Search

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.

Amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) are fatal neurodegenerative disorders that share pathological features, including the aberrant accumulation of ubiquitinated protein inclusions within motor neurons. Previously, we have shown that the sequestration of ubiquitin (Ub) into inclusions disrupts Ub homeostasis in cells expressing ALS-associated variants superoxide dismutase 1 (SOD1), fused in sarcoma (FUS) and TAR DNA-binding protein 43 (TDP-43). Here, we investigated whether an ALS/FTD-linked pathogenic variant in the CCNF gene, encoding the E3 Ub ligase Cyclin F (CCNF), also perturbs Ub homeostasis. The presence of a pathogenic CCNF variant was shown to cause ubiquitin-proteasome system (UPS) dysfunction in induced pluripotent stem cell-derived motor neurons harboring the CCNF S621G mutation. The expression of the CCNFS621G variant was associated with an increased abundance of ubiquitinated proteins and significant changes in the ubiquitination of key UPS components. To further investigate the mechanisms responsible for this UPS dysfunction, we overexpressed CCNF in NSC-34 cells and found that the overexpression of both wild-type (WT) and the pathogenic variant of CCNF (CCNFS621G) altered free Ub levels. Furthermore, double mutants designed to decrease the ability of CCNF to form an active E3 Ub ligase complex significantly improved UPS function in cells expressing both CCNFWT and the CCNFS621G variant and were associated with increased levels of free monomeric Ub. Collectively, these results suggest that alterations to the ligase activity of the CCNF complex and the subsequent disruption to Ub homeostasis play an important role in the pathogenesis of CCNF-associated ALS/FTD.

Glioblastoma multiforme (GBM) is a fatal central nervous system tumor without effective treatment. Chemotherapeutic agents are mainstays in the treatment of glioblastoma. However, the effectiveness of these is seriously hindered by poor blood-brain-barrier (BBB) penetrance and tumor targeting, together with short biological half-life. Improved chemotherapy is thus urgently needed for GBM. Multifunctional nanoparticle delivery systems offer much promise in overcoming current limitations. Accordingly, a multifunctional biomimetic nanomedicine is developed by functionalizing the surface of red blood cell membranes (RBCms) with angiopep-2 and loading pH-sensitive nanoparticles (polymer, doxorubicin (Dox), and lexiscan (Lex)) using the functionalized cell membrane to generate the novel nanomedicine, Ang-RBCm@NM-(Dox/Lex). The studies toward orthotopic U87MG human glioblastoma tumor-bearing nude mice show that the Ang-RBCm@NM-(Dox/Lex) nanomedicine has much improved blood circulation time, superb BBB penetration, superior tumor accumulation and retention. Moreover, effective suppression of tumor growth and significantly improved medium survival time are also observed after Ang-RBCm@NM-(Dox/Lex) treatment. The results show that this biomimetic nanoplatform can serve as a flexible and powerful system for GBM treatment which can be readily adapted for the treatment of other central nervous system (CNS) disorders.

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.

International audience; Metallomics is a rapidly evolving field of bio-metal research that integrates techniques and perspectives from other "-omics" sciences (e.g. genomics, proteomics) and from research vocations further afield. Perhaps the most esoteric of this latter category has been the recent coupling of biomedicine with element and isotope geochemistry, commonly referred to as isotope metallomics. Over the course of less than two decades, isotope metallomics has produced numerous benchmark studies highlighting the use of stable metal isotope distribution in developing disease diagnostics-e.g. cancer, neurodegeneration, osteoporosis-as well as their utility in deciphering the underlying mechanisms of such diseases. These pioneering works indicate an enormous wealth of potential and provide a call to action for researchers to combine and leverage expertise and resources to create a clear and meaningful path forward. Doing so with efficacy and impact will require not only building on existing research, but also broadening collaborative networks, bolstering and deepening cross-disciplinary channels, and establishing unified and realizable objectives. The aim of this review is to briefly summarize the field and its underpinnings, provide a directory of the state of the art, outline the most encouraging paths forward, including their limitations, outlook and speculative upcoming breakthroughs, and finally to offer a vision of how to cultivate isotope metallomics for an impactful future.

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease, characterised by the degeneration of motor neurons innervating skeletal muscle. The mechanisms underlying neurodegeneration in ALS are not yet fully elucidated, and with current therapeutics only able to extend lifespan by a matter of months there is a clear need for novel therapies to increase lifespan and patient quality of life. Here, we evaluated whether moderate-intensity treadmill exercise and/or treatment with metallothionein-2 (MT2), a neuroprotective protein, could improve survival, behavioural or neuropathological outcomes in SOD1G93A familial ALS mice. Six-week-old female SOD1G93A mice were allocated to one of four treatment groups: MT2 injection, i.m.; moderate treadmill exercise; neither MT2 nor exercise; or both MT2 and exercise. MT2-treated mice survived around 3% longer than vehicle-treated mice, with this mild effect reaching statistical significance in Cox proportional hazards analysis once adjusted for potential confounders. Mixed model body weight trajectories over time indicated that MT2-treated mice, with or without exercise, reached maximum body weight at a later age, suggesting a delay in disease onset of around 4% compared to saline-treated mice. Exercise alone did not significantly increase survival or delay disease onset, and neither exercise nor MT2 substantially ameliorated gait abnormalities or muscle strength loss. We conclude that neither exercise nor MT2 treatment was detrimental in female SOD1G93A mice, and further study could determine whether the mild effect of peripheral MT2 administration on disease onset and survival could be improved via direct administration of MT2 to the central nervous system.

Iron detection is one of the critical markers to diagnose multiple blood-related disorders that correspond to various biological dysfunctions. The currently available anemia detection approach can be used only for pre-treated blood samples that interfere with the actual iron level in blood. Real-time detection approaches with higher sensitivity and specificity are certainly needed to cope with the commercial level clinical analyses. Herein, we presented a novel strategy to determine the blood iron that can be easily practiced at commercial levels. The blend of well-known iron-cyanide chemistry with nanotechnology is advantageous with ultrahigh sensitivity in whole blood analysis without any pre-treatments. This approach is a combined detection system of the conventional assay (UV-visible spectroscopy) with surface-enhanced Raman scattering (SERS). Organic cyanide modified silver nanoparticles (cAgNPs) can selectively respond to Fe3+ ions and Hb protein with a detection limit of 10 fM and 0.46 μg mL-1, respectively, without being affected by matrix interfering species in the complex biological fluid. We confirmed the clinical potential of our new cAgNPs by assessing iron-status in multiple anemia patients and normal controls. Our SERS-based iron quantitation approach is highly affordable for bulk-samples, cheap, quick, flexible, and useful for real-time clinical assays. Such a method for metal-chelation has extendable features of therapeutics molecular tracking within more complex living systems at cellular levels.

Emerging applications in isotope geochemistry using stable metal isotope systems, including those in archaeological, environmental and medical sciences, along with commercial/industrial applications such as environmental source tracing and ore exploration, require high sample throughput that conventional element purification techniques—i.e. gravity flow ion exchange chromatography (IEC)—cannot match. Here, a simple and robust method for element purification from biological and geological samples has been developed, SpinChem™, wherein element purification via IEC is greatly facilitated through centrifugation. In brief, this involves adapting chromatographic columns for encapsulation within large (50 mL) centrifuge tubes, and subsequent element purification at enhanced flow rates (1.2–1.5 mL min−1) generated by centrifugal force. As proof of concept, Zn IEC purification from biological and geological materials has been detailed, demonstrating the utility of this method across the sample spectrum, and validating high yields (97 ± 4%), high separation efficiency and low contamination levels (blanks ≤1 ng). SpinChem™ IEC in the centrifuge proceeds at rates up to an order of magnitude faster than in gravity flow protocols, with 8–10 samples processed through IEC in under 2 hours (vs. 4–10 hours in gravity flow), while maintaining high subsequent MC-ICP-MS analytical precision, with average errors of 0.06‰ (2σ) or better for δ66Zn. Benchmarking this technique using an element very sensitive to endogenous and exogenous contamination (e.g. inefficient separation and external contaminants, respectively)—Zn—exemplifies the utility and high performance of this technique, and its ready adaptation to other isotope systems of interest, including Ca, Fe, Cu, Sr/Nd, Mo and Sn.

BackgroundMutations in the CCNF gene encoding cyclin F are associated with sporadic and familial amyotrophic lateral sclerosis (ALS) and frontotemporal dementia, but the underlying pathophysiological mechanisms are unknown. Proper functioning of the endoplasmic reticulum (ER) is essential for physiological cellular function. MethodsWe used human neuroblastoma SH-SY5Y and human embryonic kidney HEK293T cell lines and mouse primary neurons-overexpressing two familial ALS cyclin F mutants to examine whether mutant ALS/FTD-associated cyclin F perturbs key functions of the ER and Golgi compartments. Specific cellular assays were used to examine ER-Golgi transport (VSVGts045), the budding of vesicles from ER membranes and ER-associated degradation (ERAD). Immunocytochemistry was used to examine the morphology of the Golgi and ER-exit sites, and to detect ER stress and apoptosis. Western blotting was used to examine the content of vesicles budding from ER membranes and the interaction between Sec 31 and cyclin F. Flow cytometry was used to examine cell death.Results We demonstrated that mutant cyclin F inhibited protein transport from the ER to Golgi apparatus by a mechanism involving aberrant vesicle sorting from the ER. It also impeded ER-associated degradation, whereby misfolded ER proteins are ubiquitinated and degraded by the proteasome. This was associated with induction of ER stress and Golgi fragmentation, leading to apoptosis. Conclusion Together, these results demonstrate that ER dysfunction is a pathogenic pathway associated with ALS/FTD-variant cyclin F.

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.

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.

Precise genome editing is limited by the inefficiency of homology-directed repair (HDR) compared to the non-homologous end-joining (NHEJ) of double strand breaks (DSBs). The CRISPR (clustered regularly interspaced short palindromic repeat)/Cas9 system generates precise, locus-specific DSBs that can serve as substrates for HDR. We developed an in vivo visual reporter assay to quantify HDR-mediated events at single-cell resolution in zebrafish and used this system to identify small-molecule modulators that shift the DNA repair equilibrium in favor of HDR. By further optimizing the reaction environment and repair template, we achieved dramatic enhancement of HDR-mediated repair efficiency in zebrafish. Accordingly, under optimized conditions, inhibition of NHEJ with NU7441 enhanced HDR-mediated repair up to 13.4-fold. Importantly, we demonstrate that the increase in somatic HDR events correlates directly with germline transmission, permitting the efficient recovery of large seamlessly integrated DNA fragments in zebrafish., Aksoy et al. develop a reporter assay that allows them to quantify events mediated by homology-directed repair (HDR) in live zebrafish at single cell resolution. Using this assay, they systematically test and identify small molecule modulators that enhance the efficiency of HDR-mediated genome editing in zebrafish.

Poly(amidoamine) dendrimer (PAMAM) is well-known for its high efficiency as a drug delivery vehicle. However, the intrinsic cytotoxicity and lack of a detectable signal to facilitate tracking have impeded its practical applications. Herein, we have developed a novel label-free fluorescent and biocompatible PAMAM derivative by simple surface modification of PAMAM using acetaldehyde. The modified PAMAM possessed a strong green fluorescence, which was generated by the C=N bonds of the resulting Schiff Bases via n−π* transition, while the intrinsic cytotoxicity of PAMAM was simultaneously ameliorated. Through further PEGylation, the fluorescent PAMAM demonstrated excellent intracellular tracking in human melanoma SKMEL28 cells. In addition, our PEGylated fluorescent PAMAM derivative achieved enhanced loading and deliveryefficiency of the anticancer drug doxorubicin (DOX) compared to the original PAMAM. Importantly, the accelerated kinetics of DOX-encapsulated fluorescent PAMAM nanocomposites in an acidic environment facilitated intracellular drug release, which demonstrated comparable cytotoxicity to that of the free-form doxorubicin hydrochloride (DOX·HCl) against melanoma cells. Overall, our label free fluorescent PAMAM derivative offers a new opportunity of traceable and controlled delivery for DOX and other drugs of potential clinical importance.

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.

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.

Environmental factors inherent to animal facilities can impact on the neuro-behavioural phenotype of laboratory mice and genetic mouse models for human diseases. Many facilities have upgraded from traditional 'open filter top' cages (FT) to individually ventilated cage (IVC) systems, which have been shown to modify various behavioural responses of laboratory mice. Importantly, the impact of IVC housing on the G93A superoxide dismutase 1 mouse model of amyotrophic lateral sclerosis (ALS) is currently unknown. Male and female wild type-like (WT) and heterozygous SOD1G93A mice were group-housed in FT or IVC systems from PND 30 ± 5 onwards. Body weight and motor function were assessed weekly from 15 weeks onward. Mice were also tested for cognitive abilities (i.e., fear conditioning and social recognition memory) and sensorimotor gating (i.e., prepulse inhibition: PPI). SOD1G93A mice lost body weight, and their motor function degenerated over time compared with control littermates. Motor impairments developed faster when SOD1G93A females were housed in IVCs. Context and cue freezing were increased in SOD1G93A females compared with controls, whereas all SOD1G93A mice exhibited lower acoustic startle and PPI than WT mice. IVC housing led to an increase in cue freezing in males and reduced the severity of PPI deficits in SOD1G93A females. Overall, IVC housing impacted moderately on the SOD1G93A phenotype but central behavioural deficits were still evident across housing conditions. Nonetheless, our findings indicate the importance of assessing the effect of cage system in genetic mouse models as these systems can modulate the magnitude and onset of genotypic differences.

Machado-Joseph disease (MJD, also known as spinocerebellar ataxia-3) is a fatal neurodegenerative disease that impairs control and coordination of movement. Here we tested whether treatment with the histone deacetylase inhibitor sodium valproate (SV) prevented a movement phenotype that develops in larvae of a transgenic zebrafish model of the disease. We found that treatment with SV improved the swimming of the MJD zebrafish, increased levels of acetylated histones 3 and 4, but also increased expression of polyglutamine expanded human ataxin-3. Proteomic analysis of protein lysates generated from the treated and untreated MJD zebrafish also predicted that SV treatment had activated the sirtuin longevity signaling pathway and this was confirmed by findings of increased SIRT1 protein levels and sirtuin activity in SV treated MJD zebrafish and HEK293 cells expressing ataxin-3-84Q, respectively. Treatment with resveratrol (another compound known to activate the sirtuin pathway), also improved swimming in the MJD zebrafish. Co-treatment with SV alongside EX527, a SIRT1 activity inhibitor, prevented induction of autophagy by SV and the beneficial effects of SV on the movement in the MJD zebrafish, indicating that they were both dependent on sirtuin activity. These findings provide the first evidence of sodium valproate inducing activation of the sirtuin pathway. Further, they indicate that drugs that target the sirtuin pathway, including sodium valproate and resveratrol, warrant further investigation for the treatment of MJD and related neurodegenerative diseases.

NEW FINDINGS: What is the central question of this study? Striated muscle activator of rho signalling (STARS) is an actin-binding protein that regulates transcriptional pathways controlling muscle function, growth and myogenesis, processes that are impaired in dystrophic muscle: what is the regulation of the STARS pathway in Duchenne muscular dystrophy (DMD)? What is the main finding and its importance? Members of the STARS signalling pathway are reduced in the quadriceps of patients with DMD and in mouse models of muscular dystrophy. Overexpression of STARS in the dystrophic deficient mdx mouse model increased maximal isometric specific force and upregulated members of the actin cytoskeleton and oxidative phosphorylation pathways. Regulating STARS may be a therapeutic approach to enhance muscle health. ABSTRACT: Duchenne muscular dystrophy (DMD) is characterised by impaired cytoskeleton organisation, cytosolic calcium handling, oxidative stress and mitochondrial dysfunction. This results in progressive muscle damage, wasting and weakness and premature death. The striated muscle activator of rho signalling (STARS) is an actin-binding protein that activates the myocardin-related transcription factor-A (MRTFA)/serum response factor (SRF) transcriptional pathway, a pathway regulating cytoskeletal structure and muscle function, growth and repair. We investigated the regulation of the STARS pathway in the quadriceps muscle from patients with DMD and in the tibialis anterior (TA) muscle from the dystrophin-deficient mdx and dko (utrophin and dystrophin null) mice. Protein levels of STARS, SRF and RHOA were reduced in patients with DMD. STARS, SRF and MRTFA mRNA levels were also decreased in DMD muscle, while Stars mRNA levels were decreased in the mdx mice and Srf and Mrtfa mRNAs decreased in the dko mice. Overexpressing human STARS (hSTARS) in the TA muscles of mdx mice increased maximal isometric specific force by 13% (P

Biometals play a critical role in both the healthy and diseased brain's functioning. They accumulate in the normal aging brain, and are inherent to neurodegenerative disorders and their associated pathologies. A prominent example of this is the brain accumulation of metals such as Ca, Fe and Cu (and more ambiguously, Zn) associated with Alzheimer's disease (AD). The natural stable isotope compositions of such metals have also shown utility in constraining biological mechanisms, and in differentiating between healthy and diseased states, sometimes prior to conventional methods. Here we have detailed the distribution of the biologically relevant elements Mg, P, K, Ca, Fe, Cu and Zn in brain regions of Göttingen minipigs ranging in age from three months to nearly six years, including control animals and both a single- and double-transgenic model of AD (PS1, APP/PS1). Moreover, we have characterized the Ca isotope composition of the brain for the first time. Concentration data track rises in brain biometals with age, namely for Fe and Cu, as observed in the normal ageing brain and in AD, and biometal data point to increased soluble amyloid beta (Aβ) load prior to AD plaque identification via brain imaging. Calcium isotope results define the brain as the isotopically lightest permanent reservoir in the body, indicating that brain Ca dyshomeostasis may induce measurable isotopic disturbances in accessible downstream reservoirs such as biofluids.

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.

Nanotechnology-based RNA interference (RNAi) has shown great promise in overcoming the limitations of traditional clinical treatments for glioblastoma (GBM). However, because of the complexity of brain physiology, simple blood-brain barrier (BBB) penetration or tumor-targeting strategies cannot entirely meet the demanding requirements of different therapeutic delivery stages. Herein, we developed a charge conversional biomimetic nanoplatform with a three-layer core-shell structure to programmatically overcome persistent obstacles in siRNA delivery to GBM. The resulting nanocomplex presents good biocompatibility, prolonged blood circulation, high BBB transcytosis, effective tumor accumulation, and specific uptake by tumor cells in the brain. Moreover, red blood cell membrane (RBCm) disruption and effective siRNA release can be further triggered elegantly by charge conversion from negative to positive in the endo/lysosome (pH 5.0-6.5) of tumor cells, leading to highly potent target-gene silencing with a strong anti-GBM effect. Our study provides an intelligent biomimetic nanoplatform tailored for systemically siRNA delivery to GBM, leveraging Angiopep-2 peptide-modified, immune-free RBCm and charge conversional components. Improved therapeutic efficacy, higher survival rates, and minimized systemic side effects were achieved in orthotopic U87MG-luc human glioblastoma tumor-bearing nude mice.

Small interfering RNA (siRNA) has been considered as a highly promising therapeutic agent for human cancer treatment including glioblastoma (GBM), which is a fatal disease without effective therapy methods. However, siRNA-based GBM therapy is seriously hampered by a number of challenges in siRNA brain delivery including poor stability, short blood circulation, low blood-brain barrier (BBB) penetration, and tumor accumulation, as well as inefficient siRNA intracellular release. Herein, an Angiopep-2 (Ang) functionalized intracellular-environment-responsive siRNA nanocapsule (Ang-NCss (siRNA)) is successfully developed as a safe and efficient RNAi agent to boost siRNA-based GBM therapy. The experimental results demonstrate that the developed Ang-NCss (siRNA) displays long circulation in plasma, efficient BBB penetration capability, and GBM accumulation and retention, as well as responsive intracellular siRNA release due to the unique design of small size (25 nm) with polymeric shell for siRNA protection, Ang functionalization for BBB crossing and GBM targeting, and disulfide bond as a linker for intracellular-environment-responsive siRNA release. Such superior properties of Ang-NCss (siRNA) result in outstanding growth inhibition of orthotopic U87MG xenografts without causing adverse effects, achieving remarkably improved survival benefits. The developed siRNA nanocapsules provide a new strategy for RNAi therapy of GBM and beyond.

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 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.