Aquaporin-4 and Brain Therapy

Catalyst Volume 7

Introduction

One of the tasks of modern medicine is to address the many diseases affecting the brain. These maladies come in various forms – including neurodegenerative complications, tumors, vascular constriction, and buildup of intracranial pressure.^1,2,3 Several of these disease classes are caused, in part or in full, by a faulty waste clearance or water flux. Although a pervasive system of slow cerebrospinal fluid (CSF) movement in the brain’s ventricles is present, a rapid method for clearing solutes from the cortex’s interstitial space, which contains neural tissue and the surrounding extracellular matrix, was unknown.^4,5 Recently, Iliff et al discovered a new mechanism for the flow of CSF in the mouse brain – the “glymphatic” system.^5,6 This pathway provides an accelerated mechanism to clear dissolved materials from the interstitial space, preventing a buildup of solutes and toxins.^5,6 At the center of this system is the water transport protein aquaporin-4 (AQP4), and the extent of this water channel’s various roles are only now being identified. New perspectives on the mechanism by which the brain is “cleansed” may lead to breakthroughs in therapeutics for brain disorders such as Alzheimer’s Disease (AD), which is the sixth leading cause of death in the US each year.⁷

The Infrastructure

To understand AQP4 and its role in the brain, the environment in which it operates must be examined. As seen in Figure 1, surrounding the brain are three meninges, protective layers between the skull and the cortex.⁸ Between these layers – the dura, arachnoid, and pia maters – are cavities, including the subarachnoid space that lies just below the arachnoid layer.⁹ As the figure shows, directly underneath the pia mater are the cortex and interstitial space. Within the cortex, CSF flows through a system of chambers called the ventricles, illustrated in Figure 2.10 CSF suffuses the brain and has several vital functions, namely shock absorption, nutrient provision, and waste clearance.¹¹ CSF is produced in a mass of capillaries called the choroid plexus and flows through the ventricles into the subarachnoid space, bathing the brain and never crossing the blood-brain barrier.¹⁰ After circulating through the brain’s interstitial space and ventricles, the CSF then leaves the brain through aquaporin channels surrounding the cephalic veins.⁶

A New Plumbing System

A team of researchers has discovered an alternate pathway for CSF that clears water-soluble materials from the interstitial space.⁶ CSF in this so-called “glymphatic pathway” starts in the subarachnoid cavity and then seeps into the cortex, as seen in Figure 3.⁶ This fluid eventually leaves the brain, carrying with it the waste generated by cells. CSF enters the parenchyma from the subarachnoid cavity and travels immediately alongside the blood vessels.⁶ This route, which forms a sheath around the blood vessels, is dubbed the “paravascular” pathway, and CSF enters and exits the interstitial space through these avenues.⁶ The pia membrane guides this pathway until the artery penetrates the cortex, as seen in Figure 3. From there, the endfeet of astrocytes bind the outer wall.⁶ Astrocytes are glial cells that play structural roles in the nervous system, and endfeet are the enlarged endings of the astrocytes that contact other cell bodies and contain AQP4 proteins.^6,12

Iliff et al found that AQP4 are highly polarized at the endfeet of astrocytes, which suggested that these proteins provide a pathway for CSF into the parenchyma.⁶ To test this hypothesis, they compared wild type and Aqp4-null mice on the basis of CSF influx into the parenchyma.⁶ They injected tracers such as radiolabeled TR-d3 intracisternally, finding that that tracer influx into the parenchyma was significantly reduced in Aqp4-null mice.⁴ According to their model, AQP4 facilitates CSF flow into the parenchyma. There, CSF mixes with the interstitial fluid in the parenchyma; AQP4 then drives these fluids out and into the paravenous pathway by bulk flow.⁶ The rapid clearance of tracer in wild type mice and the significantly reduced clearance in Aqp4-null mice demonstrated the pathway’s ability to clear solutes from the brain. This finding is important because the build-up of Aβ is often associated with the onset and progression of AD.

AQP4 and Aβ

To facilitate Aβ removal, astrocytes become activated at a threshold Aβ, but undergo apoptosis at high concentrations.^13,14,15 Thus, the concentration of astrocytes has to be in a narrow window. A study by Yang et al further explored the role of AQP4 in the removal of Aβ.¹⁶ They found that AQP4 deficiency reduced the astrocytic activation in response to Aβ in mice, and Aqp4-knockout reduced astrocyte death at high Aβ levels.¹⁶ Furthermore, AQP4 expression increased as Aβ concentration increased, likely due to a protein synthesis mechanism. Further investigation demonstrated that lipoprotein receptor-related protein-1 (LRP1) is directly involved in the uptake of Aβ, and knockout of Aqp4 reduced up-regulation of LRP1 in response to Aβ.^15,16 Finally, AQP4 deficiency was found to alter the levels and time-course of MAPKs, a family of protein kinases involved in the response to astrocyte stressors.¹⁶ The role of AQP4 in cleansing the parenchyma as well as modulating astrocytic responses to Aβ thus pinpoint it as a major target for the following potential therapies: repairing defects in toxin clearance from the interstitial space, increasing expression in AQP4-deficient patients to increase astrocyte response, and knocking out Aqp4 in patients with high levels of Aβ to prevent astrocyte damage.

Sleep and Aβ Clearance

Interestingly, there is a link between Aβ clearance and sleep. Xie et al. studied Aβ clearance from the parenchyma in sleeping, anesthetized, and wakeful rodents, obtaining evidence that sleep plays a role in solute clearance from the brain. The researchers found that glymphatic CSF influx was suppressed in wakeful rodents compared to sleeping rodents.¹⁷ Glymphatic CSF influx is vital because it clears solutes from the brain in a way somewhat analogous to the way kidneys filer the blood. Real-time measurements showed that the parenchymal space was reduced in wakeful rodents, which led to increased resistance to fluid influx.¹⁷ Moreover, Aβ clearance was faster in sleeping rodents. Adrenergic signaling was hypothesized as the cause of volume reduction, implicating hormones such as norepinephrine.¹⁷ AQP4 is implicated in this phenomenon, as constricted interstitial space resists the CSF influx that this protein enables.

Aquaporin Therapy

If future treatment will target AQP4 function, then researchers must learn to manipulate its expression. However such regulatory mechanisms are not well understood. It is well-known that cells can ingest proteins in the plasma membrane and thus modulate the membrane protein landscape. Huang et al studied this phenomenon with AQP4, utilizing the fact that occluding the middle cerebral artery mimics ischemia and alters AQP4 expression in astrocyte membranes.¹⁸ They found that artery occlusion down-regulates AQP4 expression and discovered various mechanisms behind this response.¹⁸ Specifically, they determined that AQP4 co-localized in the cytoplasm with several proteins involved in membrane protein endocytosis, after the onset of ischemia.¹⁸ They posited that this co-localization indicates the internalization of AQP4.¹⁸ These correlations indicate that AQP4 is intimately connected with fluctuations in brain oxygen and nutrient levels, which are limited when blood flow is restricted.

Future Research

Aquaporin-4 is vital to many processes in the brain, but the range and details of these roles are not yet fully understood. As demonstrated, this protein is the central actor in the newly defined glymphatic system responsible for clearing solutes from CSF in the interstitial space. This function implicates AQP4 in the progression of AD and suggests other brain states and neurological conditions may have links to the protein’s function. Studies have demonstrated that AQP4 expression is dynamic, indicating that it can be regulated. The hope is that modulation of aquaporin expression or function could be used in brain therapy. Future research will no doubt focus on these mechanisms, and discoveries will aid in developing a treatment for various brain disorders.

References

1. Goetz, C., Textbook of Clinical Neurology, 3rd Edition; Saunders: Philadelphia, 2007.
2. Goldman, L. Goldman’s Cecil Medicine; Saunders Elsevier: Philadelphia, 2008.
3. Karriem-Norwood, V. Brain Diseases, WebMD. http://www.webmd.com/brain/brain-diseases (Accessed December 1, 2013).
4. Crisan, E. Ventricles of the Brain, Medscape. http://emedicine.medscape.com/article/1923254-overview#aw2aab6b3 (Accessed December 1, 2013).
5. Scientists Discover Previously Unknown Cleansing System in Brain, University of Rochester Medical Center. http://www.urmc.rochester.edu/news/story/index.cfm?id=3584 (Accessed February 11, 2014).
6. Iliff J.J. Cerebrospinal Fluid Circulation: A Paravascular Pathway Facilitates CSF Flow Through the Brain Parenchyma and the Clearance of Interstitial Solutes, Including Amyloid ß. Sci Transl Med 2012, 4, 147ra111.
7. 2012 Alzheimer’s Disease Facts and Figures. Alzheimer’s Association. http://www.alz.org/downloads/facts_figures_2012.pdf (Accessed December 6, 2013).
8. Dugdale III, D. Meninges of the Brain. MedlinePlus, National Institutes of Health. http://www.nlm.nih.gov/medlineplus/ency/imagepages/19080.htm (Accessed December 1, 2013).
9. O’Rahilly, R.; Muller, F.; Carpenter, S.; Swenson, R. Chapter 43: The Brain, Cranial Nerves, and Meninges. Basic Human Anatomy. [Online] Dartmouth Medical School: Hanover, 2008. http://www.innerbody.com/anatomy/nervous/subarachnoid-space (Accessed December 1, 2013).
10. Agamanolis, Dimitri. Chapter 14 Cerebrospinal Fluid. Neuropathology. [Online] http://neuropathology-web.org/chapter14/chapter14CSF.html (Accessed Dec. 1, 2013).
11. Cerebrospinal Fluid (CSF), National Multiple Sclerosis Society. http://www.nationalmssociety.org/about-multiple-sclerosis/what-we-know-about-ms/diagnosing-ms/cerebrospinal-fluid/index.aspx (Accessed December 1, 2013).
12. Millodot, M. Astrocytes. Dictionary of Optometry and Visual Science, 7th edition; Butterworth-Heinemann: Oxford, U.K., 2009.
13. Nielsen, H.M. et al. Glia [Online] 2010, 58, 1235-1246.
14. Kobayashi, K. J Alzheimer’s Dis [Online] 2004, 6, 623-632.
15. Arelin, K. Brain Research Molecular/Brain Research [Online] 2002, 104, 38-46.
16. Yang, W. Mol Cell Neurosci [Online] 2012, 49, 406-414.
17. Xie, L Science [Online] 2013, 342, 373-377.
18. Huang, J. Brain Research [Online] 2013, 1539, 61-72.
19. Almodovar, B. et al. Rev Cubana Me Top [Online] 2005, 57, 3, 230-232.
20. Ibe, B.C., et al. J. Tropical Pediatr. [Online] 1994, 40, 315-316.
21. Slowik, G. What Is Meningitis? eHealthMD. http://ehealthmd.com/content/what-meningitis#axzz2l3OzwGfb (Accessed Dec. 1, 2013).
22. Iadecola C. and Nedergaard M. Glial regulation of the cerebral microvasculature. Nat Neurosci [Online] 2007, 10, 1369-1376.

Farming the Unknown: The Role of the Livestock Industry in Preserving Human Health

Catalyst Volume 7

The livestock industry is a vast network of expectations. A farmer expects meat, dairy, and eggs from his animals, and a consumer expects to obtain these products from grocery stores. Industry expects profitable revenue from the sales of these products. Given the intensiveness of modern agriculture, this chain of action has been massively amplified. Meat production has doubled since the 1950s, and currently almost 10 billion animals—not including additional goods such as dairy and eggs—are consumed every year in the United States alone.¹ Due to the magnitude of this industry, even small changes can bring about large scale effects. Infections exemplify this chain of events.

Though animal infections might initially seem to be a lesser concern, their effects on human health are rapidly becoming more pronounced and pervasive. During the past few years, an increased number of food-borne disease outbreaks have been traced to products such as beef, pork, poultry, and milk.² These outbreaks are especially concerning because the pathogens involved are new strains previously harmless to humans. Rather, these pathogens have become infectious to humans due to mutations that occur in animal hosts; such diseases that jump from animals to humans are termed zoonotic. Within the food industry, zoonotic illnesses can be transmitted by consumption or through contact with animals. Crucially, zoonotic cases are much harder to treat because there is no precedent for their treatment.

How often does this transmission occur? Since 1980, 87 new human pathogens have been identified, out of which a staggering 80% are zoonotic.³ Furthermore, many of these have been found in domestic animals, which serve as reservoirs for a variety of infectious agents. The large number of zoonoses raises several key questions. Are these outbreaks the product of our management of livestock or simply a natural phenomenon? How far could zoonotic illnesses escalate in terms of human cases and mortality? What practices or perspectives should we modify to prevent further damage?

Prominent virologist and Nobel laureate in medicine Sir Frank MacFarlane Burnet provided a timeless perspective to this issue in the mid-20th century. He conceptualized infectious disease as equally fundamental to other interactions between organisms such as predation, decomposition, and competition.⁴ Taking into account how we have harnessed nature, particularly with the aim of producing more food, we can see how farming animals has also inadvertently farmed pathogens.

Treating animals as living environments that can promote pathogenic evolution and diffusion is crucial to creating proper regulations in the livestock industry that protect the safety of consumers in the long run. Current practices risk the emergence of zoonotic diseases by facilitating transmission under heavily industrialized environments and by fostering antibiotic resistance in bacteria. Cooperative action between government, producers, and educated consumers is necessary to improve current practices and preserve good health for everyone.

Influenza: Old Threats, New Fears

The flu is not exactly a stranger to human health, but we must realize that the influenza virus not only affects humans but also other species such as pigs and birds. In fact, what is known as “the flu” is not a single virus but rather a whole family of viruses. The largest family of influenza viruses, influenza A, has different strains of viruses classified with a shorthand notation for their main surface glycoproteins –H for hemagglutinin and N for neuraminidase (Figure 1). These surface glycoproteins are important because their structure and shape determines if the virus will attach to the cellular receptors of its host and infect it. For example, the influenza H7N7 virus has a structure that allows it to specifically infect horses but not humans. Trouble arises when these surface glycoproteins undergo structural changes and the virus gains the capacity to infect humans, as was the case during the 2003 avian flu and the 2009 swine flu pandemics, when the influenza virus jumped from poultry and swine to humans.

Since 2003 when it was first documented in humans, avian influenza H5N1 has been responsible for over 600 human infections and associated with a 60% mortality rate due to severe respiratory failure.⁵ The majority of these cases occurred in Asia and Africa, particularly in countries such as Indonesia, Vietnam, and Egypt, which accounted for over 75% of all cases.^5-6 Though no H5N1 cases have been reported in the U.S., there have been 17 low-pathogenicity outbreaks of avian flu in American poultry since 1997, and one highly pathogenic outbreak of H5N2 in 2004 with 7,000 chickens infected in Texas.⁵

Poultry is not the only area of livestock industry where flu viruses are a human health concern. The 2009 outbreak of influenza H1N1—popularly termed “swine flu” from its origin in pigs—was officially declared a pandemic by the WHO and the CDC. With an estimated 61 million cases and over 12,000 deaths attributed to the swine flu since 2009, H1N1 is an example of a zoonotic disease that became pandemic due to an interspecies jump that turned it from a regular pig virus to a multi-species contagion.⁷

The theory of how influenza viruses mutate to infect humans includes the role of birds and pigs as “mixing vessels” for mutant viruses to arise.⁸ In pigs, the genetic material from pig, bird, and human viruses (in any combination) reassorts within the cells to produce a virus that can be transmitted among several species. This process also occurs in birds with the mixing of human viruses and domestic and wild avian viral strains. If this theory is accurate, one can infer that a high density of pigs in an enclosed area could easily be a springboard for the emergence of new, infectious influenza strains. Thus, the “new” farms of America where pigs and poultry are stocked to minimize space and maximize production provide just the right environment for one infected pig to transfer the disease to the rest. Human handlers then face the risk of exposure to a new disease that can be as fatal as it is infectious, as the 2009 swine flu pandemic and the 2003 avian flu cases demonstrated. As consumers, adequate care of our food sources should not only be priority in avoiding disease but also in national and global health.

Feeding our Food: Antibiotic Resistance in the Food Industry

Interspecies transmission is not the only way through which new diseases can become pathogenic to humans. In the case of bacteria, new pathogenic strains can arise in animals from the action of another mechanism: antibiotic resistance. Antibiotic resistance is the result of the fundamental concept of evolutionary biology—individuals with advantageous traits that allow survival and reproduction will perpetuate these traits to their offspring. Even within the same population, antibiotic resistance varies among individual bacteria—some have a natural resistance to certain antibiotics while others simply die off when exposed. Thus, antibiotic use effectively selects bacteria with such resistance or, in some cases, total immunity. In this way, the livestock industry provides a selective environment.

The rise of these resistant strains—commonly termed “superbugs” for their extensive resistance to a variety of common antibiotics—has been a serious threat in hospitals; there, antibiotic use is widespread, and drug resistance causes almost 100,000 deaths each year from pathogens such as Methicillin-resistant Streptococcus aureus, Candida albicans, Acenitobacter baumanni, and dozens of other species.⁹ Our attention should not be exclusively focused to hospitals as sources of superbug infections, however. The widespread use of antibiotics in the livestock industry to avoid common bacterial diseases in food animals also poses the risk of emerging superbug strains, and it has not been without its share of outbreaks and casualties.

The Center for Science in the Public Interest –a non-profit organization that focuses on advocating for increased food safety in the US—has reported that antibiotic-resistant pathogens have been the cause of 55 major outbreaks since 1973, and that the majority of cases have come from dairy products, beef, and poultry. Furthermore, the same study reported that most of these pathogens exhibit resistance to over 7 different antibiotics.¹⁰ One of the main culprits identified in these outbreaks is the bacterium Salmonella typhimurium along with other Salmonella species, which account for over half of these cases. Salmonella is especially dangerous because it is so pervasive; it is able to lay dormant in a variety of livestock products such as uncooked eggs, milk, cheese, poultry, and beef until incubating in a live host for infection. Escherichia coli 0157:H7 (commonly known as E. coli), a bacterium that usually resides in the intestines of mammals, has also been implicated in a number of outbreaks related primarily to beef products. Overall, antibiotic-resistant pathogens have been the cause of over 20,500 illnesses, with over 31,000 hospitalizations and 27 deaths.¹⁰

These cases demonstrate how the widespread use of antibiotics in the food industry is perpetuating the risk of infections and damage to human health with antibiotic-resistant bacteria. Currently, the Food and Drug Administration (FDA) in the U.S. still approves of the use of antibiotics as a treatment for sick animals; furthermore, the organization allows antibiotic use in healthy animals as prevention and even as growth enhancers.¹¹ In fact, over 74% of all antibiotics produced in the United States are used in livestock animals for these reasons.^9,11 Using antibiotics in non-infected animals in this way generates a greater environmental pressure for superbugs to emerge; this type of use in particular should be restricted. Managing a proper use of antibiotics to reduce the risk of emerging strains of superbugs should be prioritized in the food industry just as it is in health care.

Hungry for a Solution

Still open to debate is the question of how many resources should be allocated to the problem of widespread antibiotic use. Currently, diseases are transmitted from animals to humans faster than they are evolving within humans. Not only that, many of these zoonotic diseases have high potential to become a pandemic due to their high infectivity, as in the case of H5N1 avian influenza. Measures to prevent the transmission of viruses among livestock animals and to reduce the rate of emergent antibiotic-resistant strains need to take into account the environmental and evolutionary nature of a zoonosis.

A more thorough surveillance of livestock animals and monitoring signs of new emerging strains are important in preventing the spread of such deadly pathogens. This strategy requires intensive molecular analysis, a larger number of professionals working in the field, and a nationwide initiative. Keeping an accurate record of where new strains arise and the number of animal and human cases would significantly improve epidemiological surveillance of infectious disease. This process requires cooperation at multiple levels to ensure that the logistics and public support for these initiatives is ongoing and effective. Additionally, educating people about the nature of zoonotic pathogens is crucial to fostering the dialogue and action necessary to secure the good health of animals, producers, and consumers.

References

1. John’s Hopkins Center for a Livable Future: Industrial Food Animal Production in America. Fall 2013. http://www.jhsph.edu/research/centers-and-institutes/johns-hopkins-center-for-a-livable-future/_pdf/research/clf_reports/CLF-PEW-for%20Web.pdf (accessed Oct 24, 2013).
2. Cleaveland, S. et al. Phil. Trans. R. Soc. B. 2001, 356, 991.
3. Watanabe, M. E. BioScience 2008, 58, 680.
4. Burnet, F. M. Biological Aspects of Infectious Disease. Macmillan: New York, 1940.
5. Centers for Disease Control and Prevention: Avian Flu and Humans. http://www.cdc.gov/flu/avianflu/h5n1-people.html. (accessed Oct 12, 2013)
6. Cumulative number of confirmed human cases of avian influenza A(H5N1) reported to WHO. http://www.who.int/influenza/human_animal_interface/H5N1_cumulative_table_archives/en/ (accessed March 14, 2013)
7. Chan, M. World Now at the Start of the 2009 Influenza Pandemic. http://www.who.int/mediacentre/news/statements/2009/h1n1_pandemic_phase6_20090611/en/ (accessed March 14, 2013).
8. Ma, W. et al. J. Mol. Genet. Med. [Online] 2009, 3, 158-164.
9. Mathew, A. G. et al. Foodborne Pathog. Dis. 2007, 4, 115-133.
10. DeWaal, C. S.; Grooters, S. V. Antibiotic Resistance in Foodborne Pathogens. http://cspinet.org/new/pdf/outbreaks_antibiotic_resistance_in_foodborne_pathogens_2013.pdf (accessed March 14, 2014).
11. Shames, L. Agencies Have Made Limited Progress Addressing Antibiotic Use in Animals http://louise.house.gov/images/user_images/gt/stories/GAO_Report_on_Antibioic_Resistance.pdf. (accessed Jan 20, 2014).