Cell & Molecular Biology Lab

Utilizing the Bacteria E. coli

E. coli is a type of bacteria that is commonly used as a “workhorse” in the laboratory. It grows very quickly, can be safely stored, and can very easily be genetically engineered. An artificial gene encoding the amino acid sequence of Amyloid-β can be expressed in E. coli from a plasmid. Using this method, we can produce large quantities of Amyloid-β very quickly. We can use this bacterially-produced Amyloid-β to screen for drug-induced changes in Amyloid-β aggregation patterns. Despite some limitations to this assay, this a has been a starting point for a number of potential therapies developed in our lab.

PCR (Polymerase Chain Reaction)

PCR has become a standard procedure used in molecular biology laboratories. All known forms of life use nucleic acid (DNA or RNA) as a way to store and pass on their genetic information. To study this information easily, scientists need a lot of it. PCR is used to amplify small amounts of DNA (or RNA) into much larger amounts that can be visualized, sequenced, or for a variety of other procedures.

In the COVID era, PCR has become the “gold-standard” in diagnostics. We can now search for extremely low concentrations of viral nucleic acid in patients, or even the sewers. As this technology moves forward, it will only become cheaper and more reliable.

PCR is already extremely sensitive. It can not only identify, but also quantify the nucleic acid in a given sample. Recently, it has become recognized that ncRNA (Non-Coding RNA) can be used as a marker for different pathologies, including Alzheimer’s. We are very interested in developing PCR based diagnostics for Alzheimer’s and other dementias.

Native and SDS-PAGE

One of the most used methods in our lab is PAGE (Polyacrylamide Gel Electrophoresis). PAGE is a well established method used to measure protein size and concentration in a sample. By applying current to a buffered solution, peptides will move through an acrylamide gel at a rate correlative to their size and charge. The resulting separation in the gel allows us to measure the size and heterogeneity of Amyloid-β (AB) aggregates and other proteins in a sample. Coupled with the use of fluorescently labeled Amyloid-β or further analysis using Western Blot, this is a powerful and inexpensive way to measure the effect of potential drugs on amyloid aggregation and degradation.

ELISA

ELISA (Enzyme-Linked Immunosorbent Assay) is a plate-reader based assay utilizing specific antibodies to detect a compound of interest. The detection is facilitated by covalently attaching an easily detectable enzyme marker to the antibody, or by using a secondary antibody with a similarly attached marker.

Well characterized antibodies can be purchased to detect Amyloid-β, secretase enzymes, or inflammatory compounds involved in Alzheimer’s pathogenesis. This provides a robust and efficient means to detect the changing concentrations of these important compounds in a number of different experimental models.

Cell Culture

Culturing eukaryotic cell lines is central to our work. This process allows us to work in an approximately physiologically relevant environment, without the need for animal or human test subjects. For example, we can much more easily test the impact of our drug on microglial activation in cell culture than we can in a mouse model. Kidney epithelial cells extracted from an african green monkey or “Vero cells” are very easily grown and can be utilized in antiviral assays if we suspect that a drug or extract might show utility in such a task. However, in our lab we are primarily concerned with the accumulation of Amyloid-β (AB) and the consequential effect that peptide has on neurons.

The production of AB is a complicated enzymatic process involving multiple different proteins. The complexity of this process also gives more opportunities, more potential targets for novel therapeutics. In particular, the beta and gamma secretase are two enzymes directly involved in AB processing. In order to screen drugs for a potential impact involving these enzymes, we needed a cell culture model with all the relevant molecular machinery in place.

The neuroblastoma cell line BE(2)-M17 has been developed as an in vitro model for exactly this purpose, and has become one of the primary models used in our lab. Described by Macias et al. (see citation below), this model carries the required enzymatic machinery to study secretase inhibition and modulation, as well as providing robust production of AB.

Macias MP, Gonzales AM, Siniard AL, et al. A cellular model of amyloid precursor protein processing and amyloid-β peptide production. J Neurosci Methods. 2014;223:114-122. doi:10.1016/j.jneumeth.2013.11.024

Bacteriophage Handling and Genetic Engineering

Bacteriophage (phage) are viruses that infect bacteria. They have a long history of use in molecular biology. Structurally they are simply nucleic acid encased in protein, but can be remarkably complex in their assembly pathways. Because of the ease in which they are produced, and the large amount of supporting literature regarding their nature, they are very accessible models for the study of protein-protein interactions.

Interestingly, there is some crossover between bacteriophage research and Alzheimer's research . At least some phage can cross the blood-brain barrier (1). The filamentous phage M13 has been shown to modulate Amyloid-β aggregation (2). Phage therapy is enjoying a kind of renaissance (3), and may be useful in treating infectious agents that contribute to Alzheimer’s pathogenesis. We look forward to seeing other future advancements.

Although phage research is only peripherally related to our Alzheimer’s research, we can appreciate the utility of phages in the lab. We keep a small library of phages as standards and controls for experiments having to do with amyloid-drug, and amyloid-protein interactions. Additionally, we have protocols ready for genetically altering phage (4) for any number of potential reasons (nanoparticle design, phage display, or antigen presentation).

Dubos, R. J., Straus, J. H., & Pierce, C. (1943). THE MULTIPLICATION OF BACTERIOPHAGE IN VIVO AND ITS PROTECTIVE EFFECT AGAINST AN EXPERIMENTAL INFECTION WITH SHIGELLA DYSENTERIAE. The Journal of experimental medicine, 78(3), 161–168.
Messing, J. (2016). Phage M13 for the treatment of Alzheimer and Parkinson disease. Gene, 583(2), 85-89.
Young, R., & Gill, J. J. (2015). Phage therapy redux—What is to be done?. Science, 350(6265), 1163-1164
Leavitt, J. C., Gilcrease, E. B., Wilson, K., & Casjens, S. R. (2013). Function and horizontal transfer of the small terminase subunit of the tailed bacteriophage Sf6 DNA packaging nanomotor. Virology, 440(2), 117-133.

A plaque assay (seen here) is used to innumerate the amount of infectious phage particles