Peer review of: Synucleinopathy alters nanoscale organization and diffusion in the brain extracellular space throu (2020)

Reviewers' comments: 

Reviewer #1 (Remarks to the Author): 

This paper uses cryo-fixed electron microscopy in combination with super-resolution tracking of 
single-walled carbon nanotubes (SWCNTs) to study the extracellular space (ECS) in normal and 
alpha-synuclein induced neurodegeneration in the mouse substantia nigra (SN). Using several 
manipulations, including Lewy body (LB) injection, hyaluronidase and chronic administration of 4-
methylumbelliferone, together with a variety of histological and analytical approaches, it is shown 
that the hyaluronan (HA) component of the extracellular matrix (ECM) plays a major role in the re-
organization of the ECS under these pathological conditions. 

The paper represents an important further advance in the exploration of the ECS on the nanoscale 
and may contribute to clarifying some of the mechanisms that underlie Parkinson’s disease. The 
use of SWCNTs is an emerging technique and is revealing information about the ECS that cannot 
be obtained with the older diffusion methods. There may be opportunities to extract additional 
information in the present study by comparison with earlier studies measuring diffusion on a 
macroscopic scale. 

The techniques are in general use but the super-resolution tracking of SWCNTS would only be 
possible in a few laboratories at this time. 

Major points 

This paper employs a multifaceted attack on the nanoscale organization of the ECS. The most 
novel aspect is the use of SWCNTs to directly probe the diffusion characteristics of the ECS. This 
approach has been used by some of the present investigators in two other recent publications. 
Cryo-fixation for electron microscopy of the ECS has been employed by other groups in a few 
studies but only now is becoming a fairly routine procedure. The numerous other techniques are 
established and appropriate. Thus one may see the study as having three aspects (a) continuing 
validation of the SWCNT technology (b) a study of the effect of HA modification on ECS structure 
(c) a contribution to understanding the increasingly complex etiology of Parkinson’s and Lewy Body 
diseases. This review will focus mainly on topic (a). 

The SWCNT method is yielding important new insights into the ECS and this study is a further 
valuable contribution. The authors show that both cryo-fixed EM and SWCNT give comparable 
distributions for the width of the ECS and cryo-fixed EM also indicated that the volume fraction in 
substantia nigra is about 20%. Most measurements of ECS volume fraction in the brain with 
macroscopic methods, such as the Real-Time Iontophoresis (RTI) method, do give values around 
20% so the present results would be validated. But the volume fractions of the SNc and SNr have 
been measured in guinea pig brain slices using the RTI method (Cragg et al., J. Neurophysiol. 85: 
1761-1771, 2001) and in both regions volume fraction was unusually high at 30%. Perhaps this 
value is unique to the guinea pig; however the same study found a volume fraction of 22% in the 
guinea pig cortex, which is comparable to values in rat and mouse. Questions will remain until RTI 
measurements in the SN of the mouse are available but in the meantime: How confident are the 
present investigators in their estimates of volume fraction? 

A second feature revealed by the SWCNT measurements is that the median relative diffusivity in 
the normal SN is 0.026 and that in the LB-exposed SN is 0.042 (page 7). Translating these values 
into tortuosity for comparison with prior studies one obtains tortuosity values of 6.2 and 4.9 
whereas the tortuosity obtained in the normal guinea pig SNc and SNr were 1.58 and 1.69 
respectively (see Cragg reference above). For comparison the guinea pig study found a tortuosity 
of 1.59 in the cortex. Crucially, those tortuosity values were obtained with the small 
tetramethylammonium molecule and are similar to numerous other measurements in brain tissue 
with this molecule (see Ref. 1). The huge values of tortuosity seen in the present study are only 
comparable to the value obtained using the Integrative Optical Imaging (IOI) method with a 
quantum dot of 35 nm diameter in the rat cortex (Thorne & Nicholson, PNAS 14: 5567-5572, 
2006). The quantum dot tortuosity was interpreted using restricted diffusion theory to indicate 
significant interaction of the particle with the walls of the ECS and this view was consistent with 
the observation that tortuosity rises as a function of molecular diameter for a range of 
macromolecules (see Ref. 7). Assuming the free diffusion values have been calculated correctly 
here (page 21), there is no doubt that the large tortuosities (small relative diffusivities) obtained 
with SWCNTs represent major hindrance of the nanotube compared to a typical small molecule. If 
this hindrance arose from interaction with walls of the ECS one might expect the values to 
decrease with an expanded ECS, which is reported here. Alternatively, the hindrance might be 
caused by interaction with the ECM and then the hindrance following modifications to the HA could 
clarify the situation. There may be an opportunity here for some useful further consideration of the 
present data. 

There is also limited comment on resolution. Figs. 2F and 6e have colored calibration bars that go 
down to 0 nm yet there has to be a practical limit to the width of a channel that SWCNT can 
explore. This is more pertinent given the large hindrance experienced by the nanotubes which 
suggests there could be regions of the ECS that nanotubes are unable to access. 

The manipulations of HA collectively make a convincing case for an important role for this 
glycosaminoglycan in structuring the ECS. Exactly how it does so is still to be determined. As the 
authors note, the present results do not completely accord with the recent study of Arranz et al 
(Ref 39), which reinforces the point that the elucidation of mechanisms is just beginning and these 
nanoscale tools are likely to play a major role. It is very important therefore to continue to refine 
and validate these tools. 

The relation of the ECS to Parkinson’s disease is more tenuous. The enhanced spread of toxic 
proteins may indeed be a factor (page 14) but it is difficult to estimate its importance without a 
quantitative model. Over the years, many studies have shown changes in ECS properties that 
correlate with pathophysiological manipulations (see Ref. 1) but strict causality has remained 
elusive. 

Minor points 

Title: Possibly change to: “Synucleinopathy in substantia nigra alters…”. 
Abstract. Line 26. “reveal” may be better than “divulge”. 
Page 3, line 49. Omit “prevailing”. 
Page 3, line 51. Change “yet” to “still”. 
Page 3 lines 46 – 47 and 52. A major function of the ECS is to provide a reservoir of ions to 
support the membrane potential and action and synaptic potentials. Thus the major constituent is 
probably NaCl. 
Page 3 line 56. It is not clear that the ECM functions as a “space-filler” because large 
macromolecules and quantum dots can diffuse through it. The precise form taken by the matrix is 
unknown but it might more resemble a “hydro-gel”. It may have a role in maintaining the patency 
of the ECS. 
Page 4, line 79 “a first”. 
Page 4, line 84. Substitute “integrated” for “integral”? 
Page 5 line 100 may be better to say ”…difficult to implement…”. 
Page 5 line116. See comment above about volume fraction of ECS in SN. 
Page 7, Line 168-170. It is not clear what aspect of “diffusion” is being compared here. Elsewhere 
it appears that there is a correlation between ECS dimensions and diffusion. The use of the word 
“anisotropy” here may be confusing; see remarks about anisotropy elsewhere in this review. 
Page 10 line 242. Define “PK”. 
Page 10, line 252. Replace “respect” with “in comparison to”? 
Page 11 line 274. Change “alter” to “alters” 
Page 12 line 296. “live brain slice” 
Page 12 line 301. Not clear what the authors want to signify by “founding stone”. “…a foundation 
for…”? 
Page 12 line 304. Volume fraction in pathological states is more extensively described in Ref 1. 
Page 12 line 313. Probably “analogous” better than “analog”. 
Page 12 line 313. The phrase used in Refs. 6 and 37 was “sheets and tunnels”. 
Page 13, line 327. If we define anisotropy as being a macroscopic property characterized by a six-
component symmetric tensor representing the effective diffusion coefficient (usually assume to 
have only three components because measurements are taken in the principal axes) then the 
brain is not always anisotropic. Much of the cerebral cortex is isotropic (hence its popularity for 
measurements); verified examples of anisotropy occur in the molecular layer of the cerebellum 
and the corpus callosum. It might be possible but challenging to measure anisotropy on the 
nanoscopic scale. What is evident here perhaps is local inhomogeneity or channeling on a scale of 
a few micrometers. 
Page 13 line 328. The more recent contribution about viscosity of the ECS from the Rusakov lab 
might also be cited here (Ref. 3). 
Page 13 lines 331-332. This is a subtle issue. If the hindrance is largely caused by interaction with 
the ECS boundaries, as may be the case for SWCNTs, then restricted diffusion theory says that 
relative diffusivity will increase as the walls move further apart. On the other hand, a small 
molecule will have negligible interaction the walls and an increase in ECS width may only bring 
about a small change in relative diffusivity. Consider the very simple case of an ECS formed by an 
ensemble of cubic cells (Tao & Nicholson, J. Theoret. Biol.229: 59 – 68, 2004). The relative 
diffusivity for a vanishingly small molecule is given by 2/(3 – alpha). Assuming that alpha tracks 
ECS width then if alpha goes from 0.2 to 0.3, representing a 50% increase, relative diffusivity only 
goes from 0.71 to 0.74 – a 4% increase. 
Page 13 line 343. For the reason noted above, there may be some confusion about the use of the 
word “anisotropic” here. The anisotropic diffusion described in both of the cited references (Refs. 1 
and 23) was of the macroscopic variety. Here it appears that the authors are talking about a local 
channeling of substances in the ECS. Note that extrasynaptic communication is not dependent on 
(macroscopic) anisotropic diffusion. It could equally well occur in a macroscopically isotropic 
medium. Even in a strongly anisotropic tissue, such as the molecular layer of the cerebellum, 
molecules do diffuse in all three axes. Bulk flow in the ECS (if it occurs) probably will be vectorial 
but even then there will be a diffusive component perpendicular to the flow. It is, however, 
intriguing to speculate about how HA or other ECM components might channel molecular 
movement on the nanoscopic scale. 
Page 14, line 353 Change “found” to “find”. 
Page 15 line 387. There is perhaps no need to introduce the term “extracellular compartment” but 
rather to go back 50 years to a remarkable volume in the Neuroscience Research Program Bulletin 
series entitled “The Brain Cell Microenvironment” (Schmitt and Sampson, NRP Bull. 7: 277 – 417, 
1969). Sadly, this publication is no longer easily available. This volume was way ahead of its time 
in focusing on the role of the ECM in brain function and coining the term “brain cell 
microenvironment”. This term has been used in a number of papers about the ECS. Perhaps “brain 
extracellular microenvironment” might be even better. 
Page 18 line 471 Change “and interactive” to “an interactive”. 
Page 19. What was the length distribution of SWCNTs? 
Page 21 lines 533-534. “…equations in which that frame participates.”? 

Figure 1. Panels f and g: ordinates mislabeled – they are cumulative frequency. 
Figure 1. Panel e: the volume fraction is based on n = 3 mice which seems a bit low for a 
statistical test. The actual measuring procedure in each mouse presumably takes place over many 
sections so the n value may be a bit misleading. Identifying the ECS in electron micrographs is 
sometimes challenging. 
Figure 1. Here in the histograms in panel i and also in Figure 2 panels d and g, as well as Figure 6, 
panels g and i, it is difficult to see the rose-colored bars against the gray. 
Figure 2. Panels c and d. Presumably the short distance over which trajectories are imaged – a few 
micrometers – ensures that the nanotubes remain in or close to the focal plane of the microscope, 
so no nanotubes are lost by going out of the plane. In panel f the dimension scale goes to 0 nm – 
what is the lower limit of the method? There are some indications in the text that it is around 50 
nm. The statement about anisotropy should be considered in the light of earlier comments in this 
review. 
Figure 3. Ordinate in panel d mislabeled: it is cumulative frequency. 
Figure 5. Second line perhaps should read “… lasts only a few days…”. 
Figure 6. Again panel e shows 0 nm scale bar value. 

Reviewed by: 

Charles Nicholson 

Professor Emeritus 
NYU School of Medicine 



Reviewer #2 (Remarks to the Author): 

This is a potentially important, ground-breaking body of work. The authors address a hitherto 
underappreciated aspect of Parkinson’s disease; the role of extracellular space and matrix (ECS 
and ECM) in the aetiology of the synuclein-driven neurodegenerative process in the substantia 
nigra. It is also a strength that they report a successful attempt to reverse the neurodegenerative 
process by interfering with these ECM mechanisms, so suggesting the potential therapeutic 
importance of the findings. 
The work employs a multi-disciplinary set of techniques to address this issue from multiple 
directions. The techniques employed, from animal model to analysis of structure and diffusion 
properties etc of ECM are the most appropriate available, being state-of-the-art and avoiding many 
limitations of alternative approaches. 
Many of the findings appear robust and overall the authors present a compelling argument to 
support their hypothesis. Thus, the data on, for instance, width and diffusion maps of the 
extracellular space, microglial activation, the degradation of the hyaluronan matrix, cytokine levels 
and neuroprotective effect of hyaluronidase are compelling. In themselves these are sufficient to 
make the manuscript of interest and could be highlighted better in the Abstract. 
However, a weakness of the work is that not all measures are evaluated with the same level of 
rigour. Some aspects of the work seem underpowered, and so conclusions are uncertain, at other 
times, the extrapolation of conclusions to broader populations of animals is uncertain. Specifically, 
• Fig 1e, a trend towards an increase in %ECS in Lewy body treated animals is seen. The analysis 
appears to only employ N=3, if a larger group were used, it is likely that a clearer conclusion could 
have been drawn. If this were significant, it would be a simple and easily appreciated measure to 
open the argument for the role of ECS changes in PD. 
• In the K-S analyses, e.g. Fig 1f and g, and elsewhere; although several hundred datapoints are 
entered into the analyses, these only come from very small numbers of animals in each treatment 
group, for instance N=3 in the case of Fig 1f. While the significance suggests that it can be stated 
that the 3 animals in one group likely had a distribution dissimilar to the 3 animals in the other 
group, this does not mean that it is so likely that populations of animals treated in this way would 
be significantly different. The sample is a sample of the two groups of three animals not a sample 
of the two treatment populations, and thus the statistics are powered for, and assess that 
comparison. 
• The data on the ECM structure in the face of chronic HA reduction are very interesting and could 
add significantly to the ability to draw conclusions as to cause and effect of ECS dysfunction in 
neurodegeneration. However, the authors relegate the data showing the effect of this chronic HA 
reduction on neurodegeneration to the Supplementary materials. This is understandable as the 
data are inconclusive, but again suffer from low N, N=4. If this aspect of the work were properly 
powered to show a change in neurodegeneration, and a clear effect of treatment were seen, or 
not, then the conclusions to be drawn would be stronger. 
The authors use the human-derived Lewy body extracts to induce synucleinopathy. This is a 
reasonable choice and one of several available models that can produce robust synucleinopathy 
and degeneration in animals. However, one limitation of the model lies in the ability of the 
community to reproduce the findings reported. This limitation stems mainly from the availability of 
LB extract, either from the same source as used in these studies or from other sources that can be 
prepared in a way that that produces verifiably-similar protein material. It would thus have been a 
significant enhancement of the work had they shown replication of some of the key findings with 
another model of synucleinopathy. Of course, an orthogonal approach to modelling PD, would have 
further demonstrated the robustness, and likely translatability, to the disease, of the conclusions. 



Reviewer #3 (Remarks to the Author): 

Soria et al have used electron microscopy in cryofixed tissue and single nanotube tracking in live 
brain tissue to study extracellular space (ECS) in healthy and pathological adult mouse brain. They 
show that alpha synuclein-induced neurodegeneration increased the length and width of local 
“pools” of ECS, increased the level of nanoscale diffusion and was linked to the levels of 
hyaluronan. The results and conclusions are clearly presented, and the findings are novel and of 
interest to many in the field. 

The manuscript would benefit from the suggested clarifications below, especially regarding the role 
of hyaluronan in ECS size. 

Major comments: 

1. Another ECM component should be analysed alongside hyaluronan to ensure that the effect 
observed is specific to hyaluronan and not a general effect on the ECM as a whole. This should 
include staining and quantification of other ECM components after synuclein-induced 
neurodegeneration and the use of another ECM-degrading enzyme for the Hyase experiments. 
Currently, the control used is injection of PBS. Injection of an ECM-degrading enzyme that targets 
another component of the matrix present would show if the neuroprotective effects observed are 
due to hyaluronan levels, or simply ECM remodelling. 
2. The authors state that small MW-HA or hyaluronan breakdown has a neuroprotective effect, as 
injection of HMW-HA did not show an effect. LMW-HA/HA fragments should also be injected to see 
if the presence of LMW-HA is sufficient for the neuroprotective effect, or if the endogenous HMW-
HA must be degraded. 
3. No neuroprotective effect was shown for 4-MU at 4 months, unlike Hyase. Is there a 
neuroprotective effect of 4-MU at an earlier time point? That is later lost due to a decrease in 
Iba1+ microglia levels over time? Levels of microglia activation should be analysed at an earlier 
stage (when 4-MU would be expected to start reducing HA levels) to assess if 4-MU alters 
microglia levels/activation as Hyase did at 72 h. The levels of other ECM components present in 
the matrix should also be analysed in these animals to see if that may explain the lack of 
neuroprotection, as should the levels of the Hyaluronidases. 

Minor comments: 

1. The authors state that the width and length of local ECS “pools” is increased, but the total 
volume of ECS space is not. This is a little contradictory. The total number of pools should also be 
quantified, as the images in Fig. 1h appear to show there are fewer, larger “pools” present after 
synuclein-induced neurodegeneration. 
2. There are several methodological details that should be included in the results. How long was 
the imaging of the SWCNTs performed for? How was the mRNA expression analysed? Including 
this information in the results as well as the methods will enable the reader to follow the 
experiments outlined more easily. 
3. Microglia levels appear to have been quantified by the area of Iba1 staining (Fig 4c), yet Iba1+ 
soma are used for another quantification (Fig 4f). Iba1+ soma should also be analysed when the 
level/number of microglia are quantified (Fig 4c, 5j). 
4. Supplementary videos of SWCNTs in synuclein-induced neurodegeneration brain tissue and the 
Hyase condition should also be included.