Investigating Amyloid Precursor Protein Expression in Alzheimer’s Diseases

Allison Zetterman, Dr. Luwen Zhang Author

04/06/2021 Added

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Enhancement of Amyloid Precursor Protein (APP) in Alzheimer's disease proteins by converting a viral polypeptide into cDNA and injecting into mice.

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[00:00:00.630]Hello. My name is Alison Zetterman
[00:00:02.810]and I am a junior Biological Sciences major
[00:00:05.460]with minors in Biochemistry and Humanities and Medicine.
[00:00:08.990]This past year, I have been investigating
[00:00:10.960]amyloid precursor protein expression
[00:00:13.030]and Alzheimer's disease proteins alongside my PI, Dr. Zhang.
[00:00:19.240]The Zika virus is a neurotropic flavivirus
[00:00:22.300]that is known to cause several diseases.
[00:00:24.520]This includes birth defects, such as microcephaly
[00:00:26.900]and congenital Zika syndrome.
[00:00:28.840]Flaviviruses or enveloped viruses
[00:00:30.900]that are positive-stranded RNA viruses
[00:00:33.550]and enter the host via receptor-mediated endocytosis
[00:00:36.948]where it then replicates in the host's genome.
[00:00:39.860]The Zika virus is predominantly transmitted
[00:00:42.200]through mosquito bites, particularly in the Aedes species.
[00:00:46.139]Alzheimer's disease is a
[00:00:47.720]progressive debilitating neurological disease
[00:00:50.210]that leads to widespread brain inflammation
[00:00:52.290]and neuron degeneration.
[00:00:54.220]It is characterized by the development
[00:00:55.830]of beta-amyloid plaques or misfolded protein aggregates
[00:00:59.450]and neurofibrillary tangles in the brain.
[00:01:03.690]Alzheimer's disease is the highest prevalent form
[00:01:05.810]of dementia accounting for approximately 57% of all cases.
[00:01:10.320]There is no known cure for Alzheimer's disease
[00:01:12.692]and most treatment plans are often focused
[00:01:15.230]on symptom management.
[00:01:16.860]In the United States,
[00:01:17.810]it is one of the top 10 leading causes of death.
[00:01:20.675]Figure A shows the difference
[00:01:22.650]between a normal healthy brain, as seen on the left,
[00:01:25.408]and a brain with Alzheimer's disease, as shown on the right.
[00:01:29.600]One of the main differences you can see is
[00:01:32.140]that the brain on the right is shrunken
[00:01:34.240]compared to the brain on the left.
[00:01:35.920]This shrunken-ness is
[00:01:36.753]due to the development of amyloid plaques,
[00:01:38.870]which are formed during the progression
[00:01:40.430]of Alzheimer's disease.
[00:01:42.390]Amyloid plaques come from the expression
[00:01:44.300]of amyloid precursor protein in the brain
[00:01:46.902]which can enter the cell
[00:01:48.310]and aggregate in the misfolded protein complexes
[00:01:51.930]forming the plaques.
[00:01:53.380]These aggregates collect in the brain
[00:01:55.030]and disrupt neuron function,
[00:01:56.510]causing the overall degradation
[00:01:57.970]of the brain and causing it to shrink.
[00:02:00.610]The plaques and tangles are clearly shown
[00:02:02.560]in the right corner of Figure A.
[00:02:05.000]You can see how the development of these structures
[00:02:08.030]can disrupt neuron function
[00:02:09.350]and overall can cause reduced brain function.
[00:02:12.920]Figure B shows how
[00:02:14.090]the amyloid precursor protein permeates the cell
[00:02:18.090]and the steps that it takes to form the plaque aggregates.
[00:02:22.986]Amyloid precursor protein, or APP, is a membrane protein
[00:02:27.880]primarily expressed within the brain
[00:02:29.610]and is implicated in
[00:02:30.610]the pathogenesis of Alzheimer's disease,
[00:02:33.210]as seen in the beta-amyloid plaques.
[00:02:36.380]Through previous research, we have found
[00:02:38.230]that APP can be enhanced in Alzheimer proteins,
[00:02:41.040]and Zika infection can also lead to an increase
[00:02:44.780]in the expression of APP.
[00:02:46.650]We were also able to identify the P2 peptide,
[00:02:49.590]this was what's converted into cDNA.
[00:02:54.010]Overall, the main objective
[00:02:55.580]of our research was to investigate the mechanism
[00:02:58.720]of the enhancement of amyloid precursor proteins
[00:03:01.130]within Alzheimer's proteins.
[00:03:05.300]The viral polypeptide shown in the first bullet was found
[00:03:08.650]to have the ability to enhance APP expression.
[00:03:12.290]The viral polypeptide was converted into cDNA
[00:03:15.373]and injected into mice.
[00:03:17.810]Western blotting analysis was used to detect
[00:03:20.090]the presence and enhancement of APP proteins in tubulin.
[00:03:23.920]A 10% acryl gel was made and ran,
[00:03:26.690]and the gel was transferred to a membrane
[00:03:28.400]where the antibodies were applied.
[00:03:30.550]The primary antibody used was a 1:2,000 APP
[00:03:34.493]with a 1:5,000 tubulin ratio.
[00:03:37.073]And the secondary antibody was
[00:03:39.640]a 1:3000 ratio Goat anti-mouse.
[00:03:45.620]For the primary antibody, two different antibodies
[00:03:48.090]were used to show the expression of two different proteins,
[00:03:50.870]in our case, APP and tubulin.
[00:03:53.252]Tubulin levels were not expected
[00:03:55.430]to vary significantly between the samples,
[00:03:57.670]which remained true with our results.
[00:04:00.200]Two samples were compared to one another.
[00:04:02.290]The membranes were transferred onto a film
[00:04:04.070]to visualize the expression,
[00:04:06.690]as exhibited on the right of the slide.
[00:04:09.780]The sample on the left contained only the cDNA vector,
[00:04:14.440]was shown to not be able to increase APP expression.
[00:04:18.260]A vector is to carry something or express something,
[00:04:21.900]so cDNA is one of the vectors we use.
[00:04:25.570]The sample on the right contained the cDNA vector
[00:04:29.190]with the P2 plasmid
[00:04:31.380]and was shown to increase APP expression.
[00:04:35.350]You can clearly see the differences
[00:04:36.970]in levels of expression between the two samples,
[00:04:39.390]showing that the P2 plasmid increased the levels
[00:04:42.060]of expression of APP within the cell.
[00:04:45.100]High levels of APP expression are linked
[00:04:47.410]to Alzheimer's disease pathogenicity,
[00:04:51.123]so understanding what factors can increase its expression
[00:04:54.110]can be crucial for preventative measures.
[00:04:57.870]In the future, we plan to convert the cDNA
[00:05:00.104]into an adeno-associated virus (AAV) with the P2 plasmid,
[00:05:05.260]which will then be injected into mouse brains
[00:05:08.210]and see if it will block APP production.
[00:05:11.040]AAV can be used for gene therapy and is fairly safe.
[00:05:15.550]AAV is theorized to get into the cell and stay in the cell
[00:05:19.380]which would help it block APP expression.
[00:05:21.860]We plan to inject the P2 adeno-associated virus
[00:05:25.360]into mouse brains and see if they're able
[00:05:27.080]to block APP expression in mice.
[00:05:30.475]We also anticipate that understanding the mechanism
[00:05:33.220]behind the enhancement of APP would be useful
[00:05:36.060]in treatments for Alzheimer's disease or designing a drug.
[00:05:40.050]Thank you very much for listening to my presentation.

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Investigating Amyloid Precursor Protein Expression in Alzheimer’s Diseases

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