C9orf72-Associated Dipeptide Repeat Expansions Perturb ER-Golgi Vesicular Trafficking, Inducing Golgi Fragmentation and ER Stress, in ALS/FTD.

Hexanucleotide repeat expansions (HREs) in the chromosome 9 open reading frame 72 (C9orf72) gene are the most frequent genetic cause of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). Both are debilitating neurodegenerative conditions affecting either motor neurons (ALS) in the brain and spinal cord or neurons in the frontal and/or temporal cortical lobes (FTD). HREs undergo repeat-associated non-ATG (RAN) translation on both sense and anti-sense strands, generating five distinct dipeptide repeat proteins (DPRs), poly-GA, -GR, -GP, -PA and -PR. Perturbed proteostasis is well-recognised in ALS pathogenesis, including processes affecting the endoplasmic reticulum (ER) and Golgi compartments. However, these mechanisms have not been well characterised for C9orf72-mediated ALS/FTD. In this study we demonstrate that C9orf72 DPRs polyGA, polyGR and polyGP (× 40 repeats) disrupt secretory protein transport from the ER to the Golgi apparatus in neuronal cells. Consistent with this finding, these DPRs also induce fragmentation of the Golgi apparatus, activate ER stress, and inhibit the formation of the omegasome, the precursor of the autophagosome that originates from ER membranes. We also demonstrate Golgi fragmentation in cells undergoing RAN translation that express polyGP. Furthermore, dysregulated ER-Golgi transport was confirmed in C9orf72 patient dermal fibroblasts. Evidence of aberrant ER-derived vesicles in spinal cord motor neurons from C9orf72 ALS patients compared to controls was also obtained. These data thus confirm that ER proteostasis and ER-Golgi transport is perturbed in C9orf72-ALS in the absence of protein over-expression. Hence this study identifies novel molecular mechanisms associated with the ER and Golgi compartments induced by the C9orf72 HRE.

The E3 Ubiquitin Ligase SCF Cyclin F Promotes Sequestosome-1/p62 Insolubility and Foci Formation and is Dysregulated in ALS and FTD Pathogenesis.

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.

A novel electrochemical sensor based on ß-cyclodextrin functionalized carbon nanosheets@carbon nanotubes for sensitive detection of bactericide carbendazim in apple juice.

Carbendazim (CBZ) abuse always causes the over-standard of pesticide residues in agricultural products, which has adverse effects on human health. Herein, a novel electrochemical sensor was firstly fabricated based on the ß-cyclodextrin (ß-CD) functionalized carbon nanosheets@carbon nanotubes (CNS@CNT) for the CBZ determination. CNS@CNT combined large surface area of CNS and excellent electrical conductivity of CNT, which significantly enhanced the electrocatalytic performance. Moreover, ß-CD possessed excellent host-gest supramolecular recognition ability, which could improve the selective recognition and enrichment capability of CBZ. Thanks to the synergistic interaction of CNS@CNT and ß-CD, the ß-CD/CNS@CNT/GCE sensor exhibited a low limit of detection of 9.4 nM in the linear CBZ concentration range of 0.03-30 µM. The fabricated sensor presented favorable stability, high sensitivity (30.86 µA µM-1 cm-2), and reliable reproducibility (RSD = 3.6%). Especially, the ß-CD/CNS@CNT/GCE sensor could show pretty practical feasibility for the detection of CBZ in apple juice with recoveries of 97.1%-99.4%.

A Robust Intrinsically Green Fluorescent Poly(Amidoamine) Dendrimer for Imaging and Traceable Central Nervous System Delivery in Zebrafish.

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.