Literature 003: Navaroli et al., PNAS, 2012, 109, 8, E471-480

Niewoehner et al. state that "The precise mechanism by which the sFab construct escapes lysosomal sorting and is released on the abluminal side is not known."

This is interesting because it essentially forms part of the frontier of the human understanding of how the cell works.

A paper by Navaroli et al. from 2012 discusses a specific internalization and sorting mechanism, the uptake of the Transferrin receptor (TfR).

As one might guess, internalization of cargo across the cell membrane is a highly complex process. It is highly regulated because the cell wants to avoid internalizing substances which are toxic or which are already present in the cell at sufficient concentrations while at the same time it wants to promote uptake of needed substances.
TfR allows the cell to internalize Transferrin (Tf) which again binds Fe. In the figure below, "receptor" would be TfR (AP-2 is the so called adapter protein type 2 and PM is the plasma membrane). Clathrin is a factor involved in vesicle formation. Conceptually, AP-2 can be understood to have a certain "directing" effect on the vesicle in a similar way as it is attempted to be established for Rabenosyn-5 in this article.

(In the following, the figure caption labels correspond to the figure captions in the article.)

http://en.wikipedia.org/wiki/Receptor-mediated_endocytosis#mediaviewer/File:Itrafig2.jpg

Rabenosyn-5 is another endosomal component implicated in early endosome fusion and, as reported in this paper, is found in the vicinity of clathrin coated cell surface regions (Fig. 2 in article).

Fig. 2: http://www.pnas.org/content/109/8/E471/F2.expansion.html
Top panels: Clathrin
Lower panels: Rabenosyn-5

TESM refers to TIRF ESL Microscopy, where TIRF and ESL stand for Total Internal Reflection Fluorescence and Epifluorescence Structured Light, respectively. The top two panels show clathrin while the lower two show Rabenosyn-5 distribution. The scale, z-position, indicates how deep within the cell interior a pixel is measured.
TIRF allows to observe fluorescent objects in the cell but can not provide information about the location relative to the cell surface because, as reported, the brightness of an object can result from either its distance to the cell surface or its size. ESL corrects for this and the overlay, TESM, shows the z-axis dependent distribution of clathrin and Rabenosyn-5 at the cell surface: most pixels are blue which means that the majority of the proteins reside at the surface and the surface is populated by both clathrin and Rabenosyn-5.

Since the question is to figure out what Rabenosyn-5 does, the next question to ask is what happens when Rabenosyn-5 is not present in the cell (Fig. 4D in article)?

Fig. 4D: http://www.pnas.org/content/109/8/E471/F4.expansion.html
Sc: Rabenosyn-5 present
Si: Rabenosyn-5 not present

Using siRNA (small interfering RNA), transcription of Rabenosyn-5 can be prevented. The white pixels are TfR. "Sc" is a control for the siRNA function (scrambled siRNA).

Considering the TIRF images (where Sc means that Rabenosyn-5 is active because the surpressing siRNA is scrambled), it is seen that the cell has a higher TfR surface concentration compared to the case where the siRNA is active (right TIRF image). The difference is reported to be a decrease of 75% in the TIRF image. The decrease measured by EPI is less pronounced. Put differently, when Rabenosyn-5 is not produced, the TfR surface amount is reduced which also means that Tf uptake is reduced in TfR depleted cells.

This observation leads to the question, how Rabenosyn-5 controls the cell surface TfR level. Does Rabenosyn-5 regulate TfR gene transcription, TfR gene translation or some other kind of post-translational mechanism? This question is answered by panel A of Fig. 6A in the article.

Fig. 6A: http://www.pnas.org/content/109/8/E471/F6.large.jpg

GADPH is used to provide a scale for the measurement.


Possibility of translational regulation. It is known that translation of TfR can be regulated by mRNA parts before and behind the sequence coding for the protein (so called untranslated regions, UTRs).
To test for translational effects of Rabenosyn-5, the host was transfected by TfR-GFP (green fluorescent protein) lacking these UTRs (that means translation of this TfR-GFP can not be regulated). As can be seen, when Rabenosyn-5 is active (Rbsn Sc column), both TfR and TfR-GFP are produced (right most panel, Sc column). However, when Rabenosyn-5 is surpressed (right most panel, Si column) both TfR-GFP (lacking the regulatory sequences) as well as native TfR are present at lower levels.

This means Rabenosyn-5 can not be having an effect on the translation of TfR, because then TfR-GFP levels would not have been reduced (since its translation can not have been regulated)- but the TfR-GFP levels are reduced. Put differently, regulation of TfR by Rabenosyn-5 must be post-translational. So how come the TfR-GFP levels be reduced when this can not be caused by translational regulation?

Possibility of transcriptional regulation. Although there is no experiement discussing this specifically, the question, if TfR levels are regulated at the transcriptional level, is answered by considering that the gene for TfR-GFP does not contain promoter sequences. Therefore it is not possible that Rabenosyn-5 has a transcriptional effect.

Now, if not at the transcriptional nor translational level, how else can Rabenosyn-5 control TfR levels?
The hypothesis is that Rabenosyn-5 acts as a sort of label for how the cell should handle internalized TfR. Once TfR is internalized, it can be directed to the lyosome, i.e. degraded or it can be recycled, that means the TfR is returned to the cell surface and reused. The explanation for the lower levels of TfR in the absence of Rabenosyn-5 is that when Rabenosyn-5 is not present, a larger proportion of TfR gets directed towards the lysosome and subsequently degraded. Evidence for this mechanism is found in that recycling rates are significantly lower in Rabenosyn-5 deactivated cells (Fig. 5D in article).

Release rates (arbitrary units) of previously internalized Tf depending on time.
http://www.pnas.org/content/109/8/E471/F5.expansion.html

Put differently, a lower proportion gets recycled to the cell surface resulting in apparent lower TfR cell surface amount. These mechanistic alternatives are illustrated by the red arrows in the figure below (Fig. 6C in article):

Fig. 6C: Alternative mechanisms of TfR sorting in presence and absence of Rabenosyn-5.
Top panel: Rabenosyn-5 labels clathrin coated TfR vesicles for recycling (and reuse of TfR) to cell surface.
Lower panel: Absence of Rabenosyn-5 results in TfR vesicles being directed to lysosome for degrading.
http://www.pnas.org/content/109/8/E471/F6.large.jpg

This is verified by comparing TfR levels between cells with activated and deactivated lysosomes (Fig. 6B in article).

Fig. 6B: Baf: Bafilomycin A1.
Sc/Si refer to scrambled and siRNA of Rabenosyn-5.
"+": Baf present, "-": Baf absent
http://www.pnas.org/content/109/8/E471/F6.large.jpg

Under the influence of Bafilomycin A1 ("Baf") an inhibitor of lysosome operation, the TfR level is higher than when the lysosome is operational: the TfR level in the column Baf+/Sc is most intensive because it reports the TfR levels on the cell surface as well as those in the disfunctional lysosomes.

An additional finding is that the absence of Rabenosyn-5 has an effect on clathrin dynamics. In the absence of Rabenosyn-5, the number of clathrin regions and the size of the clathrin regions are increased. In the absence of Rabenosyn-5, increased clathrin signals are reported in the region of the cell nucleus (Fig. 7C in article).

Fig. 7C: left panels: Rabenosyn-5 present
right panels: Rabenosyn-5 deactivated
http://www.pnas.org/content/109/8/E471/F7.expansion.html

However, as shown in Fig. 6C, the total cellular clathrin amount is not affected by the deactivation of Rabenosyn-5. It appears therefore as if the depletion of Rabenosyn-5 results in a redistribution of clathrin, which normally is also present as unassembled clathrin in the cytoplasma, to the cell surface and to the nuclear region. It is therefore reasonable to expect that such a redistribution of clathrin at the nuclear region is only observed because a specific, directing, not yet completely characterized interaction between Rabenosyn-5 and clathrin is missing in the Rabenosyn-5 deactivated cells. The authors contemplate that when Rabenosyn-5 is missing, this results in delayed movement of cathrin coated vesicles away from the plasma membrane or a delay in their uncoating and fusion with endosomes.

Summary. The paper presents evidence for the hypothesis that Rabenosyn-5 prevents TfR from getting directed to the lysosome for degradation. The mechanism of this labeling is however yet to be characterized in detail.

Editorial. I have contacted the senior author of the paper (Prof. Corvera) to ask if she agrees to calling Rabenosyn-5 a labeling agent as stated above. Prof. Corvera added that in Rabenosyn-5 deficient mice severe perinatal phenotypes are observed. Furthermore, she added that based on this work she was approached by geneticists who have found Rabensyn-5 mutations in humans with severe neurological and developmental delays.

Illustrating protein structures

PyMOL can produce really nice protein structures.

Bacillus circulans xylanase with bound substrate (center).

This rendering uses ambient occlusion. The imporant settings are

set light_count, 10
set spec_count, 1
set shininess, 10
set sphecular, 0.25
set ambient, 0
set direct, 0
set reflect, 1.5
set ray_shadow_decay_factor, 0.1
set ray_shadow_decay_range, 2
unset depth_cue

What seems a bit tricky to get are the black outlines and depends on the background color setting.
The following command sequence seems to work, assuming the background is white when you start PyMOL.

1) set ray_trace_mode, 1 (color + outlines)
2) ray (outlines will be white)
3) bg black
4) ray (outlines will be white)
5) bg white
6) ray (outlines should be black)

This swapping of background is most likely not needed if you want to plot to a black background. I'm using PyMOL 1.3 and it could also be different in other versions.

If you use these instructions in your work, please be kind enough to credit this blog post.

Literature 002: Niewoehner et al., Neuron, 2014, 81, 1, p49–60

A number of disorders affect the central nervous system (CNS) such as inflammations, viral infections, Alzheimer's disease, cancers, Parkinson's disease and many, *many* more. In case of Alzheimer, which currently is not curable, people who suffer from the disease are found to have what is called Amyloid-beta plaques in the brain, aggregates of some kind of protein. These aggregates negatively affect the brain function but can be removed if targeted with special antibodies such as Aducanumab, Bapineuzumab or Gantenerumab, possibly leading to a reversal of the disease.

The treatment of diseases of the CNS is difficult, because the body protects the CNS from the outside using a special kind of barrier. In the brain, this barrier is called blood-brain-barrier. The barrier in principle consists of special cells (brain-endothelial-cells, BECs/Endothelium in the figure below) which form the blood vessels and only permit certain substances to transpass from the vessel to the brain. For example, antibodies as those mentioned above are not as such allowed to pass the barrier.

http://clincancerres.aacrjournals.org/content/13/6/1663/F3.large.jpg

In the following, out of the shown possible passways, the focus is on (C), transferrin receptor mediated transcytosis. In this pathway, a substance first binds to the transferrin receptor (Tfr) and is then transported across the cell. This is where the research of Niewoehner et al. comes in. In short, they want to find a way of transporting an active substance such as one of the above mentioned antibodies to the brain.

In "Increased Brain Penetration and Potency ofa Therapeutic Antibody Using a Monovalent Molecular Shuttle", the researchers are able to do this by using a construct which consists of the therapeutically active antibody, a linker peptide and a Tfr active antibody fragment (Tabf). They test two versions of this construct, one with only one Tfr-active fragment, one with two such fragments. The control experiments are done using just the antibody without the linker unit or the Tfr active fragment.

http://www.cell.com/action/showImagesData?pii=S0896-6273%2813%2901035-0

Here, dFab stands for double-Fab and sFab for single-Fab. mAb31 is the monoclonal antibody 31.

In the following, the series of experiments is addressed by the questions they answer in order to establish a causal chain of reasoning.

1 Do the active parts of the construct interfere with their respective function?

http://www.cell.com/action/showImagesData?pii=S0896-6273%2813%2901035-0

In B, the solid and striped bars refer to two different coating densities used in the affinity experiment. As seen, the construct still can bind both ways (Abeta referes to Amyloid beta, the target protein aggregate). Notably, sFab and dFab have very similar binding affinity.

2 Does the construct enter the BEC?

Using a laser to highlight cells where the construct entered, the number of cells at a given fluorescence intensity are counted.

http://www.cell.com/cms/attachment/2012543313/2034747624/gr2.jpg

In red the cells with two Tabf's are counted, in green with only one. The black count corresponds to no Tabf. Apparently two Tabf's lead to a slightly better internalization of the construct.

3 What happens to the constructs (one or two Tabfs) once inside the cell?

http://www.cell.com/action/showImagesData?pii=S0896-6273%2813%2901035-0

One hour after exposure to the construct, it is measured how much of the construct is colocalized with the Lysosome associated membrane protein 2 (Lamp2). This gives an indication of where the construct is found in the cell.

The BECs have a mechanism to identify what has entered them. It is unclear how the cell identifies the dFab and moves it to the lysosome for digestion.

4 How can one be sure that the cell does not digest the sFab construct?

http://www.cell.com/action/showImagesData?pii=S0896-6273%2813%2901035-0

Red and pink are two different concentrations of dFab.

After having entered the cell, the construct has three possible ways to continue: 1) to cross the cell, 2) be digested by the cell and 3) exit the cell where it has entered (see also the first figure).

In the first case, this would mean that the overall number of receptors on the surface should remain constant and this is observed (green bars). The surface concentration of receptors associated with dFab however decreases over time, meaning they do not reappear at the surface once internalized. After 72 hours, apparently new Tfr's are expressed by the cell.

5 Do the constructs cross the cell?

An in vitro experiment is designed where a layer of human BECs separates two sections of liquid (the cells in the above experiments were from mice).

http://www.cell.com/action/showImagesData?pii=S0896-6273%2813%2901035-0

The construct is added to the Lumen compartment (model for the blood vessel inside) and allowed to enter the cells for 1 hour. Then both layers are washed, which controls for constructs entering the abluminal layer by channels inbetween the cells, and amount of construct over time in the two compartments is measured. sFab is observed to appear in both the abluminal and luminal part, while dFab is only found reappearing in the luminal part.

This is where I get confused.

The authors note that

EC50(sFab) = 3.8 nM

EC50(dFab) = 6.4 nM

but say "This suggests that the monovalent binding mode to the Tfr, and not just the reduced affinity, is the key factor for efficient transcytosis." (p. 51) If they are referring to the measured affinities for the construct towards mouse Tfr (see question 1 where dFab has 5.3-8.9 times higher affinity than sFab), then it makes sense. Otherwise I would have said that sFab has higher affinity than dFab based on these numbers.

One more aspect I find strange:

In my opinion, in (J), the yellow bars should also appear because the way it looks, it seems to indicate that the construct only returns to the lumen, i.e. it exits the cell only in one direction. Why should there be a preference for exiting the cell in only one direction?

Edit:
After discussing this issue with fellow collegues, the following explanation arose. The cell layer is incubated with sFab/dFab for one hour. Then both the luminal and abluminal compartments are flushed. Any sFab or dFab that appears in either compartment after that has to be coming from within the cells. Since the cargo enters the cell on one side, it has to travel a distance through the cell, but because this is a diffusion process, it has no preference as to what direction it takes. But because the cell has a sorting mechanism, the dFab cargo gets filtered out while it is crossing the cell. That's why no yellow bars appear for dFab. dFab however does also appear in the luminal side, that's because this distance is too short for the cell to sort it out before dFab can exit the cell again on the side it entered. That's also the reason why the luminal bars for sFab are slightly higher than for the abluminal side. The spatial distance within the cell simply requires more time to cross.

An in vivo image of the vessels after 15 min and 8 h of exposure to the construct shows what happens.

http://www.cell.com/action/showImagesData?pii=S0896-6273%2813%2901035-0

In the beginning, both constructs occupy the vessel cells (A, B). After 8 hours though, only sFab has crossed the vessel cells, i.e. they are not illuminating any longer (C), dFab has remained in the cells (D). In (C), the arrows point to accumulations of the construct on Amyloid-beta plaque aggregates.

6 Does the observed difference in BEC crossing between sFab and dFab depend on the rate at which they enter the BECs?

http://www.cell.com/action/showImagesData?pii=S0896-6273%2813%2901035-0

No.

7 Which construct binds more to Amyloid-beta plaque?

http://www.cell.com/action/showImagesData?pii=S0896-6273%2813%2901035-0

The measured increased plaque binding by sFab is therefore causal attributed to the fact that sFab binds to Tfr only by one Tabf.

8 Does sFab cover plaque faster than only mAb31 even at lower concentration?

http://www.cell.com/action/showImagesData?pii=S0896-6273%2813%2901035-0

Yes. The concentration of the antibody alone (light blue is 5 times higher than green) when not having a Tabf attached does not provide any activity. Duration of exposure does not change this, in other words the blood-brain-barrier remains closed no matter how high the concentration.

9 Is there a therapeutic effect of the construct?

Yes. The baseline shows the plaque number when the mice are 4.5 months old. Then, during 14 weeks, only the linker and the Tabf (vehicle), only the therapeutic antibody (mAb31) and the sFab are injected.

The plaque concentration increases in all mice, but when treated with sFab it increases at a lower rate.

10 Remarks

I would like to better understand the experiment from question 5. I  would expect that the next thing to do is to study dependence of therapeutic effect on amount of construct administered. What are the elimination rates of the construct? Interferences with other mechanisms? How stable is the construct? What is the exact nature of the cell sorting mechanism?
Also, in case you're interested, you can find the patent for this vehicle is WO 2014/033074.

A number of questions were raised during the discussion:
- If the cell sorting mechanism could be explained, maybe also dFab could be used as a carrier. It has some benefitial characteristics over sFab. Is this possible?
- If I was to manage this project, what would I do if it turns out that the shuttle is not linked strongly enough to the cargo, meaning it lets it's cargo go and picks up something, possibly toxic, else and carries it to the brain. I would say this shuttle is too important and therefore the engineering efforts have to be directed towards making the shuttle more strongly attached to the cargo.
- One short-coming of the study, in order to prove that it is not dependent on the type of cargo, is that no other cargo was used. This would have demonstrated independence of cargo nature. Of course, this would require a major additional effort.

Some out of context notes.

I was inspired to do this summary because I wanted to see how well I can understand and communicate a study from a totally different field. I believe it's important to be open to other fields and especially to be able to understand experimental approaches. Initially, the biology specific terms were difficult to understand but after a while I think I could identify the more important aspects.

Also, it seems sometimes that because of all the pharma-bashing, one doesn't notice that these guys actually do really amazing work.

Disclaimer: I have nothing to do with this research

Coursera Certificate

My first coursera certificate. Lot's of work, but totally worth the effort.

Papers and Thesis

CalB Paper 1 PLOS ONE:
http://www.plosone.org/article/info%3Adoi%2F10.1371%2Fjournal.pone.0049849

CalB Paper 2 PEERJ:
https://peerj.com/articles/145/

BCX Paper PEERJ:
https://peerj.com/articles/111/

BIOFET-SIM Online Paper PLOS ONE:
http://www.plosone.org/article/info%3Adoi%2F10.1371%2Fjournal.pone.0045379

BIOFET-SIM Online Chapter Nanoscale Sensors:
http://link.springer.com/chapter/10.1007/978-3-319-02772-2_3

PhD Thesis:
http://www.scribd.com/doc/213075474/MH-Thesis

PhD Defence Slides:
http://www.scribd.com/doc/213105271/Presentation-Defence