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other·ophthalmology, dry eye, nutrition, circadian biology, endocrinology·PMC10154200

Intracrine activity involving NAD-dependent circadian steroidogenic activity governs age-associated meibomian gland dysfunction

Nature Aging · 25 authors, 12 centres

AI SUMMARY

FIDELITY 100%

POPULATIONAged mice (21-24 months old) and young/aged human volunteers (29-35 vs 61-70 years); Hsd3b6 knockout and Bmal1 knockout mice

INTERVENTIONTopical eye-drop administration of nicotinamide mononucleotide (NMN) or nicotinamide riboside (NR) (5% solution, 4-6 times/day for 14-90 days)

COMPARISONVehicle-treated contralateral eye; untreated wild-type mice; Hsd3b6-/- mice; daytime vs night-time administration

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TL;DR

This study demonstrates that age-related decline in meibomian gland 3β-HSD activity, driven by reduced NAD+ availability, causes meibomian gland dysfunction (MGD) and evaporative dry eye. Topical eye-drop administration of NAD+ precursors (NMN or NR) restores local steroidogenesis, reverses gland atrophy, and improves dry eye signs in aged mice. These findings identify a mechanism-based therapeutic strategy for age-associated MGD.

Full summary

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**Background:** Meibomian gland dysfunction (MGD) is the most common cause of dry eye disease, with incidence increasing with age. The meibomian gland is known to express 3β-hydroxysteroid dehydrogenase (3β-HSD), an enzyme essential for steroid hormone biosynthesis, but the specific isozyme and its role in MGD were unknown. The concept of intracrinology—local hormone synthesis and action within the same tissue—had not been explored in the meibomian gland. This study aimed to identify the meibomian-specific 3β-HSD isozyme, investigate its role in age-associated MGD, and test whether reactivation of local steroidogenesis via NAD+ precursor eye drops could ameliorate MGD.

**Methods:** The study used human eyelid specimens (postmortem donors), young (29-35 years, n=6) and aged (61-70 years, n=6) healthy volunteers for noncontact meibography, and multiple mouse models: wild-type C57BL/6J, Hsd3b6-/- (global knockout), Hsd3b6ΔMG (conditional meibomian gland-specific knockout using K14-Cre), Bmal1-/- (circadian clock-deficient), and Per2Luc/+ reporter mice. 3β-HSD activity was measured by conversion of 3H-DHEA to 3H-androstenedione. Testosterone levels were quantified by LC-MS/MS. Meibomian gland morphology was assessed by whole-mount Sudan IV staining. Cell proliferation was measured by BrdU incorporation. NAD+ levels were measured by LC-MS/MS in laser-microdissected meibomian glands. RNA-seq was performed on FACS-purified Itgav+CD45- acinar cells. Evaporative dry eye (EDE) was scored by corneal fluorescein staining after 1 hour of imposed desiccation. Eye-drop treatments (5% NMN or NR in PBS) were administered 4-6 times/day for 14 or 90 days.

**Key Results:** Human meibomian glands expressed HSD3B1 (type I 3β-HSD), not the gonadal/adrenal HSD3B2. Mouse meibomian glands expressed Hsd3b6 (the murine counterpart). 3β-HSD activity declined ~50% in aged (24-month) vs young (6-month) mice (P=0.0056). Hsd3b6-/- mice had no detectable meibomian 3β-HSD activity, tissue testosterone reduced to ~50% of wild-type (and to ~10% after castration), and severe meibomian gland atrophy by 2 months of age (gland area significantly reduced, P<0.0001). Hsd3b6-/- mice also exhibited EDE (significantly increased fluorescein score after desiccation, P=0.0025) despite compensatory increased tear volume. RNA-seq revealed upregulation of inflammation-related genes and downregulation of morphogenic genes including Wnt2 and Adamts17 in Hsd3b6-/- mice. BrdU+ proliferating cells were significantly reduced in Hsd3b6-/- mice (P<0.05). 3β-HSD activity showed circadian rhythmicity (peak at ZT03) in wild-type but was constantly low in Bmal1-/- mice. Hsd3b6 mRNA did not cycle or decline with age, but NAD+ levels in the meibomian gland were significantly lower at ZT15 vs ZT03 (11.7±1.0 vs 15.4±1.3 pmol/mm3, P=0.0477), reduced in Bmal1-/- mice (10.8±2.0 vs 18.4±2.1 pmol/mm3, P=0.0261), and declined with age (8.2±1.3 pmol/mm3 at 24 months vs 12.3±2.2 at 20 months, P=0.0011 vs 8 months). Topical NMN or NR eye drops for 14 days significantly increased meibomian 3β-HSD activity and BrdU+ cells in aged mice (right vs left eye). Daytime administration was more effective than night-time. After 90 days, NMN and NR significantly enlarged meibomian glands (~1.14-fold, P<0.05) and reduced EDE scores. NMN had no effect in Hsd3b6-/- mice. Topical NMN increased meibomian NAD+ within 5 minutes, peaking at 1 hour, with minimal systemic effects.

**Clinical Implications:** This study identifies a novel mechanism for age-associated MGD: decline in local intracrine steroidogenesis due to reduced NAD+ availability, not loss of enzyme expression. The finding that topical NAD+ precursors (NMN or NR) can restore 3β-HSD activity, promote acinar cell proliferation, reverse gland atrophy, and improve dry eye signs in aged mice provides a potential mechanism-based therapy for MGD. The topical, non-steroidal approach may avoid systemic side effects of hormone replacement. The circadian dependence of efficacy (daytime better than night-time in nocturnal mice) suggests timing of administration may be important. Human studies are needed to confirm translational potential.

PICO

PPOPULATION

Aged mice (21-24 months old) and young/aged human volunteers (29-35 vs 61-70 years); Hsd3b6 knockout and Bmal1 knockout mice

IINTERVENTION

Topical eye-drop administration of nicotinamide mononucleotide (NMN) or nicotinamide riboside (NR) (5% solution, 4-6 times/day for 14-90 days)

OOUTCOME

Meibomian gland 3β-HSD activity, gland area (whole-mount morphometry), BrdU+ proliferating acinar cells, corneal fluorescein staining (EDE score), tear volume, meibomian NAD+ levels, Wnt2 and Adamts17 mRNA expression

STUDY TYPE

other

SPECIALTY

ophthalmology

SUMMARISED BY

AI pipeline

FIDELITY CHECK

100% · A

ORIGINAL PAPER

PMC10154200

Intracrine activity involving NAD-dependent circadian steroidogenic activity governs age-associated meibomian gland dysfunction

Nature Aging

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ABSTRACT

Abstract Canonically, hormones are produced in the endocrine organs and delivered to target tissues. However, for steroids, the concept of tissue intracrinology, whereby hormones are produced in the tissues where they exert their effect without release into circulation, has been proposed, but its role in physiology/disease remains unclear. The meibomian glands in the eyelids produce oil to prevent tear evaporation, which reduces with aging. Here, we demonstrate that (re)activation of local intracrine activity through nicotinamide adenine dinucleotide (NAD + )-dependent circadian 3β-hydroxyl-steroid dehydrogenase (3β-HSD) activity ameliorates age-associated meibomian gland dysfunction and accompanying evaporative dry eye disease. Genetic ablation of 3β-HSD nullified local steroidogenesis and led to atrophy of the meibomian gland. Conversely, reactivation of 3β-HSD activity by boosting its coenzyme NAD + availability improved glandular cell proliferation and alleviated the dry eye disease phenotype. Both women and men express 3β-HSD in the meibomian gland. Enhancing local steroidogenesis may help combat age-associated meibomian gland dysfunction. Subject terms: Ageing, Endocrine system and metabolic diseases

CITATION EXPORT

Lena Sasaki, Yuki Hamada, Daisuke Yarimizu et al.. Intracrine activity involving NAD-dependent circadian steroidogenic activity governs age-associated meibomian gland dysfunction. Nature Aging. (2022). https://doi.org/10.1038/s43587-021-00167-8

AUTHORS

25 · 12 CENTRES

Lena Sasaki, Yuki Hamada, Daisuke Yarimizu, Tomo Suzuki, Hiroki Nakamura, Aya Shimada, Khanh Tien Nguyen Pham, Xinyan Shao, Koki Yamamura, Tsutomu Inatomi, Hironobu Morinaga, Emi K Nishimura, Fujimi Kudo, Ichiro Manabe, Shogo Haraguchi, Yuki Sugiura, Makoto Suematsu, Shigeru Kinoshita, Mamiko Machida, Takeshi Nakajima, Hiroshi Kiyonari, Hitoshi Okamura, Yoshiaki Yamaguchi, Takahito Miyake, Masao Doi

1Department of Systems Biology, Graduate School of Pharmaceutical Sciences, Kyoto University, Kyoto, Japan
2Department of Ophthalmology, Kyoto Prefectural University of Medicine, Kyoto, Japan
3Department of Ophthalmology, Kyoto City Hospital, Kyoto, Japan
4Department of Ophthalmology, National Center for Geriatrics and Gerontology, Aichi, Japan
5Dpartment of Stem Cell Biology, Medical Research Institute, Tokyo Medical and Dental University, Tokyo, Japan
6Department of Disease Biology and Molecular Medicine, Graduate School of Medicine, Chiba University, Chiba, Japan
7Department of Biochemistry, Showa University School of Medicine, Tokyo, Japan
8Department of Biochemistry, Keio University School of Medicine, Tokyo, Japan
9Department of Frontier Medical Science and Technology for Ophthalmology, Kyoto Prefectural University of Medicine, Kyoto, Japan
10Senju Laboratory of Ocular Sciences, Senju Pharmaceutical Co., Kobe, Japan
11Laboratory for Animal Resources and Genetic Engineering, RIKEN Center for Biosystems Dynamics Research, Kobe, Japan
12Division of Physiology and Neurobiology, Graduate School of Medicine, Kyoto University, Kyoto, Japan