Neurochemical Differences in Spinocerebellar Ataxia Type 14 and 1.

Autosomal-dominant spinocerebellar ataxias (SCA) are neurodegenerative diseases characterized by progressive ataxia. Here, we report on neurometabolic alterations in spinocerebellar ataxia type 1 (SCA1; SCA-ATXN1) and 14 (SCA14; SCA-PRKCG) assessed by non-invasive 1H magnetic resonance spectroscopy. Three Tesla 1H magnetic resonance spectroscopy was performed in 17 SCA14, 14 SCA1 patients, and in 31 healthy volunteers. We assessed metabolites in the cerebellar vermis, right cerebellar hemisphere, pons, prefrontal, and motor cortex. Additionally, clinical characteristics were obtained for each patient to correlate them with metabolites. In SCA14, metabolic changes were restricted to the cerebellar vermis compared with widespread neurochemical alterations in SCA1. In SCA14, total N-acetylaspartate (tNAA) was reduced in the vermis by 34%. In SCA1, tNAA was reduced in the vermis (24%), cerebellar hemisphere (26%), and pons (25%). SCA14 patients showed 24% lower glutamate+glutamine (Glx) and 46% lower γ-aminobutyric acid (GABA) in the vermis, while SCA1 patients showed no alterations in Glx and GABA. SCA1 revealed a decrease of aspartate (Asp) in the vermis (62%) and an elevation in the prefrontal cortex (130%) as well as an elevation of myo-inositol (Ins) in the cerebellar hemisphere (51%) and pons (46%). No changes of Asp and Ins were detected in SCA14. Beyond, glucose (Glc) was increased in the vermis of both SCA14 (155%) and SCA1 (247%). 1H magnetic resonance spectroscopy revealed differing neurochemical profiles in SCA1 and SCA14 and confirmed metabolic changes that may be indicative for neuronal loss and dysfunctional energy metabolism. Therefore, 1H magnetic resonance spectroscopy represents a helpful tool for in-vivo tracking of disease-specific pathophysiology.

The Amyloid-beta rich CNS environment alters myeloid cell functionality independent of their origin.

Microglia, the innate immune cells of the central nervous system (CNS) survey their surroundings with their cytoplasmic processes, phagocytose debris and rapidly respond to injury. These functions are affected by the presence of beta-Amyloid (Aß) deposits, hallmark lesions of Alzheimer's disease (AD). We recently demonstrated that exchanging functionally altered endogenous microglia with peripheral myeloid cells did not change Aß-burden in a mouse model mimicking aspects of AD at baseline, and only mildly reduced Aß plaques upon stimulation. To better characterize these different myeloid cell populations, we used long-term in vivo 2-photon microscopy to compare morphology and basic functional parameters of brain populating peripherally-derived myeloid cells and endogenous microglia. While peripherally-derived myeloid cells exhibited increased process movement in the non-diseased brain, the Aß rich environment in an AD-like mouse model, which induced an alteration of surveillance functions in endogenous microglia, also restricted functional characteristics and response to CNS injury of newly recruited peripherally-derived myeloid cells. Our data demonstrate that the Aß rich brain environment alters the functional characteristics of endogenous microglia as well as newly recruited peripheral myeloid cells, which has implications for the role of myeloid cells in disease and the utilization of these cells in Alzheimer's disease therapy.

New variant of necklace fibres display peculiar lysosomal structures and mitophagy.

Here, we describe a new variant of necklace fibres with specific myopathological features that have not been described thus far. They were observed in two patients, from two independent families with identical DNM2 (dynamin 2) mutation (c.1106 G > A (p.Arg369Gln)), displaying mildly heterogeneous clinical phenotypes. The variant is characterized by lysosomal inclusions, arranged in a necklace pattern, containing homogenous material, devoid of myonuclei. The so-called necklace region has a certain characteristic distance to the sarcolemma. Electron microscopy, including three dimensional reconstructions of serial section images highlights their ultrastructural properties and relation to neighbouring organelles. This new pattern is compared to the previously reported patterns in muscle biopsies containing necklace fibres associated with MTM1- and DNM2-mutations.

Neuroimaging Biomarkers Predict Brain Structural Connectivity Change in a Mouse Model of Vascular Cognitive Impairment.

BACKGROUND AND PURPOSE: Chronic hypoperfusion in the mouse brain has been suggested to mimic aspects of vascular cognitive impairment, such as white matter damage. Although this model has attracted attention, our group has struggled to generate a reliable cognitive and pathological phenotype. This study aimed to identify neuroimaging biomarkers of brain pathology in aged, more severely hypoperfused mice. METHODS: We used magnetic resonance imaging to characterize brain degeneration in mice hypoperfused by refining the surgical procedure to use the smallest reported diameter microcoils (160 µm). RESULTS: Acute cerebral blood flow decreases were observed in the hypoperfused group that recovered over 1 month and coincided with arterial remodeling. Increasing hypoperfusion resulted in a reduction in spatial learning abilities in the water maze that has not been previously reported. We were unable to observe severe white matter damage with histology, but a novel approach to analyze diffusion tensor imaging data, graph theory, revealed substantial reorganization of the hypoperfused brain network. A logistic regression model from the data revealed that 3 network parameters were particularly efficient at predicting group membership (global and local efficiency and degrees), and clustering coefficient was correlated with performance in the water maze. CONCLUSIONS: Overall, these findings suggest that, despite the autoregulatory abilities of the mouse brain to compensate for a sudden decrease in blood flow, there is evidence of change in the brain networks that can be used as neuroimaging biomarkers to predict outcome.

Afferent Visual Pathway Affection in Patients with PMP22 Deletion-Related Hereditary Neuropathy with Liability to Pressure Palsies.

BACKGROUND: The PMP22 gene encodes a protein integral to peripheral myelin. Its deletion leads to hereditary neuropathy with liability to pressure palsies (HNPP). PMP22 is not expressed in the adult central nervous system, but previous studies suggest a role in CNS myelin development. The objective of this study was to identify potential structural and functional alterations in the afferent visual system in HNPP patients. METHODS: Twenty HNPP patients and 18 matched healthy controls (HC) were recruited in a cross-sectional study. Participants underwent neurological examination including visual acuity, visual evoked potential (VEP) examination, optical coherence tomography (OCT), and magnetic resonance imaging with calculation of brain atrophy, regarding grey and white matter, and voxel based morphometry (VBM), in addition answered the National Eye Institute's 39-item Visual Functioning Questionnaire (NEI-VFQ). Thirteen patients and 6 HC were additionally examined with magnetic resonance spectroscopy (MRS). RESULTS: All patients had normal visual acuity, but reported reduced peripheral vision in comparison to HC in the NEI-VFQ (p = 0.036). VEP latency was prolonged in patients (P100 = 103.7±5.7 ms) in comparison to healthy subjects (P100 = 99.7±4.2 ms, p = 0.007). In OCT, peripapillary retinal nerve fiber layer thickness RNFL was decreased in the nasal sector (90.0±15.5 vs. 101.8±16.5, p = 0.013), and lower nasal sector RNFL correlated with prolonged VEP latency (Rho = -0.405, p = 0.012). MRS revealed reduced tNAA (731.4±45.4 vs. 814.9±62.1, p = 0.017) and tCr (373.8±22.2 vs. 418.7±31.1, p = 0.002) in the visual cortex in patients vs. HC. Whole brain volume, grey and white matter volume, VBM and metabolites in a MRS sensory cortex control voxel did not differ significantly between patients and HC. CONCLUSION: PMP22 deletion leads to functional, metabolic and macro-structural alterations in the afferent visual system of HNPP patients. Our data suggest a functional relevance of these changes for peripheral vision, which warrants further investigation and confirmation.

Tracking CNS and systemic sources of oxidative stress during the course of chronic neuroinflammation.

The functional dynamics and cellular sources of oxidative stress are central to understanding MS pathogenesis but remain elusive, due to the lack of appropriate detection methods. Here we employ NAD(P)H fluorescence lifetime imaging to detect functional NADPH oxidases (NOX enzymes) in vivo to identify inflammatory monocytes, activated microglia, and astrocytes expressing NOX1 as major cellular sources of oxidative stress in the central nervous system of mice affected by experimental autoimmune encephalomyelitis (EAE). This directly affects neuronal function in vivo, indicated by sustained elevated neuronal calcium. The systemic involvement of oxidative stress is mirrored by overactivation of NOX enzymes in peripheral CD11b(+) cells in later phases of both MS and EAE. This effect is antagonized by systemic intake of the NOX inhibitor and anti-oxidant epigallocatechin-3-gallate. Together, this persistent hyper-activation of oxidative enzymes suggests an "oxidative stress memory" both in the periphery and CNS compartments, in chronic neuroinflammation.

Cerebellar neurochemical alterations in spinocerebellar ataxia type 14 appear to include glutathione deficiency.

Autosomal dominant ataxia type 14 (SCA14) is a rare usually adult-onset progressive disorder with cerebellar neurodegeneration caused by mutations in protein kinase C gamma. We set out to examine cerebellar and extracerebellar neurochemical changes in SCA14 by MR spectroscopy. In 13 SCA14 patients and 13 healthy sex- and age-matched controls, 3-T single-voxel brain proton MR spectroscopy was performed in a cerebellar voxel of interest (VOI) at TE = 30 ms to obtain a neurochemical profile of metabolites with short relaxation times. In the cerebellum and in additional VOIs in the prefrontal cortex, motor cortex, and somatosensory cortex, a second measurement was performed at TE = 144 ms to mainly extract the total N-acetyl-aspartate (tNAA) signal besides the signals for total creatine (tCr) and total choline (tCho). The cerebellar neurochemical profile revealed a decrease in glutathione (6.12E-06 ± 2.50E-06 versus 8.91E-06 ± 3.03E-06; p = 0028) and tNAA (3.78E-05 ± 5.67E-06 versus 4.25E-05 ± 5.15E-06; p = 0023) and a trend for reduced glutamate (2.63E-05 ± 6.48E-06 versus 3.15E-05 ± 7.61E-06; p = 0062) in SCA14 compared to controls. In the tNAA-focused measurement, cerebellar tNAA (296.6 ± 42.6 versus 351.7 ± 16.5; p = 0004) and tCr (272.1 ± 25.2 versus 303.2 ± 31.4; p = 0004) were reduced, while the prefrontal, somatosensory and motor cortex remained unaffected compared to controls. Neuronal pathology in SCA14 detected by MR spectroscopy was restricted to the cerebellum and did not comprise cortical regions. In the cerebellum, we found in addition to signs of neurodegeneration a glutathione reduction, which has been associated with cellular damage by oxidative stress in other neurodegenerative diseases such as Parkinson's disease and Friedreich's ataxia.

Intravital FRET: Probing Cellular and Tissue Function in Vivo.

The development of intravital Förster Resonance Energy Transfer (FRET) is required to probe cellular and tissue function in the natural context: the living organism. Only in this way can biomedicine truly comprehend pathogenesis and develop effective therapeutic strategies. Here we demonstrate and discuss the advantages and pitfalls of two strategies to quantify FRET in vivo-ratiometrically and time-resolved by fluorescence lifetime imaging-and show their concrete application in the context of neuroinflammation in adult mice.

Inflammatory myopathy with abundant macrophages (IMAM): the immunology revisited.

We describe a patient with a clinically atypical presentation of inflammatory myopathy with abundant macrophages (IMAM) but with convincing muscle biopsy features of this subform of inflammatory myopathy. IMAM is characterized mainly by a conspicuous infiltration of muscle and connective tissue by numerous macrophages remote from necrotic and basophilic regenerating muscle fibers. Typically few, mostly CD4(+) T helper (Th) cells are also present. Here, we report a patient with IMAM and demonstrate, that most macrophages express the macrophage mannose receptor 1 (CD206) corresponding to alternatively activated (M2) polarization. Accordingly, signal transducer and activator of transcription 6 (STAT6), involved in Th2-M2 immunity, was expressed at high levels in skeletal muscle. However, TNFα, IFNγ and STAT1, mediators of the T helper 1-classically activated (M1) response were elevated in skeletal muscle and in blood, while expression of CD206 was elevated in skeletal muscle only. Our results argue that IMAM could be a distinct entity between the inflammatory myopathies rather than a subform of dermatomyositis.

Metabolic evidence for cerebral neurodegeneration in spinocerebellar ataxia type 1.

Autosomal-dominant spinocerebellar ataxia type 1 (SCA1) is an adult-onset progressive disorder with well-characterized neurodegeneration in the cerebellum and brainstem. The objective of this study is to evaluate neurochemical changes associated with neurodegeneration in cerebral tissue in SCA1 patients compared to age- and gender-matched healthy controls. Nine patients with genetically proven SCA1 and nine gender- and age-matched healthy controls were prospectively recruited from the ataxia clinic and received clinical examination. A 1.5 T single-voxel brain proton MR spectroscopy was performed for total N-acetyl aspartate (tNAA) in cerebellum, parietofrontal lobe white matter, sensory cortex, and visual cortex. In the patients, tNAA was severely decreased in the cerebellar voxel; however, in the voxels positioned in sensory cortex, parietofrontal lobe white matter and visual cortex tNAA was reduced in comparison to controls. In addition to the profoundly affected cerebellum, we also found evidence for cerebral neurodegeneration in parietal lobe white matter, sensory cortex, and visual cortex in SCA1 patients illustrating a multisystem neurodegenerative character of the disease.

Membrane-type 1 metalloproteinase is upregulated in microglia/brain macrophages in neurodegenerative and neuroinflammatory diseases.

We previously reported that glioma cells induce the expression of membrane-type 1 metalloproteinase (MT1-MMP or MMP-14) in tumor-associated microglia/macrophages and promote tumor growth, whereas MMP-14 expression in microglia under physiological conditions is very low. Here, we show that the increase in MMP-14 expression is also found in microglia/macrophages associated with neurodegenerative and neuroinflammatory pathologies in mouse models as well as in human biopsies or post-mortem tissue. We found that microglial/macrophage MMP-14 expression was upregulated in Alzheimer's disease tissue, in active lesions of multiple sclerosis, and in tissue from stage II stroke as well as in the corresponding mouse models for the human diseases. In contrast, we observed no upregulation for MMP-14 in microglia/macrophages in the early phase of stroke or in the corresponding mouse model, in human amyotrophic lateral sclerosis (ALS) tissue or in a mouse model of ALS as well as in human cases of acute brain trauma. These data indicate that MMP-14 expression is not a general marker for activated microglia/macrophages but is upregulated in defined stages of neuroinflammatory and neurodegenerative diseases and that there is generally a good match between mouse models and human brain pathologies.

Parallelized TCSPC for dynamic intravital fluorescence lifetime imaging: quantifying neuronal dysfunction in neuroinflammation.

Two-photon laser-scanning microscopy has revolutionized our view on vital processes by revealing motility and interaction patterns of various cell subsets in hardly accessible organs (e.g. brain) in living animals. However, current technology is still insufficient to elucidate the mechanisms of organ dysfunction as a prerequisite for developing new therapeutic strategies, since it renders only sparse information about the molecular basis of cellular response within tissues in health and disease. In the context of imaging, Förster resonant energy transfer (FRET) is one of the most adequate tools to probe molecular mechanisms of cell function. As a calibration-free technique, fluorescence lifetime imaging (FLIM) is superior for quantifying FRET in vivo. Currently, its main limitation is the acquisition speed in the context of deep-tissue 3D and 4D imaging. Here we present a parallelized time-correlated single-photon counting point detector (p-TCSPC) (i) for dynamic single-beam scanning FLIM of large 3D areas on the range of hundreds of milliseconds relevant in the context of immune-induced pathologies as well as (ii) for ultrafast 2D FLIM in the range of tens of milliseconds, a scale relevant for cell physiology. We demonstrate its power in dynamic deep-tissue intravital imaging, as compared to multi-beam scanning time-gated FLIM suitable for fast data acquisition and compared to highly sensitive single-channel TCSPC adequate to detect low fluorescence signals. Using p-TCSPC, 256×256 pixel FLIM maps (300×300 µm(2)) are acquired within 468 ms while 131×131 pixel FLIM maps (75×75 µm(2)) can be acquired every 82 ms in 115 µm depth in the spinal cord of CerTN L15 mice. The CerTN L15 mice express a FRET-based Ca-biosensor in certain neuronal subsets. Our new technology allows us to perform time-lapse 3D intravital FLIM (4D FLIM) in the brain stem of CerTN L15 mice affected by experimental autoimmune encephalomyelitis and, thereby, to truly quantify neuronal dysfunction in neuroinflammation.

High-resolution intravital microscopy.

Cellular communication constitutes a fundamental mechanism of life, for instance by permitting transfer of information through synapses in the nervous system and by leading to activation of cells during the course of immune responses. Monitoring cell-cell interactions within living adult organisms is crucial in order to draw conclusions on their behavior with respect to the fate of cells, tissues and organs. Until now, there is no technology available that enables dynamic imaging deep within the tissue of living adult organisms at sub-cellular resolution, i.e. detection at the level of few protein molecules. Here we present a novel approach called multi-beam striped-illumination which applies for the first time the principle and advantages of structured-illumination, spatial modulation of the excitation pattern, to laser-scanning-microscopy. We use this approach in two-photon-microscopy--the most adequate optical deep-tissue imaging-technique. As compared to standard two-photon-microscopy, it achieves significant contrast enhancement and up to 3-fold improved axial resolution (optical sectioning) while photobleaching, photodamage and acquisition speed are similar. Its imaging depth is comparable to multifocal two-photon-microscopy and only slightly less than in standard single-beam two-photon-microscopy. Precisely, our studies within mouse lymph nodes demonstrated 216% improved axial and 23% improved lateral resolutions at a depth of 80 µm below the surface. Thus, we are for the first time able to visualize the dynamic interactions between B cells and immune complex deposits on follicular dendritic cells within germinal centers (GCs) of live mice. These interactions play a decisive role in the process of clonal selection, leading to affinity maturation of the humoral immune response. This novel high-resolution intravital microscopy method has a huge potential for numerous applications in neurosciences, immunology, cancer research and developmental biology. Moreover, our striped-illumination approach is able to improve the resolution of any laser-scanning-microscope, including confocal microscopes, by simply choosing an appropriate detector.

In vivo imaging of partially reversible th17 cell-induced neuronal dysfunction in the course of encephalomyelitis.

Neuronal damage in autoimmune neuroinflammation is the correlate for long-term disability in multiple sclerosis (MS) patients. Here, we investigated the role of immune cells in neuronal damage processes in animal models of MS by monitoring experimental autoimmune encephalomyelitis (EAE) by using two-photon microscopy of living anaesthetized mice. In the brainstem, we detected sustained interaction between immune and neuronal cells, particularly during disease peak. Direct interaction of myelin oligodendrocyte glycoprotein (MOG)-specific Th17 and neuronal cells in demyelinating lesions was associated with extensive axonal damage. By combining confocal, electron, and intravital microscopy, we showed that these contacts remarkably resembled immune synapses or kinapses, albeit with the absence of potential T cell receptor engagement. Th17 cells induced severe, localized, and partially reversible fluctuation in neuronal intracellular Ca(2+) concentration as an early sign of neuronal damage. These results highlight the central role of the Th17 cell effector phenotype for neuronal dysfunction in chronic neuroinflammation.