Concurrent axon and myelin destruction differentiates X-linked adrenoleukodystrophy from multiple sclerosis.

Cerebral disease manifestation occurs in about two thirds of males with X-linked adrenoleukodystrophy (CALD) and is fatally progressive if left untreated. Early histopathologic studies categorized CALD as an inflammatory demyelinating disease, which led to repeated comparisons to multiple sclerosis (MS). The aim of this study was to revisit the relationship between axonal damage and myelin loss in CALD. We applied novel immunohistochemical tools to investigate axonal damage, myelin loss and myelin repair in autopsy brain tissue of eight CALD and 25 MS patients. We found extensive and severe acute axonal damage in CALD already in prelesional areas defined by microglia loss and relative myelin preservation. In contrast to MS, we did not observe selective phagocytosis of myelin, but a concomitant decay of the entire axon-myelin unit in all CALD lesion stages. Using a novel marker protein for actively remyelinating oligodendrocytes, breast carcinoma-amplified sequence (BCAS) 1, we show that repair pathways are activated in oligodendrocytes in CALD. Regenerating cells, however, were affected by the ongoing disease process. We provide evidence that-in contrast to MS-selective myelin phagocytosis is not characteristic of CALD. On the contrary, our data indicate that acute axonal injury and permanent axonal loss are thus far underestimated features of the disease that must come into focus in our search for biomarkers and novel therapeutic approaches.

Three-dimensional virtual histology of the cerebral cortex based on phase-contrast X-ray tomography.

In this work, we optimize the setups and experimental parameters of X-ray phase-contrast computed-tomography for the three-dimensional imaging of the cyto- and myeloarchitecture of cerebral cortex, including both human and murine tissue. We present examples for different optical configurations using state-of-the art synchrotron instruments for holographic tomography, as well as compact laboratory setups for phase-contrast tomography in the direct contrast (edge-enhancement) regime. Apart from unstained and paraffin-embedded tissue, we tested hydrated tissue, as well as heavy metal stained and resin-embedded tissue using two different protocols. Further, we show that the image quality achieved allows to assess the neuropathology of multiple sclerosis in a biopsy sample collected during surgery.

Lack of astrocytes hinders parenchymal oligodendrocyte precursor cells from reaching a myelinating state in osmolyte-induced demyelination.

Demyelinated lesions in human pons observed after osmotic shifts in serum have been referred to as central pontine myelinolysis (CPM). Astrocytic damage, which is prominent in neuroinflammatory diseases like neuromyelitis optica (NMO) and multiple sclerosis (MS), is considered the primary event during formation of CPM lesions. Although more data on the effects of astrocyte-derived factors on oligodendrocyte precursor cells (OPCs) and remyelination are emerging, still little is known about remyelination of lesions with primary astrocytic loss. In autopsy tissue from patients with CPM as well as in an experimental model, we were able to characterize OPC activation and differentiation. Injections of the thymidine-analogue BrdU traced the maturation of OPCs activated in early astrocyte-depleted lesions. We observed rapid activation of the parenchymal NG2+ OPC reservoir in experimental astrocyte-depleted demyelinated lesions, leading to extensive OPC proliferation. One week after lesion initiation, most parenchyma-derived OPCs expressed breast carcinoma amplified sequence-1 (BCAS1), indicating the transition into a pre-myelinating state. Cells derived from this early parenchymal response often presented a dysfunctional morphology with condensed cytoplasm and few extending processes, and were only sparsely detected among myelin-producing or mature oligodendrocytes. Correspondingly, early stages of human CPM lesions also showed reduced astrocyte numbers and non-myelinating BCAS1+ oligodendrocytes with dysfunctional morphology. In the rat model, neural stem cells (NSCs) located in the subventricular zone (SVZ) were activated while the lesion was already partially repopulated with OPCs, giving rise to nestin+ progenitors that generated oligodendroglial lineage cells in the lesion, which was successively repopulated with astrocytes and remyelinated. These nestin+ stem cell-derived progenitors were absent in human CPM cases, which may have contributed to the inefficient lesion repair. The present study points to the importance of astrocyte-oligodendrocyte interactions for remyelination, highlighting the necessity to further determine the impact of astrocyte dysfunction on remyelination inefficiency in demyelinating disorders including MS.

Phase-contrast x-ray tomography of neuronal tissue at laboratory sources with submicron resolution.

Purpose: Recently, progress has been achieved in implementing phase-contrast tomography of soft biological tissues at laboratory sources. This opens up opportunities for three-dimensional (3-D) histology based on x-ray computed tomography ( µ - and nanoCT) in the direct vicinity of hospitals and biomedical research institutions. Combining advanced x-ray generation and detection techniques with phase reconstruction algorithms, 3-D histology can be obtained even of unstained tissue of the central nervous system, as shown, for example, for biopsies and autopsies of human cerebellum. Depending on the setup, i.e., source, detector, and geometric parameters, laboratory-based tomography can be implemented at very different sizes and length scales. We investigate the extent to which 3-D histology of neuronal tissue can exploit the cone-beam geometry at high magnification M using a nanofocus transmission x-ray tube (nanotube) with a 300 nm minimal spot size (Excillum), combined with a single-photon counting camera. Tightly approaching the source spot with the biopsy punch, we achieve high M ≈ 10 1 - 10 2 , high flux density, and exploit the superior efficiency of this detector technology. Approach: Different nanotube configurations such as spot size and flux, M , as well as exposure time, Fresnel number, and coherence are varied and selected in view of resolution, field of view, and/or phase-contrast requirements. Results: The data show that the information content for the cytoarchitecture is enhanced by the phase effect. Comparison of results to those obtained at a microfocus rotating-anode x-ray tomography setup with a high-resolution detector, i.e., in low- M geometry, reveals similar to slightly superior data quality for the nanotube setup. In addition to its compactness, reduced power consumption by a factor of 10 3 , and shorter scan duration, the particular advantage of the nanotube setup also lies in its suitability for pixel detector technology, enabling an increased range of opportunities for applications in laboratory phase-contrast x-ray tomography. Conclusions: The phase retrieval scheme utilized mixes amplitude and phase contrast, with results being robust with respect to reconstruction parameters. Structural information content is comparable to slightly superior to previous results achieved with a microfocus rotating-anode setup but can be obtained in shorter scan time. Beyond advantages as compactness, lowered power consumption, and flexibility, the nanotube setup's scalability in view of the progress in pixel detector technology is particularly beneficial. Further progress is thus likely to bring 3-D virtual histology to the performance in scan time and throughput required for clinical practice in neuropathology.

Pro-inflammatory activation following demyelination is required for myelin clearance and oligodendrogenesis.

Remyelination requires innate immune system function, but how exactly microglia and macrophages clear myelin debris after injury and tailor a specific regenerative response is unclear. Here, we asked whether pro-inflammatory microglial/macrophage activation is required for this process. We established a novel toxin-based spinal cord model of de- and remyelination in zebrafish and showed that pro-inflammatory NF-κB-dependent activation in phagocytes occurs rapidly after myelin injury. We found that the pro-inflammatory response depends on myeloid differentiation primary response 88 (MyD88). MyD88-deficient mice and zebrafish were not only impaired in the degradation of myelin debris, but also in initiating the generation of new oligodendrocytes for myelin repair. We identified reduced generation of TNF-α in lesions of MyD88-deficient animals, a pro-inflammatory molecule that was able to induce the generation of new premyelinating oligodendrocytes. Our study shows that pro-inflammatory phagocytic signaling is required for myelin debris degradation, for inflammation resolution, and for initiating the generation of new oligodendrocytes.

GM-CSF and CXCR4 define a T helper cell signature in multiple sclerosis.

Cytokine dysregulation is a central driver of chronic inflammatory diseases such as multiple sclerosis (MS). Here, we sought to determine the characteristic cellular and cytokine polarization profile in patients with relapsing-remitting multiple sclerosis (RRMS) by high-dimensional single-cell mass cytometry (CyTOF). Using a combination of neural network-based representation learning algorithms, we identified an expanded T helper cell subset in patients with MS, characterized by the expression of granulocyte-macrophage colony-stimulating factor and the C-X-C chemokine receptor type 4. This cellular signature, which includes expression of very late antigen 4 in peripheral blood, was also enriched in the central nervous system of patients with relapsing-remitting multiple sclerosis. In independent validation cohorts, we confirmed that this cell population is increased in patients with MS compared with other inflammatory and non-inflammatory conditions. Lastly, we also found the population to be reduced under effective disease-modifying therapy, suggesting that the identified T cell profile represents a specific therapeutic target in MS.

Microglia damage precedes major myelin breakdown in X-linked adrenoleukodystrophy and metachromatic leukodystrophy.

X-linked adrenoleukodystrophy (X-ALD) and metachromatic leukodystrophy (MLD) are two relatively common examples of hereditary demyelinating diseases caused by a dysfunction of peroxisomal or lysosomal lipid degradation. In both conditions, accumulation of nondegraded lipids leads to the destruction of cerebral white matter. Because of their high lipid content, oligodendrocytes are considered key to the pathophysiology of these leukodystrophies. However, the response to allogeneic stem cell transplantation points to the relevance of cells related to the hematopoietic lineage. In the present study, we aimed to better characterize the pathogenetic role of microglia in the above-mentioned diseases. Applying recently established microglia markers to human autopsy cases of X-ALD and MLD we were able to delineate distinct lesion stages in evolving demyelinating lesions. The immune-phenotype of microglia was altered already early in lesion evolution, and microglia loss preceded full-blown myelin degeneration both in X-ALD and MLD. DNA fragmentation indicating phagocyte death was observed in areas showing microglia loss. The morphology and dynamics of phagocyte decay differed between the diseases and between lesion stages, hinting at distinct pathways of programmed cell death. In summary, the present study shows an early and severe damage to microglia in the pathogenesis of X-ALD and MLD. This hints at a central pathophysiologic role of these cells in the diseases and provides evidence for an ongoing transfer of toxic substrates primarily enriched in myelinating cells to microglia.

BCAS1 expression defines a population of early myelinating oligodendrocytes in multiple sclerosis lesions.

Investigations into brain function and disease depend on the precise classification of neural cell types. Cells of the oligodendrocyte lineage differ greatly in their morphology, but accurate identification has thus far only been possible for oligodendrocyte progenitor cells and mature oligodendrocytes in humans. We find that breast carcinoma amplified sequence 1 (BCAS1) expression identifies an oligodendroglial subpopulation in the mouse and human brain. These cells are newly formed, myelinating oligodendrocytes that segregate from oligodendrocyte progenitor cells and mature oligodendrocytes and mark regions of active myelin formation in development and in the adult. We find that BCAS1+ oligodendrocytes are restricted to the fetal and early postnatal human white matter but remain in the cortical gray matter until old age. BCAS1+ oligodendrocytes are reformed after experimental demyelination and found in a proportion of chronic white matter lesions of patients with multiple sclerosis (MS) even in a subset of patients with advanced disease. Our work identifies a means to map ongoing myelin formation in health and disease and presents a potential cellular target for remyelination therapies in MS.