The Spike Protein and “Prion Disease”: Unveiling a Causative Mechanism

It is not Prion Aggregation per se that kills neurons, it is the presence of Prion in the cytosol, which activates – Caspase 3!

Live cell imaging of the endocytosis of SARS-CoV-2 S-RBD-sfGFP in the iPSC-CMs. (a) Time course of immunostaining and live cell imaging after replating the iPSC-CMs. Differentiated cardiomyocytes were replated on day 14 after differentiation and transduced with AAV2-DsRed-RAB5A at 1.0–2.0 × 104 vg/cell on day 21, then incubated with SARS-CoV-2 S-RBD-sfGFP for 48 h before immunostaining or 10 min before live cell imaging on day 28 after differentiation. (b) ACE2-WT-iPSC-CMs transduced with AAV2 encoding DsRed-RAB5A were incubated with SARS-CoV-2 S-RBD-sfGFP for 48 h before immunostaining on day 28 after differentiation with the indicated antibodies. (c) ACE2-WT-iPSC-CMs were incubated with SARS-CoV-2 S-RBD-sfGFP for 10 min before live-cell imaging on day 28 after differentiation and observed by confocal microscopy. The areas enclosed within the white squares are enlarged in the right panel. (d) ACE2-WT-iPSC-CMs transduced with AAV2 encoding DsRed-RAB5A were incubated with SARS-CoV-2 S-RBD-sfGFP for 10 min before live-cell imaging on day 28 after differentiation and observed by confocal microscopy. Each dot indicated by a white or yellow arrow shows the same signal of S-RBD-sfGFP co-localized with AAV2-DsRed-RAB5A.

From the beginning of the pandemic myself and many others warned that the Spike Protein would be capable of inducing Prion Disease, Unfortunately, there have been cases of CJD post COVID vaccination.

A 64-year-old woman with a past medical history of bipolar depression and anxiety presents with rapidly progressive dementia, behavioral changes, headaches, and gait disturbance approximately one week after receiving the second dose of the Pfizer-BioNTech COVID-19 vaccine.

Sporadic Creutzfeldt-Jakob Disease After Receiving the Second Dose of Pfizer-BioNTech COVID-19 Vaccine
https://scholarlycommons.hcahealthcare.com/cgi/viewcontent.cgi?article=1420&context=internal-medicine

But how?

I hypothesized that, like the Prion protein the Spike protein is a surface protein. If there is a mistake in the creation of the Prion protein it is then taken back into the cell to be degraded. This process happens all the time and, under normal conditions, the body has no problem eliminating the incorrect protein.

PrP is a plasma membrane protein that begins its journey to the cell surface in the endoplasmic reticulum (ER). Like many proteins that traffic through the ER (12, 13), a substantial fraction of PrP normally misfolds, is retrograde transported to the cytosol, and is degraded by proteasomes (14, 15). Several PrP mutants that cause familial neurodegeneration are as stable as wild-type PrP once they mature (16,17), but are more likely to misfold during maturation (18), and in the one case tested, are more frequently subject to retrograde transport (14). In the cytosol, PrP is usually degraded so rapidly it is undetectable.

Neurotoxicity and Neurodegeneration When PrP Accumulates in the Cytosol
https://www.science.org/doi/10.1126/science.1073725

It is when the body is overwhelmed and cannot destroy the incorrect proteins that the disease form of PrP appears. However, it is not the aggregates of this protein that destroy the cell, it is their presence in the cytosol itself.

However, when proteasome activity is compromised, PrP accumulates and sometimes converts to a PrPSc-like conformation (19). Proteasome inhibitors kill neuroblastoma cells more rapidly than other cultured cell types tested (14), yet the fraction of PrP that converts to a PrPSc-like form seems to have little influence on toxicity (19). Here we explore the relation between PrP misfolding, proteasome inhibition, accumulation of PrP in the cytosol, and neurotoxicity.

Neurotoxicity and Neurodegeneration When PrP Accumulates in the Cytosol
https://www.science.org/doi/10.1126/science.1073725

OK, so we know that the presence of a lot of incorrect Prion proteins in the cell causes it to die, but what is the mechanism? It turns out to be induction of apoptosis, caused by Caspase-3.

Different neurodegenerative disorders like prion disease, is caused by protein misfolding conformers. Reverse-transfected cytosolic prion protein (PrP) and PrP expressed in the cytosol have been shown to be neurotoxic. To investigate the possible mechanism of neurotoxicity due to accumulation of PrP in cytosol, a PrP mutant lacking the signal and GPI (CytoPrP) was introduced into the SH-SY5Y cell. MTT and trypan blue assays indicated that the viability of cells expressing CytoPrP was remarkably reduced after treatment of MG-132. Obvious apoptosis phenomena were detected in the cells accumulated with CytoPrP, including loss of mitochondrial transmembrane potential, increase of caspase-3 activity, more annexin V/PI-double positive-stained cells and reduced Bcl-2 level.

Cytosolic prion protein induces apoptosis in human neuronal cell SH-SY5Y via mitochondrial disruption pathway
https://www.bmbreports.org/journal/view.html?volume=42&number=7&spage=444

And, like the Prion Protein the Spike Protein (RBD) translocates from the cell surface to the cytosol.

To further visualize the endocytosis of S-RBD into the iPSC-CMs using live-cell imaging, we generated an adeno-associated virus (AAV), encoding an N-terminal DsRed-fused full-length human RAB5A sequence driven by the CMV promoter (AAV2-DsRed-RAB5A). We selected the AAV2 serotype, as AAV2 has been shown to efficiently transduce the iPSC-CMs42,43. ACE2-WT-iPSC-CMs were transduced with AAV2-DsRed-RAB5A, incubated for 5–7 days, and treated with S-RBD-sfGFP (Fig. 3a). Immunostaining confirmed that S-RBD-sfGFP signals co-localized with DsRed-RAB5A in ACE2-WT-iPSC-CMs (Fig. 3b). Sequential observation of S-RBD-sfGFP-positive cardiomyocytes using confocal microscopy demonstrated that S-RBD-sfGFP was bound to the cell membrane, co-localized with DsRed-RAB5A, and delivered to the cytosol (Fig. 3c, d and Supplementary Video).

SARS-CoV-2 spike receptor-binding domain is internalized and promotes protein ISGylation in human induced pluripotent stem cell-derived cardiomyocytes
https://www.nature.com/articles/s41598-023-48084-7

Now to complete the puzzle, yes, it is the Spike RBD that activates Caspase-3.

We also observed significantly elevated intracellular calcium levels and apoptosis rates in the pulmonary vascular endothelium upon treatment with S-RBD, reflected by increased phosphorylated calcium-dependent kinase (p-CaMKII) (Fig. 4g, h) and increased cleaved-caspase 3 immunoreactivity (Fig. 4i, j) levels in the specific endothelial cell marker CD31 positive region of pulmonary vessels.

SARS-CoV-2 spike protein receptor-binding domain perturbates intracellular calcium homeostasis and impairs pulmonary vascular endothelial cells
https://pmc.ncbi.nlm.nih.gov/articles/PMC10349149/

The Spike Protein is a great mimicker. It is able to induce the MECHANISMS of virtually all the fatal diseases of aging. As I have mentioned before, it is not so much that we are seeing an increase in autoimmune disease x, y or z, or cardiovascular disease a, b, or c, or cancer r, s or t. It is that the MECHANISMS that cause these diseases to occur are being initiated and/or revved up by the Spike Protein.

We now have a satisfactory explanation for how the Spike Protein can induce “Prion Disease.” Of course, more studies are needed but it is my hope that this article will help other researchers design studies to home in on these mechanisms.

Thank you, as always, for your continued support, readership and dialogue. Please have a blessed week.

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