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CNS & Neurological Disorders - Drug Targets, 2009, 8, 309-314

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Cell Transplantation: A Future Therapy for Narcolepsy? Oscar Arias-Carrión*,1 and Eric Murillo-Rodríguez2 1

Experimental Neurology, Phillips University. Marburg, Germany

2

Laboratorio de Neurociencias Moleculares e Integrativas, División de Ciencias de la Salud. Escuela de Medicina. Universidad Anáhuac Mayab, Mérida Yucatán, México Abstract: Narcolepsy is a sleep disorder characterized by excessive daytime sleepiness, cataplexy, hypnagogic hallucinations, and sleep-onset rapid eye movement (REM) sleep periods. Narcolepsy is now identified to be a neurodegenerative disease, as there is a massive loss of neurons containing the neuropeptide, hypocretin/orexin. Orexin neurons are solely located in the hypothalamus, particularly in its perifornical, dorsomedial and lateral portions. Orexin fibers widely project throughout the brain and generally have excitatory effects on their postsynaptic cells. Patients with narcolepsy have a severe reduction in the levels of orexins in the cerebrospinal fluid, a finding consistent with orexin neuronal loss. Experimental models have been generated in order to study the physiology of the orexin system and narcolepsy. The discovery of orexin deficiency in narcolepsy is redefining the clinical entity of narcolepsy and offering novel diagnostic procedures. This article reviews the current understanding of narcolepsy and discusses the opportunity to explore the potential use of transplants as a therapeutical tool in order to treat narcolepsy.

Keywords: hypocretin/orexin neurons, narcolepsy, sleep, lateral hypothalamus, animal models, cell transplant. 1. INTRODUCTION The orexins (hypocretins) are two neuropeptides that are encoded by a single gene that drives the synthesis of preproorexin, a gene product that is subsequently matured into the 33-amino acid orexin-A and the 28-amino acid orexin-B [1, 2]. In the rat, orexin neurons are located between the fornix and the mammillothalamic tracts in the lateral hypothalamus (LH) from whence orexin fibers project throughout the brain and spinal cord, including several areas implicated in regulations of the sleep/wake cycle [1-4]. Orexin fibers widely project throughout the brain and spinal cord and generally have excitatory effects on serotonergic [5, 6], noradrenergic [7], histaminergic [8], and cholinergic neurons in basal forebrain [9], laterodorsal tegmental nucleus [10] as well as thalamocortical neurons of thalamus [7]. Two orexin receptor subtypes, OX1R and OX2R, have been cloned [1]. They are serpentine G-protein coupled receptors that bind both orexins with low selectivity and are coupled functionally to Ca++ mobilization [1], probably through transient receptor potential channels. OX1R and OX2R mRNAs exhibit a markedly different and basically complementary distribution, indicating that these receptors have distinct physiological roles [1]. Studies in rodents, dogs and humans indicate that orexins play a part in the regulation of various functions including sleep-wake, arousal, muscle tone, locomotion, regulation of feeding behaviour, and neuroendocrine and autonomic functions. Even though the orexin neurons constitute only ~1% of the neurons in the lateral hypothalamus [3], the orexins have emerged recently as an important mode of signalling in the hypothalamus.

*Address correspondence to this author at the Experimental Neurology, Biomedical Research Center (BMFZ), Philipps University, Hans Meerwein Str., D-35043 Marburg, Germany; E-mail: [email protected]

1871-5273/09 $55.00+.00

2. OREXINS: NARCOLEPSY

PEPTIDES

ASSOCIATED

WITH

Narcolepsy is a debilitating neurological disease characterized by excessive daytime sleepiness, premature transitions to rapid eye movement sleep (named “sleep onset REM periods”) and cataplexy (sudden bilateral skeletal muscle weakness without impairment of conscience) [11]. Orexin was linked to human narcolepsy with the discovery of a mutation in the OX2R in mice [12] and dogs [13]. Once it was speculated that a deficiency in the orexin system might be the explanation for human narcolepsy, diverse groups explored and then validated the link between the orexin system and human narcolepsy. In a study of post-mortem brains of narcoleptic patients, a massive reduction in the number of orexin-containing cells (85-98%) was discovered as compared with healthy controls [14, 15]. In addition, narcoleptic patients have reduced cerebrospinal fluid (CSF) levels of orexins [16-20], a finding consistent with the loss of orexin-containing neurons as mentioned above. Measuring cerebrospinal fluid orexin-A is a definitive diagnostic test, provided that it is interpreted within the clinical context, separating narcolepsy from other sleep and neurological disorders [20]. Genetic and environmental factors both clearly play a role in narcolepsy-cataplexy. Currently, the best evidence for autoimmunity as a cause of the disorder was the discovery that the majority of patients with the disorder has unique variants of a gene called HLA-DQB1*0602 [21]. This is one of the genes that encode HLA proteins which dot cell surfaces and help the immune system identify foreign proteins. In addition to unique HLA variants, people with narcolepsy-cataplexy are also more likely to have unique variants of the T-cell receptor alpha gene, which encodes a receptor protein on the surface of T cells [21]. Changes to

© 2009 Bentham Science Publishers Ltd.

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CNS & Neurological Disorders - Drug Targets, 2009, Vol. 8, No. 4

the T cell receptor could increase the likelihood that the hypocretin/orexin cells will direct their attack against the body. 3. ANIMAL MODELS OF NARCOLEPSY

Arias-Carrión and Murillo-Rodríguez

that measured eight hours in to the inactivity period (ZT8). The rats with a 72% orexin neuronal loss did not show a significant difference between ZT0 and ZT8. We also found a significant correlation between orexin neurons and orexin levels at ZT0 [28].

There are several experimental models that mimic the sleep disorder narcolepsy including orexin-knockout mice [12], canines with a mutation in the OX2R [13] or mice with a targeted destruction of the orexin neurons [22] that exhibit symptoms of narcolepsy. Recently, our group has generated a toxin-based experimental narcolepsy model consisting of a ribosomeinactivating protein saporin (SAP) [23] that is conjugated to the orexin receptor. This toxin conjugate binds ligand orexinB (Hcrt-2) and thus lesions orexin receptor-bearing neurons. LH contains a high concentration of orexin receptor mRNA [24] as well as immunoreactivity [25], suggesting a high likelihood of orexin receptors on orexin neurons. When Hcrt-2/SAP is injected into LH of rats, the toxin lesions orexin neurons, and produces behavioural symptoms that are characteristic of narcolepsy (Fig. 1). Hcrt-2/SAP binds selectively to orexin receptorcontaining cells and produces orexin-specific effects. For instance, despite a retention of the diurnal rhytm of wakefulness (W) and slow wage sleep (SWS), Hcrt-2/SAP increases SWS over the slow period, due to an increase in sleep during the lights-off period. Total time spent in SWS and rapid eye movement sleep (REMS) was found to correlate with a decline in number of orexin neurons. Using this model, Gerashchenko et al., [26] demonstrated that Hcrt-2/SAP induced more SWS and REM sleep at during the diurnally active period and multiple periods of abnormal behavioural arrest during purposeful behaviour. Indeed, this experimental model of narcolepsy provides a method of investigating the contribution of the orexin system to the regulation of the sleep-wake cycle and its relationship with narcolepsy. Gerashchenko et al., [27] also reported that two concentrations (90ng or 490ng/0.5μl) of the Hcrt-2/SAP injected directly to the lateral hypothalamus caused a significant orexin cell lost. Narcoleptic-like sleep behaviour was produced by both concentrations of this toxin [27]. Together, these results indicate that the use of Hcrt2/SAP induced characteristics of narcolepsy, such as sleep fragmentation, sleep-onset REM sleep periods, increased SWS and REM sleep time during the normally active lightsoff period [26, 27]. Prior to these studies, the precise relationship between orexin neuronal loss and changes in levels of the peptide was poorly characterized. Using the Hcrt-2/SAP to lesion neurons in the LH, we found that there was a 50% reduction in CSF orexin levels when 73% of orexin neurons were lesioned successfully [28]. The sleep deprivation method is used to test the homeostatic mechanism of sleep regulation, a manipulation that typically increases orexin expression in the hypothalamus. In lesioned rats, the orexin levels were not increased by 6 h prolonged W, indicating that surviving neurons were not able to increase the output of orexin into CSF to compensate for the orexin neuronal loss. In the same study, orexin levels in CSF measured at the end of the wake-active period (ZT0) was 72.9% greater than

Fig (1). Experimental procedure showing the injection of the Hcrt2/SAP into lateral hypothalamus (LH). The Hcrt-2/SAP induces Hcrt neuronal loss leading a diminution in levels of orexin-A in CSF. Narcolepsy is the behavioral consequence as a result of the orexin neuronal loss and diminution levels in CSF.

Orexin neurons begin to appear on embryonic day 19 (E19) and are fully developed by postnatal day 20 [29, 30]. Orexin display a diurnal rhythm in young animals [31, 32]. Such a rhythm of orexin, however, has not been determined in aged animals. We measured CSF orexin levels at 4-hour intervals across a 24-hour period to test the hypothesis that there was a decline in orexin levels in aged rats. In agreement with previous studies in young rats [31], peak levels of orexin were found at ZT0 and lowest levels occurred at the end of the sleep period (ZT12). This profile was present in young and old rats; however, aged rats had significantly reduced orexin levels in CSF compared to the young rats [33]. As mentioned above, CSF orexin levels are increased in response to prolonged W. Thus, we tested the orexin level response to 8 hours of W. Both young and aged rats had

Cell Transplantation: A Future Therapy for Narcolepsy?


significantly increased CSF orexin levels in response to 8 hours of prolonged waking [33], a finding that is consistent with other studies [32]. The overall CSF orexin levels after 8 h of prolonged W, however, were still lower in the aged rats when compared to the young rats. While a possible explanation for this phenomenon might be a decline in orexin-A in aged rats, there were no age-related differences in the prepro-orexin mRNA levels in the posterior hypothalamus of young and aged rats by Northern blot analysis [33].

have reported clinical benefit [37, 38, 40]. Some patients have been able to withdraw from L-dopa treatment for several years and resume an independent life [37, 38]. Beneficial effects have been demonstrated, and autopsy cases have shown that many transplanted cells were able to survive in the human brain for long periods. These findings have contributed a great deal to the research in regeneration of the central nervous system.

4. CELL THERAPY TO TREAT NARCOLEPSY Patients with narcolepsy are typically treated with amphetamine-like stimulants and antidepressants [34]. Newer compounds, such as modafinil and sodium oxybate still only marginally improve the quality of life of narcoleptic patients [34]. The goal of all therapeutic approaches in narcolepsy is to control the narcoleptic symptoms and to allow the patient to continue full participation in familial and professional daily activities [34, 35]. Although these treatments are effective in early stages of the disease and modafinil can provide substantial symptomatic relief, there is a need for novel therapeutic approaches. Neural transplantation is one of the most promising approaches for the treatment of Parkinson’s disease (PD), a major neurodegenerative disorder with prevalence as frequent as that of narcolepsy [36]. Several studies using animal models have demonstrated that grafted tissue survives, integrates within the host brain, and provides functional recovery following brain inventions [37, 38]. Analogously, a recent publication demonstrated a ‘rescue’ from narcolepsy symptoms through a non-specific overexpression of orexin by non-orexin cells in the hypothalamus of orexin-knockout mice [39]. In the same way, cell therapy could be employed to implant grafts of post-mitotic orexinergic neuroblasts into the hypothalamus or even other brain regions. Neural transplantation involves implantation of living neuronal tissue into a host system. Several studies using animal models have demonstrated that grafted tissue survives, integrates within the host brain, and provides functional recovery following brain inventions [37, 38]. This type of study for PD began in the latter half of the 1970s. Initially, dopaminergic neurons from animal fetuses were used as donors, and then paraneurons such as chromaffin cells were used [40]. Based on a large number of experimental animal studies, neural transplantation has been applied clinically [37, 38, 40]. Studies in patients with PD after intrastriatal transplantation of human fetal mesencephalic tissue, rich in postmitotic dopaminergic neurons, have provided proof of the principle that neuronal replacement can work in the human brain [37, 38, 40]. The grafted neurons survive and reinnervate the striatum for as long as 10 years despite an ongoing disease process, which destroys the patient's own dopaminergic neurons [37, 38, 40]. The grafts are able to normalize striatal dopamine release and to reverse the impairment of cortical activation underlying akinesia [37, 38, 40]. Thus, grafted dopaminergic neurons can become functionally integrated into neuronal circuitries in the brain [37, 38, 40]. Several open-label trials

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Thus the possibility remains that a graft of orexincontaining neurons into a host brain could prove viable. We demonstrated that cell suspensions containing orexinergic neurons derived from posterior hypothalamus of 8-10-d old rat pups can survive when transplanted into the pons (a region of the brain that is innervated by orexin axons but where the orexin somata are not present) in adults rats [41, 42]. Well-defined orexin-immunoreactive somata with processes and varicosities were present in the graft zone 36 d after implantation of the cell suspension, suggesting that orexin neurons obtained from rat pups can be grafted into an adult host brain [41]. These somata were similar in size and appearance to adult rat orexin-immunoreactive neurons (Fig. 2). Recently, we investigated the survival time-course of grafted orexin neurons into the pons of adult rats [42]. Control rats received a transplant that consisted of cells from the cerebellum where no orexin neurons are present. All adult host rats were sacrificed 1, 3, 6, 9, 12, 24, or 36 days after grafting. Inmunohistochemestry was used to identify and count the presence of the orexin grafted neurons in the target area. The tally of orexin neurons present in the graft zone 1 day post-grafting was considered to be the baseline. From day 3 to 36 post-transplant there was a steady decline in the number of orexin neurons. We also noted that on day 36, the orexin neurons that survived in the pons had morphological features that were similar to mature orexin neurons in the adult lateral hypothalamus, suggesting that these neurons might be functionally active. Control rats that received grafts of cerebellar tissue did not show orexin neurons in the target area. These results demonstrate that there is a progressive decline in the number of transplanted neurons, but a significant percentage of orexin neurons does survive until day 36. However, this study highlights the potential use of transplants as a therapeutical tool in order to treat narcolepsy. Whether the transplants will be able to restore W or establish functional connections is still unknown. We are currently carrying out those experiments at the laboratory aimed to answer these questions. 5. CONCLUSIONS AND FUTURE DIRECTIONS Narcolepsy is a sleep disorder characterized by sleep attacks. Experimental models have been generated in order to understand the physiology of this disease. Recent evidence has concluded that narcolepsy in humans and animal models are the result of the failure of cellular signalling mediated by orexins. Given that the orexin depletion is the hallmark of narcolepsy, animal models represent the opportunity to explore the potential use of transplants as a therapeutical tool in order to treat narcolepsy.

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Fig (2). (A) Representation of the brain sections taken from donor containing the hypothalamus for tissue preparation for transplants in references 41 and 42. (B) Targeted area within the brainstem for orexin grafted neurons in references 41 and 42. (C) Microphotography of orexin survival cells in target area at 36 days after grafting (previously unreported data). Abbreviations: 3V: 3rd ventricle; 4V: 4th ventricle; Cx: cortex; CC: corpus callosum; f; fornix; LC; locus coeruleus; LH: lateral hypothalamus; PnC: pontine reticular nucleus; RPn: raphe pontis nucleus; GT: gauge track.

In this review, we have described that orexin neurons are able to be transplanted with the aim to generate an alternative therapeutical tool to treat narcolepsy. We have showed here that grafted cells express the machinery for orexin release, and possess the morphological feature of an orexin neuron. This method, however, has limitations: (i) the source of orexinergic neurons is from postnatal tissue and (ii) the survival rate of the grafts is very poor (~5% of implanted cells). The main interest is now focused on producing orexinergic neuroblasts for transplantation from stem cells. After maturation, these neurons would have the potential to demonstrate functional efficacy similar to or superior to those existing in the adult lateral hypothalamus. Should this therapeutic avenue prove fruitful, stem-cellderived cells have to fulfill the following requirements to induce recovery from narcolepsy symptoms after transplantation: (1) higher graft survival probability; (2) regulated orexin release; (3) molecular, morphological and electrophysiological properties of fully mature orexinergic neurons; (4) re-establishment of a dense, functional orexin releasing terminal network; (5) grafts that are functionally integrated into host circuitries to restore wakefulness. The

human application of stem-cell-derived orexinergic neuroblasts will be based on solid preclinical work in animal models of the disease. ACKNOWLEDGEMENTS This work was supported by Lundbeck Institute (Denmark), Grant PROMEP (103/08/2971) and Grant CONACYT (79009) given to EM-R. OA-C is supported by the German Academic Exchange Service (DAAD). ABBREVIATIONS LH

=

Lateral hypothalamus

REM

=

Rapid eye movement

CSF

=

Cerebrospinal fluid

SAP

=

Saporin

Hcrt

=

Hypocretin

W

=

Wakefulness

SWS

=

Slow wave sleep

Cell Transplantation: A Future Therapy for Narcolepsy?


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CNS & Neurological Disorders - Drug Targets, 2009, Vol. 8, No. 4 Siegel, J.M.; Boehmer, L.N. Narcolepsy and the hypocretin system-where motion meets emotion. Nat. Clin. Pract. Neurol., 2006, 2, 548-556. Longstreth, W.T.; Jr.; Koepsell, T.D.; Ton, T.G.; Hendrickson, A.F.; van Belle, G. The epidemiology of narcolepsy. Sleep, 2007, 30, 13-26. Deierborg, T.; Soulet, D.; Roybon, L.; Hall, V.; Brundin, P. Emerging restorative treatments for Parkinson's disease. Prog. Neurobiol., 2008; 85, 407-432. Lindvall, O.; Lokaia, Z.; Martinez-Serrano, A. Stem cell therapy for human neurodegenerative disorders-how to make it work. Nat. Med., 2004, 10, S42-S50. Liu, M.; Thankachan, S.; Kaur, S.; Begum, S.; Blanco-Centurion, C.; Sakurai, T.; Yanagisawa, M.; Neve, R.; Shiromani, P.J. Orexin

Received: June 2, 2009


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[42]

Revised: June 4, 2009

(hypocretin) gene transfer diminishes narcoleptic sleep behavior in mice. Eur. J. Neurosci., 2008; 28, 1382-1393. Drucker-Colín, R.; Verdugo-Díaz, L. Cell transplantation for Parkinson’s disease: present status. Cell Mol. Neurobiol., 2004, 24, 301-316. Arias-Carrión, O., Murillo-Rodriguez, E., Xu, M., BlancoCenturion, C., Drucker-Colín, R., Shiromani, P.J., Transplantation of hypocretin neurons into the pontine reticular formation: preliminary results. Sleep, 2004, 27, 1465-1470. Arias-Carrión, O.; Drucker-Colín, R.; Murillo-Rodríguez, E., Survival rates through time of hypocretin grafted neurons within their projection site. Neurosci. Lett., 2006, 404, 93-97.

Accepted: June 4, 2009