Markus Morawski

Aneuploidy and DNA Replication in the Normal Human Brain and Alzheimer's Disease

Reactivation of the cell cycle, including DNA replication, might play a major role in Alzheimers disease (AD). A more than diploid DNA content in differentiated neurons might alternatively result from chromosome mis-segregation during mitosis in neuronal progenitor cells. It was our objective to distinguish between these two mechanisms for aneuploidy and to provide evidence for a functional cell cycle in AD. Using slide-based cytometry, chromogenic in situ hybridization, and PCR amplification of alu-repeats, we quantified the DNA amount of identified cortical neurons in normal human brain and AD and analyzed the link between a tetraploid DNA content and expression of the early mitotic marker cyclin B1. In the normal brain, the number of neurons with a more than diploid content amounts to ∼10%. Less than 1% of neurons contains a tetraploid DNA content. These neurons do not express cyclin B1, most likely representing constitutional tetraploidy. This population of cyclin B1-negative tetraploid neurons, at a reduced number, is also present in AD. In addition, a population of cyclin B1-positive tetraploid neurons of ∼2% of all neurons was observed in AD. Our results indicate that at least two different mechanisms need to be distinguished giving rise to a tetraploid DNA content in the adult brain. Constitutional aneuploidy in differentiated neurons might be more frequent than previously thought. It is, however, not elevated in AD. In addition, in AD some neurons have re-entered the cell cycle and entirely passed through a functional interphase with a complete DNA replication.

Myelin and iron concentration in the human brain: A quantitative study of MRI contrast

During the last five years ultra-high-field magnetic resonance imaging (MRI) has enabled an unprecedented view of living human brain. Brain tissue contrast in most MRI sequences is known to reflect mainly the spatial distributions of myelin and iron. These distributions have been shown to overlap significantly in many brain regions, especially in the cortex. It is of increasing interest to distinguish and identify cortical areas by their appearance in MRI, which has been shown to be feasible in vivo. Parcellation can benefit greatly from quantification of the independent contributions of iron and myelin to MRI contrast. Recent studies using susceptibility mapping claim to allow such a separation of the effects of myelin and iron in MRI. We show, using post-mortem human brain tissue, that this goal can be achieved. After MRI scanning of the block with appropriate T1 mapping and T2* weighted sequences, we section the block and apply a novel technique, proton induced X-ray emission (PIXE), to spatially map iron, phosphorus and sulfur elemental concentrations, simultaneously with 1μm spatial resolution. Because most brain phosphorus is located in myelin phospholipids, a calibration step utilizing element maps of sulfur enables semi-quantitative ex vivo mapping of myelin concentration. Combining results for iron and myelin concentration in a linear model, we have accurately modeled MRI tissue contrasts. Conversely, iron and myelin concentrations can now be estimated from appropriate MRI measurements in post-mortem brain samples.

Perineuronal nets potentially protect against oxidative stress.

A specialized form of extracellular matrix (ECM) termed perineuronal nets (PNs) consisting of large aggregating chondroitin sulfate proteoglycans (CSPGs), with hyaluronan and tenascin as main components, surrounds subpopulations of neurons. The glycosaminoglycan components of perineuronal nets form highly charged structures in the direct microenvironment of neurons and thus might be involved in local ion homeostasis. The polyanionic character suggests that perineuronal nets also potentially contribute to reduce the local oxidative potential in the neuronal microenvironment by scavenging and binding redox-active iron, thus providing some neuroprotection to net-associated neurons. Here, we show that neurons ensheathed by a perineuronal net in the human cerebral cortex are less frequently affected by lipofuscin accumulation than neurons without a net both in normal-aged brain and Alzheimers disease (AD). As lipofuscin is an intralysosomal pigment composed of cross-linked proteins and lipids generated by iron-catalyzed oxidative processes, the present results suggest a neuroprotective function of perineuronal nets against oxidative stress, potentially involved in neurodegeneration.

PERINEURONAL NET FORMATION AND STRUCTURE IN AGGRECAN KNOCKOUT MICE

Perineuronal nets (PNNs) are specialized substructures of the neural extracellular matrix (ECM) which envelop the cell soma and proximal neurites of particular sets of neurons with apertures at sites of synaptic contact. Previous studies have shown that PNNs are enriched with chondroitin sulfate proteoglycans (CSPGs) and hyaluronan, however, a complete understanding of their precise molecular composition has been elusive. In addition, identifying which specific PNN components are critical to the formation of this structure has not been demonstrated. Previous work in our laboratory has demonstrated that the CSPG, aggrecan, is a key activity-dependent component of PNNs in vivo. In order to assess the contribution of aggrecan to PNN formation, we utilized cartilage matrix deficiency (cmd) mice, which lack aggrecan. Herein, we utilized an in vitro model, dissociated cortical culture, and an ex vivo model, organotypic slice culture, to specifically investigate the role aggrecan plays in PNN formation. Our work demonstrates that staining with the lectin, Wisteria floribunda agglutinin (WFA), considered a broad PNN marker, is eliminated in the absence of aggrecan, suggesting the loss of PNNs. However, in contrast, we found that the expression patterns of other PNN markers, including hyaluronan and proteoglycan link protein 1 (HAPLN1), tenascin-R, brevican, and hyaluronan are unaffected by the absence of aggrecan. Lastly, we determined that while all PNN components are bound to the surface in a hyaluronan-dependent manner, only HAPLN1 remains attached to the cell surface when neurons are treated with chondroitinase. These results suggest a different model for the molecular association of PNNs to the cell surface. Together our work has served to assess the contribution of aggrecan to PNN formation while providing key evidence concerning the molecular composition of PNNs in addition to determining how these components ultimately form PNNs.

Neurons associated with aggrecan-based perineuronal nets are protected against tau pathology in subcortical regions in Alzheimer's disease

The biological basis for the selective vulnerability of neurons in Alzheimers disease (AD) is elusive. Aggrecan-based perineuronal nets (PNs) of the extracellular matrix have been considered to contribute to neuroprotection in the cerebral cortex. In the present study, we investigated the organization of the aggrecan-based extracellular matrix in subcortical regions known to be preferentially affected by tau pathology in AD. Immunocytochemistry of aggrecan core protein was combined with detection of neurofibrillary degeneration. The results show that many regions affected by tau pathology in AD, such as the basal nucleus of Meynert, the dorsal thalamus, hypothalamic nuclei, raphe nuclei, and the locus coeruleus were devoid of a characteristic aggrecan-based extracellular matrix. Regions composed of nuclei with clearly different intensity of tau pathology, such as the amygdala, the thalamus and the oculomotor complex, showed largely complementary distribution patterns of neurofibrillary tangles and PNs. Quantification in the rostral interstitial nucleus of the longitudinal fascicle potentially affected by tau pathology in AD revealed that tau pathology was not accompanied by loss of aggrecan-based PNs. Neuro-fibrillary tangles in net-associated neurons extremely rarely occurred in the pontine reticular formation. We conclude that the low vulnerability of neurons ensheathed by PNs previously described for cortical areas in AD represents a more general phenomenon that also applies to subcortical regions. The aggrecan-based extracellular matrix of PNs may thus, be involved in neuroprotection.

Involvement of Perineuronal and Perisynaptic Extracellular Matrix in Alzheimer’s Disease Neuropathology

Brain extracellular matrix (ECM) is organized in specific patterns assumed to mirror local features of neuronal activity and synaptic plasticity. Aggrecan‐based perineuronal nets (PNs) and brevican‐based perisynaptic axonal coats (ACs) form major structural phenotypes of ECM contributing to the laminar characteristics of cortical areas. In Alzheimers disease (AD), the deposition of amyloid proteins and processes related to neurofibrillary degeneration may affect the integrity of the ECM scaffold. In this study we investigate ECM organization in primary sensory, secondary and associative areas of the temporal and occipital lobe. By detecting all major PN components we show that the distribution, structure and molecular properties of PNs remain unchanged in AD. Intact PNs occurred in close proximity to amyloid plaques and were even located within their territory. Counting of PNs revealed no significant alteration in AD. Moreover, neurofibrillary tangles never occurred in neurons associated with PNs. ACs were only lost in the core of amyloid plaques in parallel with the loss of synaptic profiles. In contrast, hyaluronan was enriched in the majority of plaques. We conclude that the loss of brevican is associated with the loss of synapses, whereas PNs and related matrix components resist disintegration and may protect neurons from degeneration.

Aggrecan, link protein and tenascin-R are essential components of the perineuronal net to protect neurons against iron-induced oxidative stress.

In Alzheimer’s disease (AD), different types of neurons and different brain areas show differential patterns of vulnerability towards neurofibrillary degeneration, which provides the basis for a highly predictive profile of disease progression throughout the brain that now is widely accepted for neuropathological staging. In previous studies we could demonstrate that in AD cortical and subcortical neurons are constantly less frequently affected by neurofibrillary degeneration if they are enwrapped by a specialized form of the hyaluronan-based extracellular matrix (ECM), the so called ‘perineuronal net’ (PN). PNs are basically composed of large aggregating chondroitin sulphate proteoglycans connected to a hyaluronan backbone, stabilized by link proteins and cross-linked via tenascin-R (TN-R). Under experimental conditions in mice, PN-ensheathed neurons are better protected against iron-induced neurodegeneration than neurons without PN. Still, it remains unclear whether these neuroprotective effects are directly mediated by the PNs or are associated with some other mechanism in these neurons unrelated to PNs. To identify molecular components that essentially mediate the neuroprotective aspect on PN-ensheathed neurons, we comparatively analysed neuronal degeneration induced by a single injection of FeCl3 on four different mice knockout strains, each being deficient for a different component of PNs. Aggrecan, link protein and TN-R were identified to be essential for the neuroprotective properties of PN, whereas the contribution of brevican was negligible. Our findings indicate that the protection of PN-ensheathed neurons is directly mediated by the net structure and that both the high negative charge and the correct interaction of net components are essential for their neuroprotective function.

Aggrecan: Beyond cartilage and into the brain.

Aggrecan is well-studied in cartilage but its expression and function in the central nervous system has only recently begun to be appreciated. Aggrecan plays an important role in the organization of the neural extracellular space by binding and organizing hyaluronan to the cell surface through interactions with link protein and tenascins forming a large aggregated quaternary complex. While all members of the lectican family to which aggrecan belongs are thought to mediate similar roles in organizing the neural matrix, aggrecan is unique in that it is the only family member found almost exclusively in an enigmatic matrix substructure called the perineuronal net. Current work has established a critical role for perineuronal nets and aggrecan in regulating developmental neural plasticity and in the recover from injury. In this review we focus on the structure, expression and function of aggrecan in the central nervous system.

AGGRECAN-BASED EXTRACELLULAR MATRIX IS AN INTEGRAL PART OF THE HUMAN BASAL GANGLIA CIRCUIT

The extracellular matrix is known to be involved in neuronal communication and the regulation of plastic changes, and also considered to protect neurons and synapses against damage. The goal of this study was to investigate how major extracellular matrix components (aggrecan, link protein, hyaluronan) constitute the pathways of the nigral system in the human basal ganglia circuit affected by neurodegeneration in Parkinsons disease. Here we show that aggrecan- and link protein-related components form clear regional distribution patterns, whereas hyaluronan is widely distributed in gray and white matter. Two predominant phenotypes of the aggrecan-based matrix can be discriminated: (1) perineuronal nets (PNs) and (2) axonal coats (ACs) encapsulating preterminal fibers and synaptic boutons. Clearly contoured PNs are associated with GABAergic projection neurons in the external and internal division of the globus pallidus, the lateral and reticular part of the substantia nigra, as well as subpopulations of striatal and thalamic inhibitory interneurons. Dopaminergic nigral neurons are devoid of PNs but are contacted to a different extent by matrix-coated boutons forming subnucleus-specific patterns. A very dense network of ACs is characteristic especially of the posterior lateral cell groups of the compact substantia nigra (nigrosome 1). In the subthalamic nucleus and the lateral thalamic nuclei numerous AC-associated axons were attached to principal neurons devoid of PNs. We conclude from the region-specific patterns that the aggrecan-based extracellular matrix is adapted to the fast processing of sensorimotor activities which are the therapeutic target of surgery and deep brain stimulation in the treatment of advanced stages of Parkinsons disease.

Neurochemical mapping of the human hippocampus reveals perisynaptic matrix around functional synapses in Alzheimer’s disease

Perineuronal matrix is an extracellular protein scaffold to shape neuronal responsiveness and survival. Whilst perineuronal nets engulf the somatodendritic axis of neurons, axonal coats are focal extracellular protein aggregates surrounding individual synapses. Here, we addressed the chemical identity and subcellular localization of both perineuronal and perisynaptic matrices in the human hippocampus, whose neuronal circuitry is progressively compromised in Alzheimer’s disease. We hypothesized that (1) the cellular expression sites of chondroitin sulphate proteoglycan-containing extracellular matrix associate with specific neuronal identities, reflecting network dynamics, and (2) the regional distribution and molecular composition of axonal coats must withstand Alzheimer’s disease-related modifications to protect functional synapses. We show by epitope-specific antibodies that the perineuronal protomap of the human hippocampus is distinct from other mammals since pyramidal cells but not calretinin+ and calbindin+ interneurons, neurochemically classified as novel neuronal subtypes, lack perineuronal nets. We find that cartilage link protein-1 and brevican-containing matrices form isolated perisynaptic coats, engulfing both inhibitory and excitatory terminals in the dentate gyrus and entorhinal cortex. Ultrastructural analysis revealed that presynaptic neurons contribute components of perisynaptic coats via axonal transport. We demonstrate, by combining biochemical profiling and neuroanatomy in Alzheimer’s patients and transgenic (APdE9) mice, the preserved turnover and distribution of axonal coats around functional synapses along dendrite segments containing hyperphosphorylated tau and in amyloid-β-laden hippocampal microdomains. We conclude that the presynapse-driven formation of axonal coats is a candidate mechanism to maintain synapse integrity under neurodegenerative conditions.