Hypocretin (orexin) cell loss in Parkinson's disease

Abstract

It has recently been reported that Parkinson's disease (PD) is preceded and accompanied by daytime sleep attacks, nocturnal insomnia, REM sleep behaviour disorder, hallucinations and depression, symptoms which are frequently as troublesome as the motor symptoms of PD. All these symptoms are present in narcolepsy, which is linked to a selective loss of hypocretin (Hcrt) neurons. In this study, the Hcrt system was examined to determine if Hcrt cells are damaged in PD. The hypothalamus of 11 PD (mean age 79 +/- 4) and 5 normal (mean age 77 +/- 3) brains was examined. Sections were immunostained for Hcrt-1, melanin concentrating hormone (MCH) and alpha synuclein and glial fibrillary acidic protein (GFAP). The substantia nigra of 10 PD brains and 7 normal brains were used for a study of neuromelanin pigmented cell loss. The severity of PD was assessed using the Hoehn and Yahr scale and the level of neuropathology was assessed using the Braak staging criteria. Cell number, distribution and size were determined with stereologic techniques on a one in eight series. We found an increasing loss of hypocretin cells with disease progression. Similarly, there was an increased loss of MCH cells with disease severity. Hcrt and MCH cells were lost throughout the anterior to posterior extent of their hypothalamic distributions. The percentage loss of Hcrt cells was minimal in stage I (23%) and was maximal in stage V (62%). Similarly, the percentage loss of MCH cells was lowest in stage I (12%) and was highest in stage V (74%). There was a significant increase (P = 0.0006, t = 4.25, df = 15) in the size of neuromelanin containing cells in PD patients, but no difference in the size of surviving Hcrt (P = 0.18, t = 1.39, df = 14) and MCH (P = 0.28, t = 1.39, df = 14) cells relative to controls. In summary, we found that PD is characterized by a massive loss of Hcrt neurons. Thus, the loss of Hcrt cells may be a cause of the narcolepsy-like symptoms of PD and may be ameliorated by treatments aimed at reversing the Hcrt deficit. We also saw a substantial loss of hypothalamic MCH neurons. The losses of Hcrt and MCH neurons are significantly correlated with the clinical stage of PD, not disease duration, whereas the loss of neuromelanin cells is significantly correlated only with disease duration. The significant correlations that we found between the loss of Hcrt and MCH neurons and the clinical stage of PD, in contrast to the lack of a relationship of similar strength between loss of neuromelanin containing cells and the clinical symptoms of PD, suggests a previously unappreciated relationship between hypothalamic dysfunction and the time course of the overall clinical picture of PD.

Fig. 1

Distribution of Hcrt cells in normal and across PD stages. The clinical stages of PD are based on Hoehn and Yahr criteria. The cell distribution and count from a section of anterior, middle and posterior part of the hypothalamus were mapped from a normal, stage III and stage V of PD brains. The cell counts are listed for each section. The number of Hcrt cells is decreased with severity of the disease. 3v—third ventricle, Fx—fornix, Mmb—mammillary body, Opt—optic tract. Scale bars—50 μm.

Fig. 2

Hcrt and MCH pathology in different stages of PD. (A) The total number of Hcrt and MCH cells in normal and PD-I, PD-II, PD-III, PD-IV and PD-V. The values are compared to cell numbers in the normal brains. (B) The size of the Hcrt, MCH and neuromelanin pigmented cells estimated by nucleator method. Hcrt and MCH cells in PD did not differ in size from those in normal brains. Neuromelanin pigmented cells showed hypertrophy (27%) compared with normal cells. (C) Hcrt and MCH cells were mapped in individual sections from anterior to posterior hypothalamus with 1200 μm section interval. One brain from a normal and one from each stage (Hoehn and Yahr, I–V) of PD were used for Neurolucida mapping. There was a generalized loss of Hcrt and MCH cells with severity of the disease. *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001, Student’s t-test.

Fig. 3

Distribution of MCH cells in normal and Parkinson stages. Cell counts are listed in each section. The number of MCH cell was decreased with severity of the disease. The abbreviations are same as in Fig. 1. Scale bars—50 μm.

Fig. 4

Distribution of alpha synuclein in the hypothalamus in different stages of PD. (A) Neurolucida mapping of alpha synuclein in PD stages with single immunostaining. (B) Mapping of Hcrt and alpha synuclein in double-labelled section. (C) Mapping of MCH and alpha synuclein in double-labelled section. Alpha synuclein was not colocalized with Hcrt and MCH cells (D and E), but it was colocalized with neuromelanin pigmented cells in substantia nigra (F). Arrows: red—alpha synuclein, green—Hcrt cell, black—MCH cells and blue—neuromelanin pigmented cell. Scale bars—50 μm.

Fig. 5

Gliosis and neuromelanin pigmented cell loss in PD. (A) The percentage loss of neuromelanin pigmented cell loss in the substantia nigra was correlated with duration of the disease. (B) The number of glial fibrillary acidic protein-labelled astrocytes (GFAP) in the thalamus and posterior hypothalamus. (C) GFAP in the hypothalamus of normal (a) and PD (b). GFAP density in the substantia nigra of normal (c) and PD (d) brain. The number of GFAP-labelled astrocytes were increased with severity of the disease. Scale bars—50 μm.