Chronic active plaque with three characteristic areas in a female patient of 48 years old: periplaque WM, border area, and inactive center plaque. Macrophages around vessels in chronic active plaque edge arrows. Chronic active plaque with remyelination in a male patient of 58 years old, shadow plaques are also observed arrowheads. The staining for myelin not only allowed us to delimit the chronic-active plaques but also to distinguish different morphological areas within lesions: Ramagopalan et al.
This area is characterized by the existence of small packages of newly formed myelinated nerve fibers crossing the demyelinated region.
In order to analyze the Apo D implication in demyelination and remyelination processes in MS, we analyzed the Apo D expression pattern in focal demyelinating lesions by immunohistochemistry techniques Figures 2a—d. Representative images of histochemical and immunohistochemical comparison of different lesion areas in a chronic active plaque. Focal active demyelinated lesion in periventricular WM a,b. Consecutive sections of a male patient, 35 years old. Expanding rim of a chronic active plaque surrounding an inactive area c,d.
Consecutive sections of a male patient, 68 years old. Immunopositive cell types in plaques. Reactive astrocytes with large cytoplasm, round nuclei, and stellate neurites e. Macrophagic cell with numerous phagosomes inside f. Many small OLGs with small round nuclei, scarce cytoplasm, and one or two projections g. Negative immunostaining in inactive plaque h. The active plaque areas show a low overall Apo D signal when we compare with the intact periplaque WM. However, reactive astrocytes and active macrophages of these areas appear intensely labeled for Apo D Figures 2e,f ; most astrocytes positives for this protein exhibit round-shaped cell bodies, hypertrophy, and numerous fibrillary processes reactive astrocytes.
In the case of chronic plaques, Apo D expression was again higher in periplaque WM than in the inactive plaque area Figures 2g,h. This fact may be due to the large amount of Apo D-positive cells present in these surrounding areas as well as the intensity of their labeling Figure 2g. When we analyze myelin of chronic MS lesions with a specific stain for this protein we found that, as it was to be expected, myelin was reduced to low levels in active areas and to very low levels in inactive ones, compared to control WM area and periplaque WM Figures 3a,d,g,j.
Interestingly, the Apo D immunosignal showed a similar pattern than that of myelin. Thus, the immunoreactivity for Apo D tended to decrease as we approached the plaque border and even more in the demyelinated plaque area Figures 3b,e,h,k. Based in the cell morphology, we observed that OLGs, macrophagic cells, and some isolated astrocytes inserts in Figures 3e,h were responsible for the expression of Apo D in all these areas Figures 3b,e,h,k,n.
In the remyelinating regions, both myelin and Apo D levels increase in line with the remyelination processes that are taking place Figures 3m,n. Control WM of male control subject of 66 years old. Female subject of 52 years old with MS d—o. Periplaque WM. Apo D containing granules were found surrounding myelin sheath.
Active area. Apo D positive macrophagic cells top insert and less OLGs can be observed in this area bottom insert. The arrowhead points to the granular content of distal processes of OLGs around myelin sheath. Inactive area. Remyelination area in a sclerosis plaque. Positive OLGs can be observed in this area arrow in the insert. Finally, co-localization studies of myelin and Apo D proteins were performed in immunohistochemistry tissue sections counterstained with LFB Figures 3c,f,i,l,o. We found that Apo D appeared preferentially in the cytoplasm and distal cell processes of OLGs in all the plaque types and patients studied.
Small granules containing Apo D could also be observed around the myelin sheath of these cells Figures 3c,f,i,o. Noteworthy, in the border of plaques, even with low levels of myelin, some remaining OLGs showed immunolabeling for Apo D Figure 3i. OLGs expressing Apo D reappeared in focal remyelination areas, specifically in those where new myelin envelopes are being formed Figure 3o.
The quantification of Apo D protein level in the most frequent and better defined chronic plaques showed clear discrepancies between the different areas under study. Since it was difficult to distinguish plaque areas in immunostaining sections for Apo D, we used the intrinsic fluorescent properties of myelin to unequivocally detect them and improve the quantification process Figures 4a—f.
The data showed statistically significant differences in the Apo D burden between inactive However, we did not find significant differences when we compared active with remyelination areas Figure 4g. Representative image of remyelination area in a sclerosis plaque. Immunohistochemistry for Apo D visualized under light microscopy a,c,e , and same area observed with fluorescent microscopy to visualize autofluorescence of myelin sheaths b,d,f.
Histogram showing densitometric quantification of Apo D g. Our results clearly demonstrated a decrease in the Apo D content from the active area to the chronic inactive one in parallel with the loss of myelin in all the plaque types and patients studied. However, an important issue still to be determined is the exact relationship between Apo D levels and the number of OLGs present in these areas. For this purpose, first we tested different OLG markers and we found that crystallin Pountney et al. Second and in order to confirm that crystallin is expressed specifically in OLGs, a double immunohistochemical technique for crystallin and the specific astrocytic marker GFAP was performed.
Our results clearly demonstrated no colocalization between these two markers and, consequently, that crystallin immunoreactive cells are in virtually all case OLGs Supplementary Figure S1. In fact, only a very few astrocytes were positives for crystallin so these cells have not been taken into account in our studies. Double immunostaining for Apo D and crystallin is showed in Figure 5 , in both representative images Figures 5a—d , and the quantification of OLGs negative for Apo D only positive for crystallin and positive for Apo D colocalization between crystallin and Apo D signals Figure 5e.
Myelocortical Multiple Sclerosis: Neurodegeneration Without White Matter Demyelination
A statistical significant reduction in the number of OLGs expressing Apo D colocalization signal in the active area In fact, we have found that not all the OLGs are Apo D positives, which becomes more evident in active demyelinating areas in which there also seems to be more colocalization Figures 5a—d. Representative image of active plaque area a,c. Representative image of periplaque WM area b,d. Histogram showing number of OLGs positive for crystallin gray bars and also labeled for Apo D black bars; colocalization signal e. In the light of the described results and the neuroprotective role suggested for Apo D, we used a double immunostaining for Apo D and NF to study whether the apolipoprotein expression could be related with axonal preservation.
As we expected, the expression pattern for both proteins was similar. In fact, the immunolabeling for Apo D and NF statistically significant decreases in the chronic inactive plaque areas Notably, we have observed that the amount of Apo D and neuronal NF decreased, in a statistical significant way, in parallel from the edge Of note is that many axons conserved immunostaining of NF in active demyelination areas even in absence of Apo D expression Figure 6B. In remyelination areas, both signals were again found Double immunohistochemical technique for Apo D blue and NF brown.
The representative micrographs show a chronic plaque where it can be clearly distinguish the surveyed areas: periplaque WM area a , active area b , inactive area c , and remyelination area d. Histogram showing densitometric quantification of Apo D e. Histogram showing densitometric quantification of NF f. AU: arbitrary units. While much progress has been achieved on the involvement of Apo D in some neurodegenerative diseases in the past years, little is known about its role in MS. The immunohistochemical characterization of Apo D expression pattern in the anatomopathological classical features of MS, sclerosis plaques, is a first step necessary to address this goal.
In this work, we have observed that Apo D expression is further reduced in MS lesions respect to the unaffected WM, in all the plaque types and patients studied. In this sense, an interesting finding is that there are labeling differences within the plaque, significantly lower Apo D expression in the inactive area than in the active one which increases again in remyelination areas. Remarkably, the immunohistochemical approaches reveal a possible relationship between OLGs, myelin, and Apo D expression in MS plaques.
As it was expected, myelin protein levels decrease in demyelination plaques from the periplaque WM to the border of the lesions an even more to the center. In fact, this loss of myelin sheets wrapping myelinated nerve fibers is closely related with OLGs degeneration that characterize MS pathophysiology Zindler and Zipp, ; Popescu et al.
The Apo D expression pattern in both active and chronic plaques was similar than the one found for myelin. Bearing in mind these data, one could think that the Apo D downregulation observed in sclerosis plaques is consistent with OLGs death caused by an exacerbated autoimmune reaction. However, we have demonstrated that not all OLGs express Apo D in periplaque intact regions, which becomes more evident in demyelinating areas. The OLG degeneration only could explain, in our opinion, one part of the reduced Apo D levels, what pathological processes of MS compromise, directly or indirectly, the expression of Apo D by the intact OLGs could explain the rest Patel and Balabanov, In this sense, Apo D is significantly up-regulated in Schwann cells and macrophages of sciatic nerve after crush injury Boyles et al.
In addition, Apo D gene is among the regeneration-associated genes whose expression is up-regulated in rat dorsal root ganglia following chronic constriction injury of the sciatic nerve Kim et al. Apo D is expressed in the development of the CNS and increases during aging and in some neurodegenerative diseases Navarro et al. Gene profiling analysis of ischemic and stroke rat brain tissues showed the up-regulation of genes related to lipid transport, including Apo D, mainly in peri-infarct and WM areas in cells identified as mature OLGs and reactive astrocytes Rickhag et al.
It has been proposed that Apo D may exert a neuroprotective influence in the acute stroke phase and may also support neuronal regeneration and remyelination in the extended post-stroke recovery phase Rickhag et al. Demyelination, neuronal growth, and remyelination are among the multiple events that take place in MS brains and that require a synthesis and recruitment of lipids to form plasma membranes and myelin sheaths.
Noteworthy, we have found that the expression of Apo D, one of the most important glial-derived proteins in the myelin dynamics, decreases in the sclerosis plaques of MS brains. On one hand, we have observed Apo D label in cytoplasm and distal processes of OLGs but no colocalization with myelin itself. On the other hand, the decrease in Apo D protein is not only due to the lower number of OLGs inside the plaques but many of these OLGs are not able to express it.
Activation of the inflammatory cascade in MS ultimately compromise, directly or indirectly, OLGs function and viability Patel and Balabanov, Also of interest is highlighting that Apo D disappears in demyelinated plaques but returns when remyelination occurs. According with our findings, a correlation between Apo D and loss of axonal processes in the sclerosis plaques could be ruled out. Although more studies are needed in this respect, one plausible explanation may place Apo D in the molecular and cellular mechanism that control spontaneous remyelination after damage.
As mentioned above, Apo D is expressed mainly by mature OLGs as wells as OPCs in the brain which, in turn, are ultimately responsible for the restoration of new myelin sheaths in demyelinated axons in MS Hu et al. Indeed, it seems that adult OPCs react to damage and are able to proliferate, migrate, and differentiate in response to mitogens and pro-migratory factors secreted by astrocytes and microglial cells to replace lost OLGs Clemente et al. Some of the neuroprotective functions of Apo D in this context could be related with recent findings that suggest that it may regulate the migratory behavior of different motile cells in association to growth factors Dentelli et al.
The data showed that Apo D may act as neurotrophic factor that promotes neurite outgrowth and synaptogenesis in dorsal root ganglion neurons Kosacka et al. Interestingly, it has been also shown that Apo D seems to mediate neuronal differentiation in a retinoic acid RA -dependent manner in various cell lines; Apo D may act as RA carrier Ruiz et al. Accordingly, we have observed that some reactive astrocytes in active plaques show an intense immunosignal for Apo D but, surprisingly, astrocytes in the edge of plaques or even within the glial scar in the chronic plaques show a much lower signal.
The Myelin Regeneration Project | Weill Cornell Medicine Multiple Sclerosis Center
Since one of the postulated role for Apo D is to act as a neuroprotective and antioxidant molecule, one could expect that Apo D expression would be higher in periplaque and active areas of MS plaques. This apparent contradiction could be explained again by the influence of MS local levels of cytokines in the astrocyte protein expression profiles Rassart et al. In summary, the dual function of Apo D, as a tissue specific lipid carrier and an antioxidant and neuroprotective molecule, makes it a potential versatile player in MS pathology.