ESRS 2024 Short-Term Research Grant Report by Jingru Zhou

The primary aim of this ESRS-funded research visit was to investigate the role of corticotropin-releasing hormone (CRH) in the pathophysiology of narcolepsy type 1 (NT1), using well-established mouse models. This project was designed to build upon a recent human post-mortem study that reported a significant reduction of CRH-immunoreactive neurons in the paraventricular nucleus (PVN) of NT1 patients. Our goal was to determine whether this striking finding could be recapitulated in animal models of the disease and to explore potential mechanisms underlying CRH alterations in NT1.
The visit also served a key training purpose, allowing me to gain in-depth laboratory experience in quantitative molecular techniques and to participate directly in collaborative translational research with the Sleep Team at CRNL. This collaboration has been instrumental to the progression of my PhD project, which focuses on neuropeptidergic dysfunction in central hypersomnia disorders.
In addition to the scientific and technical goals, the visit also fostered valuable intercultural exchange and professional development. I was able to engage with researchers from different disciplinary backgrounds, attend group seminars, and learn how different European institutions structure their translational neuroscience research. This broader exposure has deepened my perspective on how animal models can be used to inform human pathophysiology, particularly in rare neurological disorders such as narcolepsy.

2. Experimental Design and Personal Contributions
Over the course of the project, we studied a total of 54 adult mice, comprising three distinct NT1 models and their corresponding controls:
Orexin-KO mice (n = 12) and wild-type littermates (n = 9)
Orexin-DTR mice (n = 8) and saline-injected controls (n = 7)
Orexin-HA mice with CD8+ T-cell transfer (n = 9) and non-injected controls (n = 9)
These models allowed us to compare the impact of different orexin loss mechanisms: genetic deletion, toxin-induced neuronal ablation, and immune-mediated destruction with neuroinflammation.
During the visit, I was directly responsible for multiple core aspects of the experimental workflow:

a. Tissue Dissection: I dissected the hypothalamus and bilateral amygdala from frozen brain sections using anatomical landmarks from the Paxinos & Franklin atlas. This required meticulous technique to isolate the regions of interest without contamination.
b. RNA Extraction and Quality Control: Using the RNeasy Plus Mini Kit, I extracted total RNA from each sample and verified purity and integrity using spectrophotometric methods (A260/A280 > 1.8).
c. cDNA Synthesis and qPCR: I conducted reverse transcription and performed absolute quantitative PCR using Rotor-Gene Q cyclers. I implemented the use of synthetic mRNA spike-in controls to ensure technical robustness and reproducibility across all qPCR assays.
d. Primer Selection and Data Normalisation: I helped optimise primers for CRH, orexin, melanin-concentrating hormone (MCH), and histidine decarboxylase (HDC), and performed copy number quantification through standard curve analysis.
e.Statistical Analysis: I carried out group comparisons using Mann–Whitney U tests and explored correlations between neuropeptide expression levels using Spearman correlation and linear regression in R Studio and GraphPad Prism.

Beyond the lab, I actively engaged in scientific discussions, presented preliminary data during internal lab meetings, and received valuable feedback on experimental interpretation and manuscript preparation.
I also had the opportunity to shadow senior researchers and observe complementary methodologies, including brain immunostaining and cryosectioning protocols used in the lab. Discussions with the team helped me refine my scientific writing and data presentation, and I received guidance on how to best structure our findings for publication. This mentorship has had a lasting impact on my confidence and independence as a young scientist.

3. Key Findings (Preliminary and Unpublished)
Although the project is still ongoing and the data have not yet been formally published, preliminary findings across all three NT1 mouse models consistently revealed that CRH mRNA levels remain unchanged in both the hypothalamus and amygdala, despite robust loss of orexin neurons and, in one model, evident hypothalamic neuroinflammation.
This absence of change in CRH gene expression contrasts with recent post-mortem human data showing an 88% reduction in CRH-immunoreactive neurons in the PVN. Similarly, no significant alterations were detected in MCH or HDC expression, supporting the specificity of orexin neuron loss in current NT1 models and raising important questions about species-specific or time-dependent mechanisms.
A full manuscript is currently under review in a peer-reviewed journal.

4. Impact and Future Directions
This visit has greatly strengthened my technical expertise in molecular neurobiology and enhanced my understanding of translational models in sleep research. The experience of working within a leading European sleep research group allowed me to establish long-term collaborations, gain new methodological competencies, and contribute meaningfully to an international publication.

The data collected during this visit form a critical component of my PhD thesis, and future directions will include immunohistochemical validation and potential analyses in post-mortem human amygdala tissue to further investigate CRH neuron vulnerability in NT1.
The visit has also laid the foundation for future joint research activities between our institutions. We are currently planning follow-up experiments that will examine additional neuropeptidergic pathways and their relevance to sleep-wake regulation. I also intend to present these findings at upcoming international conferences, where I hope to share insights gained from this collaboration and gather feedback from other researchers in the field.

5. Acknowledgement
I sincerely thank the European Sleep Research Society for supporting this visit through the ESRS Travel Grant. This opportunity has advanced both my technical development and collaborative research capacity, and I am grateful for the chance to contribute to the broader understanding of narcolepsy pathophysiology.

Best Regards,

Jingru Zhou
PhD student at Leiden University Medical Centre
Leiden, The Netherlands