α-Synuclein Preformed Fibrils Bind to β-Neurexins and Impair β-Neurexin-Mediated Presynaptic Organization

Round 1

Reviewer 1 Report

This is a well written and well executed manuscript. The topic is interesting and results are solid and clearly designed. The authors:

I) Screened synaptic organizing molecules for interaction with α-synuclein preformed fibrils and isolated neurexin 1β 

II)Performed cell surface protein binding assays confirming that α-synuclein PFFs, but not α-synuclein monomers, bind directly to neurexin 1β

III) Identified the β-isoforms (but not the alpha) of neurexin as α-synuclein PFFs interactors

IV)Identified the N-terminal histidine-rich domain of β-neurexins as responsible for the binding of α-synuclein PFFs 

V) Confirmed that neuronal overexpression of NRX1β enhanced the binding of α-syn PFFs on the axon surface in an NRX1β HRD-dependent manner

VI) Functionally evaluated the effect of α-syn PFF treatment on β-NRX-mediated presynaptic organization.  

This manuscript was a pleasure to read and should be accepted in the present form.

Author Response

First, we thank all the reviewers for their careful reviewing with many positive comments and very helpful and constructive suggestions. We performed additional experiments to answer the reviewers’ comments and to strengthen our conclusions. These new data have been integrated into our revised manuscript. Below are point-by-point responses in bold font with the reviewers’ comments in italic font.

Reviewer 1: Comments and Suggestions for Authors

This is a well written and well executed manuscript. The topic is interesting and results are solid and clearly designed. The authors:

1. Screened synaptic organizing molecules for interaction with α-synuclein preformed fibrils and isolated neurexin 1β 
2. II)Performed cell surface protein binding assays confirming that α-synuclein PFFs, but not α-synuclein monomers, bind directly to neurexin 1β

• III) Identified the β-isoforms (but not the alpha) of neurexin as α-synuclein PFFs interactors

1. IV)Identified the N-terminal histidine-rich domain of β-neurexins as responsible for the binding of α-synuclein PFFs 
2. V) Confirmed that neuronal overexpression of NRX1β enhanced the binding of α-syn PFFs on the axon surface in an NRX1β HRD-dependent manner
3. VI) Functionally evaluated the effect of α-syn PFF treatment on β-NRX-mediated presynaptic organization.  

This manuscript was a pleasure to read and should be accepted in the present form.

Response: We thank the reviewer very much for the careful review of our manuscript and very positive comments.

Reviewer 2 Report

In this paper, Feller et al. has described α-synuclein preformed fibrils can bind to β-neurexins and impair β-neurexin-mediated presynaptic organization. It is interesting, but there are some following problems.

1.     Is there a larger magnification in Figure 1A? Because the fiber structure after sonication is not clear enough to see the structure clearly. In addition, what is the size of the ruler?

2.     In Figure 1B, "," should be ".". In addition, what is the value of PDI?

3.     In Figure 1D, it is suggested to supplement the results of WB to further verify the increase of phosphorylated α-synuclein.

4.     Does biotin labeling of α-syn PFFs affect the structure and characteristics of α-syn PFFs? Has the author tested the α-syn PFFs structure after labeling?

5.     Will the binding of α-syn PFFs and NRX1β be affected by biotin labeling? In Figure 2, it is suggested to further use immunoprecipitation (IP) to verify the interaction between α-sy PFFs and NRX1β. In addition, it is suggested to further verify the interaction between α-sy PFFs and NRX1β by using the primary neurons with endogenous expression of NRX1β.

6.     In the result description part, the author mentioned that the primary hippocampal neurons were used for the experiment, but the results of primary hippocampal neurons were not seen in Figure 7. Moreover, the results of primary hippocampal neurons will be more convincing.

7.     In Figure 7, the author is suggested to further supplement the WB results of VGAT and VGLUT1.

8.     From the morphology of COS-7 cells in the result figures, the morphology of cells is abnormal. Did the author conduct nuclear staining? What is the shape of the nucleus? Has the author carried out the detection and analysis of cell activity? Does α-sy PFFs damage cells first and then bind to NRX1βon the damaged cells? Or does it bind to NRX1β on normal cells first and then play its role？

Author Response

Please see the attachment.

Author Response File: Author Response.pdf

Reviewer 3 Report

Manuscript entitled “α-synuclein preformed fibrils bind to β-neurexins and impair 2 β-neurexin-mediated presynaptic organization” It is an interesting manuscript, which is describing the importance of presynaptic adhesion molecules B-NRXNs in the pathophysiology of Parkinson’s and Alzheimer’s disease. However I still have minor suggestions.

Authors should include the graphical abstract for a better and more comprehensive understanding.

 

In the discussion section authors also add some information about how NRXN-1B affects not only NLGNs but also the other interacting partners like glutamate delta receptors.

Author Response

First, we thank all the reviewers for their careful reviewing with many positive comments and very helpful and constructive suggestions. We performed additional experiments to answer the reviewers’ comments and to strengthen our conclusions. These new data have been integrated into our revised manuscript. Below are point-by-point responses in bold font with the reviewers’ comments in italic font.

Reviewer 3: Comments and Suggestions for Authors

Manuscript entitled “α-synuclein preformed fibrils bind to β-neurexins and impair 2 β-neurexin-mediated presynaptic organization” It is an interesting manuscript, which is describing the importance of presynaptic adhesion molecules B-NRXNs in the pathophysiology of Parkinson’s and Alzheimer’s disease. However, I still have minor suggestions. 

1. Authors should include the graphical abstract for a better and more comprehensive understanding.

Response 1: We agree that a graphical abstract would be useful for a better and more comprehensive understanding at glance, however we decided not to add such a graphical abstract since sometimes it can overestimate presented data and/or mislead readers to biased data interpretation. Instead, we added a schematic illustration showing the binding of α-syn PFFs with NRX1β through its HRD in Figure 5G.

 

2. In the discussion section authors also add some information about how NRXN-1B affects not only NLGNs but also the other interacting partners like glutamate delta receptors.

Response 2: In the discussion section at page 19-20, we added the following additional description about whether and how NRX1b affects other NRX-interesting synaptic organizers. 

“In addition, for their physiological roles, β-NRXs bind not only NLGNs but also some other synaptic organizers such as LRRTM1/2 and cerebellin-glutamate delta receptor complexes [17,21,82,83]. Each distinct β-NRX-interacting organizer has some shared and distinct roles in synaptic functions such as the regulation of AMPA (α-amino-3-hydroxy5-methyl-4-isoxazole pro-pionic acid) and NMDA (N-methyl-d-aspartate) receptors in synaptic transmission and plasticity [84-86]. Given that α-syn PFF treatment impaired excitatory synaptic transmission and plasticity [55,78,87], future studies will be important to address whether and how α-syn PFFs affect the physiological roles of these β-NRX-interacting synaptic organizers.”

Round 2

Reviewer 2 Report

The author has answered most of the reviewer's questions.