Human endogenous retrovirus-K contributes to motor neuron disease

Abstract

The role of human endogenous retroviruses (HERVs) in disease pathogenesis is unclear. We show that HERV-K is activated in a subpopulation of patients with sporadic amyotrophic lateral sclerosis (ALS) and that its envelope (env) protein may contribute to neurodegeneration. The virus was expressed in cortical and spinal neurons of ALS patients, but not in neurons from control healthy individuals. Expression of HERV-K or its env protein in human neurons caused retraction and beading of neurites. Transgenic animals expressing the env gene developed progressive motor dysfunction accompanied by selective loss of volume of the motor cortex, decreased synaptic activity in pyramidal neurons, dendritic spine abnormalities, nucleolar dysfunction, and DNA damage. Injury to anterior horn cells in the spinal cord was manifested by muscle atrophy and pathological changes consistent with nerve fiber denervation and reinnervation. Expression of HERV-K was regulated by TAR (trans-activation responsive) DNA binding protein 43, which binds to the long terminal repeat region of the virus. Thus, HERV-K expression within neurons of patients with ALS may contribute to neurodegeneration and disease pathogenesis.

Fig. 1.. HERV-K expression in brain tissue from ALS patients.

(A) HERV-K genome showing regions amplified by PCR. (B) All HERV-K genes showed increased expression in ALS patients compared to healthy controls (ALS, n = 11; control, n = 16). Brain tissue sample information is listed in table S1. Values represent means ± SEM. Significance was determined by unpaired Student’s t test. Variances were significantly different between groups. (C) Pearson correlation analyses revealed positive correlations between mRNA expression of HERV-K env, pol, and gag from autopsy brain cortical tissues. Pearson’s correlation coefficients were used to quantify the linear relationship between two variables. (D) Representative images show HERV-K env-immunoreactive neurons in the frontal cortex of a patient with ALS. (E) A higher-magnification image of the boxed area in the image shows focal accumulation at the cell membrane of cortical neurons. (F) Anterior horn motor neurons in the lumbar spinal cord of an ALS patient were also immunoreactive for HERV-K env. (G) The white matter of a patient with ALS. (H and I) Cortical neurons of an individual with a normal brain who died in a motor vehicle accident (H) and cortical neurons from a patient with Alzheimer’s disease showing no immunoreactivity for HERV-K env (I). Scale bars, 50 μm (D to I).

Fig. 2.. HERV-K env-induced neuronal toxicity in vitro.

(A) The HERV-K env gene or the entire HERV-K genome was transfected into pluripotent stem cell-derived human neurons expressing tdTomato (a fluorescent marker to label neurons) and monitored for morphological changes. pcDNA was used as a control. Scale bars, 200 μm; 50 μm. (B and C) At 24 hours after transfection, (B) total cell counts and (C) neurite length were significantly decreased. Values represent means ± SEM from three independent experiments. Significance was determined by one-way analysis of variance (ANOVA) followed by Newman-Keuls post hoc comparison. Variances were significantly different between groups. (D and E) Endogenous HERV-K expression was induced using the CRISPR/Cas9 system. Stem cell–derived human neurons were transduced with a lentiviral construct encoding Cas9 fused to the transcription activation domain VP64 for 24 hours. Cells were either mock-treated (Cas9 alone) or transduced with guide RNA targeting the HERV-K promoter (sgRNA 8). Forty-eight hours after sgRNA delivery, (D) total cell counts and (E) mean neurite fiber length were significantly decreased. Values represent means ± SEM from three independent experiments. Significance was determined by unpaired Student’s t test.

Fig. 3.. HERV-K-induced neuronal toxicity in vivo.

(A) Coronal sections of wild-type (WT) and HERV-K env transgenic (Tg) mice were immunostained for HERV-K env (red) and glial fibrillary acidic protein (GFAP) (purple). DAPI, 4′,6-diamidino-2-phenylindole (blue). (B) Enlarged images of cerebral cortex. (C to G) Golgi impregnated mouse pyramidal neurons show that (C) total dendrite length, (D) mean branch number, (E) dendritic complexity by Sholl analysis, and (F) spine density were significantly reduced in transgenic mice with (G) extensive dendritic beading. Values represent means ± SEM. The number of animals used for quantification was three animals per group. Significance was determined by unpaired Student’s t test. Scale bars, 500 μm (A); 50 μm (B and G). (H to J) Immunostaining for NeuN as a marker for neurons (H), Ctip2 immunostaining as a marker for corticospinal motor neurons (I), and Satb2 immunostaining as a marker for callosal projection neurons (J) in layer V of the motor cortex of wild type (n = 4) and transgenic (n = 3) mice. Values represent means ± SEM. Significance was determined by unpaired Student’s t test. Scale bars, 50 μm. Magnetic resonance images for wild type (n = 5) and transgenic (n = 5) mice were acquired on a 14-Tesla magnetic resonance imaging scanner. (K and L) Regional analysis revealed a reduction in cortical thickness (K) and volume (L) of the rostral part of the motor cortex. (M to O) There was no significant difference between wild-type and transgenic mice for the volume of the cingulate cortex (M), corpus callosum (N),and hippocampus (O). Values represent means ± SEM. Significance was determined by unpaired Student’s t test. The number of animals used for quantification was five animals in each group.

Fig. 4.. HERV-K env expression and injury to lower motor neurons.

(A to C) Representative photomicrographs show HERV-K env immunoreactivity in the cervical (A), thoracic (B), and lumbar (C) spinal cord of transgenic mice. (D) This panel shows lack of HERV-K env immunoreactivity in the thoracic spinal cord of WT mice. (E to H) ChAT immunoreactivity is shown in the cervical (E), thoracic (F), and lumbar (G) spinal cord of transgenic mice and in the thoracic spinal cord of WT mice at 6 months of age (H). Intense ChAT signal seen in the motor neurons of the ventral horn in the thoracic spinal cord of WT mice (H) contrasts with very few ChAT-positive cell bodies in the ventral horn of the thoracic spinal cord of transgenic mice (F). Scale bars, 50 μm (A to H). (I) Sections of the tibialis anterior muscle from 6-month-old animals show a normal mosaic distribution of type I (green), type IIb (red), and type IIa (unstained) fibers in WT mice and fiber type grouping in the transgenic mice. Skeletal muscles from WT (n = 5) and transgenic (n = 6) mice were isolated and immunostained. MyHC, myosin heavy chain. (J) γH2A.X-positive foci in immunostained entorhinal cortex from 6-month-old WT (n = 4) and transgenic (n = 4) mice. Numbers of cells with γH2A.X-positive foci were increased in motor cortex of transgenic mice. Values represent means ± SEM. Significance was determined by unpaired Student’s t test. Scale bar, 20 μm. (K) Fluorescence micrographs showing the localization of nucleophosmin (NPM) in cells in the motor cortex of WT (n = 4) and transgenic (n = 3) mice. Numbers of cells with nucleophosmin localized to the cytoplasm were increased in the motor cortex of transgenic mice. Values represent means ± SEM. Significance was determined by unpaired Student’s t test. Scale bar, 10 μm.

Fig. 5.. HERV-K-induced alterations in behavioral and functional analysis of mouse phenotype.

(A) Open-field testing showed that the transgenic mice were less active than WT animals as determined by decreased path length traveled, increased periods of immobility, decreased line crossings, decreased number of rearings, and decreased number of entries into the center of the field. Representative tracings are shown. There was a progressive decrease in activity overtime [n= 16(WT)and n = 15 (transgenic) at 3 months; n = 26 (WT) and n = 24 (transgenic) at 6 months]. (B) Transgenic mice spent less time on an accelerating rotarod [n = 18 (WT) and n = 17 (transgenic) at 3 and 6 months; n = 18 (WT) and n = 9 (transgenic) at 9 months]. The sample size declined at 9 months due to increased death at that age. (C) Transgenic mice showed an increased clasping reaction in the tail suspension test [n = 18 (WT) and n = 17 (transgenic)]. (D) A Y maze test shows that spontaneous alteration was not different between WT and transgenic mice [n = 18 (WT) and n = 17 (transgenic) at 3 and 6 months; n = 18 (WT) and n = 9 (transgenic) at 9 months]. (E) The time to notice adhesive tapes sticking on the palms of the hind paws in transgenic mice was not significantly different from WT mice [n = 10 (WT) and n = 10 (transgenic)]. (F) When placed on a 45° angle slope, the time to turn was not different between WT and transgenic mice [n = 8 (WT) and n = 12 (transgenic)]. (G) A cohort of transgenic animals declined from 30 to 15 animals over 10 months. Insert shows a 9-month-old terminally ill transgenic mouse with a hunched back. (H and I) Electrophysiological properties of mouse cortical pyramidal neurons [n = 3, WT (blue); n = 3, transgenic (red)]. (H) Voltage traces evoked by −160, −40,40, and 80 pA current steps (1 s). Scale bar, 200 ms/20 mV. Amplitude of the steady-state membrane potentials is plotted against each injected current step. (I) Traces of sEPSCs (scale, 5 s/100 pA) and average sEPSCs (scale, 10 pA/5 s). (J) Change in membrane potential (ΔVm) and number of action potentials evoked for a range of current injections. (K) Pooled data values of sEPSC frequency (left) and amplitude (right). Values represent means ± SEM and were analyzed by the Mann-Whitney nonparametric test.

Fig. 6.. HERV-K activation by TDP-43 and identification of binding sites on LTR.

(A and B) Stem cell-derived neurons were transfected with either pcDNA control or TDP-43 expression construct. (A) Forty-eight hours after transfection, cells were fixed with PFA and stained for HERV-K env protein. Scale bar, 50 μm. (B) Twenty-four hours after transfection, cells were collected for RNA extraction, and quantitative RT-PCR was used to measure HERV-K transcripts. (C and D) HERV-K plasmid was cotransfected with chloramphenicol acetyltransferase (CAT) (control), Tat, TDP-43, or Tat and TDP-43 in HeLa cells, and 24 hours after transfection, reverse transcriptase activity (HERV-K RT) was measured in culture supernatants by the product-enhanced reverse transcriptase (PERT) assay. (D) The levels of HERV-K transcripts were measured using RT-PCR and expressed as fold change compared to CAT control. (E) HERV-K LTR-MetLuc plasmid was cotransfected with CAT, Tat, TDP-43, or Tat and TDP-43, and luciferase activity was measured. RLU, relative light units. (F) Knockdown of endogenous TDP-43 with siRNA reduced HERV-K expression. (G) Putative TDP-43 binding sites in HERV-K LTR reported relative to the first base of the LTR. (H) Binding of TDP-43 to biotinylated oligonucleotides derived from the putative binding sites under low- or high-salt conditions. (I) Quantification of (H) indicating binding affinity. Values represent means ± SEM from three independent experiments. Significance was determined by unpaired Student’s t test, nt, nucleotide.