Megalencephalic leukoencephalopathy with subcortical cysts is a developmental disorder of the gliovascular unit
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Evaluation Summary:
This study shows how astrocytic MCL1 can contribute to postnatal maturation of the brain vascular system. Since the development and physiological roles of perivascular astrocyte coverage are not well understood, this manuscript provides potentially important frame works and should be of interest to the broad fields of neuroscientists.
(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #1, Reviewer #2 and Reviewer #3 agreed to share their names with the authors.)
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Abstract
Absence of the astrocyte-specific membrane protein MLC1 is responsible for megalencephalic leukoencephalopathy with subcortical cysts (MLC), a rare type of leukodystrophy characterized by early-onset macrocephaly and progressive white matter vacuolation that lead to ataxia, spasticity, and cognitive decline. During postnatal development (from P5 to P15 in the mouse), MLC1 forms a membrane complex with GlialCAM (another astrocytic transmembrane protein) at the junctions between perivascular astrocytic processes. Perivascular astrocytic processes along with blood vessels form the gliovascular unit. It was not previously known how MLC1 influences the physiology of the gliovascular unit. Here, using the Mlc1 knock-out mouse model of MLC, we demonstrated that MLC1 controls the postnatal development and organization of perivascular astrocytic processes, vascular smooth muscle cell contractility, neurovascular coupling, and intraparenchymal interstitial fluid clearance. Our data suggest that MLC is a developmental disorder of the gliovascular unit, and perivascular astrocytic processes and vascular smooth muscle cell maturation defects are primary events in the pathogenesis of MLC and therapeutic targets for this disease.
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Author Response:
Reviewer #1 (Public Review):
In this manuscript by Gilbert et al., the authors found that MLC1 is required for postnatal maturation of perivascular astrocyte coverage. Through various detailed experiments, they further found that Mlc1 KO mice showed a number of defects, including the reduced VSMC contractility, neurovascular coupling and parenchymal CSF flow. The data is well presented, however there are several points that need to be addressed to strengthen the manuscript.
- Since many PvAP proteins showed the normal expression after P60 in Mlc1 KO mice, it is also possible that many of the phenotype that the authors presented, such as the reduced VSMC contractility, neurovascular coupling and parenchymal CSF flow, can be recovered after P60. This would be important as well to understand the prime pathological cause …
Author Response:
Reviewer #1 (Public Review):
In this manuscript by Gilbert et al., the authors found that MLC1 is required for postnatal maturation of perivascular astrocyte coverage. Through various detailed experiments, they further found that Mlc1 KO mice showed a number of defects, including the reduced VSMC contractility, neurovascular coupling and parenchymal CSF flow. The data is well presented, however there are several points that need to be addressed to strengthen the manuscript.
- Since many PvAP proteins showed the normal expression after P60 in Mlc1 KO mice, it is also possible that many of the phenotype that the authors presented, such as the reduced VSMC contractility, neurovascular coupling and parenchymal CSF flow, can be recovered after P60. This would be important as well to understand the prime pathological cause of megalencephalic leukoencephalopathy induced by MLC1 deletion.
Although some protein levels are normal in Mlc1 KO mice, PvAP coverage and gliovascular unit morphology are altered at P60. We also now show (using TEM) that PvAPs are swollen in 1-year-old Mlc1 KO mice (the new Fig. S4) - indicating that edema develops progressively in Mlc1 KO mice. Myelin vacuolation starts at 3 months and progressively worsens (Dubey et al., 2015). Taken as a whole, these data show that MLC is a degenerative disease. Under these conditions, the recovery of gliovascular unit function after P60 is very unlikely.
All the molecular morphological and functional changes in gliovascular unit described in our manuscript precede myelin degradation, which starts at the age of 3 months in Mlc1 KO mice (Dubey et al., 2015). Moreover, our previous study (Gilbert et al., 2019) demonstrated that MLC1 expression starts around P5 and that the MLC1/GlialCAM complex is only mature at P15. Taken together, these results strongly suggest that gliovascular unit alterations are the primary pathological events in MLC.
Dubey M, Bugiani M, Ridder MC, Postma NL, Brouwers E, Polder E, Jacobs JG, Baayen JC, Klooster J, Kamermans M, et al. 2015. Mice with megalencephalic leukoencephalopathy with cysts: a developmental angle. Ann Neurol 77: 114-131.10.1002/ana.24307. Gilbert A, Vidal XE, Estevez R, Cohen-Salmon M, and Boulay AC. 2019. Postnatal development of the astrocyte perivascular MLC1/GlialCAM complex defines a temporal window for the gliovascular unit maturation. Brain Struct Funct 224: 1267-1278.10.1007/s00429-019-01832-w.
- Does CBF get differed only after neuronal stimulation in Mlc1 KO mice? It is unclear whether the basal CBF/neurovascular coupling level is disrupted as well in Mlc KO brains and how this defect is related to the reduced vasoconstriction in these mice.
The baselines of the functional ultrasound experiments were aligned prior to stimulation. This technique measures the percentage increase in blood flow after neuronal stimulation (here, whisker movement) but does not measure the basal flow and does not enable one to distinguish between an abnormal basal cerebral blood flow (as suggested by the reduction of the arterial diameter) and the loss of vascular contractility - both of which probably contribute to the defect in neurovascular coupling.
- The reduced cohesiveness of PvAPs and the associated neuronal fibers to the vessel in Mlc1 KO brains should be validated with additional experimental approach.
To strengthen our analysis, we now give the results of a parallel quantitative immunofluorescence analysis of purified brain vessels (presented in a new figure, Fig.5). The results show that part of the Aqp4 and NF-M perivascular immunolabeling is absent in the Mlc1 KO. Taken as a whole, our data demonstrate that PvAPs and the associated neuronal fibers (which normally remain attached to brain vessels during mechanical purification) are lost during the purification process in Mlc1 KO mice but not in the WT. In conclusion, the absence of MLC1 reduces the mechanical cohesiveness of PvAPs and the associated neuronal fibers.
- The defective polarity of astrocytes should be better described by using other markers other than GFAP. The distribution of Aquaporin4, Cx43 or several glutamate transporters in the specific compartment of astrocytes can be examined.
GFAP is the marker typically used to analyze the astrocytes’ overall morphology and polarity. Nevertheless, we agree that it is of interest to study the molecular polarity of PvAPs. Indeed, morphological changes in the PvAPs and astrocytes and changes in polarity in Mlc1 KO might all influence the localization of molecules in PvAPs. To address this question, we performed a quantitative stimulated emission depletion (STED) analysis of protein localization in PvAPs. Our results indicate that the perivascular localization of aquaporin 4 was not affected. However, the density and size of Cx43 puncta were greater - indicating that the gap junctions in PvAPs are not organized in the same way in the Mlc1 KO as in the WT. This observation is consistent with our electron microscopy observations of perivascular astrocytic processes stacked on the top of each other and linked by extended gap junctions.
We have also added results for Kir4.1, a potassium channel that is expressed preferentially in PvAPs. The Kir4.1 expression level in Mlc1 KO was lower at all stages of development, indicating that perivascular potassium homeostasis was probably perturbed. These results are interesting because (i) epilepsy is a significant component of megalencephalic leukoencephalopathy (Dubey et al., 2018; Yalcinkaya et al., 2003), and (ii) Kir4.1 deletion or downregulation is associated with greater susceptibility to epilepsy (Sibille et al., 2014). These points are now discussed.
Dubey M, Brouwers E, Hamilton EMC, Stiedl O, Bugiani M, Koch H, Kole MHP, Boschert U, Wykes RC, Mansvelder HD, et al. 2018. Seizures and disturbed brain potassium dynamics in the leukodystrophy megalencephalic leukoencephalopathy with subcortical cysts. Ann Neurol 83: 636- 649.10.1002/ana.25190. Sibille J, Pannasch U, and Rouach N. 2014. Astroglial potassium clearance contributes to short-term plasticity of synaptically evoked currents at the tripartite synapse. J Physiol 592: 87- 102.jphysiol.2013.261735 [pii] 10.1113/jphysiol.2013.261735. Yalcinkaya C, Yuksel A, Comu S, Kilic G, Cokar O, and Dervent A. 2003. Epilepsy in vacuolating megalencephalic leukoencephalopathy with subcortical cysts. Seizure 12: 388-396.10.1016/s1059-1311(02)00350-3.
- The authors provide interesting observations such that the formation of perivascular astrocyte coverages is required for the dissociation of the contacts between neuronal components and the vessel during development. The authors need to discuss more about potential regulation and implication of this phenomenon.
This is indeed a fascinating phenomenon. The postnatal period is also an intense synaptogenic phase in the mouse brain (Chung et al., 2015), during which astrocytes and neurons might compete for the perivascular space. In the absence of MLC1 and thus PvAPs, the neurons might expand into the free space. We now comment on this point.
Chung WS, Allen NJ, and Eroglu C. 2015. Astrocytes Control Synapse Formation, Function, and Elimination. Cold Spring Harb Perspect Biol 7: a020370.cshperspect.a020370 [pii] 10.1101/cshperspect.a020370.
- It is interesting that DOTA-Gd tracer shows different traces in Mlc1 KO brains. However, it is unclear how MLC1 deletion affects glymphatic system. Does the tracer normally enter to the perivascular spaces in Mlc KO brains? Does the tracer leak out more from the perivascular spaces in Mlc1 KO mice? Is the general clearance or drainages of the tracer impaired in Mlc1 KO mice? Would these defects be originated by the reduced perivascular astrocyte coverage or the reduced vasoconstriction itself?
Paravascular transport (as revealed by the injection of a tracer into the CSF) depends mainly on dispersion of the tracer in the subarachnoid space (SAS), the cisternae, and the parenchyma (including the interstitial and perivascular spaces). The uneven, slow dispersion of the tracer within the SAS (compared with dispersion in the blood) means that the tracer’s kinetics in the parenchyma are regiondependent. These differences can be accentuated by regional differences in the anatomy of the brain’s vasculature, i.e. the presence or absence of a perivascular space and the vessel’s topology. Lastly, the amount of DOTA-Gd available for diffusion within the parenchyma depends directly on its local concentration in the SAS. This can be seen on our contrast concentration maps (see Fig. 8), where the highest DOTA-Gd concentrations are found near the injection site (the cisterna magna), in line with previous reports (Iliff et al., 2012). In the Mlc1 KO model, dispersion of DOTA-Gd is presumably affected in the SAS and the parenchyma.
With regard to tracer dispersion in the SAS and the cisternae, our anatomical MRI showed that the brain volume is greater in the Mlc1 KO mouse than in the WT (see Fig. 1). These variations in the geometry of the SAS may account for much of the difference between the Mlc1 KO mice and WT mice. Although tracer concentrations appear to be similar in the cerebellum (close to the injection site), they are much lower in the more distant septal area of Mlc1 KO mice - suggesting that tracer transport within the SAS is restricted.
With regard to parenchymal dispersion, we showed that MLC1 is essential for the position of the astrocytes’ perivascular endfeet. Thus, in Mlc1 KO mice, the formation of the perivascular space (as a conduit for solute distribution) is likely to be deficient. This aspect is revealed by the slope of the tracer’s concentration-time curve, which indicate slower kinetics in Mlc1 KO mice; this might be due to poor integrity of the perivascular space. The higher volume of fluid in the Mlc1 KO parenchyma (reflected by the increased apparent diffusion coefficient (ADC); Fig. 1 and S1) might also be involved in this phenotype.
The heart beat is the main driver of CSF circulation in the perivascular space (Iliff et al., 2013). The heart rate is very rapid and so the heart exerts a much greater driving force on the CSF than the vasodilation of the vessels induced by neuronal activity. Alterations in vascular contractility observed in Mlc1 KO mice might be involved in the impaired CSF flux but this is unlikely.
All these points are now discussed in the revised version of the manuscript.
Iliff JJ, Lee H, Yu M, Feng T, Logan J, Nedergaard M, and Benveniste H. 2013. Brain-wide pathway for waste clearance captured by contrast-enhanced MRI. Journal of Clinical Investigation 123: 1299-1309.10.1172/jci67677. Iliff JJ, Wang M, Liao Y, Plogg BA, Peng W, Gundersen GA, Benveniste H, Vates GE, Deane R, Goldman SA, et al. 2012. A paravascular pathway facilitates CSF flow through the brain parenchyma and the clearance of interstitial solutes, including amyloid beta. Sci Transl Med 4:147ra111.10.1126/scitranslmed.3003748.
**Reviewer #2 (Public Review): **
This very interesting manuscript by Gilbert and colleagues uncovers that the astrocyte specific membrane protein MLC1, the mutation of which causes a rare disease called megalencephalic leukoencephalopathy with subcortical Cysts (MLC), plays a fundamental role in the postnatal development of the gliovascular unit and the organization of the perivascular astrocyte processes, in particular. To reach this conclusion, the authors used an elegant multiscale approach including in vivo MRI, in vivo functional ultrasound, ex vivo analysis of vascular constriction, anatomical approaches at the light and electron microscopic level, and molecular characterization of the gliovascular unit from isolated microvessels. The manuscript is very well-written although it uses too many (unnecessary) abbreviations, which prevents a fluid reading of the manuscript, results are well illustrated and convincing and the discussion is reasonable.
I have a major concern regarding the results reported in Figure 4D, which seem somewhat contradictory to those shown in Figure 6A-F. Indeed, the authors report in Figure 4D that there is less Neurofilament-M protein around isolated microvessels in MLC1 KO mice, whereas Figure 6A-F shows that these animals have more neuronal processes in contact with the vessels than in wiltypes. How can the authors explain this?
The two situations are not comparable. On one hand, we observed the structure of the gliovascular unit in situ in fixed tissues. On the other, we mechanically purified microvessels. The detachment of astrocytic processes and associated neuronal fibers (linked to the mechanical dissociation of microvessels in the Mlc1 KO mouse) was clearly not counterbalanced by the presence of neuronal fibers contacting the vessels.
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Evaluation Summary:
This study shows how astrocytic MCL1 can contribute to postnatal maturation of the brain vascular system. Since the development and physiological roles of perivascular astrocyte coverage are not well understood, this manuscript provides potentially important frame works and should be of interest to the broad fields of neuroscientists.
(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #1, Reviewer #2 and Reviewer #3 agreed to share their names with the authors.)
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Reviewer #1 (Public Review):
In this manuscript by Gilbert et al., the authors found that MLC1 is required for postnatal maturation of perivascular astrocyte coverage. Through various detailed experiments, they further found that Mlc1 KO mice showed a number of defects, including the reduced VSMC contractility, neurovascular coupling and parenchymal CSF flow. The data is well presented, however there are several points that need to be addressed to strengthen the manuscript.
Since many PvAP proteins showed the normal expression after P60 in Mlc1 KO mice, it is also possible that many of the phenotype that the authors presented, such as the reduced VSMC contractility, neurovascular coupling and parenchymal CSF flow, can be recovered after P60. This would be important as well to understand the prime pathological cause of megalencephalic …
Reviewer #1 (Public Review):
In this manuscript by Gilbert et al., the authors found that MLC1 is required for postnatal maturation of perivascular astrocyte coverage. Through various detailed experiments, they further found that Mlc1 KO mice showed a number of defects, including the reduced VSMC contractility, neurovascular coupling and parenchymal CSF flow. The data is well presented, however there are several points that need to be addressed to strengthen the manuscript.
Since many PvAP proteins showed the normal expression after P60 in Mlc1 KO mice, it is also possible that many of the phenotype that the authors presented, such as the reduced VSMC contractility, neurovascular coupling and parenchymal CSF flow, can be recovered after P60. This would be important as well to understand the prime pathological cause of megalencephalic leukoencephalopathy induced by MLC1 deletion.
Does CBF get differed only after neuronal stimulation in Mlc1 KO mice? It is unclear whether the basal CBF/neurovascular coupling level is disrupted as well in Mlc KO brains and how this defect is related to the reduced vasoconstriction in these mice.
The reduced cohesiveness of PvAPs and the associated neuronal fibers to the vessel in Mlc1 KO brains should be validated with additional experimental approach.
The defective polarity of astrocytes should be better described by using other markers other than GFAP. The distribution of Aquaporin4, Cx43 or several glutamate transporters in the specific compartment of astrocytes can be examined.
The authors provide interesting observations such that the formation of perivascular astrocyte coverages is required for the dissociation of the contacts between neuronal components and the vessel during development. The authors need to discuss more about potential regulation and implication of this phenomenon.
It is interesting that DOTA-Gd tracer shows different traces in Mlc1 KO brains. However, it is unclear how MLC1 deletion affects glymphatic system. Does the tracer normally enter to the perivascular spaces in Mlc KO brains? Does the tracer leak out more from the perivascular spaces in Mlc1 KO mice? Is the general clearance or drainages of the tracer impaired in Mlc1 KO mice? Would these defects be originated by the reduced perivascular astrocyte coverage or the reduced vasoconstriction itself?
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Reviewer #2 (Public Review):
This very interesting manuscript by Gilbert and colleagues uncovers that the astrocyte specific membrane protein MLC1, the mutation of which causes a rare disease called megalencephalic leukoencephalopathy with subcortical Cysts (MLC), plays a fundamental role in the postnatal development of the gliovascular unit and the organization of the perivascular astrocyte processes, in particular. To reach this conclusion, the authors used an elegant multiscale approach including in vivo MRI, in vivo functional ultrasound, ex vivo analysis of vascular constriction, anatomical approaches at the light and electron microscopic level, and molecular characterization of the gliovascular unit from isolated microvessels. The manuscript is very well-written although it uses too many (unnecessary) abbreviations, which prevents …
Reviewer #2 (Public Review):
This very interesting manuscript by Gilbert and colleagues uncovers that the astrocyte specific membrane protein MLC1, the mutation of which causes a rare disease called megalencephalic leukoencephalopathy with subcortical Cysts (MLC), plays a fundamental role in the postnatal development of the gliovascular unit and the organization of the perivascular astrocyte processes, in particular. To reach this conclusion, the authors used an elegant multiscale approach including in vivo MRI, in vivo functional ultrasound, ex vivo analysis of vascular constriction, anatomical approaches at the light and electron microscopic level, and molecular characterization of the gliovascular unit from isolated microvessels. The manuscript is very well-written although it uses too many (unnecessary) abbreviations, which prevents a fluid reading of the manuscript, results are well illustrated and convincing and the discussion is reasonable.
I have a major concern regarding the results reported in Figure 4D, which seem somewhat contradictory to those shown in Figure 6A-F. Indeed, the authors report in Figure 4D that there is less Neurofilament-M protein around isolated microvessels in MLC1 KO mice, whereas Figure 6A-F shows that these animals have more neuronal processes in contact with the vessels than in wiltypes. How can the authors explain this?
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Reviewer #3 (Public Review):
Astrocytes unique to the central nervous system such as the brain is known to play a role in maintaining BBB integrity by surrounding brain vessels tightly vis their end-feet structure. However, how astrocytes form end-feet along brain vessels and how they regulate gliovascular function are largely unknown. The present manuscript by Gilbert et al. describes how perivascular astrocytic processes are established during postnatal development and pinpoint a player involved. Moreover, it revealed dysmorphic astrocytes could affect neurovascular coupling and CSF transport. They find that MLC1 deletion resulted in disorganized perivascular astrocytic processes and defective contractility of vascular smooth muscle cells, thereby leading to impaired neurovascular coupling and intraparenchymal fluid clearance.
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