Research Articles: Neurobiology of Disease Sleeping sickness disrupts the sleep- regulating adenosine system https://doi.org/10.1523/JNEUROSCI.1046-20.2020 Cite as: J. Neurosci 2020; 10.1523/JNEUROSCI.1046-20.2020 Received: 30 April 2020 Revised: 28 September 2020 Accepted: 11 October 2020 This Early Release article has been peer-reviewed and accepted, but has not been through the composition and copyediting processes. The final version may differ slightly in style or formatting and will contain links to any extended data. Alerts: Sign up at www.jneurosci.org/alerts to receive customized email alerts when the fully formatted version of this article is published. Copyright © 2020 the authors 1 Title: Sleeping sickness disrupts the sleep-regulating adenosine system 2 3 Short title: Sleeping sickness and adenosine 4 5 Filipa Rijo-Ferreira*1,2, Theresa E. Bjorness*3,4, Kimberly H. Cox1, Alex Sonneborn1,3, 6 Robert W. Greene1,3, and Joseph S. Takahashi1,2,# 7 8 1 Department of Neuroscience, Peter O’Donnell Jr. Brain Institute, University of Texas 9 Southwestern Medical Center, Dallas, TX 75390-9111, USA 10 2 Howard Hughes Medical Institute, University of Texas Southwestern Medical Center, 11 Dallas, TX 75390-9111, USA 12 3 Department of Psychiatry, University of Texas Southwestern Medical Center, Dallas, 13 TX 75390-9111, USA 14 4Research Service, VA North Texas Health Care System, Dallas, TX 75126 15 16 * co-authors 17 18 # Correspondence: [email protected] 19 20 21 Number of pages: 32 22 23 Number of figures: 7 24 25 Number of tables: 1 26 27 Number of words for abstract: 130 28 29 Number of words for introduction: 588 30 31 Number of words for discussion: 1259 32 33 Conflict of Interest Statement: The authors have nothing to declare. 34 35 Acknowledgements: We would like to thank Nelly Garduño and Iza Kornblum for their 36 technical assistance and Fernando Augusto for artwork. J.S.T. is an Investigator and 37 F.R-F. is a Postdoctoral Research Associate in the Howard Hughes Medical Institute. 38 This research was supported by NIH grant NIGMS K99GM132557 to F.R-F., VA 39 BLR&D CDA grant IK2BX002531 to T.E.B., NIH grant R01MH080297 and WPI program 40 for IIIS to R.W.G., as well as funding from Howard Hughes Medical Institute to J.S.T. 41 The contents of this manuscript do not represent the views of the U.S. Department of 42 Veterans Affairs or the United States Government. 43 44 ABSTRACT 45 46 Patients with sleeping sickness, caused by the parasite, Trypanosoma brucei, have 47 disruptions in both sleep timing and sleep architecture. However, the underlying cause 48 of these sleep disturbances is not well understood. Here we assessed the sleep 49 architecture of male mice infected with T. brucei and found that infected mice had 50 drastically altered sleep patterns. Interestingly, T. brucei infected mice also had a 51 reduced homeostatic sleep response to sleep deprivation, a response modulated by the 52 adenosine system. We found that infected mice had a reduced electrophysiological 53 response to an adenosine receptor antagonist and increased adenosine receptor gene 54 expression. Although the mechanism by which T. brucei infection causes these changes 55 remains to be determined, our findings suggest that the symptoms of sleeping sickness 56 may be due to alterations in homeostatic adenosine signaling. 57 58 59 SIGNIFICANCE STATEMENT 60 61 Sleeping sickness is a fatal disease that disrupts the circadian clock, causes disordered 62 temperature regulation, and induces sleep disturbance. To examine the neurological 63 effects of infection in the absence of other symptoms, in this study we used a mouse 64 model of sleeping sickness in which the acute infection was treated but brain infection 65 remained. Using this model, we evaluated the effects of the sleeping sickness parasite, 66 T. brucei, on sleep patterns in mice, under both normal and sleep-deprived conditions. 67 Our findings suggest that signaling of adenosine, a neuromodulator involved in 68 mediating homeostatic sleep drive, may be reduced in infected mice. 1 69 INTRODUCTION 70 Sleeping sickness (human African trypanosomiasis) is a disease endemic to sub- 71 Saharan Africa. It is caused by the parasite Trypanosoma brucei, which is transmitted to 72 humans by the tsetse fly (Franco et al., 2014). Sleeping sickness occurs in two phases. 73 In the first, hemolymphatic phase, T. brucei parasites are primarily found in the 74 bloodstream, interstitial spaces of peripheral organs, and lymphatic system (Caljon et 75 al., 2016; Capewell et al., 2016; Trindade et al., 2016). The main symptoms during this 76 first phase of the disease include fever, fatigue, and pain. In the second, meninge- 77 encephalitic stage, the parasites invade the central nervous system and cause 78 psychiatric symptoms as well as the progressive disruption of circadian rhythms, 79 including altered hormonal secretion, body temperature rhythms, daytime somnolence, 80 and nighttime insomnia (Buguet et al., 2001; Blum et al., 2006; Tesoriero et al., 2019). 81 In humans, sleep can be divided into 4 stages (3 sub-stages of non-rapid eye 82 movement [NREM] sleep (stage 3-4 also called Slow Wave Sleep [SWS]) and Rapid 83 Eye Movement [REM] sleep. These stages can be delineated by differences in brain 84 activity as measured by electroencephalography (EEG) and other physiological 85 parameters, such as muscle and eye movements (Jafari and Mohsenin, 2010). In 86 addition to changes in sleep timing, sleeping sickness patients have altered sleep 87 architecture, with REM episodes occurring immediately after sleep onset (SOREM) 88 instead of after NREM (Buguet et al., 2001; Buguet et al., 2005). This change in sleep 89 structure is similar to what is seen with narcolepsy (Nishino and Mignot, 1997; Fujiki et 90 al., 2009). Interestingly, while neither sleeping sickness nor narcolepsy causes 91 increased total sleep across the 24 h period (Dantz et al., 1994; Buguet et al., 2001), 2 92 narcoleptic patients do not have altered circadian rhythms (Dantz et al., 1994). T. brucei 93 is also distinct from other infections, which tend to increase sleep (Krueger and Opp, 94 2016; Tesoriero et al., 2019). How the T. brucei parasite produces these specific 95 alterations to the sleep system remains an open question (Lundkvist et al., 2004). 96 Sleep is regulated by interactions of the circadian system with a complex 97 homeostatic network that balances sleep need with arousal (Borbely et al., 2016). 98 Although there are many signals involved in maintaining arousal, an increase in 99 extracellular adenosine during wakefulness is thought to be a primary driver of both 100 homeostatic sleep need (Porkka-Heiskanen et al., 1997; Saper et al., 2005; Bjorness et 101 al., 2016; Bringmann, 2018) and motivation-related arousal (Lazarus et al., 2019). In 102 addition, allostatic mechanisms, such as the production of somnogens during infection, 103 also promote sleep (Bringmann, 2018; Toda et al., 2019). We previously showed that T. 104 brucei infection shortens the period of circadian behavioral activity in mice and disrupts 105 the molecular circadian clock within the suprachiasmatic nucleus (SCN) of the 106 hypothalamus (Rijo-Ferreira et al., 2018). However, the effects of T. brucei on 107 homeostatic drivers of sleep are unclear. 108 The purpose of the present study was to examine the effects of T. brucei 109 infection on sleep architecture in a mouse model of sleeping sickness. We found that, 110 similarly to the human infection, T. brucei infected mice had altered sleep architecture. 111 Interestingly, infection reduced the ability of mice to achieve or maintain REM sleep. In 112 addition, infected mice lacked a response to sleep deprivation, which may indicate a 113 change in their ability to achieve sleep or reduced sleep drive in the face of homeostatic 114 challenge. Electrophysiological experiments and quantification of gene expression 3 115 suggested that these behavioral changes were not due solely to inflammation induced 116 by infection, but rather may be caused by altered homeostatic adenosine signaling. 117 118 MATERIALS AND METHODS 119 Ethics statement 120 All animal care and experimental procedures were performed in accordance with 121 University of Texas Southwestern Medical Center (UTSW) IACUC guidelines, approved 122 by the Ethical Review Committee at the University of Southwestern Medical Center. 123 Housing and Surgical Procedures 124 Adult (8-12-week-old) male C57BL/6J mice from the UT Southwestern Medical 125 Center Mouse Breeding Core Facility were housed in standard mouse cages with food 126 and water provided ad libitum. Cages were in a temperature-controlled environment in a 127 T24 (12:12) LD cycle. To determine sleep/waking activity, EEG and EMG electrodes 128 were surgically implanted as described previously (Bjorness et al., 2009). Briefly, mice 129 were anesthetized with isoflurane and the scalp was sheared and cleaned. Mice were 130 then placed in a stereotax (Kopf, Tujunga, CA) and four holes were drilled in the skull. 131 Two EEG electrodes (Plastics One, Roanoke, VA) were placed bilaterally over the 132 frontal cortex (AP +1.7 mm, ML +/-1.77mm) and two electrodes were placed bilaterally 133 over the parietal cortex (AP -1.7 mm, ML +/-2mm). Two EMG electrodes (Plastics One, 134 Roanoke, VA) were placed bilaterally into the nuchal musculature. Electrode pins were 135 threaded into a 6-pin pedestal and affixed to the skull with dental adhesive (Fisher, 136 Pittsburgh, PA). After the surgery, the implant base was coated with antibiotic and 4 137 animals were given Buprenex (0.1 ml, IP) for analgesia. Animals were given 14 days to 138 recover from surgery prior to tether adaption and were housed singly following surgery. 139 Infection and Suramin Treatment 140 Infections were performed by intraperitoneal (i.p.) injection of 2000 Trypanosoma 141 brucei brucei AnTat 1.1E parasites between ZT9-12 (Zeitgeber time, ZT, 3-hour interval 142 before lights are off) (Rijo-Ferreira et al., 2018).