Research Rundown: Mouse study to find link between MT-1 protein and Alzheimer’s-associated factors.

Alzheimer’s Disease is a form of neurodegenerative disease thought to be caused by the formation of brain-lesioning plaques of amyloid-β oligomers and tangles of tau proteins accumlati. For a more in-depth look at this process, visit my summary of Alzheimer’s Disease here.

For tips on reading scientific literature, look at my previous blog post on that topic here. These concepts will be applied in the Research Rundown posts.


Article Information:

Influence of Transgenic Metallothionein-1 on Gliosis, CA1 Neuronal Loss, and Brain Metal Levels of the Tg2576 Mouse Model of Alzheimer’s Disease – DOI

Implications:

  • Risk for Alzheimer’s Disease may be influenced by production of proteins that mediate zinc (Zn) and copper (Cu) levels in the brain.
  • Potential new target for Alzheimer’s therapeutic agent.

 

Abstract and Introduction

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The first step to trudging your way through any research article is to skim the Abstract and read the Introduction. This will help set up the contextual framework formed by asking fundamental questions about the research.

Why is this research being done?
Alzheimer’s is a debilitating disease that affects millions of people just in the U.S. Any research done to clue us into its mechanism of pathogenesis offers up new potential targets for therapeutics and, perhaps, a cure.

What are the researchers hoping to accomplish?
The researchers are looking for a link between the protein Mt-1 and Alzheimer’s-associated neural death or factors that may contribute to neural cell death.

How is this relevant to me… to the world?
Any research that brings us closer to understanding the nuances of Alzheimer’s Disease pathogenesis, brings us closer to finding a more effective and reliable solution. I may one day struggle with Alzheimer’s disease, as will millions of people globally, but perhaps a solution will be found before that time.

Now that those questions are answered, we have an idea of what the researchers intend to find out, and why their findings are important. Trying to read a scientific article without knowing the intention of the researchers is like being asked to find a specific location in a city you’ve never visited, with no map and no one to ask for directions. You can see how that would be difficult.

Additionally, in reading the Abstract and Introduction, we may come across some terms that we have never heard before. In research articles, scientific jargon is necessary for the sake of concision, but often bogs down the unknowing reader. This can be addressed by writing down words and acronyms we might not know and looking them up with a quick Google or Wikipedia search.


Expression: the process wherein proteins are synthesized using DNA as the instructions. If proteins are a chocolate chip cookie, then the DNA is the recipe and expression is the process of baking.

Phenotype: observable characteristics as a result of an organism’s genetic material. 

Glial (GLEE-uhl) Cells: cells that play a supportive role to neurons.

Gliosis (glee-OH-sis): change in glial cells as a response to damage. Thank you Wikipedia.

Transgenic: Refers to the quality of being produced by a ‘swapping’ of genetic material between species. See below

Tg2576 Mice: Mice that have been genetically modified to express human APP.

Amyloidosis (am-uh-loi-DO-sis): resulting condition of a buildup of amyloid, aggregates of misshapen proteins.

Amyloid (AM-uh-loid) Precursor Protein (APP): a protein commonly expressed and displayed on the outside (extracellular) surface of neuronal cell bodies. It is thought to be involved in synaptic formation and repair. A region of this protein is cleaved on the outside to form Amyloid-β. More here.

Amyloid-β(beta) (Aβ): the product of cleaved APP. The normal function of Aβ is not fully understood. Aβ is thought to be one of the causative agents in Alzheimer’s Disease, as when it aggregates in the brain, it causes lesion-forming plaques associated with cognitive loss. More here.

Metallothionein (meh-TA-lo-THY-0-neen) Proteins (MT): a family of small proteins whose function dependent on heavy metals like zinc (Zn) and copper (Cu). They are involved in heavy metal storage, transport, and detoxification. A specific MT protein, MT-1, may play a role in amyloidosis. More here.

Antibody: a molecule involved with the immune system that binds to only one type of protein that it is specific for.

Immunostaining (im-MYUN-no-staining): the process of chemically staining a protein by using a labeled antibody that is specific to that protein. Imagine you want to find your car keys, but they’re lost in a pile of a bunch of other keys. You also, strangely enough, have a bunch of red magnets that only stick to your keys. You bombard the key pile with red magnets and many of them clump to your keys. This is immunostaining. Also associated with immunohistochemistry (im-MYUN-o-HIST-o-chemistry). More here.

Hippocampus: the area of the brain associated with emotion and memory.

CA1: a specific region of the hippocampus.

Tau Protein: a protein that is thought to be involved in the pathogenesis of Alzheimer’s Disease when it becomes hyperphosphorylated (see below) and forms tangles with itself inside of the cell.

Phosphorylation (fos-FOR-uh-LAY-shun): the process wherein a phosphate (PO43-) molecule is added to another molecule. This has many regulatory uses.

Hyperphosphorylation (hyper-fos-FOR-uh-LAY-shun): the process by which a molecule becomes fully saturated (all phosphorylation sites used) with phosphate molecules. This also has many regulatory uses.

Oxidative Stress: an imbalance in the body’s production of reactive oxygen species (superoxide, hydroxyl radical, and hydrogen peroxide) and the body’s ability to remove these agents. More here.

Isoform: describes different forms of proteins.

Electrophoresis (ee-LEC-tro-for-EE-sis): process by which molecules (such as proteins) are separated by driving them through a gel matrix with electrical current. The molecules separate by weight, the lighter molecules better able to pass through the matrix.

Western Blotting: process by which proteins that have been separated by electrophoresis are labeled using chromatically labeled, fluorescence labeled, or radiolabeled antibodies.


Now that we’ve defined some terms and checked those definitions within the context of the study, we can really dig into the article.

The Introduction reveals that the researchers already know a few key facts about the protein they are studying, Metallothionein-1/2 (two separate isoforms of the metallothionein protein).

  1. Excess levels of metals such as copper (Cu), zinc (Zn), and iron (Fe) contribute to oxidative stress as well as aggregation and precipitation of Aβ in the brain.
  2. MT-1/2 is expressed in greater quantity in someone with Alzheimer’s Disease.
  3. MT-1/2 levels are increased in regions with amyloid plaques in mouse models.
  4. MT-1/2 isoforms are involved in the formation of amyloid plaques. How they do so is not known.

The Introduction also reveals what is not known – things that the researchers intend to find out:

  1. The effects of MT-1/2 on gliosis, neuronal survival, and Zn and Cu levels.
  2. Information as to how MT-1/2 contributes to plaque formation.

Materials and Methods

I have to admit – this is where the article I chose is somewhat unique. The authors chose to report their Results and Discussion prior to reporting their Materials and Methods. This isn’t typical so I opted to stick to the more common layout in this Research Rundown. This will be okay because the Materials and Methods section is really only read in detail in situations where you have to replicate or build on the experiment (or if you have to read it for a research seminar class). Here are the highlights:

Animals

The researchers used three types of mouse strains: C57BL/6JOlaHsd as the wild-type (WT, normal, control mice), TgMT as the mice that will produce extra metallothionien, and Tg2576 as the Alzheimer’s disease mouse model that produces extra human APP. These mice were then crossed (strategically bred) such that they produced mice of the following groups: WT (WTxWT), TgMT (TgMTxTgMT), APPWT (APPxWT), and APPTgMT (APP x TgMT). This new generation should take on characteristics of their parents (i.e. the APPTgMT mice will express increase human APP and will also produce extra metallothionien). The article also provides information about when the mice were killed (6 and 14 months, and their deaths were for science!), how they were fed, and how they simulated day/night cycles. They also outline the procedure for harvesting the brains of the mice for the study.

Immunohistochemistry (IHC) and Histochemistry (HC)

These practices have to do with the dying of molecules using chromatically labeled antibodies specific for said molecules. The researchers give information for mounting and dying the harvested brain tissue to analyze for MT-1/2 presence as well as different types of gliosis.

Western Blotting

Information for the Western blotting procedure to assess the presence of markers associated with gliosis.

Inductively Coupled Plasma-Mass Spectrometry (ICP-MS)

This was used to assess presence of Zn and Cu. Learn more about ICP-MS here.

Statistical Analysis

Information as to how the researchers went about performing the statistics behind their experimental models. This process is important as it indicates that the results were indeed a reflection of reality and not mere happenstance.

Results

This is a good point to remind you to thoroughly analyze all figures and captions. In them, By and large, is the majority of a research article’s ‘important’ information. The information that makes the research.

MT-1/2 Immunostaining is Dramatically Increased in TgMT Mice

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Figure 1. Effect of Mt1 overexpression on MT-1/2 and Congo Red staining in the cortex. (A) Representative brain MT-1/2 immunostaining in wild-type (WT) (top) and TgMT (bottom) mice; (B) Quantification of MT-1/2 IHC of the different genotypes in the cortex showed a dramatic increase in Mt1-expressing (TgMT and APPTgMT) mice (★ p at least ≤0.05 vs. WT or APP mice, respectively) with a prominent caudal-frontal gradient. As revealed by the significant interaction between APP expression and Mt1 overexpression (♠ p < 0.05 in male caudal region; the rest was not significant), APP expression tended towards an increase in MT-1/2 in WT mice; and the opposite was true in TgMT mice; (C) The greatest accumulation of dense amyloid plaques (stained with Congo Red) was localized in the medial area in both sexes. Results are mean ± SEM (n = 7–11). Scale bar: 400 µm. a.u., arbitrary units. 

This figure tells us what was found during the immunohistochemistry portion of the experiment – namely, the level of production of MT-1/2 isoforms in the cortex. The researchers note that even though the dye used in the experiment stains both isoforms of MT, the increase in stain intensity is a result of increased MT-1 as Mt1 is the gene that is being overexpressed in the transgenic mice.

In Figure 1A, we see a photo of the histochemically stained MT-1/2 in the WT mouse and TgMT mouse cortices. Remember, the WT mice are the control and the TgMT mice are intended to produce mot MT-1, which is seen clearly by the increased amount of staining.

Figure 1B tells us about the production of MT-1/2 in different regions of the mouse cortex (caudal (or rear), medial, and frontal) for male and female mice of all categories (genotypes). This data is gathered via immunohistochemistry and the quantities are measured by the relative intensity of the staining. Deeper stain, more MT. The researchers note a gradient in MT-1/2 production, with the greatest amount of production in the caudal region and least amount in the frontal region. Bars with a symbol (♠ or ★) above them are statistically significant, the rest are not. Figure 1B also tells us that MT+ mice (TgMT and APPTgMT) produce much more MT-1 than MT- mice (WT and APP). This makes sense because the MT+ mice have an increased capability to do so. Interestingly, it was found that there was an increase in levels of MT-1/2 in APPWT  mice relative to WT mice, but the opposite trend for APPTgMT mice relative to TgMT mice (TgMT mice have, theoretically, have 2x the MT-1/2 producing capability as APPTgMT mice due to their extra copy of the metallothionein gene. Why would APPWT mice have increased levels of MT-1/2 despite there being no explicit way for them to make more MT-1/2 (such as the TgMT mice have)?

Figure 1C gives us information as to the level of amyloid production, which they measured, again, by intensity. Intensity, not of immunostaining, but of a Congo Red dye that allows the researchers to see production of dense amyloid plaques. This measurement was only done on APP+ mice (APP and APPTgMT). It shows that the greatest production of amyloid plaques was in the medial region of the mouse cortex. Why would there be a relatively lower amount of MT-1/2 in the medial region of the cortex despite there being a greater amount of amyloid plaque?

ijms-18-00251-g002.png

Figure 2. Effect of Mt1 overexpression on MT-1/2 staining in the hippocampus. (A) Representative immunostaining for MT-1/2 counterstained with Congo Red in the hippocampus of APPTgMT mice (left); scale bar: 200 µm. A higher magnification of the black lined square area is shown at the right to better demonstrate plaques stained with Congo Red dye (arrow); scale bar: 50 µm; (B) Quantification of total MT-1/2 immunohistochemistry (IHC) produced similar results to the cortex, with dramatic increases in TgMT and APPTgMT mice (★ p < 0.001 vs. WT or APP mice, respectively). An opposing trend of APP expression was again seen between WT and TgMT male mice (♠ p < 0.05 interaction); (C) Comparison of MT-1/2 levels associated with plaques to those not associated with plaques indicated an increased immunostaining in the vicinity of the amyloid plaques only in male mice (◆ p < 0.05 vs. staining associated to plaques). Results are mean ± SEM (n = 7–11). a.u., arbitrary units.

Figure 2 involves the staining and quantification of MT-1/2 in the mouse hippocampus. Figure 2A is an immunostaining of the mouse hippocampus wherein the box on the right is a magnified image of the region contained within the box on the left. This magnified image clearly shows the amyloid plaque (arrow) dyed with the Congo Red dye. Like the results from the cortex staining, Figure 2B shows an increased production of MT-1/2 in MT+ male and female mice. Figure 2C shows a statistically significant difference between MT-1/2 associated with amyloid plaques and MT-1/2 not associated with amyloid plaques in male mice only. This was not seen significantly in female mice. Note the error bars.

Mt1 Overexpression Has Only Minor Effects on the Gliosis Elicited by Amyloid Plaques

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Figure 3. Effect of Mt1 overexpression on gliosis in the hippocampus. (A,C) Representative immunostaining for GFAP (astrocytes) and Iba-1 (microglia), respectively, counterstained with Congo Red, in the hippocampus of APPTgMT mice (left); scale bar: 200 µm. On the right, a higher magnification of the black lined square area from left panel shows astroglia and microglia surrounding dense plaques; scale bar: 50 µm; (B,D) Quantification of GFAP and Iba-1 IHC indicated a dramatic increase in the vicinity of the plaques. Results are mean ± SEM (n = 11–18); ◆ p < 0.001 vs. plaque-associated staining. a.u., arbitrary units.

While the previous few figures referred to MT-1/2 production in regions of the mouse brain, the following figures refer to another aspect of the study: the assocation between Mt1 (gene) overexpression and the process of gliosis initially elicited by amyloid plaques in the hippocampus. No discernable trends were seen when this process was done in the cortex

Figure 3A shows the immunostaining of Glial fibrillary acidic protein (GFAP), a protein expressed in astrocytes (a type of glial cell). Again, the panel on the right is the magnified region in the box on the left. Figure 3B shows us, for male and female mice, the increased amount of glial (GFAP) staining, thus increased levels of, glial cells in the vicinity of the amyloid plaques as compared to areas without plaques. This trend is seen in microglial cells as well (Figure 3C&D) via the staining of the protein Iba-1, found in microglial cells. This shows that the amyloid plaques are, in fact, eliciting gliosis in the mouse brain.

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Figure 4. Effect of Mt1 overexpression on hippocampal gliosis as measured by western blot (WB). Total hippocampal and cortex homogenates were assayed by WB to further characterize gliosis. (A) Representative band pattern of the WB (in an autoradiographic film) of old male hippocampus using antibodies for GFAP, Iba-1, and Actin; (B) Quantification of hippocampal GFAP and Iba-1 levels in young and old APPWT and APPTgMT mice. Iba-1 levels were increased by Mt1 overexpression in old male mice but decreased in young males; the latter also showed decreased GFAP levels. Data are mean ± SEM (n = 10–11). ★ p at least ≤0.05 vs. APPWT mice. a.u., arbitrary units.

This figure uses western blot to quantify cortical and hippocampal levels of glial cell-associated proteins Iba-1 (microglia) and GFAP (astrocytes) in male and female mice, both young and old. Using the western blot in 4A, the researchers retrieved the data in 4B. Figure 4B shows three statistically significant results: First, old, male APPTgMT mice showed increased Iba-1 levels, with decreased levels for young, male APPTgMT mice. The third result is that young, male, APPTgMT showed decreased GFAP levels.

Mt1 Overexpression Does Not Affect Hippocampal CA1 Neuronal Loss

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Figure 5. Effect of Mt1 overexpression on hippocampal CA1 neurons. (A) Representative histochemistry of Nissl body staining of neurons in hippocampal CA1 of WT and APPWT mice. Scale bar: 20 μm; (B) Quantification of the thickness of the CA1 layer indicated a significant decrease in APPWT and APPTgMT mice in both sexes, whereas no significant effects of Mt1 overexpression were observed. Results are mean ± SEM (n = 11–18); ▲ p < 0.01 vs. APP negative mice.

Remember that CA1 is a region of the hippocampus, thus, CA1 neuronal death leads to hippocampal damage.

Although neuronal loss was observed by the researchers in male and female TgMT mice, they note that this was not due to Mt1 overexpression. Figure 5A shows immunostaining of CA1 hippocampal region in WT and APPWT mice, indicating neuronal loss in the APPWT mice as compared to the WT. Significant losses in CA1 layer thickness were observed in both APPWT and APPTgMT (Figure 5B), but no significant difference was found in losses between the two, indicating that presence of MT-1/2 did not play a significant role in neuronal death. 

Mt1 Overexpression Has Only Minor Effects on Zinc and Copper Levels

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Figure 6. Effects of Mt1 overexpression on Zn and Cu levels. Total hippocampal (top) and cortical (bottom) homogenates from young (~6 months) and old (~14 months) mice were analyzed by ICP-MS. In the hippocampus, copper and zinc levels were increased and decreased by aging, respectively; both metals were increased in the cortex. APP and Mt1 expression showed different effects depending on the metal and brain area. Results are mean ± SEM (n = 7–11); ▲ p at least ≤0.05 vs. APP negative mice. ♠ p < 0.05 interaction between APP and TgMT.
Figure 6 is telling a lot of information. It is ultimately concerned with levels of Zn and Cu in the hippocampus and cortex of young and old, male and female mice in all 4 categories (WT, TgMT, APP, and APPTgMT). These levels are described by micro-grams of metal per gram of tissue (▲ and ♠ denote statistical significance). Results are as follows (this is taken straight from the literature, read each one and locate it in the figure):
  • Hippocampal copper levels were increased by aging in both sexes (increases most with APP+).
  • Mt1 overexpression decreased the effect of APP expression on copper levels of young female mice.
  • Aging had a significant decreasing effect on hippocampal zinc levels, Mt1 overexpression did not.
  • Both cortical zinc and copper levels were moderately increased by aging, a trend opposed by APP expression in male mice.
  • Mt1 overexpression tended to increase cortical zinc levels in a statistically insignificant way.

Take away: APP and Mt1 overexpression show different effects depending on metal and brain region.

Discussion & Conclusion

The conclusions and justifications are as follows:

  1. Caudal-frontal gradient observed in cortex likely due to MT-1/2 production in astrocytes as they show a similar gradient when expressing GFAP.
  2. Immunostaining decreased in cortical and hippocampal tissue of APPTgMT mice as compared to TgMT mice, an opposing trend to what was expected. This may be due to differences between the regulation of normal MT-1/2 genes and the transgenic MT-1/2 genes placed in the TgMT mouse genome or by some mechanism related to the amyloid plaques themselves.
  3. Astrocytes were more reactive in the vicinity of amyloid plaques but Mt1 overexpression did not significantly alter cortical or hippocampal GFAP levels.
  4. Microgliosis was not altered significantly by Mt1 overexpression.
  5. By Mt1 overexpression, hippocampal Iba-1 levels were increased in old male mice likely due to increased amyloid plaque burden. Combined with evidence in a previous study wherein MT-1/2 knockout mice (mice where the gene for MT-1/2 is removed) show decreased burden, it is concluded that MT-1/2 control amyloid plaque deposition which drives microglial reactivity (mechanism unknown).
  6. Conversely, in young male mice, Mt1 overexpression results in cortical and hippocampal microgliosis and astrogliosis. This suggests that MT-1 may have an inhibitory role in controlling microglia that is overridden by an activating role in APP+ mice due to its effects of plaque formation as plaques are not typically found in young mice.
  7. Mt1 overexpression did not influence neuronal survival, though it is not known why this is the case. Though Metallothionien proteins may play a protective role in APP+ mice as they are known to aid in the detoxification of metals and reactive oxygen species that are involved in amyloid plaque formation.

The MT-1/2 protein isoforms are involved in the deposition of amyloid plaques, but show only modest effects on glial activation, neuronal survival, and metal accumulation. The MT-1/2 proteins may show promise as a target for therapeutics but more research is certainly required for this implication to be realized. 


Closing

Research like this is needed for the discovery of potential, new drug targets. Sure, not all of the results of this study are flashy and exciting, as some of the associations the researchers were hoping to find were modest if not nonexistent, but that doesn’t mean that information isn’t useful. Negative results are still able to tell use about the reality of these processes. We learned what not to look for and perhaps what processes a therapeutic agent aimed at the MT-1/2 protein isoforms would not be affected.

In closing, know that this is obviously the first Rundown I’ve posted and I, like the researchers in this study, am finding out what works and what doesn’t. This process will likely go on for a little while as I begin refining my process for these posts and coming up with new ideas

Bear with me and please leave comments, questions, suggestions, and corrections (in the spirit of collaboration and fidelity).


References For This Post

Comes, G., Manso, Y., Escrig, A., Fernandez-Gayol, O., Sanchis, P., Molinero, A., … & Hidalgo, J. (2017). Influence of Transgenic Metallothionein-1 on Gliosis, CA1 Neuronal Loss, and Brain Metal Levels of the Tg2576 Mouse Model of Alzheimer’s Disease. International Journal of Molecular Sciences, 18(2), 251.

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