Pro-Inflammatory Signaling Upregulates a Neurotoxic Conotoxin-Like Protein Encrypted Within Human Endogenous Retrovirus-K

Motor neuron degeneration and spinal cord demyelination are hallmark pathological events in Amyotrophic Lateral Sclerosis (ALS). Endogenous retrovirus-K (ERVK) expression has an established association with ALS neuropathology, with murine modeling pointing to a role for the ERVK envelope (env) gene in disease processes. Here, we describe a novel viral protein cryptically encoded within the ERVK env transcript, which resembles two distinct cysteine-rich neurotoxic proteins: conotoxin proteins found in marine snails and the Human Immunodeficiency Virus (HIV) Tat protein. Consistent with Nuclear factor-kappa B (NF-κB)-induced retrotransposon expression, the ERVK conotoxin-like protein (CTXLP) is induced by inflammatory signaling. CTXLP is found in the nucleus, impacting innate immune gene expression and NF-κB p65 activity. Using human autopsy specimens from patients with ALS, we further showcase CTXLP expression in degenerating motor cortex and spinal cord tissues, concomitant with inflammation linked pathways, including enhancement of necroptosis marker mixed lineage kinase domain-like (MLKL) protein and oligodendrocyte maturation/myelination inhibitor Nogo-A. These findings identify CTXLP as a novel ERVK protein product, which may act as an effector in ALS neuropathology.

NPV CTXLP domain (red). Structure alignment is based on sequence alignment and was prepared using UCSF Chimera software [3]. CTXLP sequence and the ERVK Consensus sequence found in the ERVK envelope transgenic mice [5]. Note the similarity and retention of key cysteine motif.

Bioinformatics
Open reading frames (ORFs) in the ERVK genome were predicted on both the sense and antisense strands using CLCbio software. Any amino acid-encoding codon was accepted as an ORF start, although each ended with a stop codon. All ORFs identified were searched using NCBI Conserved domains

RNA seq analysis
To evaluate the significance of CTXLP in disease, we evaluated the expression of CTXLP encoding ERVK loci in publicly available RNA-Seq datasets in the Sequence Read Archive (SRA) (Figure S6). These loci were identified by searching the SRA by disease affiliation and then evaluating each potential study based on sample size, tissue, and sequencing

Orthologues and paralogues analysis
The most recent genomic assembly for each primate species was searched for CTXLP in the same manner as the human genome. panTro5 and gorGor5 were retrieved from UCSC, BLAST matches of whole retroelement entries and their flanking 1,000 bp, which mostly correspond to entire ERVs, but which sometimes were fragments. Three and four-way orthology was determined from pairwise orthology. A Heatmap was generated by superheat from frames 0 (CTXLP) and 1 (Envelope) of the human and gorilla orthologs ORFs, which were both positive for Toxin_18 (CTXLP).

Ethics statement
All research involving human autopsy tissue was approved by the University of

Diagnosis and demographics of patient samples
Pathologic examination was used to confirm the clinical diagnosis of ALS. The postmortem interval of all patients was <24 h. Table S3 indicates the individual patient diagnosis, location of brain tissue sampling, age, gender, and post-mortem interval (PMI in hours) of the samples used in this study. The brain regions analyzed were the motor cortex (Brodmann areas, BA4 & BA6) and cervical and lumbar spinal cord (CC, LC).

Immunohistochemistry of autopsy tissue
To determine the extent of ERVK CTXLP and cellular target expression patterns in the CNS of ALS patients, immunohistochemistry was performed to detect the levels and localization of these target proteins in autopsy human cortical brain tissue, as previously described [9]. Primary antibodies used are described in Table S4. Tissues were also counterstained with DAPI. Free-floating tissues were mounted onto slides and stained in a 0.1% solution of Sudan Black B. Slides were rinsed, and coverslips mounted using ProLong Gold anti-fade reagent (Molecular Probes). All samples were batch stained with case-control matched tissues. Immunostained tissues were imaged with Olympus FV1200 laser scanning confocal microscope fitted with the Olympus Fluoview version 4.0B software suite. This software was used to outline cellular as well as nuclear boundaries of ERVK + neurons in neuro-normal and ALS specimens. Quantification of micrographs was done using ImageJ to perform the intensity, puncta, and colocalization measurements. GraphPad Prism was used to perform statistical analysis of micrograph data between neuro-normal and ALS patient groups.

Solochrome cyanine staining
Free-floating cervical and lumbar spinal cord sections (60 μm) in ethylene glycol-sucrose solution from human neuro-normal (NN) and amyotrophic lateral sclerosis (ALS) patient tissues (n = 3 for each group and cord region) were used for solochrome cyanine staining.
Sections were transferred to well plates and rinsed three times for five minutes with Trisbuffered saline (TBS) and 0.05% Triton X100. After washing, sections were carefully mounted onto Superfrost+ slides and dried before staining. Histochemical staining was performed for solochrome cyanine and counterstaining with eosin for visualization of myelin, as previously described [10], using erichrome cyanine R (J.T. Baker #L128-05) and 10% Iron Alum (ferric ammonium sulfate, Fisher #I75-500) solutions for staining and Eosin Y (Fisher #E511-25) solution for counterstaining. Sections were mounted with Permount, and slides were coverslipped and dried before imaging. Low magnification scanning of slides was performed to obtain whole spinal cord section images of ALS and NN tissue for comparison of myelin staining.

Cell culture and treatments
The SVGA cell line is derived from immortalized human fetal astrocytes [11] and was maintained in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% Fetal Bovine Serum (HyClone). ReNcell CX cells (Millipore #SCC007) are immortalized human neural progenitor cells (hNPCs) [12], and were maintained in a proprietary ReNcell neural stem cell medium (Millipore) supplemented with 20 ng/mL human epidermal growth factor (EGF; PeproTech #AF10015) and 20 ng/mL human basic fibroblast growth factor (bFGF; PeproTech #AF10018B). T47D cells were maintained as previously described [13]. All cell lines were maintained in a 37 °C incubator containing 5% CO2. SVGA cells were seeded into six-well plates and onto glass coverslips at a density of 300,000 cells/mL and 30,000 cells/mll, respectively, for 24 h. To differentiate HNPCs into neurons, ReNcells CX were seeded in laminin (20 μg/mL; Millipore #CC095) coated six-well plates at a density of 50,000 cells/mL for 24 h. Adhered cells were rinsed with 1X PBS and allowed to differentiate in the presence of ReNcell medium lacking growth factors for 10 days.

Immunoprecipitation and cell treatments
Immunoprecipitation (IP) beads (BioRad Surebeads, USA) were prepared by transferring 100 μL of beads into a 1.5 mL Eppendorf tube and washing three times with 1 mL 0.1% PBS-Tween. Next, 10 µL of protein-specific antibody in PBS is added to the tube and incubated for 10 min at room temperature while rotating. The mixture was then centrifuged briefly, and beads are washed three times with 1 mL 0.1% PBS-Tween. Next, 200 µL 0.4% paraformaldehyde was added to beads, and the mixture was incubated for 10 min at room temperature while rotating. Centrifugation and wash steps were repeated. Beads were then incubated for 1 h at room temperature, rotating, with 500 ug of whole-cell extracts in PBS. Centrifugation and wash steps were repeated, and beads were transferred to a new tube before the last wash was removed. Beads were incubated a final time with 20 µL 0.1M glycine, pH 2.85, for 5 min at room temperature. The supernatant was then transferred into a new 1.5 mL Eppendorf tube containing 2 µL 0.5 M NaOH.
Cells were dosed by volume of IP product (5 μL standard dose), as there was no reliable way to measure the concentration of the protein in the IP product, as protein concentration was vanishingly below the sensitivity of our in-house BCA assay (20 µg/mL).

Immunocytochemistry
For the cleaved caspase-3 assays, SVGA astrocytes were cultured in complete Dulbecco's modified eagle's medium in 12 well plates until 80% cell confluency was reached.  was directly imaged using Protein Simple FluorChem M imager. Image Lab software (BioRad, USA) was used to determine the molecular weight and relative density (normalized to β-actin) of each band. GraphPad Prism was used to compare the patient groups through the Mann-Whitney t-test. Correlation analysis was performed using the Pearson rank correlation coefficient.

Quantitative PCR
QPCR was performed on cDNA generated from transfected 293T cells using SYBR Green detection to amplify inflammatory genes, as previously described [14]. Primers were NF-

Chromatin immunoprecipitation (ChIP)
SVGAs were seeded in 10 cm dishes at an approximate density of 3 × 10 6 cells/dish for 24 h at 37 °C and 5% CO2. Laminin-coated dishes were used to seed ReNcell CX cells at a density of 3 × 10 5 cells/dish for 24 h at 37 °C and 5% CO2. The culture media on adhered ReNcell CX cells was then replaced with that lacking EGF and bFGF growth factors, and cells were allowed to differentiate into neurons for 10 days. SVGAs and neurons were treated with 10 ng/ml human TNFα (PeproTech) or human LIGHT (PeproTech) for 8 h, fixed with 4% paraformaldehyde, and harvested. Untreated cells were used as the negative control. Chromatin Immunoprecipitation (ChIP) was performed using the Pierce Magnetic ChIP kit (Thermo Scientific #26157) as per manufacturer's instructions.
CTXLP bound DNA segments were isolated using 5 µg of rabbit anti-CTXLP (custom) antibody. Immunoprecipitation with IgG antibody was used as negative control. QPCR was performed on the immunoprecipitated DNA using SYBR Green detection to amplify the ISREs in the ERVK 5′ LTR. Primers for the first ISRE (nt. 380-392) were F: 5′-TCACCACTCCCTAATCTCAAGT-3′ and R: 5′-TCAGCACAGACCCTTTACGG-3′ and for second ISRE (nt. 563-575) were F: 5′-CTGAGATAGGAGAAAAACCGCCT-3′ and R: 5′-GGAGAGGGTCAGCAGACAAA-3′, as previously described [14]. Data were analyzed using the ΔΔCt method and normalized relative to the input and IgG controls for each condition. All data were graphed as mean ± standard error of measurement. Statistical analyses were performed in GraphPad PRISM using Two-Way ANOVA and Tukey's multiple comparisons test.

NF-κB
Nuclear factor kappa B NLS Nuclear localization sequence NN Neuro-normal Nogo-A Neurite outgrowth inhibitor NPV Nuclear polyhedrosis virus OL Oligodendrocyte Olig1 Oligodendrocyte transcription factor 1 Olig2 Oligodendrocyte