic signs of apoptosis and cell death. We furthermore illustrate a targeted microglia response to the induction site as part of a physiological response within the zebrafish spinal cord. Our data demonstrate the successful implementation of KR mediated ROS toxicity in motor neurons in vivo and has important implications for studying the effects of ROS in a variety of conditions within the central nervous system, including aging and age-related neurodegenerative diseases, such as Alzheimer's disease, Parkinson's disease and amyotrophic lateral sclerosis., Graphical abstract fx1, Highlights • Motor neurons can be targeted for oxidative stress using optogenetics in zebrafish. • KillerRed expressing neurons undergo characteristic sequence of neurodegeneration. • Targeted neurons show microglial activation as part of the physiological response. • ROS toxicity has important implications for mechanisms driving neurodegeneration.

Amyotrophic lateral sclerosis (ALS) is a rapidly progressing and fatal disease characterized by muscular atrophy because of loss of upper and lower motor neurons. Histopathologically, most patients with ALS have abnormal cytoplasmic accumulation and aggregation of the nuclear RNA-regulating protein TAR DNA-binding protein 43 (TDP-43). Pathogenic mutations in the TARDBP gene that encode TDP-43 have been identified in familial ALS. We have previously reported transgenic mice with neuronal expression of human TDP-43 carrying the pathogenic A315T mutation (iTDP-43A315T mice), presenting with early-onset motor deficits in adolescent animals. Here, we analyzed aged iTDP-43A315T mice, focusing on the spatiotemporal profile and progression of neurodegeneration in upper and lower motor neurons. Magnetic resonance imaging and histologic analysis revealed a differential loss of upper motor neurons in a hierarchical order as iTDP-43A315T mice aged. Furthermore, we report progressive gait problems, profound motor deficits, and muscle atrophy in aged iTDP-43A315T mice. Despite these deficits and TDP-43 pathologic disorders in lower motor neurons, stereological analysis did not show cell loss in spinal cords. Taken together, neuronal populations in aging iTDP-43A315T mice show differential susceptibility to the expression of human TDP-43A315T.

Transactivating DNA-binding protein-43 (TDP-43) deposits represent a typical finding in almost all ALS patients, more than half of FTLD patients and patients with several other neurodegenerative disorders. It appears that perturbation of nucleo-cytoplasmic transport is an important event in these conditions but the mechanistic role and the fate of TDP-43 during neuronal degeneration remain elusive. We have developed an experimental system for visualising the perturbed nucleocytoplasmic transport of neuronal TDP-43 at the single-cell level in vivo using zebrafish spinal cord. This approach enabled us to image TDP-43-expressing motor neurons before and after experimental initiation of cell death. We report the formation of mobile TDP-43 deposits within degenerating motor neurons, which are normally phagocytosed by microglia. However, when microglial cells were depleted, injury-induced motor neuron degeneration follows a characteristic process that includes TDP-43 redistribution into the cytoplasm, axon and extracellular space. This is the first demonstration of perturbed TDP-43 nucleocytoplasmic transport in vivo, and suggests that impairment in microglial phagocytosis of dying neurons may contribute towards the formation of pathological TDP-43 presentations in ALS and FTLD. Electronic supplementary material The online version of this article (10.1007/s00401-018-1875-2) contains supplementary material, which is available to authorized users.

Cannabinoids exert dynamic control over many physiological processes including memory formation, cognition and pain perception. In the central nervous system endocannabinoids mediate negative feedback of quantal transmitter release following postsynaptic depolarization. The influence of cannabinoids in the peripheral nervous system is less clear and might have broad implications for the therapeutic application of cannabinoids. We report a novel cannabinoid effect upon the mouse neuromuscular synapse: acutely increasing synaptic vesicle volume and raising the quantal amplitudes. In a mouse model of myasthenia gravis the cannabinoid receptor agonist WIN 55,212 reversed fatiguing failure of neuromuscular transmission, suggesting future therapeutic potential. Our data suggest an endogenous pathway by which cannabinoids might help to regulate transmitter release at the neuromuscular junction.

A remaining challenge in the treatment of glioblastoma multiforme (GBM) is surmounting the blood-brain barrier (BBB). Such challenge prevents the development of efficient theranostic approaches that combine reliable diagnosis with targeted therapy. In this study, we developed brain-targeted near-infrared Ⅱb (NIR-Ⅱb) aggregation-induced emission (AIE) nanoparticles via rational design that involved twisting the planar molecular backbone with steric hindrance. The resulting nanoparticles can balance competing responsiveness demands for radiation-mediated NIR fluorescence imaging at 1550 nm and non-radiation NIR photothermal therapy (NIR-PTT). The brain targeting peptide apolipoprotein E peptide (ApoE) is grafted onto these nanoparticles (termed as ApoE-Ph NPs) to target glioma and promote efficient BBB traversal. A long imaging wavelength 1550 nm band-pass filter was utilized to monitor the in vivo biodistribution and accumulation of our nanoparticles in a model of orthotopic glioma, which overcame previous limitations in wavelength range and equipment. The results demonstrate that our ApoE-Ph NPs have a higher PTT efficiency and significantly enhanced survival of mice bearing orthotopic GBM with moderate irradiation (0.5 W cm-2 ). Collectively, our work highlights the smart design of a brain-targeted NIR-Ⅱ AIE theranostic approach that opens new diagnosis and treatment options in the photonic therapy of GBM. This article is protected by copyright. All rights reserved.

Glioblastoma (GBM) is the most common and fatal form of malignant brain tumor. Despite intensive effort, there is still no effective GBM treatment. Therefore, novel and more effective GBM therapeutic approaches are highly desired. In this study, we combined polymeric nanotechnology with microRNA (miRNA) regulation technology to develop a targeted polymeric nanoparticle to co-deliver anti-miR-21 and miR-124 into the brain to effectively treat GBM. The polymeric nanoparticle decorated with Angiopep-2 peptide not only can encapsulate miRNA via triple-interaction (electrostatic, hydrogen bond and hydrophobic bonding) to protect miRNA against enzyme degradation in the blood, but also is capable of crossing blood brain barrier (BBB) and allowing targeted delivery of miRNAs to GBM tissue due to the dual-targeting function of Angiopep-2. Moreover, the co-delivered anti-miR-21 and miR-124 simultaneously regulated the mutant RAS/PI3K/PTEN/AKT signaling pathway in tumor cells, consequently achieving combinatorial GBM therapy. This combinatorial effect was confirmed by our results showing that these miRNA nanomedicines can effectively reduce tumor cell proliferation, migration and invasion as well as reducing tumor angiogenesis. Consequently, effective suppression of tumor growth and significantly improved medium survival time are observed when these miRNA nanomedicines were assessed in an orthotopic GBM xenograft model. This work indicated that our new polymeric nanoparticles successfully mediate inhibition of miR-21 and miR-124 supplementation to significantly reduce tumorigenesis, and may have strong potential in GBM therapy.

Central nervous system (CNS) disorders feature the progressive and selective loss of normal brain functions. CNS disorders often include an irreversible physiological and anatomical loss of neurons that can lead to dysfunction in various parts of the brain and eventually death. Glioblastoma multiforme (GBM) and neurodegenerative diseases such as Alzheimer's disease (AD) and Parkinson's disease (PD) are hard to be diagnosed at an early stage for the prevention of disease propagation. Such diagnosis is vital for the timely commencement of actual treatments. Nanotechnology brings new diagnosis hope for CNS disorders as it provides ultrasensitive detection for more specific biomarkers. Herein, the recent progress in techniques development for detecting pathological biomarkers for GBM, AD, and PD is summarized, in particular, the principles that govern the design of these sensors, blood–brain barrier (BBB) dysfunction, and its integrity during disease development. Finally, a perspective on future directions to further advance and improve the early‐stage diagnosis of CNS disorders is presented.

Amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) are fatal neurodegenerative disorders that have common molecular and pathogenic characteristics, such as aberrant accumulation and ubiquitylation of TDP-43; however, the mechanisms that drive this process remain poorly understood. We have recently identified CCNF mutations in familial and sporadic ALS and FTD patients. CCNF encodes cyclin F, a component of an E3 ubiquitin–protein ligase (SCFcyclin F) complex that is responsible for ubiquitylating proteins for degradation by the ubiquitin–proteasome system. In this study, we examined the ALS/FTD-causing p.Ser621Gly (p.S621G) mutation in cyclin F and its effect upon downstream Lys48-specific ubiquitylation in transfected Neuro-2A and SH-SY5Y cells. Expression of mutant cyclin FS621G caused increased Lys48-specific ubiquitylation of proteins in neuronal cells compared to cyclin FWT. Proteomic analysis of immunoprecipitated Lys48-ubiquitylated proteins from mutant cyclin FS621G-expressing cells identified proteins that clustered within the autophagy pathway, including sequestosome-1 (p62/SQSTM1), heat shock proteins, and chaperonin complex components. Examination of autophagy markers p62, LC3, and lysosome-associated membrane protein 2 (Lamp2) in cells expressing mutant cyclin FS621G revealed defects in the autophagy pathway specifically resulting in impairment in autophagosomal–lysosome fusion. This finding highlights a potential mechanism by which cyclin F interacts with p62, the receptor responsible for transporting ubiquitylated substrates for autophagic degradation. These findings demonstrate that ALS/FTD-causing mutant cyclin FS621G disrupts Lys48-specific ubiquitylation, leading to accumulation of substrates and defects in the autophagic machinery. This study also demonstrates that a single missense mutation in cyclin F causes hyper-ubiquitylation of proteins that can indirectly impair the autophagy degradation pathway, which is implicated in ALS pathogenesis.

NEW FINDINGS What is the central question of this study? Non-invasive muscle function tests have not been validated for use in the study of muscle performance in high-fat-fed mice. What is the main finding and its importance? This study shows that grip strength, hang wire and four-limb hanging tests are able to discriminate the muscle performance between chow-fed and high-fat-fed mice at different time points, with grip strength being reliable after 5, 10 and 20 weeks of dietary intervention. Non-invasive tests are commonly used for assessing muscle function in animal models. The value of these tests in obesity, a condition where muscle strength is reduced, is unclear. We investigated the utility of three non-invasive muscle function tests, namely grip strength (GS), hang wire (HW) and four-limb hanging (FLH), in C57BL/6 mice fed chow (chow group, n = 48) or a high-fat diet (HFD group, n = 48) for 20 weeks. Muscle function tests were performed at 5, 10 and 20 weeks. After 10 and 20 weeks, HFD mice had significantly reduced GS (in newtons; mean ± SD: 10 weeks chow, 1.89 ± 0.1 and HFD, 1.79 ± 0.1; 20 weeks chow, 1.99 ± 0.1 and HFD, 1.75 ± 0.1), FLH [in seconds per gram body weight; median (interquartile range): 10 weeks chow, 2552 (1337-4964) and HFD, 1230 (749-1994); 20 weeks chow, 2048 (765-3864) and HFD, 1036 (717-1855)] and HW reaches [n; median (interquartile range): 10 weeks chow, 4 (2-5) and HFD, 2 (1-3); 20 weeks chow, 3 (1-5) and HFD, 1 (0-2)] and higher falls [n; median (interquartile range): 10 weeks chow, 0 (0-2) and HFD, 3 (1-7); 20 weeks chow, 1 (0-4) and HFD, 8 (5-10)]. Grip strength was reliable in both dietary groups [intraclass correlation coefficient (ICC) = 0.5-0.8; P

Currently there is a lack in fundamental understanding of disease progression of most neurodegenerative diseases, and, therefore, treatments and preventative measures are limited. Consequently, there is a great need for adaptable, yet robust model systems to both investigate elementary disease mechanisms and discover effective therapeutics. We have generated a Tol2 Gateway-compatible toolbox to study neurodegenerative disorders in zebrafish, which includes promoters for astrocytes, microglia and motor neurons, multiple fluorophores, and compatibility for the introduction of genes of interest or disease-linked genes. This toolbox will advance the rapid and flexible generation of zebrafish models to discover the biology of the nervous system and the disease processes that lead to neurodegeneration.

Kupffer cells are liver-resident macrophages that play an important role in mediating immune-related functions in mammals and humans. They are well-known for their capacity to phagocytose large amounts of waste complexes, cell debris, microbial particles and even malignant cells. Location, appearance and functional aspects are important features used to identify these characteristic cells of the liver sinusoid. To-date, there is limited information on the occurrence of macrophages in zebrafish liver. Therefore, we aimed to characterise the ultrastructural and functional aspects of liver-associated macrophages in the zebrafish model by taking advantage of the latest advances in zebrafish genetics and multimodal correlative imaging. Herein, we report on the occurrence of macrophages within the zebrafish liver exhibiting conventional ultrastructural features (e.g. presence of pseudopodia, extensive lysosomal apparatus, a phagolysosome and making up ∼3% of the liver volume). Intriguingly, these cells were not located within the sinusoidal vascular bed of hepatic tissue but instead resided between hepatocytes and lacked phagocytic function. While our results demonstrated the presence and structural similarities with liver macrophages from other experimental models, their functional characteristics were distinctly different from Kupffer cells that have been described in rodents and humans. These findings illustrate that the innate immune system of the zebrafish liver has some distinctly different characteristics compared to other animal experimental models. This conclusion underpins our call for future studies in order to have a better understanding of the physiological role of macrophages residing between the parenchymal cells of the zebrafish liver.

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease commonly treated with riluzole, a small molecule that may act via modulation of glutamatergic neurotransmission. However, riluzole only modestly extends lifespan for people living with ALS and its precise mechanisms of action remain unclear. Most ALS cases are characterised by accumulation of cytoplasmic TAR DNA binding protein of 43 kDa (TDP-43), and understanding the effects of riluzole in models that closely recapitulate TDP-43 pathology may provide insights for development of improved therapeutics. We therefore investigated the effects of riluzole in transgenic mice that inducibly express nuclear localisation sequence (NLS)-deficient human TDP-43 in neurons (NEFH-tTA/tetO-hTDP-43ΔNLS, ‘rNLS’, mice). Riluzole treatment from the first day of hTDP-43ΔNLS expression did not alter disease onset, weight loss or performance on multiple motor behavioural tasks. Riluzole treatment also did not alter TDP-43 protein levels, solubility or phosphorylation. Although we identified a significant decrease in GluA2 and GluA3 proteins in the cortex of rNLS mice, riluzole did not ameliorate this disease-associated molecular phenotype. Likewise, riluzole did not alter the disease-associated atrophy of hindlimb muscle in rNLS mice. Finally, riluzole treatment beginning after disease onset in rNLS mice similarly had no effect on progression of late-stage disease or animal survival. Together, we demonstrate specific glutamatergic receptor alterations and muscle fibre-type changes reminiscent of ALS in rNLS mice, but riluzole had no effect on these or any other disease phenotypes. Future targeting of pathways directly related to accumulation of TDP-43 pathology may be needed to develop better treatments for ALS.Significance StatementAccumulation of cytoplasmic TDP-43 protein is the hallmark pathology of ALS. Riluzole is the most widely used drug for ALS treatment, but provides only a short extension of lifespan. We demonstrate here in the rNLS mouse model, which mimics TDP-43 pathology, that riluzole does not ameliorate progressive alterations in motor strength and coordination, muscle atrophy, glutamate receptor levels, or TDP-43 protein levels and solubility, and does not prolong animal survival. Riluzole similarly did not affect decreased levels of glutamate receptor subunits GluA2/GluA3 in rNLS mice. The inability of riluzole to rescue pathological or phenotypic changes in this TDP-43 model provides further impetus for the discovery of improved therapies targeting the key drivers of ALS pathogenesis.

KEY POINTS: Adeno-associated viral vector was used to elevate the expression of muscle specific kinase (MuSK) and rapsyn (a cytoplasmic MuSK effector protein) in the tibialis anterior muscle of wild-type and dystrophic (mdx) mice. In mdx mice, enhanced expression of either MuSK or rapsyn ameliorated the acute loss of muscle force associated with strain injury. Increases in sarcolemmal immunolabelling for utrophin and β-dystroglycan suggest a mechanism for the protective effect of MuSK in mdx muscles. MuSK also caused subtle changes to the structure and function of the neuromuscular junction, suggesting novel roles for MuSK in muscle physiology and pathophysiology. ABSTRACT: Muscle specific kinase (MuSK) has a well-defined role in stabilizing the developing mammalian neuromuscular junction, but MuSK might also be protective in some neuromuscular diseases. In the dystrophin-deficient mdx mouse model of Duchenne muscular dystrophy, limb muscles are especially fragile. We injected the tibialis anterior muscle of 8-week-old mdx and wild-type (C57BL10) mice with adeno-associated viral vectors encoding either MuSK or rapsyn (a cytoplasmic MuSK effector protein) fused to green fluorescent protein (MuSK-GFP and rapsyn-GFP, respectively). Contralateral muscles injected with empty vector served as controls. One month later mice were anaesthetized with isoflurane and isometric force-producing capacity was recorded from the distal tendon. MuSK-GFP caused an unexpected decay in nerve-evoked tetanic force, both in wild-type and mdx muscles, without affecting contraction elicited by direct electrical stimulation of the muscle. Muscle fragility was probed by challenging muscles with a strain injury protocol consisting of a series of four strain-producing eccentric contractions in vivo. When applied to muscles of mdx mice, eccentric contraction produced an acute 27% reduction in directly evoked muscle force output, affirming the susceptibility of mdx muscles to strain injury. mdx muscles overexpressing MuSK-GFP or rapsyn-GFP exhibited significantly milder force deficits after the eccentric contraction challenge (15% and 14%, respectively). The protective effect of MuSK-GFP in muscles of mdx mice was associated with increased immunolabelling for utrophin and β-dystroglycan in the sarcolemma. Elevating the expression of MuSK or rapsyn revealed several distinct synaptic and extrasynaptic effects, suggesting novel roles for MuSK signalling in muscle physiology and pathophysiology.

Research in the field of gastroenterology is increasingly focused on the use of alternative nonrodent model organisms to provide new experimental tools to study chronic diseases. The zebrafish is a particularly valuable experimental platform to explore organ and cell structure-function relationships under relevant biological and pathobiological settings. This is due to its optical transparency and its close-to-human genetic makeup. To-date, the structure-function properties of the GIS of the zebrafish are relatively unexplored and limited to histology and fluorescent microscopy. Occasionally those studies include EM of a given subcellular process but lack the required full histological picture. In this work, we employed a novel combined biomolecular imaging approach in order to cross-correlate 3D ultrastructure over different length scales (optical-, X-ray micro-CT, and high-resolution EM). Our correlated imaging studies and subsequent data modelling provide to our knowledge the first detailed 3D picture of the zebrafish larvae GIS. Our results provide unequivocally a limit of confidence for studying various digestive disorders and drug delivery pathways in the zebrafish.

Amyotrophic lateral sclerosis (ALS) is a rapidly progressive, fatal neurodegenerative disease characterised by the death of upper and lower motor neurons. Approximately 10% of cases have a known family history of ALS and disease-linked mutations in multiple genes have been identified. ALS-linked mutations in CCNF were recently reported, however the pathogenic mechanisms associated with these mutations are yet to be established. To investigate possible disease mechanisms, we developed in vitro and in vivo models based on an ALS-linked missense mutation in CCNF. Proteomic analysis of the in vitro models identified the disruption of several cellular pathways in the mutant model, including caspase-3 mediated cell death. Transient overexpression of human CCNF in zebrafish embryos supported this finding, with fish expressing the mutant protein found to have increased levels of cleaved (activated) caspase-3 and increased cell death in the spinal cord. The mutant CCNF fish also developed a motor neuron axonopathy consisting of shortened primary motor axons and increased frequency of aberrant axonal branching. Importantly, we demonstrated a significant correlation between the severity of the CCNF-induced axonopathy and a reduced motor response to a light stimulus (photomotor response). This is the first report of an ALS-linked CCNF mutation in vivo and taken together with the in vitro model identifies the disruption of cell death pathways as a significant consequence of this mutation. Additionally, this study presents a valuable new tool for use in ongoing studies investigating the pathobiology of ALS-linked CCNF mutations.

Although liver transport routes have been extensively studied in rodents, live imaging under in situ and in vivo conditions of large volumes is still proven to be difficult. In this study, we took advantage of the optical transparency of zebrafish and their small size to explore their usefulness for correlative imaging studies and liver transport experimentations. First, we assessed the micro-architecture of the zebrafish liver and compared its fine structure to the rodent and humans' literature. Next, we investigated the transport routes and cellular distribution of albumin using combined and correlative microscopy approaches. These methods permitted us to track the injected proteins at different time points through the process of liver uptake and clearance of albumin. We demonstrate strong structural and functional resemblance between the zebrafish liver and its rodents and humans' counterparts. In as short as 5 min post-injection, albumin rapidly accumulated within the LSECs. Furthermore, albumin entered the space of Disse where it initially accumulated then subsequently was taken up by the hepatocytes. We propose the zebrafish as a viable alternative experimental model for hepatic transport studies, allowing swift multimodal imaging and direct quantification on the hepatic distribution of supramolecular complexes of interest.