TDP-43 proteinopathy in ALS is triggered by loss of ASRGL1 and associated with HML-2 expression.

TAR DNA-binding protein 43 (TDP-43) proteinopathy in brain cells is the hallmark of amyotrophic lateral sclerosis (ALS) but its cause remains elusive. Asparaginase-like-1 protein (ASRGL1) cleaves isoaspartates, which alter protein folding and susceptibility to proteolysis. ASRGL1 gene harbors a copy of the human endogenous retrovirus HML-2, whose overexpression contributes to ALS pathogenesis. Here we show that ASRGL1 expression was diminished in ALS brain samples by RNA sequencing, immunohistochemistry, and western blotting. TDP-43 and ASRGL1 colocalized in neurons but, in the absence of ASRGL1, TDP-43 aggregated in the cytoplasm. TDP-43 was found to be prone to isoaspartate formation and a substrate for ASRGL1. ASRGL1 silencing triggered accumulation of misfolded, fragmented, phosphorylated and mislocalized TDP-43 in cultured neurons and motor cortex of female mice. Overexpression of ASRGL1 restored neuronal viability. Overexpression of HML-2 led to ASRGL1 silencing. Loss of ASRGL1 leading to TDP-43 aggregation may be a critical mechanism in ALS pathophysiology.

Microcapillary cell extrusion deposition with picolitre dispensing resolution.

Extrusion-based cell deposition has become a prominent technique for expanding bioprinting applications. However, the associated print resolution in the order of nanolitre or above has been a limiting factor. The demand for improving print resolution towards the scale of a single cell has driven the development of precision nozzle extrusion, although the benefits gained remain ambiguous. Here, aided by in situ imaging, we investigated the dynamics of cell organisation through an extrusion-based microcapillary tip with picolitre precision through in-air or immersion deposition. The microcapillary extrusion setup, termed 'Picodis', was demonstrated by generating droplets of colouring inks immersed in an immiscible medium. Next, using 3T3 fibroblast cells as an experimental model, we demonstrated the deposition of cell suspension, and pre-aggregated cell pellets. Then, the dynamic organisation of cells within the microcapillary tip was described, along with cell ejection and deposition upon exiting the tip opening. The vision-assisted approach revealed that when dispersed in a culture medium, the movements of cells were distinctive based on the flow profiles and were purely driven by laminar fluid flow within a narrow tip. The primary process limitations were cell sedimentation, aggregation and compaction, along with trapped air bubbles. The use of picolitre-level resolution microcapillary extrusion, although it provides some level of control for a small number of cells, does not necessarily offer a reliable method when a specified number of cells are required. Our study provides insights into the process limitations of high-resolution cell ink extrusion, which may be useful for optimising biofabrication processes of cell-laden constructs for biomedical research. Supplementary information: The online version contains supplementary material available at 10.1007/s42242-022-00205-3.

Antibody Response to HML-2 May Be Protective in Amyotrophic Lateral Sclerosis.

OBJECTIVES: Reactivation of HERV-K(HML-2) has been found in subsets of individuals with amyotrophic lateral sclerosis (ALS). This study examines the antibody response against HML-2 in ALS and analyzes its clinical relevance. METHODS: Antibodies to HML-2 envelope (env) were analyzed using a peptide array for epitope mapping and by a peptide enzyme-linked immunosorbent assay (ELISA) in 242 healthy donors, and 243 ALS and 85 multiple sclerosis (MS) individuals. Extracellular levels of HML-2 were analyzed by digital polymerase chain reaction (PCR). RESULTS: Antibodies in the sera of ALS individuals recognized more HML-2 env peptides compared to healthy controls (p < 0.0001). ALS individuals had higher levels of HML-2 than healthy donors (p = 0.02) and higher antibody levels against a select HML-2 env peptide compared to healthy donors or individuals with multiple sclerosis (p < 0.0001). 55.14% of ALS compared to 21.16% of healthy donors and 13.10% of MS individuals had antibodies against the HML-2 peptide (AUC = 0.769, p < 0.0001). Levels of extracellular HML-2 DNA in serum (p = 0.02) and the number of HML-2 env peptides recognized by ALS sera (p = 0.02) correlated with disease duration. Among ALS individuals, lower levels of HML-2 antibodies were associated with a definite diagnosis per EL Escorial criteria (p = 0.03), and with a lower predicted (p = 0.02) and observed survival (p = 0.03). INTERPRETATION: There is a differential antibody response against specific epitopes of HML-2 env in ALS and controls, suggesting epitope spreading, likely due to persistent antigenic exposure following reactivation of the viral genes. Low levels of antibodies to HML-2 env in ALS are associated with poor prognosis and decreased survival probability. ANN NEUROL 2022;92:782-792.

SMARCB1 deletion in atypical teratoid rhabdoid tumors results in human endogenous retrovirus K (HML-2) expression.

Atypical Teratoid Rhabdoid Tumor (AT/RT) is a rare pediatric central nervous system cancer often characterized by deletion or mutation of SMARCB1, a tumor suppressor gene. In this study, we found that SMARCB1 regulates Human Endogenous Retrovirus K (HERV-K, subtype HML-2) expression. HML-2 is a repetitive element scattered throughout the human genome, encoding several intact viral proteins that have been associated with stem cell maintenance and tumorigenesis. We found HML-2 env expression in both the intracellular and extracellular compartments in all AT/RT cell lines (n = 4) and in 95% of AT/RT patient tissues (n = 37) evaluated. SMARCB1 knock-down in neural stem cells (NSCs) led to an upregulation of HML-2 transcription. We found that SMARCB1 binds adjacent to the HML-2 promoter, repressing its transcription via chromatin immunoprecipitation; restoration of SMARCB1 expression in AT/RT cell lines significantly downregulated HML-2 expression. Further, targeted downregulation of HML-2 transcription via CRISPR-dCas9 coupled with suppressor proteins led to cellular dispersion, decreased proliferation, and cell death in vitro. HML-2 knock-down with shRNA, siRNA, and CRISPR-dCas9 significantly decreased Ras expression as measured by qRT-PCR, suggesting that HML-2 modulates MAPK/ERK signaling in AT/RT cells. Overexpression of NRAS was sufficient to restore cellular proliferation, and MYC, a transcription factor downstream of NRAS, was bound to the HERV-K LTR significantly more in the absence of SMARCB1 expression in AT/RT cells. We show a mechanism by which these undifferentiated tumors remain pluripotent, and we demonstrate that their formation is aided by aberrant HML-2 activation, which is dependent on SMARCB1 and its interaction with MYC.

Guided Assembly and Patterning of Intrinsically Fluorescent Amyloid Fibers with Long-Range Order.

Fibrillar amyloids exhibit a fascinating range of mechanical, optical, and electronic properties originating from their characteristic ß-sheet-rich structure. Harnessing these functionalities in practical applications has so far been hampered by a limited ability to control the amyloid self-assembly process at the macroscopic scale. Here, we use core-shell electrospinning with microconfinement to assemble amyloid-hybrid fibers, consisting of densely aggregated fibrillar amyloids stabilized by a polymer shell. Up to centimeter-long hybrid fibers with micrometer diameter can be arranged into aligned and ordered arrays and deposited onto substrates or produced as free-standing networks. Properties that are characteristic of amyloids, including their high elastic moduli and intrinsic fluorescence signature, are retained in the hybrid fiber cores, and we show that they fully persist through the macroscopic fiber patterns. Our findings suggest that microlevel confinement is key for the guided assembly of amyloids from monomeric proteins.

Broad Bandwidth, Self-Powered Acoustic Sensor Created by Dynamic Near-Field Electrospinning of Suspended, Transparent Piezoelectric Nanofiber Mesh.

Freely suspended nanofibers, such as spider silk, harnessing their small diameter (sub-micrometer) and spanning fiber morphology, behave as a nonresonating acoustic sensor. The associated sensing characteristics, departing from conventional resonant acoustic sensors, could be of tremendous interest for the development of high sensitivity, broadband audible sensors for applications in environmental monitoring, biomedical diagnostics, and internet-of-things. Herein, a low packing density, freely suspended nanofiber mesh with a piezoelectric active polymer is fabricated, demonstrating a self-powered acoustic sensing platform with broad sensitivity bandwidth covering 200-5000 Hz at hearing-safe sound pressure levels. Dynamic near-field electrospinning is developed to fabricate in situ poled poly(vinylidene fluoride-co-trifluoroethylene) (P(VDF-TrFE)) nanofiber mesh (average fiber diameter ≈307 nm), exhibiting visible light transparency greater than 97%. With the ability to span the nanomesh across a suspension distance of 3 mm with minimized fiber stacking (≈18% fiber packing density), individual nanofibers can freely imitate the acoustic-driven fluctuation of airflow in a collective manner, where piezoelectricity is harvested at two-terminal electrodes for direct signal collection. Applications of the nanofiber mesh in music recording with good signal fidelity are demonstrated.