Spectral Fluorescence Pathology of Protein Misfolding Disorders.

Protein misfolding has been extensively studied in the context of neurodegenerative disorders and systemic amyloidoses. Due to misfolding and aggregation of proteins being highly heterogeneous and generating a variety of structures, a growing body of evidence illustrates numerous ways how the aggregates contribute to progression of diseases such as Alzheimer's disease, Parkinson's disease, and prion disorders. Different misfolded species of the same protein, commonly referred to as strains, appear to play a significant role in shaping the disease clinical phenotype and clinical progression. The distinct toxicity profiles of various misfolded proteins underscore their importance. Current diagnostics struggle to differentiate among these strains early in the disease course. This review explores the potential of spectral fluorescence approaches to illuminate the complexities of protein misfolding pathology and discusses the applications of advanced spectral methods in the detection and characterization of protein misfolding disorders. By examining spectrally variable probes, current data analysis approaches, and important considerations for the use of these techniques, this review aims to provide an overview of the progress made in this field and highlights directions for future research.

Amyloid dye pairs as spectral sensors for enhanced detection and differentiation of misfolded proteins.

Protein misfolding with subsequent formation of cross-ß-sheet-rich fibrils is a well-known pathological hallmark of various neurodegenerative conditions, including Alzheimer's disease (AD). Recent evidence suggests that specific protein conformations may be the primary drivers of disease progression, differentiation of which remains a challenge with conventional methods. We have previously described a unique phenomenon of light-induced fluorescence enhancement and spectral changes of the amyloid dyes K114 and BSB, and demonstrated its utility in characterizing different amyloid fibrils. In this study, we further characterize and explore the potential of photoconversion, coupled with dual-probe staining, for improved detection of heterogeneity of amyloids using silk fibers and 5xFAD mouse brain sections. BSB and K114 were paired with either Nile Red or MCAAD-3, aiming to increase the sensitivity and specificity of staining and misfolded protein detection via complementary binding and FRET. Principal component analysis of spectral data revealed significant differences between various amyloids, and was able to detect subtle amyloid pathology in the 5xFAD mouse background brain parenchyma.

Spectral focusing-based stimulated Raman scattering microscopy using compact glass blocks for adjustable dispersion.

Spectral focusing is a well-established technique for increasing spectral resolution in coherent Raman scattering microscopy. However, current methods for tuning optical chirp in setups using spectral focusing, such as glass rods, gratings, and prisms, are very cumbersome, time-consuming to use, and difficult to align, all of which limit more widespread use of the spectral focusing technique. Here, we report a stimulated Raman scattering (SRS) configuration which can rapidly tune optical chirp by utilizing compact adjustable-dispersion TIH53 glass blocks. By varying the height of the blocks, the number of bounces in the blocks and therefore path length of the pulses through the glass can be quickly modulated, allowing for a convenient method of adjusting chirp with almost no necessary realignment. To demonstrate the flexibility of this configuration, we characterize our system's signal-to-noise ratio and spectral resolution at different chirp values and perform imaging in both the carbon-hydrogen stretching region (MCF-7 cells) and fingerprint region (prostate cores). Our findings show that adjustable-dispersion glass blocks allow the user to effortlessly modify their optical system to suit their imaging requirements. These blocks can be used to significantly simplify and miniaturize experimental configurations utilizing spectral focusing.

Quantitation of Tissue Amyloid via Fluorescence Spectroscopy Using Controlled Concentrations of Thioflavin-S.

Amyloids are misfolded proteins that aggregate into fibrillar structures, the accumulation of which is associated with the pathogenesis of many neurodegenerative diseases, such as Alzheimer's disease (AD). Early, sensitive detection of these misfolded aggregates is of great interest to the field, as amyloid deposition begins well before the presentation of clinical symptoms. Thioflavin-S (ThS) is a fluorescent probe commonly used to detect amyloid pathology. Protocols for ThS staining vary, but they often use high staining concentrations followed by differentiation, which causes varying levels of non-specific staining and potentially leaves more subtle amyloid deposition unidentified. In this study, we developed an optimized ThS staining protocol for the sensitive detection of ß-amyloids in the widely used 5xFAD Alzheimer's mouse model. Controlled dye concentrations together with fluorescence spectroscopy and advanced analytical methods enabled not only the visualization of plaque pathology, but also the detection of subtle and widespread protein misfolding throughout the 5xFAD white matter and greater parenchyma. Together, these findings demonstrate the efficacy of a controlled ThS staining protocol and highlight the potential use of ThS for the detection of protein misfolding that precedes clinical manifestation of disease.

Detection and Typing of Renal Amyloidosis by Fluorescence Spectroscopy Using the Environmentally Sensitive Fluorophore K114.

PURPOSE: To demonstrate that spectral analysis using the K114 fluorophore can detect and differentiate AL and AA renal amyloidosis. PROCEDURES: Kidney biopsies from patients with AL amyloidosis, AA amyloidosis, and normal samples with no evident pathology were stained with Congo Red and K114. The specimens were imaged on a spectral confocal microscope. RESULTS: Congo Red displayed homogeneous spectra across the three tissue types while K114 chromatically distinguished between normal tissue, AL amyloid, and AA amyloid. Additionally, Congo Red displayed an increased risk of false positive staining compared to K114. Spectral phasors computed from K114-stained tissue sections quantitatively differentiated the three tissue types. K114-stained amyloid deposits displayed a significantly greater increase in brightness after 50 images acquired in rapid succession compared to normal tissue. Quantitative analysis of intensity changes in the background of diseased tissue also differentiated AL and AA amyloid samples, suggesting widespread amyloid deposition. Both amyloid and the backgrounds of diseased samples red-shifted while normal tissue blue-shifted in response to repeated imaging, supporting this theory. CONCLUSIONS: K114 staining of renal biopsies is a promising technique to detect and differentiate types of renal amyloidosis. Due to the advantages this method has over traditional Congo Red staining, the techniques presented here warrant further development for potential use in clinical settings.

Dual-probe fluorescence spectroscopy for sensitive quantitation of Alzheimer's amyloid pathology.

Protein misfolding is a prominent pathological hallmark of neurodegenerative disorders, including Alzheimer's disease (AD). Studies have shown that the diversity of ß sheet-rich protein deposits (such as amyloid ß plaques and neurofibrillary tangles), present across different brain regions, might underlie different disease phenotypes and only certain types of aggregates might be associated with cognitive decline. Conformationally sensitive fluorescent amyloid probes have the ability to report different structures of protein aggregates by virtue of their shifting emission spectra. Here we defined the binding affinity of the fluorescent amyloid probes BSB and MCAAD to disease-relevant protein aggregates, and combined the two probes to examine formalin-fixed paraffin-embedded mouse and human brain samples. Coupled with quantitative spectral phasor analysis, the dual-probe staining approach revealed remarkable heterogeneity of protein aggregates across the samples. Distinct emission spectra were consistent with certain types of deposits present in the mouse and human brain sections. The sensitivity of this staining, imaging and analysis approach outperformed conventional immunohistochemistry with the detected spectral differences between the greater parenchyma of cognitively normal and AD cases indicating a subtle yet widespread proteopathy associated with disease. Our method offers more sensitive, objective, and quantitative examination of protein misfolding pathology using conventional tissue sections.

Cuprizone-induced Demyelination in Mouse Brain is not due to Depletion of Copper.

SUMMARY STATEMENT: The demyelinating effects of CPZ are not due to Cu deficiency but are instead consistent with acute toxicity of a CPZ + Cu complex.

Copper ions, prion protein and Aß modulate Ca levels in central nervous system myelin in an NMDA receptor-dependent manner.

As in neurons, CNS myelin expresses N-Methyl-D-Aspartate Receptors (NMDARs) that subserve physiological roles, but have the potential to induce injury to this vital element. Using 2-photon imaging of myelinic Ca in live ex vivo mouse optic nerves, we show that Cu ions potently modulate Ca levels in an NMDAR-dependent manner. Chelating Cu in the perfusate induced a substantial increase in Ca levels, and also caused significant axo-myelinic injury. Myelinic NMDARs are shown to be regulated by cellular prion protein; only in prion protein KO optic nerves does application of NMDA + D-serine induce a large Ca increase, consistent with strong desensitization of these receptors in the presence of prion protein limiting Ca overload. Aß1-42 peptide induced a large Ca increase that was also Cu-dependent, and was blocked by NMDAR antagonism. Our results indicate that like in neurons, myelinic NMDARs permeate potentially injurious amounts of Ca, and are also potently regulated by micromolar Cu and activated by Aß1-42 peptides. These findings shed mechanistic light on the important primary white matter injury frequently observed in Alzheimer's brain.

Subcellular localization of hippocampal ryanodine receptor 2 and its role in neuronal excitability and memory.

Ryanodine receptor 2 (RyR2) is abundantly expressed in the heart and brain. Mutations in RyR2 are associated with both cardiac arrhythmias and intellectual disability. While the mechanisms of RyR2-linked arrhythmias are well characterized, little is known about the mechanism underlying RyR2-associated intellectual disability. Here, we employed a mouse model expressing a green fluorescent protein (GFP)-tagged RyR2 and a specific GFP probe to determine the subcellular localization of RyR2 in hippocampus. GFP-RyR2 was predominantly detected in the soma and dendrites, but not the dendritic spines of CA1 pyramidal neurons or dentate gyrus granular neurons. GFP-RyR2 was also detected within the mossy fibers in the stratum lucidum of CA3, but not in the presynaptic terminals of CA1 neurons. An arrhythmogenic RyR2-R4496C+/- mutation downregulated the A-type K+ current and increased membrane excitability, but had little effect on the afterhyperpolarization current or presynaptic facilitation of CA1 neurons. The RyR2-R4496C+/- mutation also impaired hippocampal long-term potentiation, learning, and memory. These data reveal the precise subcellular distribution of hippocampal RyR2 and its important role in neuronal excitability, learning, and memory.

Diagnosing Alzheimer's Disease from Circulating Blood Leukocytes Using a Fluorescent Amyloid Probe.

BACKGROUND: Toxic amyloid-ß (Aß) peptides aggregate into higher molecular weight assemblies and accumulate not only in the extracellular space, but also in the walls of blood vessels in the brain, increasing their permeability, and promoting immune cell migration and activation. Given the prominent role of the immune system, phagocytic blood cells may contact pathological brain materials. OBJECTIVE: To develop a novel method for early Alzheimer's disease (AD) detection, we used blood leukocytes, that could act as "sentinels" after trafficking through the brain microvasculature, to detect pathological amyloid by labelling with a conformationally-sensitive fluorescent amyloid probe and imaging with confocal spectral microscopy. METHODS: Formalin-fixed peripheral blood mononuclear cells (PBMCs) from cognitively healthy control (HC) subjects, mild cognitive impairment (MCI) and AD patients were stained with the fluorescent amyloid probe K114, and imaged. Results were validated against cerebrospinal fluid (CSF) biomarkers and clinical diagnosis. RESULTS: K114-labeled leukocytes exhibited distinctive fluorescent spectral signatures in MCI/AD subjects. Comparing subjects with single CSF biomarker-positive AD/MCI to negative controls, our technique yielded modest AUCs, which improved to the 0.90 range when only MCI subjects were included in order to measure performance in an early disease state. Combining CSF Aß42 and t-Tau metrics further improved the AUC to 0.93. CONCLUSION: Our method holds promise for sensitive detection of AD-related protein misfolding in circulating leukocytes, particularly in the early stages of disease.

Quantitative detection of grey and white matter amyloid pathology using a combination of K114 and CRANAD-3 fluorescence.

BACKGROUND: Alzheimer's disease (AD) is a neurodegenerative disease that exacts a huge toll on the patient, the healthcare system and society in general. Abundance and morphology of protein aggregates such as amyloid ß plaques and tau tangles, along with cortical atrophy and gliosis are used as measures to assess the changes in the brain induced by the disease. Not all of these parameters have a direct correlation with cognitive decline. Studies have shown that only particular protein conformers can be the main drivers of disease progression, and conventional approaches are unable to distinguish different conformations of disease-relevant proteins. METHODS AND RESULTS: Using the fluorescent amyloid probes K114 and CRANAD-3 and spectral confocal microscopy, we examined formalin-fixed paraffin-embedded brain samples from different control and AD cases. Based on the emission spectra of the probes used in this study, we found that certain spectral signatures can be correlated with different aggregates formed by different proteins. The combination of spectral imaging and advanced image analysis tools allowed us to detect variability of protein deposits across the samples. CONCLUSION: Our proposed method offers a quicker and easier neuropathological assessment of tissue samples, as well as introducing an additional parameter by which protein aggregates can be discriminated.

Spectral photokinetic conversion of the fluorescent probes BSB and K114 for improved detection of amyloid assemblies.

Cross-ß-sheet-rich protein fibrils are infamous for their accumulation in the brains of patients diagnosed with a number of neurodegenerative diseases, including Alzheimer's disease (AD). Disease-relevant fibrils are a result of deviation of the proteins from their native structure to a misfolded state resulting in aggregation and formation of fibrils. In this study, we explored the phenomenon of light-induced fluorescence enhancement of amyloid assemblies stained with two amyloid probes (BSB and K114) using Bombyx mori silk and human AD brain sections. The photoconversion effect, accompanied by an increase in fluorescence intensity and spectral blue-shift, was highly dependent on the chemical structures of the dyes, pH, presence of glycerol and the type of amyloid. The degree of intensity and spectral change over time in response to high laser exposure were quantified and analyzed using custom-written analysis tools. Our findings provide further insight into possible mechanisms of amyloid-mediated photoconversion kinetics of K114 and BSB, and may provide more insight into the molecular nature of various amyloid assemblies.

Abnormalities in normal-appearing white matter from which multiple sclerosis lesions arise.

Normal-appearing white matter is far from normal in multiple sclerosis; little is known about the precise pathology or spatial pattern of this alteration and its relation to subsequent lesion formation. This study was undertaken to evaluate normal-appearing white matter abnormalities in brain areas where multiple sclerosis lesions subsequently form, and to investigate the spatial distribution of normal-appearing white matter abnormalities in persons with multiple sclerosis. Brain MRIs of pre-lesion normal-appearing white matter were analysed in participants with new T2 lesions, pooled from three clinical trials: SYNERGY (NCT01864148; n = 85 with relapsing multiple sclerosis) was the test data set; ASCEND (NCT01416181; n = 154 with secondary progressive multiple sclerosis) and ADVANCE (NCT00906399; n = 261 with relapsing-remitting multiple sclerosis) were used as validation data sets. Focal normal-appearing white matter tissue state was analysed prior to lesion formation in areas where new T2 lesions later formed (pre-lesion normal-appearing white matter) using normalized magnetization transfer ratio and T2-weighted (nT2) intensities, and compared with overall normal-appearing white matter and spatially matched contralateral normal-appearing white matter. Each outcome was analysed using linear mixed-effects models. Follow-up time (as a categorical variable), patient-level characteristics (including treatment group) and other baseline variables were treated as fixed effects. In SYNERGY, nT2 intensity was significantly higher, and normalized magnetization transfer ratio was lower in pre-lesion normal-appearing white matter versus overall and contralateral normal-appearing white matter at all time points up to 24 weeks before new T2 lesion onset. In ASCEND and ADVANCE (for which normalized magnetization transfer ratio was not available), nT2 intensity in pre-lesion normal-appearing white matter was significantly higher compared to both overall and contralateral normal-appearing white matter at all pre-lesion time points extending up to 2 years prior to lesion formation. In all trials, nT2 intensity in the contralateral normal-appearing white matter was also significantly higher at all pre-lesion time points compared to overall normal-appearing white matter. Brain atlases of normal-appearing white matter abnormalities were generated using measures of voxel-wise differences in normalized magnetization transfer ratio of normal-appearing white matter in persons with multiple sclerosis compared to scanner-matched healthy controls. We observed that overall spatial distribution of normal-appearing white matter abnormalities in persons with multiple sclerosis largely recapitulated the anatomical distribution of probabilities of T2 hyperintense lesions. Overall, these findings suggest that intrinsic spatial properties and/or longstanding precursory abnormalities of normal-appearing white matter tissue may contribute to the risk of autoimmune acute demyelination in multiple sclerosis.

Mutation of copper binding sites on cellular prion protein abolishes its inhibitory action on NMDA receptors in mouse hippocampal neurons.

We have previously reported that cellular prion protein (PrPC) can down-regulate NMDA receptor activity and in a copper dependent manner. Here, we employed AAV9 to introduce murine cellular prion protein into mouse hippocampal neurons in primary cultures from PrP null mice to determine the role of the six copper binding motifs located within the N-terminal domain of PrPC. The results demonstrate that viral expression of wild type PrPC lowers NMDAR activity in PrP null mouse hippocampal neurons by reducing the magnitude of non-desensitizing currents. Elimination of the last two copper binding sites alone, or in combination with the remaining four attenuates this protective effect. Thus our data suggest that copper ion interactions with specific binding sites on PrPC are critical for PrPC dependent modulation of NMDA receptor function.

Mechanistic underpinning of an inside-out concept for autoimmunity in multiple sclerosis.

The neuroinflammatory disease multiple sclerosis is driven by autoimmune pathology in the central nervous system. However, the trigger of the autoimmune pathogenic process is unknown. MS models in immunologically naïve, specific-pathogen-free bred rodents support an exogenous trigger, such as an infection. The validity of this outside-in pathogenic concept for MS has been frequently challenged by the difficulty to translate pathogenic concepts developed in these models into effective therapies for the MS patient. Studies in well-validated non-human primate multiple sclerosis models where, just like in humans, the autoimmune pathogenic process develops from an experienced immune system trained by prior infections, rather support an endogenous trigger. Data reviewed here corroborate the validity of this inside-out pathogenic concept for multiple sclerosis. They also provide a plausible sequence of events reminiscent of Wilkin's primary lesion theory: (i) that autoimmunity is a physiological response of the immune system against excess antigen turnover in diseased tissue (the primary lesion) and (ii) that individuals developing autoimmune disease are (genetically predisposed) high responders against critical antigens. Data obtained in multiple sclerosis brains reveal the presence in normally appearing white matter of myelinated axons where myelin sheaths have locally dissociated from their enwrapped axon (i.e., blistering). The ensuing disintegration of axon-myelin units potentially causes the excess systemic release of post-translationally modified myelin. Data obtained in a unique primate multiple sclerosis model revealed a core pathogenic role of T cells present in the normal repertoire, which hyper-react to post-translationally modified (citrullinated) myelin-oligodendrocyte glycoprotein and evoke clinical and pathological aspects of multiple sclerosis.

Label-free assessment of myelin status using birefringence microscopy.

BACKGROUND: Label-free methods for quantifying myelination can reduce expense, time, and variability in results when examining tissue white matter pathology. NEW METHOD: We sought to determine whether the optical birefringent properties of myelin could be exploited to determine myelination status of white matter in tissue sections. Sections of forebrains of mice (normal, and treated with cuprizone to cause demyelination) were examined by birefringence using a birefringence imaging system (Thorlabs LCC7201), and results compared with sections stained using Luxol Fast Blue. RESULTS: Quantitative birefringence analysis of myelin was not only reliable in detecting demyelination, but also showed abnormalities that preceded myelin loss in cuprizone-treated mice. COMPARISON WITH EXISTING METHODS: Subtle myelin pathology visible with electron microscopy but not with conventional histopathological staining was readily detected with birefringence microscopy. CONCLUSIONS: Birefringence imaging provides a rapid, label-free method of analyzing the myelin content and nanostructural status in longitudinal white matter structures, being sensitive to subtle myelin changes that precede overt pathological damage.

Complex Photophysical Properties of K114 Make for a Versatile Fluorescent Probe for Amyloid Detection.

Protein aggregation is a hallmark of Alzheimer's disease (AD) and many other neurodegenerative disorders. Small organic fluorophores such as Congo Red preferentially bind to cross-ß-sheet-rich deposits and have been used to label amyloid plaques and tau tangles in histological samples. However, distinguishing between different conformations of protein aggregates is not trivial. Using silkworm and spider silks (prototypical amyloids) and transgenic AD mouse (5XFAD) and human AD brain samples, we report how spectral confocal microscopy allowed for improved detection and differentiation of protein aggregates based on the unexpected photophysical behavior of the amyloid-specific dye K114. The pH and excitation power had pronounced effects on the emission spectrum and intensity of amyloid-bound K114 fluorescence. When bound to ß-sheet-rich assemblies, the emission spectrum of K114 was governed by the local pH of the binding pockets much more than by the pH of the mounting medium, likely due to ionization of titratable phenols. Unexpectedly, exposure to high excitation power caused a permanent increase in fluorescence intensity and a spectral blue-shift. These light-induced fluorescence changes were dependent in a complex manner on laser power, exposure time, pH, and amyloid type examined. The above-mentioned phenomena were observed in silk fibers and Alzheimer brain sections from mouse and human, indicating that this may be a general characteristic of K114 when bound to tightly aggregated macromolecules. Potential mechanisms are discussed, likely involving photoinduced electron transfer. Our findings illustrate how the complex photophysical behavior of amyloid-bound K114 can be exploited for improved detection and differentiation of protein aggregates.

Nile Red fluorescence spectroscopy reports early physicochemical changes in myelin with high sensitivity.

The molecular composition of myelin membranes determines their structure and function. Even minute changes to the biochemical balance can have profound consequences for axonal conduction and the synchronicity of neural networks. Hypothesizing that the earliest indication of myelin injury involves changes in the composition and/or polarity of its constituent lipids, we developed a sensitive spectroscopic technique for defining the chemical polarity of myelin lipids in fixed frozen tissue sections from rodent and human. The method uses a simple staining procedure involving the lipophilic dye Nile Red, whose fluorescence spectrum varies according to the chemical polarity of the microenvironment into which the dye embeds. Nile Red spectroscopy identified histologically intact yet biochemically altered myelin in prelesioned tissues, including mouse white matter following subdemyelinating cuprizone intoxication, as well as normal-appearing white matter in multiple sclerosis brain. Nile Red spectroscopy offers a relatively simple yet highly sensitive technique for detecting subtle myelin changes.

Axon-Myelin Unit Blistering as Early Event in MS Normal Appearing White Matter.

OBJECTIVE: Multiple sclerosis (MS) is a chronic neuroinflammatory and neurodegenerative disease of unknown etiology. Although the prevalent view regards a CD4+ -lymphocyte autoimmune reaction against myelin at the root of the disease, recent studies propose autoimmunity as a secondary reaction to idiopathic brain damage. To gain knowledge about this possibility we investigated the presence of axonal and myelinic morphological alterations, which could implicate imbalance of axon-myelin units as primary event in MS pathogenesis. METHODS: Using high resolution imaging histological brain specimens from patients with MS and non-neurological/non-MS controls, we explored molecular changes underpinning imbalanced interaction between axon and myelin in normal appearing white matter (NAWM), a region characterized by normal myelination and absent inflammatory activity. RESULTS: In MS brains, we detected blister-like swellings formed by myelin detachment from axons, which were substantially less frequently retrieved in non-neurological/non-MS controls. Swellings in MS NAWM presented altered glutamate receptor expression, myelin associated glycoprotein (MAG) distribution, and lipid biochemical composition of myelin sheaths. Changes in tethering protein expression, widening of nodes of Ranvier and altered distribution of sodium channels in nodal regions of otherwise normally myelinated axons were also present in MS NAWM. Finally, we demonstrate a significant increase, compared with controls, in citrullinated proteins in myelin of MS cases, pointing toward biochemical modifications that may amplify the immunogenicity of MS myelin. INTERPRETATION: Collectively, the impaired interaction of myelin and axons potentially leads to myelin disintegration. Conceptually, the ensuing release of (post-translationally modified) myelin antigens may elicit a subsequent immune attack in MS. ANN NEUROL 2021;89:711-725.

Autofluorescence spectroscopy as a proxy for chronic white matter pathology.

BACKGROUND: The balance of tissue injury and repair ultimately determines outcomes of chronic neurological disorders, such as progressive multiple sclerosis (MS). However, the extent of pathology can be difficult to detect, particularly when it is insidious and/or offset by tissue regeneration. OBJECTIVES: The objective of this research is to evaluate whether tissue autofluorescence-typically a source of contamination-provides a surrogate marker of white matter injury. METHODS: Tissue autofluorescence in autopsied specimens both experimental and clinical was characterized by spectral confocal microscopy and correlated to severity and chronicity as determined by standard histopathology. RESULTS: Months after cuprizone (CPZ)-induced demyelination, despite robust remyelination, autofluorescent deposits progressively accumulated in regions of prior pathology. Autofluorescent deposits (likely reflecting myelin debris remnants) were conspicuously localized to white matter, proportional to lesion severity, and displayed differential fluorescence over time. Strikingly, similar features were apparent also in autopsied MS tissue. CONCLUSION: Autofluorescence spectroscopy illuminates prior and ongoing white matter injury. The accumulation of autofluorescence in proportion to the extent of progressive atrophy, despite robust remyelination in the CPZ brain, provides important proof-of-concept of a phenomenon (insidious ongoing damage masked by mechanisms of tissue repair) that we hypothesize is highly relevant to the progressive phase of MS.