Summary and Critical Analysis - Sample 1

As part of my Molecular and Cell Biology course work, I have to routinely write summaries and critical analyses of scientific articles. Though not perfect, these few samples may give you an idea of what to put down.

Paper 1: http://www.pubmedcentral.nih.gov/articlerender.fcgi?artid=1299076

Summary and Critical Analysis: Expression of the Alzheimer protease BACE1 is suppressed via its 5’- untranslated region

Summary:
BACE1 or Beta site amyloid precursor protein (APP) cleaving enzyme 1 is a membrane protein involved with the secretory pathway, and hence is localized to endosomes, trans golgi apparatus and the plasma membrane. BACE1 is expressed primarily in the brain and the pancreas. Previous research has shown an interesting link between BACE1 and Alzheimer’s disease. It was observed that Alzheimer’s disease patients expressed elevated levels of BACE1 protease. Alzheimer’s disease is presumed to be caused by the formation and aggregation of amyloid β peptides (Aβ). Studies have shown that Aβ peptides are formed by the cleavage of APP by BACE1. It was also noted that despite elevated protein levels, mRNA levels of BACE1 were not affected in AD patients. In attempt to analyze the cause for an elevation in BACE1 levels among Alzheimer’s disease patients, Lammich et al have proposed that the 5’ untranslated region plays a regulatory role in normal adults. Once conclusively proven that 5’ UTR is the defective portion in Alzheimer’s disease, they contend that a cure for the disease can be attempted.
In order to study the role of the 5’UTR in BACE1 expression, the authors first characterized the region and conformed that it is 446bp long, has 77% GC content and contains 3 upstream ORFs. They also presume that the 5’UTR plays an important role as a major part is highly conserved in humans, mice and rats. In their initial experiments, the authors transfected HEK293 cells with BACE1 ORF, BACE1 + 5’UTR and BACE1 + 3’UTR. They observed that BACE1 protein levels were significantly less in cells transfected with BACE1 + 5’UTR. 3’UTR did not seem to play a significant role in BACE1 inhibition. In order to determine the regulatory role of the 5’UTR region, they transfected HEK293 cells with the lucferase ORF and luciferase containing the 5’UTR of BACE1. They observed that cells containing the 5’UTR expressed very low levels of luciferase indicating that the 5’UTR indeed played a role in repressing the expression of a downstream ORF.
In the next portion of their study, Lammich et al proposed that the BACE1 5’UTR affected translation and not transcription. In order to prove this, they first studied the levels of BACE1 proteins in transfected HEK293 cells by immunoblotting and also studied the levels of mRNA by northern blotting. They observed that levels of mRNA were not affected even though BACE1 proteins levels in those cells was reduced up to 40 fold. To further confirm their finding, they repeated the experiment in nuclease treated rabbit reticulocyte lysate and obtained similar results.
In order to show that the results obtained were not cell-type dependant, the experiments were repeated in African green monkey COS7 cells and also in human neuroglioma H4 cells. Similar results were obtained in all the cell types confirming that the regulatory effect of the 5’UTR was cell-type independent. In order to prove that the results were not promoter dependent, the experiments were repeated with two other promoters CMV and EF1α and similar results were obtained.
In order to determine the exact portion of the 5’UTR playing a repressive role, they conducted various experiments with different portions of the 5’UTR deleted. They observed that though deletions in the three uORFs showed increased BACE1 levels, the increase were not as significant as when the 5’UTR was completely absent. Hence, Lammich et al propose that the stable secondary structure of the BACE1 5’UTR rich in GC forms many stem loops which inhibit translation in vitro.
Critical Analysis:
CDC estimates that Alzheimer’s disease is a seriously debilitating illness affecting about 4 million Americans. Being a leading cause of dementia in older adults, there is an urgent need to understand and find a cure to Alzheimer’s. In attempting to solve this problem, Lammich et al have indeed chosen a very important issue.
The paper is very concise and to the point, with ample illustrations of results obtained. Though very clear and concise in most parts, I would have appreciated further explanation about Fig 1B, which I found very difficult to analyze.
The author’s use of COS7 cells and H4 cells in addition to HEK293 cells helps reduce the probability of cell-type influence on the results, but I am unsure if they are sufficient to eliminate all possibility of cell-type effects. More experiments are probably required, using other cell types, like pancreatic cells, to confirm the results obtained. Further studies to analyze the actual structure of the 5’UTR and the mechanism of repression of translation would also prove to be very beneficial.
Another key question not answered in this paper is the role of BACE1 in normal brain cells. The authors mention that BACE1 levels are primarily elevated in the brain and in pancreas, and they also mention a possible secretory role for BACE1, which does not explain the higher occurrence of BACE1 in healthy brain cells.
In conclusion, I believe that this paper is very informative but further research is needed to get closer to understanding BACE1 and the 5’UTR of BACE1 and their role in the prevention and cure of Alzheimer’s disease.