How solutes and macromolecules are removed from brain tissue is of central importance in normal brain physiology and in how toxic protein aggregates are cleared in neurodegenerative conditions, including Alzheimer's disease (AD). Conventionally, solute transport in the narrow and tortuous extracellular space in brain parenchyma has been thought to be primarily diffusive and nondirectional. The recently proposed "glymphatic" (glial-lymphatic) hypothesis posits that solute clearance is convective and driven by active fluid transport from para-arterial to paravenous spaces though aquaporin-4 water channels in astrocyte endfeet. Glymphatic, convective solute clearance has received much attention because of its broad implications for AD and other brain pathologies and even the function of sleep. However, the theoretical plausibility of glymphatic transport has been questioned, and recent data have challenged its experimental underpinnings. A substantiated mechanism of solute clearance in the brain is of considerable importance because of its implications for pathogenic mechanisms of neurologic diseases and delivery of therapeutics.-Smith, A. J., Verkman, A. S. The "glymphatic" mechanism for solute clearance in Alzheimer's disease: game changer or unproven speculation?

Aquaporin-4 (AQP4) is a water transporting protein expressed at the plasma membrane of astrocytes throughout the central nervous system (CNS). Analysis of AQP4 knockout mice has suggested its broad involvement in brain water balance, neuroexcitation, glial scarring, neuroinflammation, and even neurodegenerative and neuropsychiatric disorders. Broad clinical utility of AQP4 modulators has been speculated. Area covered: This review covers the biology of AQP4, evidence for its roles in normal CNS function and neurological disorders, and progress in AQP4 drug discovery. Expert opinion: Critical examination of available data reduces the lengthy potential applications list to AQP4 inhibitors for early therapy of ischemic stroke and perhaps for reduction of glial scarring following CNS injury. Major challenges in identification and clinical development of AQP4 inhibitors include the apparent poor druggability of AQPs, the many homologous AQP isoforms with broad tissue distribution and functions, technical issues with water transport assays, predicted undesired CNS and non-CNS actions, and the need for high blood-brain barrier permeation. To date, despite considerable effort, validated small-molecule AQP4 inhibitors have not been advanced. However, a biologic ('aquaporumab') is in development for neuromyelitis optica, an autoimmune inflammatory demyelinating disease where CNS pathology is initiated by binding of anti-AQP4 autoantibodies to astrocyte AQP4.

BackgroundAquaporin-4-immunoglobulin G (AQP4-IgG) seropositive neuromyelitis optica spectrum disorder (herein called NMO) is an autoimmune disease of the central nervous system in which AQP4-IgG binding to AQP4 on astrocytes results in complement-dependent astrocyte injury and secondary inflammation, demyelination, and neuron loss. We previously reported evidence for a complement bystander mechanism for early oligodendrocyte injury in NMO. Herein, we tested the hypothesis that complement bystander injury, which involves diffusion to nearby cells of activated soluble complement components from complement-injured astrocytes, is a general phenomenon that may contribute to neuronal injury in NMO.MethodsPrimary cocultures of rat astrocytes and cortical neurons were established to study complement-dependent cell death after exposure to AQP4-IgG and complement. In animal experiments, AQP4-IgG was delivered to adult rats by intracerebral injection. Cell cultures and rat brain were studied by immunofluorescence.ResultsIn primary astrocyte-neuron cocultures, addition of AQP4-IgG and complement resulted in death of neurons nearby astrocytes. Deposition of complement membrane attack complex C5b-9 was seen on neurons nearby astrocytes, whereas C1q, the initiating protein in the complement pathway, was seen only on astrocytes. Neuron death was not seen with a complement inhibitor, with C1q- or C6-depleted complement, in pure neuron cultures exposed to AQP4-IgG and complement or in cocultures exposed to an astrocyte toxin. Intracerebral injection in rats of AQP4-IgG and a fixable dead cell fluorescent marker produced death of neurons near astrocytes, with C5b-9 deposition. Neuron death was not seen in rats receiving a complement inhibitor or in AQP4-IgG-injected AQP4 knockout rats.ConclusionThese results support a novel mechanism for early neuron injury in NMO and provide evidence that complement bystander injury may be a general phenomenon for brain cell injury following AQP4-IgG-targeted astrocyte death.Electronic supplementary materialThe online version of this article (10.1186/s12974-018-1333-z) contains supplementary material, which is available to authorized users.

Neuromyelitis optica spectrum disorder (herein called NMO) is an autoimmune inflammatory disease of the central nervous system in which immunoglobulin G antibodies against astrocyte water channel aquaporin-4 (AQP4-IgG) cause demyelination and neurological deficit. Injury to oligodendrocytes, which do not express AQP4, links the initiating pathogenic event of AQP4-IgG binding to astrocyte AQP4 to demyelination. Here, we report evidence for a complement ‘bystander mechanism’ to account for early oligodendrocyte injury in NMO in which activated, soluble complement proteins following AQP4-IgG binding to astrocyte AQP4 result in deposition of the complement membrane attack complex (MAC) on nearby oligodendrocytes. Primary cocultures of rat astrocytes and mature oligodendrocytes exposed to AQP4-IgG and complement showed early death of oligodendrocytes in close contact with astrocytes, which was not seen in pure oligodendrocyte cultures, in cocultures exposed to AQP4-IgG and C6-depleted serum, or when astrocytes were damaged by a complement-independent mechanism. Astrocyte-oligodendrocyte cocultures exposed to AQP4-IgG and complement showed prominent MAC deposition on oligodendrocytes in contact with astrocytes, whereas C1q, the initiating protein in the classical complement pathway, and C3d, a component of the alternative complement pathway, were deposited only on astrocytes. Early oligodendrocyte injury with MAC deposition was also found in rat brain following intracerebral injection of AQP4-IgG, complement and a fixable dead-cell stain. These results support a novel complement bystander mechanism for early oligodendrocyte injury and demyelination in NMO.

Cellular injury in AQP4-IgG seropositive neuromyelitis spectrum disorder (herein called NMO) involves AQP4-IgG binding to astrocytes, resulting in astrocyte injury by complement-dependent cytotoxicity (CDC) and antibody-dependent cellular cytotoxicity (ADCC) mechanisms. The rapid disease progression, severe tissue damage, and abundant leukocyte infiltration seen in some NMO patients suggest a more direct mechanism for demyelination and neurologic deficit than secondary injury from astrocyte loss. Here, we report evidence for an ‘ADCC bystander mechanism’ in NMO involving injury to nearby cells by leukocytes following their activation by AQP4-bound AQP4-IgG on astrocytes. In model cocultures containing AQP4-expressing and null CHO cells, AQP4-IgG and complement killed bystander null cells to ~ 100 μm away from AQP4-expressing cells; AQP4-IgG and NK cells produced bystander killing to ~ 300 μm, with perforin deposition seen on injured null cells. Bystander cytotoxicity was also seen with neutrophil-mediated ADCC and in astrocyte-neuron cocultures. Mechanistic studies, including real-time imaging, suggested that leukocytes activated by an AQP4-dependent ADCC mechanism injure bystander cells by direct targeted exocytosis on neighboring cells and not by diffusion of soluble granule contents. In support of this conclusion, ADCC bystander injury was preferentially reduced by an RGDS peptide that inhibits integrin adhesion. Evidence for ADCC bystander injury to oligodendrocytes and neurons was also found in mice following intracerebral injection of AQP4-IgG and NK cells, which was inhibited by RGDS peptide. These results establish a novel cellular pathogenesis mechanism in AQP4-IgG seropositive NMO and provide evidence that inflammatory mechanisms can cause widespread tissue damage in NMO independently of the secondary effects from astrocyte loss. Electronic supplementary material The online version of this article (10.1186/s40478-019-0766-7) contains supplementary material, which is available to authorized users.