High fat diet induces specific pathological changes in hypothalamic orexin neurons in mice.

Loss of orexin neurons in the hypothalamus is a prominent feature of narcolepsy and several other neurological conditions. We have recently demonstrated that sleep deprivation stimulates local nitric oxide (NO) production by neuronal NO synthase in the lateral hypothalamus, which leads to selective degeneration of orexin neurons accompanied by formation of orexin-immunoreactive aggregates. Here we analyzed whether lifestyle-related conditions other than sleep deprivation could trigger similar pathological changes in orexin neurons. Four-week-old male C57BL/6 mice were fed with high fat diet (HFD) for 8 weeks. Immunohistochemical analysis revealed that the number of orexin-immunopositive neurons was significantly decreased by HFD intake, whereas the number of melanin-concentrating hormone-immunopositive neurons was unchanged. In addition, HFD promoted formation of intracellular orexin-immunoreactive aggregates in a subset of orexin neurons. We also confirmed that expression of inducible NO synthase (iNOS) in the hypothalamus was upregulated in response to HFD intake. Notably, loss of orexin-immunopositive neurons and formation of orexin-immunoreactive aggregates were not observed in iNOS knockout mice fed with HFD. These results indicate that inappropriate dietary conditions could trigger specific neuropathological events in orexin neurons in an iNOS-dependent manner.

Nitric oxide mediates selective degeneration of hypothalamic orexin neurons through dysfunction of protein disulfide isomerase.

We addressed the role of nitric oxide (NO) in orexin neuron degeneration that has been observed under various pathological conditions. Administration of an NO donor NOC18 (50 nmol) into the third ventricle of mice resulted in a significant decrease of orexin-immunoreactive (-IR) neurons, in contrast to a modest change in melanin-concentrating hormone-IR neurons. In addition, NOC18 promoted formation of orexin-A-IR aggregates within orexin neurons. An endoplasmic reticulum stress inducer tunicamycin replicated the effect of NOC18 with regard to decrease of orexin-IR neurons and formation of aggregates. We also found that NOC18 caused an increase in S-nitrosation of protein disulfide isomerase (PDI) and a decrease in PDI activity in hypothalamic tissues. Moreover, PDI inhibitors, such as cystamine and securinine, caused a selective decrease of orexin neurons and promoted formation of orexin-A-IR aggregates. Aggregate formation in orexin-IR neurons was also induced by local injection of small interfering RNA targeting PDI. Interestingly, sleep deprivation for 7 consecutive days induced a selective decrease of orexin-IR neurons, which was preceded by aggregate formation in orexin-IR neurons and an increase in S-nitrosated PDI in the hypothalamus. Activity of neuronal NO synthase (nNOS)-positive neurons in the lateral hypothalamus as assessed by c-Fos expression was elevated in response to sleep deprivation. Finally, sleep deprivation-induced decrease of orexin-IR neurons, formation of aggregates, and S-nitrosation of PDI were not observed in nNOS knock-out mice. These results indicate that nNOS-derived NO may mediate specific pathological events in orexin neurons, including neuropeptide misfolding via S-nitrosation and inactivation of PDI.