The prokaryotic SPHINX 1.8 REP protein is tissue-specific and expressed in human germline cells

Abstract

Small circular DNAs of 1.8 and 2.4 kb were initially discovered in highly infectious Creutzfeldt‐Jakob Disease (CJD) and scrapie particles from mammalian brain and cultured cells. Surprisingly, these protected cytoplasmic "SPHINX" DNAs contained replication (REP) initiation sequences resembling those of Acinetobacter phage viruses. An antibody was generated against a REP peptide encoded by the SPHINX 1.8 open reading frame (ORF) that was not present in mammals. It bound to a 41kd “spx1” protein on Western blots. Cytologically, spx1 concentrated in spinal cord synapses and pancreatic islet, but not exocrine cells. We hypothesized that circular SPHINX DNAs are ancient symbiotic elements that can participate in functional differentiation and neurodegeneration. Cell and tissue‐specific patterns of spx1 expression shown below implicate somatic cell‐to‐cell communication and differentiation functions that would favor conservation of SPHINX 1.8 in evolution. Remarkably, primary human oocytes and spermatogonia, but not mature sperm, displayed intense cytoplasmic spx1 signals that underscore the maternal inheritance of SPHINX 1.8. These findings should encourage investigations of unexplored networks of incorporated environmental infectious agents that can be key actors in progressive neurodegeneration, immunity, and cancer.

Prokaryotic SPHINX replication sequences are conserved in mammalian brain and participate in neurodegeneration

“…The highest specific titer serum was affinity purified. On Western blots, this immunoglobulin G showed a strong 41 kDa signal at 1:5000 dilution that was completely blocked by the REP peptide in Western blots, cell culture, and peripheral tissue sections as reported previously . Because large anterior motor horn neurons appeared to receive synaptic boutons with high concentrations of spx by light microscopy, it was important to extend and substantiate this at the ultrastructural level.…”

“…The overall architectural pattern of spx, with high concentrations in rosette synapses on GC dendrites and in numerous GC synapses surrounding Purkinje dendrites in the molecular layer was consistently seen in all the mammals examined (human, GP, hamster, rat, and mouse). As in previous studies using procedures to enhance tissue penetration, neither 2 M GdnHCl that can loosen protein binding to nucleic acids nor other unmasking procedures, altered the pattern, specificity of spx labeling or its peptide blocking. The strongly positive mossy fiber synapses in the GC layer contrasts with minimally labeled cytoplasm of GC cell bodies and other adjacent fibers (see C and E).…”

“…Archival human and TSE experimental animal tissue were sectioned and dewaxed as previously for detection of spx using a previously characterized affinity purified rabbit antiserum . Briefly, sections were blocked in 10% normal goat serum and 3% fish gelatin in Tris‐buffered saline (TBS) at 22°C for 1 hour and then exposed to Rabbit anti‐spx at 1:5000 in TBS‐0.1% Tween 20 (TBS‐T) at 4°C overnight.…”

“…TBS‐T washes were followed by Goat anti‐rabbit biotin in TBS‐T at 1:5000 for 1.5 hours at 37°C, TBS‐T washes and then exposed for 30 minutes to strepavidin‐alkaline phosphatase and developed with Vector Red Laboratories per kit instructions. For enhanced penetration of very old formalin fixed brain, heat‐citrate autoclaving as described or 2 M GdnHCl exposure for 30 minutes was done after dewaxing and these treatments did not affect spx localization or peptide blocking of the anti‐spx antibody as previously shown . A Bio‐rad confocal microscope was used to collect fluorescent optical slices, which were then deconvolved using the Bio‐rad software, and some stacks were then rotated using commercial Zen software with the help of Dr. Larry Rizzolo.…”

“…Unrecognized environmental, toxic, and microbial agents can initiate and contribute to the development of major common diseases such as Alzheimer's Disease (AD). The current strong focus on end‐stage amyloid and a few other fibril‐forming proteins have diverted investigation of unsuspected microbial causes of late‐onset neurodegeneration and these have some precedent. Influenza viruses have caused late‐onset neurofibrillary tangles (NFTs) in Parkinson's neurodegeneration in a “hit and run” pattern, as in the 1918 epidemic, and activated subacute myxo‐paramyxoviral infections can induce NFTs .…”