Brain Advance Access originally published online on April 30, 2007
Brain 2007 130(6):1577-1585; doi:10.1093/brain/awm090
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Hypocretin (orexin) loss in Parkinson's disease
1Department of Neurology, Leiden University Medical Center, Leiden, 2Netherlands Institute for Neurosciences, Amsterdam ZO, 3Department of Neurology, Radboud University Nijmegen Medical Center, Nijmegen, 4Department of clinical Chemistry and 5Department of Pathology, Leiden University Medical Center, Leiden, The Netherlands
Corresponding to: Prof. Dick Swaab, MD, Netherlands Institute for Neuroscience, Meibergdreef 33, 1105 AZ Amsterdam, The Netherlands E-mail: d.f.swaab{at}nin.knaw.nl
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The hypothalamic hypocretin (orexin) system plays a central role in the regulation of various functions, including sleep/wake regulation and metabolism. There is a growing interest in hypocretin function in Parkinson's disease (PD), given the high prevalence of non-motor symptoms such as sleep disturbances in this disorder. However, studies measuring CSF hypocretin levels have yielded contradictory results. In PD patients and matched controls, we (i) estimated the number of hypocretin neurons in post-mortem hypothalami using immunocytochemistry and an image analysis system (ii) quantified hypocretin levels in post-mortem ventricular CSF and (iii) prefrontal cortex using a radioimmunoassay. Furthermore, presence of Lewy bodies was verified in the hypothalamic hypocretin cell area. Data are presented as median (25th–75th percentile). We showed a significant decrease between PD patients and controls in (i) the number of hypocretin neurons (PD: 20 276 (13 821–31 229); controls: 36 842 (32 546–50 938); P = 0.016); (ii) the hypocretin-1 concentration in post-mortem ventricular CSF (PD: 365.5 pg/ml (328.0–448.3); controls: 483.5 (433.5–512.3); P = 0.012) and (iii) the hypocretin-1 concentrations in prefrontal cortex (PD: 389.6 pg/g (249.2–652.2); controls: 676.6 (467.5–883.9); P = 0.043). Hypocretin neurotransmission is affected in PD. The hypocretin-1 concentration in the prefrontal cortex was almost 40% lower in PD patients, while ventricular CSF levels were almost 25% reduced. The total number of hypocretin neurons was almost half compared to controls.
Key Words: narcolepsy; hypocretin; orexin; sleep; Parkinson's disease; hypothalamus
Abbreviations: CSF, Cerebrospinal fluid; EDS, Excessive Daytime Sleepiness; IR, Immunoreactive; NBB, Netherlands Brain Bank; PD, Parkinson's disease; PMD, Post-mortem delay; REM-sleep, Rapid eye movement sleep; RIA, Radioimmunoassay
Received October 23, 2006. Revised February 28, 2007. Accepted March 22, 2007.
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Introduction |
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Parkinson's disease (PD) is a neurodegenerative disorder in which motor symptoms such as hypokinesia, tremor and rigidity are the most well known. However, there is a growing interest in the non-motor symptoms, such as autonomic dysfunction and cognitive disturbances (Chaudhuri et al., 2006
). Of these, sleep disorders are one of the most striking (Arnulf et al., 2002; Rye et al., 2004; Arnulf, 2005). Sleep disturbances occur often in patients with PD and can even precede the motor symptoms. Excessive daytime sleepiness with frequent naps and so-called ‘sleep-attacks’ have been reported in 15–50% of patients (Hobson et al., 2002; Brodsky et al., 2003). Furthermore, there are clear nighttime sleep disturbances, such as fragmented nocturnal sleep, rapid eye movement (REM)-sleep behaviour disorder and periodic leg movements, as well as daytime sleep-onset REM periods (Arnulf et al., 2002; Gagnon et al., 2002, Rye et al., 2004). The combination of these symptoms suggest a common aetiology with narcolepsy (Arnulf et al., 2000).
Narcolepsy is a primary sleep-wake disorder characterized by excessive daytime sleepiness and REM-sleep dissociation phenomena such as cataplexy. Moreover, there are core symptoms of narcolepsy that resemble the night-time sleep disturbances commonly seen in PD, most notably fragmented nocturnal sleep and REM-sleep behaviour disorder (Overeem et al., 2001). Furthermore, sleep-onset REM periods form one of the neurophysiological characteristics of narcolepsy. Narcolepsy is caused by a loss of hypocretin (orexin) producing neurons, reflected in undetectable CSF levels (Peyron et al., 2000). Hypocretin neurons are exclusively located in the lateral hypothalamus and project widely throughout the central nervous system (Peyron et al., 1998), where they have an excitatory effect on several autonomic, metabolic, neuro-endocrine and arousal systems (Siegel, 2004).
In PD, there is a progressive and irreversible degeneration of dopaminergic neurons projecting from the substantia nigra to the striatum. In addition, there are degenerative changes in many other parts of the brain, including the hypothalamus (Jellinger, 2001). Lewy bodies, the pathophysiological hallmark of PD, have been found in various brain regions, again including the hypothalamus (Langston and Forno, 1978). These observations suggest that there are hypothalamic changes in PD, thus possibly involving the hypocretin system.
Several studies have been conducted to detect damage to the hypocretin system in PD. However, these only assessed CSF hypocretin levels. Moreover, results have been conflicting. One study reported decreased levels in ventricular CSF in late stage PD patients (Drouot et al., 2003), but three other groups found normal concentrations in spinal CSF (Ripley et al., 2001; Overeem et al., 2002; Baumann et al., 2005).
Here we used a combination of approaches in three brain compartments to detect whether the hypocretin system is affected in PD. First, we measured hypocretin levels in post-mortem ventricular CSF. Second, hypocretin content was determined in peptide extracts from cerebral cortex, as this has been shown to be a more sensitive technique compared to CSF measurements (Peyron et al., 2000). Third, we directly counted the total number of hypocretin neurons in the lateral hypothalamus of PD patients versus matched controls.
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Materials and methods |
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Post mortem material
Hypothalami and ventricular CSF were provided by The Netherlands Brain Bank. Frozen prefrontal cortex tissue was obtained from the Leiden PD Brain Bank. Permission was obtained for a brain autopsy and for the use of human material and clinical information for research purposes. The controls were matched for age, sex and Alzheimer Braak stage (for both groups
2) (Braak et al., 1993). Clinicopathological details are given in Tables 1 and 2. Exclusion criteria for control subjects were use of corticosteroids and primary neurological or psychiatric disease, unless stated otherwise. This was verified by a systematic neuropathological analysis (van de Nes et al., 1998; Braak et al., 2004). In all PD patients the clinical diagnosis was confirmed by a systematic neuropathological examination (Braak et al., 2004); all patients were late-stage PD. No direct mentioning of the occurrence of EDS, sleep onset REMs or cataplexy could be found in the medical records of PD patients.
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Immunocytochemistry
Hypothalami from nine PD patients and nine matched controls were used. Tissues were fixed in 10% PBS (pH 7.4) formalin at room temperature and were paraffin-embedded and serially sectioned at 6 µm from rostral to caudal. Every 100th section in the expected hypocretin cell area, from the level where the fornix touches the paraventricular nucleus to the level where the fornix reaches the corpora mammillaria, was stained using a hypocretin-1 antibody (Phoenix Pharmaceuticals, Inc., Belmont, CA, USA; catalog no. H-003-30). The specificity of this antibody was confirmed in a previous study (Fronczek et al., 2005
). Antibody binding was visualized according to the avidin-biotin complex method using diaminobenzidinenickel solution to finish the staining as described previously by Goldstone et al. (2002). If these sections did not cover the whole hypocretin area, extra sections were added at equal distances, both rostral and caudal, until no more hypocretin cells were present.
For each subject, three sections were taken from the middle of the verified hypocretin cell area and double-stained for hypocretin-1 and Lewy Bodies, using a cocktail of the aforementioned hypocretin-1 antibody and an 
-synuclein antibody (Zymed, Carlsbad, CA, USA; catalog no. 32-8100). Hypocretin-1 antibody binding was visualized using the avidin–biotin complex method described earlier, while -synuclein antibody binding was visualized using the alkaline-phosphatase blue method (Mason et al., 1978; Panayotacopoulou et al., 1994).
Immunocytochemistry quantification
An estimate of the total number of hypocretin-1 immunoreactive (IR) cells was made using an image analysis system (ImagePro version 5.1, Media Cybernetics, Silver Spring) connected to a camera (JVC KY-F55 3CCD) and plane objective microscope (Zeiss Axioskop with Plan-NEOFLUAR Zeiss objectives, Carl Zeiss GmbH, Jena, Germany). Randomly selected fields were counted in every section, covering in total 15% of a manually outlined area containing the hypocretin-1 IR cells. This was done by one person while blinded for the diagnosis. To prevent influence of cell size, only positively stained cell profiles containing a nucleolus (
2 µm) were counted. This counting procedure, which was judged to be the best for the thin (6 µm) sections used, is based on the principle that nucleoli can be considered as hard particles that will not be sectioned by a microtome knife but, instead, are pushed either in or out of the paraffin when hit by the knife (Jones, 1937; Chung et al., 2002; Bao et al., 2005). Calculation of the total number of hypocretin-1 IR neurons was performed by a conversion program based upon multiplication of the neuronal counts by sample frequency of the sections, as was described previously by Goldstone et al. (2002). Mean (±SD) number of sections needed to cover the complete hypocretin area was 9.7 ± 3.1 per subject. The coefficient of variation (SD/mean x 100%) of this method was 7.3% (calculated by counting one complete subject five times). Reliability and completeness of the cell counting was further confirmed by graphically presenting the actual numbers of neurons counted in every section from rostral to caudal to review the distribution pattern (see sample control and patient in Fig. 1).
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Hypocretin-1 measurements in cortex and CSF
One gram of frozen pre-frontal cortex was used from nine (5 male) PD patients and 16 (4 male) healthy controls. We used the most rostral part of the pre-frontal cortex, as this cortical region is densely innervated by hypocretin neurons resulting in high hypocretin concentrations (Peyron et al., 2000
). Diced tissue samples were boiled for 10 min in 10.0 ml of MilliQ water, cooled to room temperature, acidified using glacial acetic acid and HCl (final concentration: 1.0 M and 20.0 mM, respectively), homogenized and centrifuged. The supernatant was acidified again with an equal volume of 0.1% trifluoracetic acid (TFA) and vacuum dried. Samples were re-suspended in 500 µl of RIA buffer before measurements. Ventricular CSF was available from eight Parkinson's patients and eight matched controls (all these subjects were also included in the immunocytochemistry study). After collection, ventricular CSF was centrifuged at 2500 rpm for 10 min and the supernatant immediately stored at –80°C until measurements.
Hypocretin-1 levels were measured using a commercially available radioimmunoassay (RIA) (Phoenix Pharmaceuticals, Belmont, USA). All measurements were conducted in duplicate 100 µl aliquots in a single assay run. The detection limit was 50 pg/ml and intra-assay variability was <5%. We used a validated reference sample to adjust levels to previously reported values (Peyron et al., 2000).
Statistics
All data are given as median (25th–75th percentile). Group differences were analysed using the Mann–Whitney U and the chi-square test. Correlations between hypocretin-1 tissue concentration, CSF concentration, cell number, post-mortem delay, fixation time and age were evaluated using Spearman correlation. All reported P-values are two-sided, with 0.05 as the significance threshold.
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Hypocretin-1 histochemistry
The location and staining intensity of the hypocretin-1 IR cell bodies was similar in controls and PD patients (Fig. 2A and B). Hypocretin-1 IR neurons showed the same distribution pattern as described before (Fronczek et al., 2005
): cell bodies were restricted to the perifornical region of the lateral hypothalamus. On the level where the fornix crosses the paraventricular nucleus, some hypocretin neurons started to appear in the supraoptic area. In the subsequent levels, the fornix migrated to the corpora mammillaria while passing through an area with a high number of hypocretin cells. When the fornix reached the corpora mammillaria, there were still many hypocretin-1 IR cell bodies visible. In all PD patients, Lewy bodies were abundantly present in the perifornical region of the lateral hypothalamus (Fig. 2C and D), while only a few Lewy bodies could be discerned in one control patient (NBB #00-320). However, hypocretin neurons that contained a Lewy body (Fig. 3) were rare and only 1–2 double-stained neurons could be discerned in sections that contained numerous hypocretin neurons.
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In one PD patient (NBB #00-102, Table 1), the area showing hypocretin-1 IR cell bodies was not completely present in the available hypothalamic material. Therefore, the total counts of this patient and the matched control (NBB #98-127) were not included in the final analysis.
Hypocretin-1 cell number
There were no significant differences in age, sex, post-mortem delay (PMD) and fixation time between groups (all P > 0.43). Furthermore, there was no significant correlation of these variables with hypocretin-1 cell number in PD patients (all P > 0.55), controls (all P > 0.14) or the combined group (all P > 0.33).
In PD, the total number of hypocretin neurons was almost half compared with controls (PD: 20 276 (13 821–31 229); controls: 36 842 (32 546–50 938); P = 0.016, Fig. 4).
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Ventricular CSF hypocretin-1 content
Post-mortem ventricular CSF was not available for one PD patient (NBB #93-064) and one control (NBB #00-022). There were no significant differences in age, sex, PMD and fixation time between groups (all P > 0.51). Furthermore, there was no significant correlation of these variables with hypocretin-1 CSF content in PD patients (all P > 0.13), controls (all P > 0.44) or the combined group (all P > 0.44).
There was a significant reduction in hypocretin-1 ventricular CSF content in PD patients compared with controls (PD: 365.5 pg/ml (328.0–448.3); controls: 483.5 (433.5–512.3); P = 0.012, Fig. 4).
Relation between hypocretin cell number and CSF levels
As hypocretin cell counts and CSF levels were available in the same subjects, we were able to correlate these two variables directly. There was a significant correlation between cell number and ventricular CSF content in the combined group (n = 15, r = 0.62, P = 0.010, Fig. 5), but not within the separate groups (controls: P = 0.23; PD: P = 0.70).
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Hypocretin-1 concentration in prefrontal cortex
There was no significant correlation between age or sex and hypocretin-1 concentrations in PD patients (all P > 0.19), controls (all P > 0.37) or the combined group (all P > 0.34). The effect of Braak grade could not be evaluated, since all PD subjects were late-stage. Hypocretin-1 concentration in controls was 676.6 (467.5–883.9) pg/gram of wet brain tissue, comparable to previously reported values (Peyron et al., 2000
). Hypocretin levels were almost 40% lower in PD patients (389.6 pg/g (249.2–652.2); P = 0.042; Fig. 6).
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Discussion |
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In this study, we show that the hypocretin system is affected in PD by examining three brain compartments. Hypocretin-1 tissue concentrations in the prefrontal cortex were almost 40% lower in PD patients, while ventricular CSF levels were almost 25% reduced. The total number of hypocretin neurons was almost half compared with controls. Lewy bodies were abundantly present in the perifornical hypothalamus as a sign of an active disease process in that region. Hypocretin neurons that contained a Lewy body were discernable in every PD patient, but the majority of hypocretin neurons did not show this colocalization.
These results convincingly show that the hypocretin system is damaged in PD and are thus in line with and extend upon one of the previous CSF studies, in which low hypocretin-1 levels were found in ventricular CSF in patients with late-stage PD (Drouot et al., 2003). In that study, an inverse correlation between hypocretin-1 levels and disease severity was reported. We could not correlate hypocretin concentrations with disease severity, since all our subjects were late-stage PD.
Studies using spinal CSF have all found normal hypocretin-1 levels. Even PD patients who were selected because of clear sleep abnormalities did not show lowered hypocretin-1 concentrations in spinal CSF (Overeem et al., 2002; Baumann et al., 2005). The discrepancies between the measurements in spinal CSF and ventricular CSF could be due to the fact that the results of Drouot et al. were obtained in much more advanced PD patients than the studies using spinal CSF. Another explanation could be that CSF hypocretin-1 concentrations are more representative in the area around the hypothalamus, where hypocretin-1 is produced and released by fibres protruding into the lumen of the ventricles, as was shown in the rat (Chen et al., 1999). However, in one human study, hypocretin-1 was measured in six subsequent fractions of spinal CSF, using up to 12 ml, and no clear gradient between ventricular and spinal CSF levels was found (Ripley et al., 2001).
It has been shown that hypocretin levels in spinal CSF can be decreased in subjects with acute brain pathology, such as head trauma or a vascular event (Baumann et al., 2005). Two of the control subjects included in the brain tissue measurement in our study died of stroke (#93-303 and #93-194). However, the hypocretin concentrations in their prefrontal cortex were well within the control range.
There was one control subject with a relatively low number of hypocretin neurons (#93-193). As a specific HLA subtype, DQB10602 is an almost invariably necessary factor to develop the sporadic form of narcolepsy with cataplexy. Therefore, an intriguing explanation for this finding could be HLA*DQB0602 positivity of this subject. Regrettably, we could not obtain frozen brain tissue of the subjects that were included in the cell counts. It was thus not possible for us to determine HLA subtypes. However, HLA*DQB0602 positivity could be a factor involved in a lower hypocretin cell number, even in the normal population, and this should be explored in future studies.
We used the same hypocretin-1 RIA that has been used by many authors (Ripley et al., 2001; Mignot et al., 2002; Overeem et al., 2002; Baumann et al., 2005). It is a well-known fact that the inter-assay variability of this particular RIA is not optimal. However, the intra-assay variability is very low. This stresses the importance of running all samples in a single assay in these types of study, which we did. To compare values with previous reported results, we included a standard reference sample to correct for inter-assay variability (Mignot et al., 2002). In all tested samples, hypocretin-1 levels were well above the detection limit, and therefore measured reliably.
Hypocretin measurements in CSF have been widely used as a reflection of hypocretin function. However, the relation between actual hypocretin cell number and CSF concentrations was not known as of yet. In a recent rodent study, lesioning 15% of hypocretin cells did not alter CSF hypocretin-1 levels, but a loss of >70% of neurons resulted in a 50% decline in CSF levels (Gerashchenko et al., 2003). Apparently, in young adult rats it is possible to loose a substantial number of hypocretin cells without changes in CSF levels. This is the first human study that shows a possible correlation between hypocretin cell number and ventricular CSF levels.
Whether our findings fully explain the sleep symptoms in PD remains an intriguing question. Due to the retrospective character of this brain bank study, we had no clinical data on sleep disturbances. However, between one third and half of all PD patients have been reported to experience excessive daytime sleepiness and during sleep registrations a narcolepsy-like phenotype, including sleep-onset REM periods and fragmented nocturnal sleep, is found on a regular basis (Arnulf et al., 2002; Rye et al., 2004; Arnulf, 2005). This implicates that a significant proportion of the cases we studied would have suffered from sleep disturbances. In a recent rodent study, microinjection of prepro-hypocretin short interfering RNA's (siRNA) in the perifornical hypothalamus resulted in a 60% reduction of prepro-orexin mRNA and a persistent increase in the amount of REM-sleep (Chen et al., 2006). In the aforementioned rodent study by Gershchenko and colleagues, where 70% of hypocretin neurons were lesioned, an increase in REM-sleep was seen as well (Gerashchenko et al., 2003). Although these results were obtained in rodents, it is not improbable that the reduction in hypocretin neurotransmission found in our human study contributes to the sleep problems commonly seen in PD.
It is likely that the loss of hypocretin neurons is not limited to this cell group in the hypothalamus. Many cell types are affected in PD throughout the brain, but vulnerability seems to be different. To gain more insight into the specificity of the reduction in hypocretin neurons, it would be of interest to count melanin concentrating hormone (MCH) neurons in the peri-fornical region in future studies. Deficiencies in other neurotransmitters besides hypocretin have been proposed as an explanation for the sleepiness in PD. For example, Rye et al. mention of the possible involvement of midbrain dopaminergic and noradrenergic neurons that influence sleep/wake state through thalamocortical pathways (Rye et al., 2004). Both the loss of dopamine and hypocretin neurons can thus contribute to sleep disturbances in PD.
Although sleep-onset REM and REM-sleep behaviour disorder are described frequently in PD (Arnulf et al., 2000), there are no reports about cataplexy. Cataplexy is the essential feature of narcolepsy with cataplexy, which is characterized by REM sleep abnormalities and undetectable levels of hypocretin in the spinal CSF (Overeem et al., 2001). In contrast, hypocretin is usually detectable in narcolepsy without cataplexy, where REM sleep disturbances occur without cataplexy, comparable to the findings in PD (2005). It has been proposed that narcolepsy without cataplexy may be caused by a milder form of hypocretin deficiency compared with the almost complete loss of hypocretin in narcolepsy with cataplexy (Ebrahim et al., 2003). Indeed, Thannickal et al. described the highest number of surviving hypocretin neurons in the brain of a narcoleptic patient that did not suffer from cataplexy (Thannickal et al., 2000). Our findings may support this hypothesis, since we found a reduction in number of hypocretin neurons but not a complete loss in PD, possibly leading to REM sleep disturbances and sleep/wake abnormalities, but not to cataplexy.
To conclude, our data shows that the disease process in PD also affects the hypothalamic hypocretin system. It is now important to establish the correlation between hypocretin impairment and the occurrence of the various sleep disturbances. Furthermore, our findings implicate that in the future, hypocretin agonists may have a place in the treatment of PD.
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Acknowledgements |
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We thank Dr S. Nishino from Stanford University for valuable suggestions on validating our tissue measurements and F.W.C. Roelandse for his work on the radioimmuno assay. S. O. is supported by a Veni grant from the Netherlands Organization for Scientific Research (grant no. 916.56.103).
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T. C. Thannickal, Y.-Y. Lai, and J. M. Siegel Hypocretin (orexin) and melanin concentrating hormone loss and the symptoms of Parkinson's disease Brain, January 1, 2008; 131(1): e87 - e87. [Full Text] [PDF]
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