Neuroprotective effects of p62(SQSTM1)-engineered lactic acid bacteria in Alzheimer's disease: a pre-clinical study.

Alzheimer's disease (AD) is a progressive neurodegeneration characterized by neuron death ending in memory and cognitive decline. A major concern in AD research is the identification of new therapeutics that could prevent or delay the onset of the disorder, with current treatments being effective only in reducing symptoms. In this perspective, the use of engineered probiotics as therapeutic tools for the delivery of molecules to eukaryotic cells is finding application in several disorders. This work introduces a new strategy for AD treatment based on the use of a Lactobacilluslactis strain carrying one plasmid (pExu) that contains a eukaryotic expression cassette encoding the human p62 protein. 3xTg-AD mice orally administered with these bacteria for two months showed an increased expression of endogenous p62 in the brain, with a protein delivery mechanism involving both lymphatic vessels and neural terminations, and positive effects on the major AD hallmarks. Mice showed ameliorated memory, modulation of the ubiquitin-proteasome system and autophagy, reduced levels of amyloid peptides, and diminished neuronal oxidative and inflammatory processes. Globally, we demonstrate that these extremely safe, non-pathogenic and non-invasive bacteria used as delivery vehicles for the p62 protein represent an innovative and realistic therapeutic approach in AD.

Cellular Aging Characteristics and Their Association with Age-Related Disorders.

Different molecular signaling pathways, biological processes, and intercellular communication mechanisms control longevity and are affected during cellular senescence. Recent data have suggested that organelle communication, as well as genomic and metabolic dysfunctions, contribute to this phenomenon. Oxidative stress plays a critical role by inducing structural modifications to biological molecules while affecting their function and catabolism and eventually contributing to the onset of age-related dysfunctions. In this scenario, proteins are not adequately degraded and accumulate in the cell cytoplasm as toxic aggregates, increasing cell senescence progression. In particular, carbonylation, defined as a chemical reaction that covalently and irreversibly modifies proteins with carbonyl groups, is considered to be a significant indicator of protein oxidative stress and aging. Here, we emphasize the role and dysregulation of the molecular pathways controlling cell metabolism and proteostasis, the complexity of the mechanisms that occur during aging, and their association with various age-related disorders. The last segment of the review details current knowledge on protein carbonylation as a biomarker of cellular senescence in the development of diagnostics and therapeutics for age-related dysfunctions.

p62-DNA-encoding plasmid reverts tumor grade, changes tumor stroma, and enhances anticancer immunity.

Previously, we reported that the administration of a p62/SQSTM1-encoding plasmid demonstrates high safety and signs of clinical benefits for human cancer patients. The treatment also suppressed tumor growth and metastasis in dogs and mouse models. Here we investigated some mechanistic aspects of these effects. In mammary tumors bearing-dogs, i.m. injections of p62 plasmid reduced tumor sizes and their aggressive potential in 5 out of 6 animals, with one carcinoma switching to adenoma. The treatment increased levels of smooth muscle actin in stroma cells and type III collagen in the extracellular matrix, which correlate with a good clinical prognosis. The p62 treatment also increased the abundance of intratumoral T-cells. Because of the role of adaptive immunity cannot be tested in dogs, we compared the protective effects of the p62 plasmid against B16 melanoma in wild type C57BL/6J mice versus their SCID counterpart lacking lymphocytes. The plasmid was only protective in the wild type strain. Also, p62 plasmid amplified the anti-tumor effect of T-cell transfer from tumor-bearing animals to animals challenged with the same tumors. We conclude that the plasmid acts via re-modeling of the tumor microenvironment, making it more favorable for increased anti-cancer immunity. Thus, the p62-encoding plasmid might be a new adjuvant for cancer treatments.

Plasmid DNA-coding p62 as a bone effective anti-inflammatory/anabolic agent.

We recently reported that a DNA plasmid coding p62-SQSTM1 acts as an effective anti tumor vaccine against both transplantable mouse tumors and canine spontaneous mammary neoplasms. Here we report the unexpected finding that intramuscular delivery of p62 DNA exerts a powerful anti-osteoporotic activity in a mouse model of inflammatory bone loss (i.e, ovariectomy) by combining bone-sparing and osteo-synthetic effects. Notably, the suppression of osteoporosis by p62DNA was associated with a sharp down-regulation of master inflammatory cytokines, and up-regulation of endogenous p62 protein by bone-marrow stromal cells. The present data provide a solid rational to apply p62 DNA vaccine as a safe, new therapeutic for treatment of inflammatory related bone loss diseases.

Pilot study of p62 DNA vaccine in dogs with mammary tumors.

Our previous data demonstrated profound anti-tumor and anti-metastatic effects of p62 (sqstm1) DNA vaccine in rodents with various types of transplantable tumors. Testing anti-cancer medicine in dogs as an intermediary step of translational research program provides two major benefits. First, clinical data collected in target animals is required for FDA/USDA approval as a veterinary anti-cancer drug or vaccine. It is noteworthy that the veterinary community is in need of novel medicine for the prevention and treatment of canine and feline cancers. The second more important benefit of testing anti-cancer vaccines in dogs is that spontaneous tumors in dogs may provide invaluable information for human trials. Here, we evaluated the effect(s) of p62 DNA vaccine on mammary tumors of dogs. We found that p62 DNA vaccine administered i.m. decreased or stabilized growth of locally advanced lesions in absence of its overall toxic effects. The observed antitumor activity was associated with lymphocyte infiltration and tumor encapsulation via fibrotic reaction. This data justifies both human clinical trials and veterinary application of p62 DNA vaccine.

Plasmids encoding protein aggregation domains act as molecular adjuvants for DNA vaccines.

BACKGROUND: DNA vaccines provide high tolerability and safety but commonly suffer from suboptimal immunogenicity. We previously reported that a plasmid vector (pATRex), encoding the DNA sequence for the von Willebrand I/A domain of the tumor endothelial marker-8 (TEM8) when given in combination with plasmid-encoded tumor antigens acted as a powerful molecular adjuvant enhancing immunity against breast and melanoma tumors. AIMS: In the present study we addressed two unsolved issues; would the adjuvant action of pATRex extend to a DNA vaccine against infectious disease and, if so, what is the mechanistic basis for pATRex adjuvant action? RESULTS: Here we show in a murine malaria vaccine model that co-administration of pATRex potentiates antibody production elicited by an intramuscular injection of plasmid encoding Plasmodium yoelii merozoite surface protein 4/5 (PyMSP4/5). pATRex enhanced the B-cell response and induced increased IgG1 production consistent with TH2 polarization of the DNA vaccine response. To explore the mechanism of adjuvant action, cells were transfected in vitro with pATRex and this resulted in formation of insoluble intracellular aggregates and apoptotic cell death. Using a structural modeling approach we identified a short peptide sequence (α3-ß4) within ATRex responsible for protein aggregation and confirmed that transfection of cells with plasmid encoding this self-assembling peptide similarly triggered intracellular aggregates, caspase activation and cell death. CONCLUSION: Plasmids encoding aggregation-promoting domains induce formation of insoluble intracellular aggregates that trigger caspase activation and apoptotic cell death leading to activation of the innate immune system thereby acting as genetic adjuvants.

Antibody and CD8+ T cell responses against HER2/neu required for tumor eradication after DNA immunization with a Flt-3 ligand fusion vaccine.

PURPOSE: HER2/neu is frequently overexpressed in breast cancer. In a mouse model, vaccination with HER2/neu DNA elicits antibodies that confer partial protection against tumor challenge. EXPERIMENTAL DESIGN: To enhance antitumor immunity, we fused cDNA encoding Flt-3 ligand (FL) to the rat HER2/neu extracellular domain (neu), generating a chimeric FLneu molecule. FLneu and neu DNA vaccines were compared for immunogenicity and their ability to protect mice from tumor challenge. RESULTS: The neu vaccine generated a HER2/neu-specific antibody response. In contrast, vaccination with FLneu induced CD8+ T cells specific for HER2/neu but a negligible anti-HER2/neu antibody response. The switch from an antibody-mediated to T cell-mediated response was due to different intracellular localization of neu and FLneu. Although the neu protein was secreted, the FLneu protein was retained inside the cell, co-localizing with the endoplasmic reticulum, facilitating processing and presentation to T cells. The neu and FLneu vaccines individually conferred only weak tumor immunity. However, efficient tumor rejection was seen when neu and FLneu were combined, inducing both strong anti-HER2/neu-specific antibody and T cell responses. Adoptive transfer of both immune CD8+ T cells and immune sera from immunized mice was required to confer tumor immunity in naïve hosts. CONCLUSIONS: These results show that active induction of both humoral and cellular immunity to HER2/neu is required for efficient tumor protection, and that neither response alone is sufficient.