Group Prof. Dr. Dr. Markus Morawski

The influence of perineuronal nets on the aggregation and spreading of Tau protein in Alzheimer's disease​​

The role of extracellular matrix in dystonia and dyskinesia​

Dystonia is a common movement disorder, characterized by involuntary and extended muscle contraction resulting in repetitive movements and abnormal postures. Dystonic movements are typically patterned and twisting, and may be tremulous. Dystonia is often initiated or worsened by voluntary action and associated with overflow muscle activation.

Although Dystonia is regarded as the third most common disorder movement in humans, it is often misdiagnosed because of the heterogeneity of etiology and clinical presentation. Recently, a severe paroxysmal movement disorder, the episodic falling syndrome was discovered to be caused by a deletion of the BCAN gene, encoding the brain-specific extracellular matrix (ECM) proteoglycan brevican (Gill et al., 2012). Brevican in this regard is a substantial part of the perineuronal nets (PNs) representing a special type of the brain extracellular matrix(ECM). PNs are mainly constructed by hyaluronan polymer chains, chontroitin sulfate proteoglycans (CSPGs like aggrecan, brevican and neurocan ), link-proteins and tenascin-R, altogether they form a quaternary complex surrounding the neurons. This neuronal ECM of PNs is well known to be associated with GABAergic projection neurons in the basal ganglia especially with subpopulations of striatal inhibitory interneuron (Brückner et al., 2008; Brauer et al., 1995). There is evidence from studies in human patients and animal models that reduced inhibition by striatal parvalbumin interneurons could be involved in basal ganglia disorders, including dystonia (Richter and Richter, 2014). In the dtsz mutant hamster, a model of paroxysmal dystonic choreoathetosis, an age-dependent reduction of PV-positive interneurons (Hamann et al., 2007) was found. Interestingly, the reduction of the striatal interneurons in the dtsz mutant hamster occurs at the maximum expression of dystonia, around 30 days of age, which is the exact time point of fully maturation of PNs (Avchalumov et al., Morawski et al., 2014).

Clarifying the relationship between the neuronal ECM proteins of PNs and the genesis of dystonia and dyskinesia will be the main aspect of this project. Therefore, established immunohistochemical markers and protein biochemical analysis will be used to characterize the spatiotemporal distribution and expression of the neuronal ECM in context of the occurrence of paroxysmal dystonia in the dtsz mutant hamster.​

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Extracellular matrix of perineuronal nets in the auditory system: from structure to function​

The central nervous system (CNS) is mainly composed of two types of cells, neurons and glia cells, which form most of the CNS volume. Still, about 20% of CNS volume is generated by extracellular matrix (ECM) which is composed of specific macromolecules that fill the extracellular space and remarkably contribute to the cellular and structural organization of the CNS. Perineuronal nets (PNs), in this regard are classified as a specialized composition of ECM molecules which surrounds the soma, dendrites and the axon initial segment of particular neurons in the CNS.

PNs are mainly composed of chondroitin sulfate proteoglycans (CSPGs), including aggrecan, brevican and neurocan. The CSPGs determine the physico-chemical properties of PNs by generating a strong, negative charge, thus establishing an anionic milieu in the immediate vicinity of neurons. The function of the PNs is not fully understood but it is - among others - assumed that PNs contribute to stabilization of synaptic contacts and further control neuronal activity and synaptic transmission, e.g. through buffering of physiologically relevant cations such as potassium, sodium and calcium. However, there are hardly any reports providing convincing experimental evidence.

The auditory system (especially the nuclei of the auditory brainstem) is the main target of our research since it is characterized by an exceptionally high density of PN-positive neurons. Surprisingly, these auditory nuclei have never been considered in any of the earlier studies addressing the functional role of PNs (Sonntag et al., 2015).

Our research is focused on a detailed immunohistochemical and histological analysis of the structure and composition of PNs in the auditory system of (i) humans in comparison to mouse and of (ii) animal species with exceptional auditory capabilities (including almost deaf blind-mole rat and infrasound hearing elephant). These data enable to estimate potential adaptations in PN structure depending on the anatomy of the auditory pathway and the respective hearing abilities. In addition, we use electrophysiological, biochemical, electronmicroscopical and molecular biology techniques to study the function of the proteoglycans in the auditory system, especially (iii) the role in developmental processes and (iv) the impact on neuronal activity and synaptic transmission, primarily in ECM transgenic mouse models (deficient for aggrecan, brevican and neurocan).​

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Quantification and subcellular localisation of iron in the substantia nigra in Parkinson's disease​

Iron plays due to its versatile function a key role in cell viability. In contrast, high concentrations of free iron raise the level of oxidative stress within the cell via the Fenton-Haber-Weiss-reaction and promote the formation of toxic radicals. Resulting reactive species lead ultimately to cell death.

Therefore, iron plays a fundamental role in neurodegenerative diseases such as Parkinson´s disease (PD). It is well known that the iron concentration is increased in the Substantia nigra pars compacta (SNpc) in Parkinson's disease (PD). By contrast, there is only little knowledge about the cellular distribution and quantitative iron concentrations in the different cell types such as microglia, astroglia or oligodendroglia (myelin).​

This project aims at examining the cellular and subcellular localization as well as the concentration of iron in the main cell types in the nigrosom 1 of the SNpc in post mortem brain tissue of patients with PD and healthy age-matched controls. Therefore, human brainstem is marked immunohistochemically for different cellular markers (antibodies against neurone, microglia, astroglia, oligodendroglia and myelin with nickel-diaminobenzidine-enhancement).

Additionally, histochemical methods like Perls and Turnbull stain were performed in order to identify the cellular localization of non-chelated Fe2+ - and Fe3+ - ions.

Furthermore, a differential quantitative analysis of iron by ion beam microscopy (PIXE, Particle Induced X-Ray emission) represents one of the main purposes of this project. Therefore, PIXE method can be combined with classic immunohistochemistry. The ultrapure nickel (Ni-DAB-enhancement) may be used as a respecting cell marker and offers hereby an immense variety of cellular and subcellular markers in order to determine the localization and concentration of iron in requested cell types and subcellular fractions.

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The comprehensive microstructural human connectome (COMIC): from long-range to short-association fibers​

In this DFG funded project PIs and their teams from three different institutions (MPI-CBS, UKE, PFI) will work closely together on developing models, methods and techniques to create a better map of the human microstructural connectome from long-range to short-association fibers using in vivo and ex vivo MRI as well as histology.​

This interdisciplinary project combines three work packages to integrate information from in vivo and ex vivo MRI with advanced histology to release a life span comprehensive microstructural connectome. In the first work package (WP1), a neuroanatomical connectome of short association fibers is constructed via ex vivo histology. In the second work package (WP2), novel biophysical models for short association fibers are developed. In the third work package (WP3), a comprehensive human microstructural connectome across the adult life span is generated.

Background​

The human neocortex is organized in specialized regions integrated in synchronized networks connected by long- and short-range association fibres. The microstructural composition of the fibres optimizes the speed of communication within the physical metabolic limits. These connections, their microstructural properties (e.g. axon diameters and their myelination), and their interactions can be summarized as the microstructural connectome. Most current estimates of the human structural connectome are based on Diffusion-Weighted Magnetic Resonance Imaging (DWI) and suffer from incompleteness and method-related biases. Particularly, short association connections, playing a central role in small-world-like brain networks, are underrepresented. In the first funding period, we generated a human microstructural connectome by projecting microstructural information along the long-range fibres. However, it is problematic that the short cortico-cortical connections (including U-fibers) are underrepresented in current connectomes.​

Goal​

In this project, we will generate the most comprehensive human microstructural connectome across the adult life span by additionally including the short cortico-cortical fibres and their microstructural properties.​

Methods​

To this end, we will use a powerful multidisciplinary approach that is built on three pillars (Figure). The first pillar (WP1) investigates the microstructural composition, length, and shapes of short association fibers using advanced ex vivo histology with large-scale ultra-high-resolution imaging within two specific brain networks, the visual and the sensory-motor system. The second pillar (WP2) provides deep-learning tools that quantify microstructural information (e.g. distribution of axon diameters or axonal g-ratios) from large-scale ultra-high-resolution microscopy images (WP1). Moreover, it devises biophysical models (WP2) that are validated and informed by the aforementioned quantified histological microstructural information. Finally, it implements robust methods for tractography of short- and long-range fibres based on in vivo and ex vivo MRI. The last pillar (WP3) uses the newly developed methods to robustly estimate microstructural information across short- and long-range fibres from in vivo MRI within a cohort of healthy subjects spanning the entire adult life span.​

Impact

This will generate a comprehensive life span connectome that, for the first time, incorporates both short cortico-cortical and long-range connections together with their microstructural information. Additionally, intra-cortical microstructural measures will be derived and integrated based on established cortical myelin and iron mapping approaches.​

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Human microstructural connectomics: Computational modelling and validation with histology and CLARITY (MICO-MRI)​

Long-range connections comprise only about 10% of total CNS connections. Their arrangement, length, and microstructural properties are of utmost importance for the functional organization of the CNS, because they determine how information is distributed across the brain. In particular, axon diameter and g-ratio (the ratio between axonal body and axon plus myelin sheath) is related to the conduction velocity and thus relevant for the information transfer rate in a functional CNS network.

To date, diffusion magnetic resonance imaging (dMRI)-based tractography is the only in vivo technique for mapping the structural long-range connections in the human brain. However, mapping from diffusion to fiber pathways is still ill-posed. Tractography algorithms can take "wrong turns" and produce a number of false connections.

To address this significant limitation, microstructure-informed tractography has been suggested. Microstructure-informed tractography is an emerging computational framework that associates each computed fiber tract with microstructural properties, e.g., metrics for axon diameter or density, using the dMRI technique. However, in its current form microstructure-informed tractography suffers from three major problems:

1. to estimate these metrics biophysical models are used, which in turn aim at solving another ill-posed task: relate the macroscopic MR signal to microscopic tissue properties,
2. to stabilize the ill-posed biophysical model a lower spatial resolution is required and thus coarse spatial resolution is applied thereby increasing the number of voxels with ambiguous fiber configurations,
3. it relies on the untested neuroanatomical hypothesis that two adjacent fiber pathways show less within-fiber than across-fiber variation.

In this highly inter-disciplinary project, we will develop a computational framework for microstructure-informed tractography that addresses the aforementioned limitations using multi-modal quantitative MRI at an unprecedented ultra-high spatial resolution (660 microns). Moreover, we will develop an advanced ex vivo histology analysis strategy, using complementary 2-D (high-resolution semithin and ultrathin sectioning) and 3-D (CLARITY) techniques. We will fuse gold-standard ex vivo histology with MRI to validate the proposed model at central junctions of long-range fiber pathways within the well characterized human voluntary motor control network. By emphasizing the close integration of multi-modal computational biophysical models, advanced MRI technology (the German-wide unique combination of a CONNECTOM & 7T MRI system), and advanced histological approaches (CLARITY in human tissue), this project aims at a paradigm shift in in vivo MRI-based computational models for in vivo tractography.​

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