Peer review of: A nanobody-based fluorescent reporter reveals human α-synuclein in the cell cytosol (2019)

Reviewers' comments: 
 
Reviewer #1 (Remarks to the Author): 
 
The authors show that the nanobody with specificity towards α-synuclein has a cross reactivity to 
an amino acid stretch that also occurs in Rpn10, a protein at the entry of the proteasome. As a 
consequence this nanobody tagged with fluorescent protein (FP) is only short lived in cells (rapidly 
degraded by the proteasome) unless α-syn is also present in these cells (or if proteasomal activity 
is blocked with MG132). 
This finding is exploited to generate a sensor for scoring the intracellular presence of human α-syn 
either produced inside the cells or transmitted inside the cell, by various pathways. The sensor 
(reporter cell) is specific for the α-syn (rat or human) as the epitope seems to be conserved as 
well as the conserved epitope of Rpn10 that cross reacts with this nanobody. These reporter cells 
also detect any transmittable form of the human α-syn, such as that in human CSF. A minor 
criticism is at its place: the correlation among the amount of α-syn in the CSF and the signal in the 
reporter cells is not obvious from the manuscript. 
Furthermore, although the findings descried in these report are of general interest, it is not clear 
whether this reporter or screening system could be broadened to other targets beside α-syn (by 
making use of bispecific nanobody constructs). Probably other monomeric nanobodies with other 
epitope specificity will not work as they will not cross react to the rpn10 or any other proteasome 
epitope. 
Finally, a discussion is missing on the general sensitivity of intracellular nanobodies to degradation 
if their target is absent. It was indeed shown by Keller BM (group of Rothbauer U; papers in Mol 
Cell Proteimoics and Antibodies) that intracellular nanobodies are rapidly degraded in the cytosol 
(probably independent on targeting the Rpn10 peptide). 
 
 
 
Reviewer #2 (Remarks to the Author): 
 
Gerdes et al describe a nanobody-based reporter that they have developed for the detection of 
monomeric forms of alpha-synuclein. They show that NbSyn87 may be stabilized by binding to 
human aSyn, preventing its degradation in the proteosome, to which it is otherwise recruited by 
weak binding to Rpn10. 
 
I have two major comments, one related to the use cases for this reporter, and one related to the 
experiments reported in this paper. 
 
1. As shown in Figure 6, FluoReSyn detects primarily monomeric forms of human aSyn (no signal 
with introduction of haSyn fibrils). It is not clear exactly what the use case of such a reporter 
might be – in most disease contexts, it is the detection of pathological forms of haSyn that is most 
valuable. 
 
2. With respect to the experiments reported here, the only context in which any type of dose curve 
is shown is in Figure 1, where variable amounts of haSyn plasmid are transfected into HEK293 
cells, and higher levels appear to result in a higher % of positive reporter cells. However, as shown 
in Figure 6c, variability under the same conditions is pretty large – replicates treated with haSyn 
and iMAX show anywhere from 10% to 35% FluoReSyn+ neurons. This assay is really only useful 
if there is some ability to quantify aSyn, which is minimally shown by the authors (with some data 
suggesting that in most use contexts, variability is too high for meaningful quantification). 
 
 
 
 

Reviewer #3 (Remarks to the Author): 
 
This is a very interesting paper, with a significant number of strong experiments to support the 
conclusion that a fluorescent fusion of the NbSyn87 nanobody can serve as a reporter for the 
presence of human a-Syn in cellular cytoplasm. The range of assays used to measure both target 
that is over-produced in transfections, as well as penetration of exogenously introduced protein, 
form a convincing series. Methods descriptions seem very detailed. 
 
The authors have acknowledged in the discussion that there are multiple variables of cell lines, 
culture conditions, and protocols used to produce a-Syn for the extracellular experiments. It might 
be worthwhile to explore these questions in a bit more detail, since they are very critical to the 
interpretation of the results. Not all readers will have the time or access to look up the cited 
papers. The 7-line final sentence makes it clear that the authors plan to do major optimizations, 
presumably including much more neuronal cell lines as more faithful reporters. Their more explicit 
expert opinion on which directions to prioritize could be a useful addition to the field. 
 
The only experiment that seems to have been incompletely reported is the one testing the human 
CSF from a range of disorders. While it is clear that the HEK293-derived reporter line is more proof 
of general concept than definitive, it would still be useful to see the actual distribution of the 
different diseases in this assay. Since the data are presented individually, it would seem possible 
to do this, in which case the scatter plot would become a regular, rather than a supplemental, 
figure.