Zeppenfeld DM, Simon M, Haswell JD, D'Abreo D, Murchison C, Quinn JF, Grafe MR, Woltjer RL, Kaye J, Iliff JJ. Association of Perivascular Localization of Aquaporin-4 With Cognition and Alzheimer Disease in Aging Brains. JAMA Neurol. 2017 Jan 1;74(1):91-99. PubMed.

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  1. Aquaporin-4 (Aqp4) is a major water channel in the human brain, located mainly in the astrocytic end-feet around cerebral blood vessels. Aqp4 plays a number of physiological roles in brain, including regulation of extracellular space volume, and ion and water homeostasis (Nagelhus and Ottersen, 2013). Here, Zeppenfeld et al. sought to determine whether Aqp4 expression is changed in normal aging and Alzheimer’s disease. They performed immunohistochemistry and western blot analysis in postmortem frontal cortical brain tissue from cognitively normal young, cognitively normal old, and AD patients. Using immunofluorescence staining, the authors found that total Aqp4 immunoreactivity levels are increased in AD compared to cognitively normal young and old subjects. Interestingly, perivascular (PV) Aqp4 was decreased in AD compared to age-matched controls. Their representative images of aged Aqp4 shows a strikingly different expression pattern compared to young, eldest, and AD subjects.

    It would be intriguing to find out why Aqp4 distribution would change so drastically in aged subjects and then revert back to look like the young subject in eldest and AD subjects. A previous study, however, did not show this altered distribution in aged control subjects (Pérez et al., 2007). Upon measuring Aqp4 levels by western blot analysis, Zeppenfeld et al. found no significant changes among all groups in total Aqp4. But they did find that the ratio between Aqp4 isoforms (full-length Aqp4-M1 and truncated Aqp4-M23) is changed.

    Unfortunately, it appears that the authors have mislabeled Aqp4-M1 and Aqp4-M23 in their representative blot in eFigure 3; thus currently their results reflect this mislabeling. The authors state that Aqp4-M1 significantly declined in aged cortex, but their representative blot actually shows that Aqp4-M1 significantly increased in aged and AD cortex. It would be intriguing to find out the functional significance of these findings that is not discussed in the paper. Furthermore, the importance of this altered ratio of Aqp4 isoforms during aging and AD remains to be determined. Altogether, the authors suggest that loss of perivascular expression of Aqp4 in the aging brain may affect clearance of Aβ from brain interstitial fluid by the glymphatic system.

    Several studies have previously investigated Aqp4 level changes in human AD brain tissue (Hoshi et al., 2012; Moftakhar et al., 2010; Pérez et al., 2007; Potokar et al., 2016; Wilcock et al., 2009). Pérez et al. performed quantitative real-time RT-PCR and western blot of total brain homogenates of frontal cortex from control and AD subjects and found no significant difference in Aqp4 expression between groups (Pérez et al., 2007), as similarly found by Zeppenfeld et al. Wilcock et al. performed quantitative RT-PCR and immunohistochemistry on medial temporal lobe cortical tissue from control subjects and AD subjects with mild, moderate, or severe cerebral amyloid angiopathy (CAA) (Wilcock et al., 2009). They found that there were no major changes in Aqp4 mRNA or expression levels along blood vessels from AD subjects with mild AD (Wilcock et al., 2009). However, Aqp4 mRNA and expression levels were reduced with moderate and severe CAA, and less Aqp4 was observed along blood vessels (Wilcock et al., 2009). Hoshi et al. performed Aqp4 and Aβ immunohistochemistry on temporal lobe tissue from control, sporadic AD, and familial AD subjects and found that Aqp4 is variably distributed near Aβ40 and Aβ42 senile plaques (Hoshi et al., 2012). Moftakhar et al. investigated Aqp4 using immunohistochemistry in frontal, temporal, and occipital cortex to assess expression of Aqp4 levels in control and AD subjects with varying degrees of CAA (Moftakhar et al., 2010). They found that Aqp4 expression was enhanced in AD and was prominent at the CSF fluid and brain interfaces including subpial, subependymal, pericapillary, and periarteriolar spaces (Moftakhar et al., 2010). 

    Although the jury is still out, the “glymphatic system” has attracted a lot of interest and attention in the field. It has revived the importance of the perivascular fluid flow hypothesis in overall brain homeostasis, with important potential implications to disease states. The term “glymphatic system” was originally coined to better reflect the concept that the brain does not have a proper lymphatic system. However, recent work from other groups has convincingly demonstrated the existence of the lymphatic system in the brain and connections of brain interstitial fluid (ISF) and cerebrospinal fluid (CSF) pathways with lymphatic meningeal vessels and the lymph nodes in the neck (Aspelund et al., 2015; Louveau et al., 2015).

    These recent discoveries raise an important question. What, then, would be the postulated role of the glymphatic system if a normal lymphatic system already exists in the brain? Of note, the perivascular drainage pathway connected to brain lymphatic system renamed later as a “glymphatic system” was discovered in the late 1970s and early 1980s, when astrocyte regulation of the ISF flow via this pathway had not been known.

    The question in the field remains, however, whether the direction of the ISF flow in the brain is from brain ISF to CSF to serve for clearance of brain metabolic waste products such as Alzheimer’s Aβ, as claimed by Carare-Weller’s hypothesis and several earlier studies from Bradbury, Davson, Segal, and other groups, or from CSF recycling back to brain, as claimed by the authors of the glymphatic system hypothesis. It is important to remember that the majority of Aβ is cleared from brain under physiological conditions by transvascular clearance across the blood-brain barrier (Tarasoff-Conway et al., 2015).

    Future studies should investigate what happens to the brain lymphatic system in Alzheimer’s disease and other neurodegenerative diseases, and interactions with perivascular clearance, and how this is influenced by volume changes in astrocytes mediated by Aqp4 that is central to the so-called glymphatic regulation of flow. 

    References:

    Aspelund A, Antila S, Proulx ST, Karlsen TV, Karaman S, Detmar M, Wiig H, Alitalo K. A dural lymphatic vascular system that drains brain interstitial fluid and macromolecules. J Exp Med. 2015 Jun 29;212(7):991-9. Epub 2015 Jun 15 PubMed.

    Hoshi A, Yamamoto T, Shimizu K, Ugawa Y, Nishizawa M, Takahashi H, Kakita A. Characteristics of aquaporin expression surrounding senile plaques and cerebral amyloid angiopathy in Alzheimer disease. J Neuropathol Exp Neurol. 2012 Aug;71(8):750-9. PubMed.

    Louveau A, Smirnov I, Keyes TJ, Eccles JD, Rouhani SJ, Peske JD, Derecki NC, Castle D, Mandell JW, Lee KS, Harris TH, Kipnis J. Structural and functional features of central nervous system lymphatic vessels. Nature. 2015 Jul 16;523(7560):337-41. Epub 2015 Jun 1 PubMed.

    Moftakhar P, Lynch MD, Pomakian JL, Vinters HV. Aquaporin expression in the brains of patients with or without cerebral amyloid angiopathy. J Neuropathol Exp Neurol. 2010 Dec;69(12):1201-9. PubMed.

    Nagelhus EA, Ottersen OP. Physiological roles of aquaporin-4 in brain. Physiol Rev. 2013 Oct;93(4):1543-62. PubMed.

    Pérez E, Barrachina M, Rodríguez A, Torrejón-Escribano B, Boada M, Hernández I, Sánchez M, Ferrer I. Aquaporin expression in the cerebral cortex is increased at early stages of Alzheimer disease. Brain Res. 2007 Jan 12;1128(1):164-74. PubMed.

    Potokar M, Jorgačevski J, Zorec R. Astrocyte Aquaporin Dynamics in Health and Disease. Int J Mol Sci. 2016 Jul 13;17(7) PubMed.

    Tarasoff-Conway JM, Carare RO, Osorio RS, Glodzik L, Butler T, Fieremans E, Axel L, Rusinek H, Nicholson C, Zlokovic BV, Frangione B, Blennow K, Ménard J, Zetterberg H, Wisniewski T, de Leon MJ. Clearance systems in the brain-implications for Alzheimer disease. Nat Rev Neurol. 2015 Aug;11(8):457-70. Epub 2015 Jul 21 PubMed.

    Wilcock DM, Vitek MP, Colton CA. Vascular amyloid alters astrocytic water and potassium channels in mouse models and humans with Alzheimer's disease. Neuroscience. 2009 Mar 31;159(3):1055-69. Epub 2009 Jan 19 PubMed.

    View all comments by Berislav Zlokovic

  2. Age-associated accumulation of Aβ peptides and hyperphosphorylated tau is a central event in the pathogenesis of Alzheimer’s disease. The misaggregation of these toxic proteins has been hypothesized to result from an imbalance between their production and clearance (Ross et al., 2004). Thus, it is critical to understand how toxic metabolites are cleared from the brain, which might contribute to developing promising strategies for delaying or even halting AD onset (Tarasoff-Conway et al., 2015).

    Findings from the past few years suggest that a brain-wide perivascular pathway, termed the glymphatic system, contributes to a larger portion of clearance of interstitial solutes, including Aβ and tau, from the brain (Xie et al., 2013; Louveau et al., 2015; Aspelund et al., 2015; Iliff et al., 2015). Moreover, Iliff and colleagues have provided a series of evidence suggesting that the efficient clearance of interstitial solutes is dependent on the astroglial water channel aquaporin-4 (AQP4), which is localized primarily to perivascular astrocytic end-feet and astrocytic processes of the glial limitans (Iliff et al., 2012; Iliff et al., 2014; Kress et al., 2014; Peng et al., 2016). Loss of perivascular AQP4 polarization is associated with glymphatic pathway impairment in aged mice and AD mouse models Kress et al., 2014; Peng et al., 2016). In the present study, the largest clinical case series to date, they further provided quantitative evidence showing that increasing AQP4 expression is a feature of the aging human brain, and AQP4 mislocalization is related to the glymphatic dysfunction and AD pathology (Zeppenfeld et al., 2016). 

    Further studies are necessary to clarify whether AQP4 mislocalization in AD is a cause or a consequence of pathology, or merely a parallel event, and how to find specific and nontoxic molecular markers and imaging techniques to detect glymphatic clearance function and abnormal expression of AQP4 in the brain (Iliff et al., 2013; Eide et al., 2015). These potential findings will contribute to establishing AQP4-dependent glymphatic clearance as a novel therapeutic target for the treatment of neurodegenerative diseases associated with accumulation of misfolded protein aggregates.

    References:

    Ross CA, Poirier MA. Protein aggregation and neurodegenerative disease. Nat Med. 2004 Jul;10 Suppl:S10-7. PubMed.

    Tarasoff-Conway JM, Carare RO, Osorio RS, Glodzik L, Butler T, Fieremans E, Axel L, Rusinek H, Nicholson C, Zlokovic BV, Frangione B, Blennow K, Ménard J, Zetterberg H, Wisniewski T, de Leon MJ. Clearance systems in the brain-implications for Alzheimer disease. Nat Rev Neurol. 2015 Aug;11(8):457-70. Epub 2015 Jul 21 PubMed.

    Xie L, Kang H, Xu Q, Chen MJ, Liao Y, Thiyagarajan M, O'Donnell J, Christensen DJ, Nicholson C, Iliff JJ, Takano T, Deane R, Nedergaard M. Sleep drives metabolite clearance from the adult brain. Science. 2013 Oct 18;342(6156):373-7. PubMed.

    Louveau A, Smirnov I, Keyes TJ, Eccles JD, Rouhani SJ, Peske JD, Derecki NC, Castle D, Mandell JW, Lee KS, Harris TH, Kipnis J. Structural and functional features of central nervous system lymphatic vessels. Nature. 2015 Jul 16;523(7560):337-41. Epub 2015 Jun 1 PubMed.

    Aspelund A, Antila S, Proulx ST, Karlsen TV, Karaman S, Detmar M, Wiig H, Alitalo K. A dural lymphatic vascular system that drains brain interstitial fluid and macromolecules. J Exp Med. 2015 Jun 29;212(7):991-9. Epub 2015 Jun 15 PubMed.

    Iliff JJ, Goldman SA, Nedergaard M. Implications of the discovery of brain lymphatic pathways. Lancet Neurol. 2015 Oct;14(10):977-9. PubMed.

    Iliff JJ, Wang M, Liao Y, Plogg BA, Peng W, Gundersen GA, Benveniste H, Vates GE, Deane R, Goldman SA, Nagelhus EA, Nedergaard M. A paravascular pathway facilitates CSF flow through the brain parenchyma and the clearance of interstitial solutes, including amyloid β. Sci Transl Med. 2012 Aug 15;4(147):147ra111. PubMed.

    Kress BT, Iliff JJ, Xia M, Wang M, Wei HS, Zeppenfeld D, Xie L, Kang H, Xu Q, Liew JA, Plog BA, Ding F, Deane R, Nedergaard M. Impairment of paravascular clearance pathways in the aging brain. Ann Neurol. 2014 Dec;76(6):845-61. Epub 2014 Sep 26 PubMed.

    Peng W, Achariyar TM, Li B, Liao Y, Mestre H, Hitomi E, Regan S, Kasper T, Peng S, Ding F, Benveniste H, Nedergaard M, Deane R. Suppression of glymphatic fluid transport in a mouse model of Alzheimer's disease. Neurobiol Dis. 2016 Sep;93:215-25. Epub 2016 May 24 PubMed.

    Zeppenfeld DM, Simon M, Haswell JD, D'Abreo D, Murchison C, Quinn JF, Grafe MR, Woltjer RL, Kaye J, Iliff JJ. Association of Perivascular Localization of Aquaporin-4 With Cognition and Alzheimer Disease in Aging Brains. JAMA Neurol. 2017 Jan 1;74(1):91-99. PubMed.

    Iliff JJ, Lee H, Yu M, Feng T, Logan J, Nedergaard M, Benveniste H. Brain-wide pathway for waste clearance captured by contrast-enhanced MRI. J Clin Invest. 2013 Mar 1;123(3):1299-309. PubMed.

    Eide PK, Ringstad G. MRI with intrathecal MRI gadolinium contrast medium administration: a possible method to assess glymphatic function in human brain. Acta Radiol Open. 2015 Nov;4(11):2058460115609635. Epub 2015 Nov 17 PubMed.

    View all comments by Ming Xiao

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