Like all types of loss of memory, Alzheimer’s disease is caused by brain cell death. It is a neuro-degenerative disease, which means there is progressive brain cell death that happens over time. In a person with Alzheimer’s, the tissue has fewer and fewer nerve cells and connections. Alzheimer’s disease (AD) affects 30% of people above the age of 80 yrs. The factors which produce or pathogenesis of this dementia is poorly understood, but showing of symptoms is associated with amyloid plaque, neurofibrillary tangles and neuronal loss mainly in temporal lobe and neocortex of the brain. The core of extracellular plaque is a derivative of amyloid precursor protein (APP) also referred to as beta/A4 and contains amino acid residues. The source of APP and the relationship of its accumulation to the neuropathology of AD is unknown. 

What is an amyloid plaque: Amyloid plaques are hard, insoluble accumulations of beta-amyloid proteins that clump together between the nerve cells (neurons) in the brains of Alzheimer’s disease patients.

What are neurofibrillary tanglesNeurofibrillary tangles are insoluble twisted fibers found inside the brain’s cells. These tangles consist primarily of a protein called tau, which forms part of a structure called a microtubule. The microtubule helps transport nutrients and other important substances from one part of the nerve cell to another.

What is neuronal lossNeurodegeneration is the progressive loss of structure or function of neurons, including death of neurons.

RELATION BETWEEN APP AND AMYLOID FORMATION

So here what we do, find the relationship between APP overexpression and amyloid formation, so the scientist has developed a vector to direct expression in neurons of C-terminal fragment of APP that contains the beta/A4 region by using a transgenic mouse to insert and express this construct. 

Figure 1 shows the construction of neuron-specific expression vector that we use in the transgenic mice this construct consists of 3.6 kilobases of 5’ flanking DNA of the human Thy-1 gene, which expression will get in the central nervous system. And link this to DNA fragment encoding the C terminus of APP. We choose this APP fragment because it may be toxic to separate neurons in culture. 

How neuro-specific vector is generated by mutagenesis
FIGURE 1: Diagram of strategy for constructing the neuro-specific expression vector containing the C-terminal region of APP. To obtain the Thy-1 element, an EcoRI 5’ fragment that included the translation initiation codon was sub-cloned into M13-derived vector, which is digested with EcoRI and BamHI. 

We use site-directed mutagenesis was used to alter two bases such that ATG was destroyed and HindIII site was created. The mutated fragment was then removed from the vector by EcoRI and HindIII digestion, and used to replace Beta-actin promoter of the expression vector LK444. The vector was digested at a unique pvu1 site for microinjection and partially with EcoR1 to obtain a 6.0 kb fragment. In the map of LK444 the red region represents the replace Beta-actin promoter, and the blue region represents the SV40 intron and polyadenylation signal.

BETA ACTIN ROLE IN NEURITE CELLS 

 Beta-Actin is a highly conserved protein involved in cell growth, cytoskeletal and extracellular support structures and cell migration. Because beta-Actin is everywhere expressed in all eukaryotic cells, it is frequently used as a loading control for assays involving protein detection, such as Western blots.

Beta-actin has also been shown to localize in areas associated with neuronal growth and difference in both developing and mature neuronal cell types. Beta-actin is primarily found in the growth cones, filopodia (slender cytoplasmic extension), cell bodies and axonal tracks of developing cerebellar cultures. In mature cultures, beta-actin is found to be primarily localized in dendritic spines, structures that are involved in flexibility in mature cells.

HOW SCIENTIST FOUND ALZHEIMER RELATED TO APP 

Four transgenic founder mice were produced and two of three surviving lines expressed the gene line 1 and 2. To determine the cell type that expressed the foreign gene in situ hybridization(In situ hybridization is used to reveal the location of specific nucleic acid sequences on chromosomes or in tissues )was performed on the brain using a labelled antisense cRNA probe specific for the 3’ end of human APP gene. And also, show a high concentration of APP mRNA in neurons in the brains of transgenic animals 

INTERPRETATION:

  • At four months of age transgenic mice from both lines had extracellular amyloid deposits as large as 100micrometer in diameter and found in the hippocampal area of brain visualized by anti-beta/A4 specific immunocytochemistry used to assess the presence of a specific protein or antigen in cells (cultured cells, cell suspensions) by use of a specific antibody, which binds to it, thereby allowing visualization and examination under a microscope. 
  • Negative controls tissue of brain did not stain and show no immunoreactivity on transgenic tissue and no amyloid deposits could be seen after the Bielschowsky silver staining (A silver stain to demonstrate neurofibrillary tangles, nerve fibers and plaques in Alzheimer’s disease) silver staining revealed dense -core plaques, neurofibrillary tangles and neurotic dystrophy
  • After 8 months old, line1, which showed higher transgene expression during fetal development show severe pathology. 

And from this mouse model by immunocytochemical technique shows us that these plaques and tangles strongly resembled those found in human brain with AD: this technique involves staining with and anti-beta/A4 specific antibodies show immunoreactivity with tangles and neurotic plaques. Thioflavin S-positive profiles were abundant in the hippocampus and cortex region in the brain and also in the white matter of the brain. 

In our mouse older animals of line 1 showed more neuronal cell loss as compared with either 4-month-old transgenics mice. Scientist repeatedly found 40% loss in neural cells in one part of hippocampus and specifically the pyramidal neurons of hippocampus and also punkinje cells that found in the cerebellum

RESULT

  • neuronal degeneration in the cerebellum was not associated with the presence of amyloid plaques or neurofibrillary tangles. 
  • These transgenic mice demonstrate that a single disturbance, over-production of carboxy terminal portion of APP can lead to major neuropathological features observed in AD.
  •  It is interesting to determine the precise portion of APP C terminus that is contained in the plaques of mice a transgene encodes a peptide that extends beyond the C-terminal limit of beta/A4.
  • Thy-1-APP transgenic mice have all of these neuropathological features of AD and show us the importance of rodent model for the disease. These animals can be used to provide a test system for therapeutic uses.

In down’s syndrome, where AD develops in all individuals over 30 years old, deposition of beta/A4 as diffuse plaques seen early because People with Down syndrome are born with an extra copy of chromosome 21, which carries the APP gene. This gene produces a specific protein called amyloid precursor protein (APP).

Reference

Kawabata, S., Higgins, G.A. and Gordon, J.W., 1991. Amyloid plaques, neurofibrillary tangles and neuronal loss in brains of transgenic mice overexpressing a C-terminal fragment of human amyloid precursor protein. Nature354(6353), pp.476-478.

This Post Has One Comment

  1. Sachin

    Great work 👍

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