Jens Kopatz

Neurodegeneration by activation of the microglial complement-phagosome pathway

Systemic inflammatory reactions have been postulated to exacerbate neurodegenerative diseases via microglial activation. We now demonstrate in vivo that repeated systemic challenge of mice over four consecutive days with bacterial LPS maintained an elevated microglial inflammatory phenotype and induced loss of dopaminergic neurons in the substantia nigra. The same total cumulative LPS dose given within a single application did not induce neurodegeneration. Whole-genome transcriptome analysis of the brain demonstrated that repeated systemic LPS application induced an activation pattern involving the classical complement system and its associated phagosome pathway. Loss of dopaminergic neurons induced by repeated systemic LPS application was rescued in complement C3-deficient mice, confirming the involvement of the complement system in neurodegeneration. Our data demonstrate that a phagosomal inflammatory response of microglia is leading to complement-mediated loss of dopaminergic neurons.

Sialic Acid on the Neuronal Glycocalyx Prevents Complement C1 Binding and Complement Receptor-3-Mediated Removal by Microglia

Microglial cells are professional phagocytes of the CNS responsible for clearance of unwanted structures. Neuronal processes are marked by complement C1 before they are removed in development or during disease processes. Target molecules involved in C1 binding and mechanisms of clearance are still unclear. Here we show that the terminal sugar residue sialic acid of the mouse neuronal glycocalyx determines complement C1 binding and microglial-mediated clearance function. Several early components of the classical complement cascade including C1q, C1r, C1s, and C3 were produced by cultured mouse microglia. The opsonin C1q was binding to neurites after enzymatic removal of sialic acid residues from the neuronal glycocalyx. Desialylated neurites, but not neurites with intact sialic acid caps, were cleared and taken up by cocultured microglial cells. The removal of the desialylated neurites was mediated via the complement receptor-3 (CR3; CD11b/CD18). Data demonstrate that mouse microglial cells via CR3 recognize and remove neuronal structures with an altered neuronal glycocalyx lacking terminal sialic acid.

Siglec‐h on activated microglia for recognition and engulfment of glioma cells

Sialic‐acid‐binding immunoglobulin‐like lectin‐h (Siglec‐h) is a recently identified mouse‐specific CD33‐related Siglec that signals via DAP12/TYROBP. Expression of Siglec‐h has been observed on plasmacytoid dendritic cells and microglia, but the ligand and the function of Siglec‐h remained elusive. Here, we demonstrate gene transcription and protein expression of Siglec‐h by mouse microglia after interferon‐γ treatment or polarization into a M1‐subtype. Microglial Siglec‐h acted as phagocytosis receptor since targeting of microsphere beads to Siglec‐h triggered their uptake into the microglia. The extracellular domain of Siglec‐h protein bound to mouse glioma lines, but not to astrocytes or other normal mouse cells. Microglial cells stimulated to express Siglec‐h engulfed intact glioma cells without prior induction of apoptosis and slightly reduced glioma cell number in culture. Phagocytosis of glioma cells by activated microglia was dependent on Siglec‐h and its adapter molecule DAP12. Thus, data show that M1‐polarized microglial cells can engulf glioma cells via a DAP12‐mediated Siglec‐h dependent mechanism.

Siglec functions of microglia.

Microglia are the resident immune cells of the central nervous system. They can sense intact or lesioned cells and then respond in an appropriate way. Therefore, microglia need recognition receptors that lead to either the activation or the inhibition of the immune response pathways. Most Siglecs contain an immunoreceptor tyrosine based inhibition motif and its signaling leads to the termination of signals emerging from immunoreceptor tyrosine-based activation motif-signaling receptors. Pro-inflammatory immune responses and phagocytosis are turned down in microglia by inhibitory Siglec signaling. Recently, it was demonstrated that inhibitory Siglecs have neuroprotective effects on cultured neurons by preventing the phagocytosis-associated oxidative burst. Furthermore, microglial mouse Siglec-E and human Siglec-11 have been shown to prevent neurotoxicity via interaction with sialic acid exposed on the neuronal glycocalyx. Thus, Siglecs sensing the intact glycocalyx of neighboring cells keep microglia in a silent homeostatic status.

Anti-inflammatory activity of low molecular weight polysialic acid on human macrophages

Oligosialic and polysialic acid (oligoSia and polySia) of the glycocalyx of neural and immune cells are linear chains, in which the sialic acid monomers are α2.8-glycosidically linked. Sialic acid-binding immunoglobulin-like lectin-11 (SIGLEC-11) is a primate-lineage specific receptor of human tissue macrophages and microglia that binds to α2.8-linked oligoSia. Here, we show that soluble low molecular weight polySia with an average degree of polymerization 20 (avDP20) interacts with SIGLEC-11 and acts anti-inflammatory on human THP1 macrophages involving the SIGLEC-11 receptor. Soluble polySia avDP20 inhibited the lipopolysaccharide (LPS)-induced gene transcription and protein expression of tumor necrosis factor-α (Tumor Necrosis Factor Superfamily Member 2, TNFSF2). In addition, polySia avDP20 neutralized the LPS-triggered increase in macrophage phagocytosis, but did not affect basal phagocytosis or endocytosis. Moreover, polySia avDP20 prevented the oxidative burst of human macrophages triggered by neural debris or fibrillary amyloid-β1–42. In a human macrophage-neuron co-culture system, polySia avDP20 also reduced loss of neurites triggered by fibrillary amyloid-β1–42. Thus, treatment with polySia avDP20 might be a new anti-inflammatory therapeutic strategy that also prevents the oxidative burst of macrophages.

CD14 is a key organizer of microglial responses to CNS infection and injury.

Microglia, innate immune cells of the CNS, sense infection and damage through overlapping receptor sets. Toll‐like receptor (TLR) 4 recognizes bacterial lipopolysaccharide (LPS) and multiple injury‐associated factors. We show that its co‐receptor CD14 serves three non‐redundant functions in microglia. First, it confers an up to 100‐fold higher LPS sensitivity compared to peripheral macrophages to enable efficient proinflammatory cytokine induction. Second, CD14 prevents excessive responses to massive LPS challenges via an interferon β‐mediated feedback. Third, CD14 is mandatory for microglial reactions to tissue damage‐associated signals. In mice, these functions are essential for balanced CNS responses to bacterial infection, traumatic and ischemic injuries, since CD14 deficiency causes either hypo‐ or hyperinflammation, insufficient or exaggerated immune cell recruitment or worsened stroke outcomes. While CD14 orchestrates functions of TLR4 and related immune receptors, it is itself regulated by TLR and non‐TLR systems to thereby fine‐tune microglial damage‐sensing capacity upon infectious and non‐infectious CNS challenges. GLIA 2016;64:635–649.

Loss of the smallest subunit of cytochrome c oxidase, COX8A, causes Leigh-like syndrome and epilepsy

Isolated cytochrome c oxidase (complex IV) deficiency is one of the most frequent respiratory chain defects in humans and is usually caused by mutations in proteins required for assembly of the complex. Mutations in nuclear-encoded structural subunits are very rare. In a patient with Leigh-like syndrome presenting with leukodystrophy and severe epilepsy, we identified a homozygous splice site mutation in COX8A, which codes for the ubiquitously expressed isoform of subunit VIII, the smallest nuclear-encoded subunit of complex IV. The mutation, affecting the last nucleotide of intron 1, leads to aberrant splicing, a frame-shift in the highly conserved exon 2, and decreased amount of the COX8A transcript. The loss of the wild-type COX8A protein severely impairs the stability of the entire cytochrome c oxidase enzyme complex and manifests in isolated complex IV deficiency in skeletal muscle and fibroblasts, similar to the frequent c.845_846delCT mutation in the assembly factor SURF1 gene. Stability and activity of complex IV could be rescued in the patients fibroblasts by lentiviral expression of wild-type COX8A. Our findings demonstrate that COX8A is indispensable for function of human complex IV and its mutation causes human disease.

Polysialic acid blocks mononuclear phagocyte reactivity, inhibits complement activation, and protects from vascular damage in the retina

Age‐related macular degeneration (AMD) is a major cause of blindness in the elderly population. Its pathophysiology is linked to reactive oxygen species (ROS) and activation of the complement system. Sialic acid polymers prevent ROS production of human mononuclear phagocytes via the inhibitory sialic acid‐binding immunoglobulin‐like lectin‐11 (SIGLEC11) receptor. Here, we show that low‐dose intravitreal injection of low molecular weight polysialic acid with average degree of polymerization 20 (polySia avDP20) in humanized transgenic mice expressing SIGLEC11 on mononuclear phagocytes reduced their reactivity and vascular leakage induced by laser coagulation. Furthermore, polySia avDP20 prevented deposition of the membrane attack complex in both SIGLEC11 transgenic and wild‐type animals. In vitro, polySia avDP20 showed two independent, but synergistic effects on the innate immune system. First, polySia avDP20 prevented tumor necrosis factor‐α, vascular endothelial growth factor A, and superoxide production by SIGLEC11‐positive phagocytes. Second, polySia avDP20 directly interfered with complement activation. Our data provide evidence that polySia avDP20 ameliorates laser‐induced damage in the retina and thus is a promising candidate to prevent AMD‐related inflammation and angiogenesis.

Polysialic acid production using Escherichia coli K1 in a disposable bag reactor

Polysialic acid (polySia), consisting of α‐(2,8)‐linked N‐acetylneuraminic acid monomers plays a crucial role in many biological processes. This study presents a novel process for the production of endogenous polySia using Escherichia coli K1 in a disposable bag reactor with wave‐induced mixing. Disposable bag reactors provide easy and fast production in terms of regulatory requirements as GMP, flexibility, and can easily be adjusted to larger production capacities not only by scale up but also by parallelization. Due to the poor oxygen transfer rate compared to a stirred tank reactor, pure oxygen was added during the cultivation to avoid oxygen limitation. During the exponential growth phase the growth rate was 0.61 h−1. Investigation of stress‐related product release from the cell surface showed no significant differences between the disposable bag reactor with wave‐induced mixing and the stirred tank reactor. After batch cultivation a cell dry weight of 6.8 g L−1 and a polySia concentration of 245 mg L−1 were reached. The total protein concentration in the supernatant was 132 mg L−1. After efficient and time‐saving downstream processing characterization of the final product showed a protein content of below 0.04 mgprotein/gpolySia and a maximal chain length of ∼90 degree of polymerization.

Single-use membrane adsorbers for endotoxin removal and purification of endogenous polysialic acid from Escherichia coli K1

Graphical abstract