Astrocyte-Derived Tissue Transglutaminase Interacts with Fibronectin: A Role in Astrocyte Adhesion and Migration?

An important neuropathological feature of neuroinflammatory processes that occur during e.g. Multiple Sclerosis (MS) is the formation of an astroglial scar. Astroglial scar formation is facilitated by the interaction between astrocytes and extracellular matrix proteins (ECM) such as fibronectin. Since there is evidence indicating that glial scars strongly inhibit both axon growth and (re)myelination in brain lesions, it is important to understand the factors that contribute to the interaction between astrocytes and ECM proteins. Tissue Transglutaminase (TG2) is a multifunctional enzyme with an ubiquitous tissue distribution, being clearly present within the brain. It has been shown that inflammatory cytokines can enhance TG2 activity. In addition, TG2 can mediate cell adhesion and migration and it binds fibronectin with high affinity. We therefore hypothesized that TG2 is involved in astrocyte-fibronectin interactions. Our studies using primary rat astrocytes show that intracellular and cell surface expression and activity of TG2 is increased after treatment with pro-inflammatory cytokines. Astrocyte-derived TG2 interacts with fibronectin and is involved in astrocyte adhesion onto and migration across fibronectin. TG2 is involved in stimulating focal adhesion formation which is necessary for the interaction of astrocytes with ECM proteins. We conclude that astrocyte-derived TG2 contributes to the interaction between astrocytes and fibronectin. It might thereby regulate ECM remodeling and possibly glial scarring

Vascular basement membranes as pathways for the passage of fluid into and out of the brain

In the absence of conventional lymphatics, drainage of interstitial fluid and solutes from the brain parenchyma to cervical lymph nodes is along basement membranes in the walls of cerebral capillaries and tunica media of arteries. Perivascular pathways are also involved in the entry of CSF into the brain by the convective influx/glymphatic system. The objective of this study is to differentiate the cerebral vascular basement membrane pathways by which fluid passes out of the brain from the pathway by which CSF enters the brain. Experiment 1: 0.5 µl of soluble biotinylated or fluorescent Aβ, or 1 µl 15 nm gold nanoparticles was injected into the mouse hippocampus and their distributions determined at 5 min by transmission electron microscopy. Aβ was distributed within the extracellular spaces of the hippocampus and within basement membranes of capillaries and tunica media of arteries. Nanoparticles did not enter capillary basement membranes from the extracellular spaces. Experiment 2: 2 µl of 15 nm nanoparticles were injected into mouse CSF. Within 5min, groups of nanoparticles were present in the pial-glial basement membrane on the outer aspect of cortical arteries between the investing layer of pia mater and the glia limitans. The results of this study and previous research suggest that cerebral vascular basement membranes form the pathways by which fluid passes into and out of the brain but that different basement membrane layers are involved. The significance of these findings for neuroimmunology, Alzheimer's disease, drug delivery to the brain and the concept of the Virchow-Robin space are discussed

Molecular Structure of Amyloid Fibrils Controls the Relationship between Fibrillar Size and Toxicity

According to the prevailing view, soluble oligomers or small fibrillar fragments are considered to be the most toxic species in prion diseases. To test this hypothesis, two conformationally different amyloid states were produced from the same highly pure recombinant full-length prion protein (rPrP). The cytotoxic potential of intact fibrils and fibrillar fragments generated by sonication from these two states was tested using cultured cells.For one amyloid state, fibril fragmentation was found to enhance its cytotoxic potential, whereas for another amyloid state formed within the same amino acid sequence, the fragmented fibrils were found to be substantially less toxic than the intact fibrils. Consistent with the previous studies, the toxic effects were more pronounced for cell cultures expressing normal isoform of the prion protein (PrP(C)) at high levels confirming that cytotoxicity was in part PrP(C)-dependent. Silencing of PrP(C) expression by small hairpin RNAs designed to silence expression of human PrP(C) (shRNA-PrP(C)) diminished the deleterious effects of the two amyloid states to a different extent, suggesting that the role of PrP(C)-mediated and PrP(C)-independent mechanisms depends on the structure of the aggregates.This work provides a direct illustration that the relationship between an amyloid's physical dimension and its toxic potential is not unidirectional but is controlled by the molecular structure of prion protein (PrP) molecules within aggregated states. Depending on the structure, a decrease in size of amyloid fibrils can either enhance or abolish their cytotoxic effect. Regardless of the molecular structure or size of PrP aggregates, silencing of PrP(C) expression can be exploited to reduce their deleterious effects

Calcineurin Inhibition at the Clinical Phase of Prion Disease Reduces Neurodegeneration, Improves Behavioral Alterations and Increases Animal Survival

Prion diseases are fatal neurodegenerative disorders characterized by a long pre-symptomatic phase followed by rapid and progressive clinical phase. Although rare in humans, the unconventional infectious nature of the disease raises the potential for an epidemic. Unfortunately, no treatment is currently available. The hallmark event in prion diseases is the accumulation of a misfolded and infectious form of the prion protein (PrPSc). Previous reports have shown that PrPSc induces endoplasmic reticulum stress and changes in calcium homeostasis in the brain of affected individuals. In this study we show that the calcium-dependent phosphatase Calcineurin (CaN) is hyperactivated both in vitro and in vivo as a result of PrPSc formation. CaN activation mediates prion-induced neurodegeneration, suggesting that inhibition of this phosphatase could be a target for therapy. To test this hypothesis, prion infected wild type mice were treated intra-peritoneally with the CaN inhibitor FK506 at the clinical phase of the disease. Treated animals exhibited reduced severity of the clinical abnormalities and increased survival time compared to vehicle treated controls. Treatment also led to a significant increase in the brain levels of the CaN downstream targets pCREB and pBAD, which paralleled the decrease of CaN activity. Importantly, we observed a lower degree of neurodegeneration in animals treated with the drug as revealed by a higher number of neurons and a lower quantity of degenerating nerve cells. These changes were not dependent on PrPSc formation, since the protein accumulated in the brain to the same levels as in the untreated mice. Our findings contribute to an understanding of the mechanism of neurodegeneration in prion diseases and more importantly may provide a novel strategy for therapy that is beneficial at the clinical phase of the disease

Microglial activation and chronic neurodegeneration

Microglia, the resident innate immune cells in the brain, have long been implicated in the pathology of neurode-generative diseases. Accumulating evidence points to activated microglia as a chronic source of multiple neurotoxic factors, including tumor necrosis factor-α, nitric oxide, interleukin-1β, and reactive oxygen species (ROS), driving progressive neuron damage. Microglia can become chronically activated by either a single stimulus (e.g., lipopolysaccharide or neuron damage) or multiple stimuli exposures to result in cumulative neuronal loss with time. Although the mechanisms driving these phenomena are just beginning to be understood, reactive microgliosis (the microglial response to neuron damage) and ROS have been implicated as key mechanisms of chronic and neurotoxic microglial activation, particularly in the case of Parkinson’s disease. We review the mechanisms of neurotoxicity associated with chronic microglial activation and discuss the role of neuronal death and microglial ROS driving the chronic and toxic microglial phenotype

Selective presynaptic degeneration in the synaptopathy associated with ME7-induced hippocampal pathology

Intrahippocampal injection of the murine modified scrapie (ME7) induces a model of prion disease in vivo. Animals inoculated with ME7 brain homogenate were compared to controls at 8, 12 and 21 weeks. The data show that the accumulation of misfolded prion (PrPSc) coincided with selective reduction in presynaptic protein expression early in disease. This loss is independent of a change in the number of cell bodies in CA3 that provide the major presynaptic input to the stratum radiatum. Electron microscopy of the stratum radiatum independently evidenced a progressive decrease in the number of synapses during disease. Further, the number of postsynaptic specializations lacking an intact presynaptic specialization increased from 12 to 21 weeks. This suggests that the presynaptic compartment is selectively disrupted when the previously reported first behavioural deficits are observed in this model. This synaptic pathology or “synaptopathy” may represent the earliest neuronal dysfunction in this and other protein misfolding induced neurodegenerative diseases

Fibronectin attenuates process outgrowth in oligodendrocytes by mislocalizing MMP-9 activity

The extension of multiple oligodendroglial branched processes towards axons is an important event during the early stages of myelination that likely requires remodeling of the extracellular matrix (ECM) micro-environment via matrix metalloproteinases (MMPs). Here we investigated whether fibronectin-mediated inhibition of myelin sheet formation in oligodendrocytes correlated with an altered MMP activity. Our data reveal that fibronectin enhanced, in a PKC-dependent manner, the net activity of MMP-9, but not its expression, in conditioned medium of oligodendrocytes. Residual cellular MMP-9 activity on fibronectin was confined to the cell body, whereas MMP-9 activity on laminin-2 was localized along extending processes of oligodendrocytes. The mislocalization of MMP-9 activity on fibronectin correlated with a perturbed outgrowth of oligodendroglial processes. In conclusion, our findings suggest that ECM molecules influence both the net activity of secreted MMP and the spatial distribution of cell-associated MMP activity, and thereby morphological oligodendrocyte differentiation. (C) 2009 Elsevier Inc. All rights reserved

Alpha-synuclein deficiency in the C57BL/6JOlaHsd strain does not modify disease progression in the ME7-model of prion disease

We previously detailed how intrahippocampal inoculation of C57BL/6J mice with murine modified scrapie (ME7) leads to chronic neurodegeneration (Cunningham C, Deacon R, Wells H, Boche D, Waters S, Diniz CP, Scott H, Rawlins JN, Perry VH (2003) Eur J Neurosci 17:2147–2155.). Our characterization of the ME7-model is based on inoculation of this murine modified scrapie agent into C57BL/6J mice from Harlan laboratories. This agent in the C57BL/6J host generates a disease that spans a 24-week time course. The hippocampal pathology shows progressive misfolded prion (PrPSc) deposition, astrogliosis and leads to behavioural dysfunction underpinned by the early synaptic loss that precedes neuronal death. The Harlan C57BL/6J, although widely used as a wild type mouse, are a sub-strain harbouring a spontaneous deletion of ?-synuclein with the full description C57BL/6JOlaHsd. Recently ?-synuclein has been shown to ameliorate the synaptic loss in a mouse model lacking the synaptic chaperone CSP-?. This opens a potential confound of the ME7-model, particularly with respect to the signature synaptic loss that underpin the physiological and behavioural dysfunction. To investigate if this strain-selective loss of a candidate disease modifier impacts on signature ME7 pathology, we compared cohorts of C57BL/6JOlaHsd (?-synuclein negative) with the founder strain from Charles Rivers (C57BL/6JCrl, ?-synuclein positive). There were subtle changes in behaviour when comparing control animals from the two sub-strains indicating potentially significant consequences for studies assuming neurobiogical identity of both strains. However, there was no evidence that the absence of ?-synuclein modifies disease. Indeed, accumulation of PrPSc, synaptic loss and the behavioural dysfunction associated with the ME7-agent was the same in both genetic backgrounds. Our data suggest that ?-synuclein deficiency does not contribute to the compartment specific processes that give rise to prion disease mediated synaptotoxicity and neurodegeneration