Soluble Fibrinogen Triggers Non-cell Autonomous ER Stress-Mediated Microglial-Induced Neurotoxicity.

Aberrant or chronic microglial activation is strongly implicated in neurodegeneration, where prolonged induction of classical inflammatory pathways may lead to a compromised blood-brain barrier (BBB) or vasculature, features of many neurodegenerative disorders and implicated in the observed cognitive decline. BBB disruption or vascular disease may expose the brain parenchyma to "foreign" plasma proteins which subsequently impact on neuronal network integrity through neurotoxicity, synaptic loss and the potentiation of microglial inflammation. Here we show that the blood coagulation factor fibrinogen (FG), implicated in the pathogenesis of dementias such as Alzheimer's disease (AD), induces an inflammatory microglial phenotype as identified through genetic microarray analysis of a microglial cell line, and proteome cytokine profiling of primary microglia. We also identify a FG-mediated induction of non-cell autonomous ER stress-associated neurotoxicity via a signaling pathway that can be blocked by pharmacological inhibition of microglial TNFα transcription or neuronal caspase-12 activity, supporting a disease relevant role for plasma components in neuronal dysfunction.

A 3D in vitro model reveals differences in the astrocyte response elicited by potential stem cell therapies for CNS injury.

AIM: This study aimed to develop a 3D culture model to test the extent to which transplanted stem cells modulate astrocyte reactivity, where exacerbated glial cell activation could be detrimental to CNS repair success. MATERIALS & METHODS: The reactivity of rat astrocytes to bone marrow mesenchymal stem cells, neural crest stem cells (NCSCs) and differentiated adipose-derived stem cells was assessed after 5 days. Schwann cells were used as a positive control. RESULTS: NCSCs and differentiated Schwann cell-like adipose-derived stem cells did not increase astrocyte reactivity. Highly reactive responses to bone marrow mesenchymal stem cells and Schwann cells were equivalent. CONCLUSION: This approach can screen therapeutic cells prior to in vivo testing, allowing cells likely to trigger a substantial astrocyte response to be identified at an early stage. NCSCs and differentiated Schwann cell-like adipose-derived stem cells may be useful in treating CNS damage without increasing astrogliosis.

Engineering an integrated cellular interface in three-dimensional hydrogel cultures permits monitoring of reciprocal astrocyte and neuronal responses.

This study reports a new type of three-dimensional (3D) tissue model for studying interactions between cell types in collagen hydrogels. The aim was to create a 3D cell culture model containing separate cell populations in close proximity without the presence of a mechanical barrier, and demonstrate its relevance to modeling the axon growth-inhibitory cellular interfaces that develop in the central nervous system (CNS) in response to damage. This provides a powerful new tool to determine which aspects of the astroglial scar response and subsequent neuronal regeneration inhibition are determined by the presence of the other cell types. Astrocytes (CNS glia) and dissociated dorsal root ganglia (DRG; containing neurons and peripheral nervous system [PNS] glia) were seeded within collagen solution at 4 °C in adjacent chambers of a stainless steel mould, using cells cultured from wild-type or green fluorescent protein expressing rats, to track specific populations. The divider between the chambers was removed using a protocol that allowed the gels to integrate without mixing of the cell populations. Following setting of the gels, they were maintained in culture for up to 15 days. Reciprocal astrocyte and neuronal responses were monitored using confocal microscopy and 3D image analysis. At DRG:astrocyte interfaces, by 5 days there was an increase in the number of astrocytes at the interface followed by hypertrophy and increased glial fibrillary acidic protein expression at 10 and 15 days, indicative of reactive gliosis. Neurons avoided crossing DRG:astrocyte interfaces, and neuronal growth was restricted to the DRG part of the gel. By contrast, neurons were able to grow freely across DRG:DRG interfaces, demonstrating the absence of a mechanical barrier. These results show that in a precisely controlled 3D environment, an interface between DRG and astrocyte cultures is sufficient to trigger reactive gliosis and inhibition of neuronal regeneration across the interface. Different aspects of the astrocyte response could be independently monitored, providing an insight into the formation of a glial scar. This technology has wide potential for researchers wishing to maintain and monitor interactions between adjacent cell populations in 3D culture.

Alignment of astrocytes increases neuronal growth in three-dimensional collagen gels and is maintained following plastic compression to form a spinal cord repair conduit.

After injury to the spinal cord, reactive astrocytes form a glial scar consisting of highly ramified cell processes that constitute a major impediment to repair, partly due to their lack of orientation and guidance for regenerating axons. In some nonmammalian vertebrates, successful central nervous system regeneration is attributed to the alignment of reactive glia, which guide axons across the lesion site. Here, a three-dimensional mammalian cell-seeded collagen gel culture system was used to explore the effect of astrocyte alignment on neuronal growth. Astrocyte alignment was mapped within tethered rectangular gels and was significantly greater at the edge and middle of the gels compared to the control unaligned regions. When neurons were seeded on and within astrocyte gels, neurite length was greatest in the areas of astrocyte alignment. There was no difference in expression of astrocyte reactivity markers between aligned and control areas. Having established the potential utility of astrocyte alignment, the aligned gels were plastic compressed, transforming them into mechanically robust implantable devices. After compression, astrocytes remained viable and aligned and supported neurite outgrowth, yielding a novel method for assembling aligned cellular constructs suitable for tissue engineering and highlighting the importance of astrocyte alignment as a possible future therapeutic intervention for spinal cord repair.

A versatile 3D culture model facilitates monitoring of astrocytes undergoing reactive gliosis.

A major impediment to CNS repair is the glial scar, which forms following damage and is composed mainly of ramified, 'reactive' astrocytes that inhibit neuronal regrowth. The transition of astrocytes into this reactive phenotype (reactive gliosis) is a potential therapeutic target, but glial scar formation has proved difficult to study in monolayer cultures because they induce constitutive astrocyte activation. Here we demonstrate a 3D collagen gel system in which primary rat astrocytes were maintained in a persistently less reactive state than comparable cells in monolayer, resembling their status in the undamaged CNS. Reactivity, proliferation and viability were monitored and quantified using confocal, fluorescence and time-lapse microscopy, 3D image analysis, RT-PCR and ELISA. To assess the potential of this system as a model of reactive gliosis, astrocytes in 3D were activated with TGFbeta1 to a ramified, reactive phenotype (elevated GFAP, Aquaporin 4, CSPG, Vimentin and IL-6 secretion). This provides a versatile system in which astrocytes can be maintained in a resting state, then be triggered to undergo reactive gliosis, enabling real-time monitoring and quantitative analysis throughout and providing a powerful new tool for research into CNS damage and repair.

A role for the plasminogen activator system in inflammation and neurodegeneration in the central nervous system during experimental allergic encephalomyelitis.

Early signs of inflammatory demyelination include entry of fibrin(ogen) into the central nervous system (CNS), which is normally excluded by the blood-brain barrier, and up-regulation of components of the plasminogen activator system. Using mice deficient in tissue-type plasminogen activator (tPA-/-) and urokinase plasminogen activator receptor (uPAR-/-), we investigated the involvement of the PA system on the clinical and pathological features of experimental allergic encephalomyelitis, an animal model of multiple sclerosis. tPA-/- mice suffered an early and a more severe acute disease characterized by incomplete recovery when compared to wild-type controls, with significantly higher CNS levels of plasminogen activator inhibitor-1. This correlated with fibrin accumulation, which co-localized with nonphosphorylated neurofilament on thickened axons in experimental allergic encephalomyelitis tissue. In contrast, uPAR-/- mice had a delayed, less acute disease reflected in delayed infiltration of inflammatory cells. These animals developed chronic disease as a result of steadily increased inflammation, increased levels of urokinase-type plasminogen activator (uPA), and greater degree of demyelination. Thus, the plasminogen activator system can modulate both inflammatory and degenerative events in the CNS through the respective effects of tPA and uPAR on fibrinolysis and cell adhesion/migration, manipulation of which may have therapeutic implications for multiple sclerosis.

Autoimmune tolerance eliminates relapses but fails to halt progression in a model of multiple sclerosis.

To date there has been poor translation of immunotherapies from rodent models to treatment of progressive multiple sclerosis (MS). In the robust, relapsing Biozzi ABH mouse model of MS, using a combination of a transient deletion of T cells followed by intravenous (i.v.) myelin antigen administration, established relapsing disease in EAE can be effectively silenced. However, when treatment was initiated in late stage chronic-relapsing disease, despite inhibition of further relapses, mice demonstrated evidence of disease progression shown by a deterioration in mobility and development of spasticity and indicates that targeting relapsing, immunological components of MS alone is unlikely to be sufficient to control progression in the late stages of MS.