Sunja Kim

Caspase blockade induces RIP3-mediated programmed necrosis in Toll-like receptor-activated microglia

Microglia are the resident immune cells in the central nervous system and key players against pathogens and injury. However, persistent microglial activation often exacerbates pathological damage and has been implicated in many neurological diseases. Despite their pivotal physiological and pathophysiological roles, how the survival and death of activated microglia is regulated remains poorly understood. We report here that microglia activated through Toll-like receptors (TLRs) undergo RIP1/RIP3-dependent programmed necrosis (necroptosis) when exposed to the pan caspase inhibitor zVAD-fmk. Although zVAD-fmk and the caspase-8 inhibitor IETD-fmk had no effect on unstimulated primary microglia, they markedly sensitized microglia to TLR1/2,3,4,7/8 ligands or TNF treatment, triggering programmed necrosis that was completely blocked by R1P1 kinase inhibitor necrostatin-1. Interestingly, necroptosis induced by TLR ligands and zVAD was restricted to microglial cells and was not observed in astrocytes, neurons or oligodendrocytes even though they are known to express certain TLRs. Deletion of genes encoding TNF or TNFR1 failed to prevent lipopolysaccharide- and poly(I:C)-induced microglial necroptosis, unveiling a TNF-independent programmed necrosis pathway in TLR3- and TLR4-activated microglia. Microglia from mice lacking functional TRIF were fully protected against TLR3/4 activation and zVAD-fmk-induced necrosis, and genetic deletion of rip3 also prevented microglia necroptosis. Activation of c-jun N-terminal kinase and generation of specific reactive oxygen species were downstream signaling events required for microglial cell death execution. Taken together, this study reveals a robust RIP3-dependent necroptosis signaling pathway in TLR-activated microglia upon caspase blockade and suggests that TLR signaling and programmed cell death pathways are closely linked in microglia, which could contribute to neuropathology and neuroinflammation when dysregulated.

Astrocytes promote TNF‐mediated toxicity to oligodendrocyte precursors

J. Neurochem. (2011) 116, 53–66.

Aberrant Upregulation of Astroglial Ceramide Potentiates Oligodendrocyte Injury

Oligodendroglial injury is a pathological hallmark of many human white matter diseases, including multiple sclerosis (MS) and periventricular leukomalacia (PVL). Critical regulatory mechanisms of oligodendroglia destruction, however, remain incompletely understood. Ceramide, a bioactive sphingolipid pivotal to sphingolipid metabolism pathways, regulates cell death in response to diverse stimuli and has been implicated in neurodegenerative disorders. We report here that ceramide accumulates in reactive astrocytes in active lesions of MS and PVL, as well as in animal models of demyelination. Serine palmitoyltransferase, the rate‐limiting enzyme for ceramide de novo biosynthesis, was consistently upregulated in reactive astrocytes in the cuprizone mouse model of demyelination. Mass spectrometry confirmed the upregulation of specific ceramides during demyelination, and revealed a concomitant increase of sphingosine and a suppression of sphingosine‐1‐phosphate, a potent signaling molecule with key roles in cell survival and mitogenesis. Importantly, this altered sphingolipid metabolism during demyelination was restored upon active remyelination. In culture, ceramide acted synergistically with tumor necrosis factor, leading to apoptotic death of oligodendroglia in an astrocyte‐dependent manner. Taken together, our findings implicate that disturbed sphingolipid pathways in reactive astrocytes may indirectly contribute to oligodendroglial injury in cerebral white matter disorders.

A Microchip for Quantitative Analysis of CNS Axon Growth under Localized Biomolecular Treatments

Growth capability of neurons is an essential factor in axon regeneration. To better understand how microenvironments influence axon growth, methods that allow spatial control of cellular microenvironments and easy quantification of axon growth are critically needed. Here, we present a microchip capable of physically guiding the growth directions of axons while providing physical and fluidic isolation from neuronal somata/dendrites that enables localized biomolecular treatments and linear axon growth. The microchip allows axons to grow in straight lines inside the axon compartments even after the isolation; therefore, significantly facilitating the axon length quantification process. We further developed an image processing algorithm that automatically quantifies axon growth. The effect of localized extracellular matrix components and brain-derived neurotropic factor treatments on axon growth was investigated. Results show that biomolecules may have substantially different effects on axon growth depending on where they act. For example, while chondroitin sulfate proteoglycan causes axon retraction when added to the axons, it promotes axon growth when applied to the somata. The newly developed microchip overcomes limitations of conventional axon growth research methods that lack localized control of biomolecular environments and are often performed at a significantly lower cell density for only a short period of time due to difficulty in monitoring of axonal growth. This microchip may serve as a powerful tool for investigating factors that promote axon growth and regeneration.

Microfluidic systems for axonal growth and regeneration research

Damage to the adult mammalian central nervous system (CNS) often results in persistent neurological deficits with limited recovery of functions. The past decade has seen increasing research efforts in neural regeneration research with the ultimate goal of achieving functional recovery. Many studies have focused on prevention of further neural damage and restoration of functional connections that are compromised after injury or pathological damage. Compared to the peripheral nervous system, the failure of the adult CNS to regenerate is largely attributed to two basic aspects: inhibitory environmental influences and decreased growth capabilities of adult CNS neurons. Since early demonstration of successful growth of injured CNS axons into grafted peripheral nerve (David and Aguayo, 1981), multiple CNS axonal growth inhibitory factors have been identified and are mainly associated with degenerating CNS myelin (such as Nogo, MAG, OMgp) and with glial scar (such as chondroitin sulfate proteoglycans, CSPGs) (Yiu and He, 2006). However, blockade of these extracellular inhibitory signals alone is often insufficient for the majority of injured axons to achieve long-distance regeneration, as intrinsic regenerative capacity of mature CNS neurons is also a critical determinant for axon re-growth(Sun et al., 2011). Combinatory strategies that enhance neuronal growth and in the meantime overcome environmental inhibitory cues appear to confer better axonal regeneration and neural repair (Wang et al., 2012). While animal models are instrumental and indispensable to our understanding of CNS responses to injury and investigation of intervention strategies and functional regeneration, in vitro models have been designed to address specific and unique questions due to their accessibilities to experimental manipulations and relatively low cost. For instance, early findings from in vitro culture experiments formed the basis for the concept of CNS myelin-associated inhibitory molecules such as Nogo (Schwab and Thoenen, 1985; Chen et al., 2000; GrandPre et al., 2000). Attempts to identify compounds that overcome the inhibition of CNS myelin and CSPGs on neurite outgrowth in culture have revealed key neuronal signaling components that mediate the inhibitory effects of myelin and CSPGs (Sivasankaran et al., 2004). However, conventional culture system often has to use neuronal cultures at low cell density and for only a short period of time due to technical difficulties in monitoring and quantifying axonal growth. To develop effective CNS regenerative strategies, fast and reliable assessment of axonal growth would be critical not only to identify and select potentially interesting candidate molecules that promote axon extension over inhibitory molecules, but also to rule out poor ones. Equally important are considerations that neurons are highly polarized cells and that damaged axon could be extending far away from the cell body and encountering drastically different microenvironment than that of the soma. As such, signaling events elicited by extrinsic factors are most likely spatially regulated and may have different functional outputs. Over the years, compartmentalized neuronal culture systems have been developed. Now the emergence of microfluidics technology offers many advantages and versatilities for axonal growth and regeneration studies (Figure 1). Figure 1 Schematic illustrations of compartmentalized neuron culture platforms for axon isolation.

Axon length quantification microfluidic culture platform for growth and regeneration study.

To fully understand how external biomolecular environment influences axon growth, a method that can easily quantify the extent of axon growth as well as locally control their biomolecular environment is critically needed. Here, we describe a microfluidic culture platform capable of isolating CNS axons from neuronal somata for localized biomolecular manipulation as well as providing linearly guided axon growths for simple and easy quantification of the axon growth length. The axon isolation and guidance capability combined with the multi-compartment configuration make this platform ideal for investigating and screening drugs or other molecular factors that promote axon growth as well as regeneration.