An Exploratory Clinical Study on Autophagy and Multi-level Molecular Profiling During Spermidine Supplementation

An Exploratory Clinical Study on Autophagy and Multi-level Molecular Profiling During Spermidine Supplementation

Autophagy Characterization and Multi-level Molecular Profiling of Spermidine Supplementation: a Clinical Study

Recently, the autophagy inducing caloric restriction mimic spermidine became available. Autophagy is essential for energy and cellular homeostasis through protein catabolism and dysregulation results in compromised proteostasis, stress-coping behavior, and in excessive secretion of signaling molecules and inflammatory cytokines. Antidepressants for example effect autophagy dependent pathways to exert their beneficial effects. It can therefore be hypothesized that autophagy induction through spermidine supplementation also shows beneficial clinical effect, particularly in the field of psychiatric conditions. It would be safe, low cost and easy to implement in relay to psychotropic medication in the treatment of psychiatric patients.Therefore, the aim of the project is to analyze clinical effects of spermidine supplementation in correlation to the underlying, multi-level molecular profiling.

Recently, the autophagy inducing caloric restriction mimic spermidine-rich wheat germ extract (spermidineLIFE ®, from here onwards: spermidine) was approved by the European Food Safety Authority (EFSA) and became commercially available for use. Spermidine is safe, well tolerated and as caloric restriction mimetic an easy alternative if fasting is too challenging, e.g. for psychiatric patients. Research on spermidine in animal models is limited, but a study with mice overexpressing spermidine/spermine N1-acetyltransferase (SSAT) an enzyme of spermidine catabolism, suggests that these mice may be more prone to stress. An association between spermidine supplementation and improved memory performance as well as reduced mortality has been shown in an epidemiological correlation. So far laboratory and molecular assessments are missing. It is therefore of great interest to perform broad multidisciplinary studies of behavioral changes with plasma spermidine levels, the quantification of autophagic flux, and protein acetylation levels as well as molecular signaling in a longitudinal fashion to establish an epidemiological triangulation between spermidine, autophagy and (mental) health. This study is a monocentric, randomized, double-blind, placebo-controlled trial in which a 3-week spermidine-based nutritional supplementation (6 mg/d; target intervention) will be compared to 3-weeks of placebo administration (control intervention). Recruitment of 40 healthy individuals and 40 individuals with diagnosed depressive disorder is planned, who will be allocated to one of the two study arms (n = 20 per intervention). At different time points (baseline, intervention day 7, 14 and 21, as well as one week follow up after the last intervention day) serval psychometrical questionnaires will be gathered and blood will be collected. Sleep quality will be additionally assessed by actigraphy. At selected days blood will be collected. Following, autophagy activity will be assessed by Western Blot analysis, and mass spectrometry based proteomics, phosphoproteomics, metabolomics and lipidomics will be performed. Bioinformatic analysis, statistical evaluation, quality control, and in silico pathway analyses will then specifically identify factors and cascades of relevance. Furthermore it is of great interest, whether epigenetic changes take place during spermidine supplementation and whether these are stable throughout the follow up analysis. The aim of the project is to analyze clinical effects of spermidine supplementation in correlation to the underlying, multi-level molecular profiling. Longitudinal multi-omic profiling including proteome, metabolome, lipidome, and epigenetic changes will reveal time-series analysis of thousands of molecular changes and an orchestrated composition of autophagy depended signaling. The resulting findings will advance the role of autophagy in the development of psychiatric disorders, investigate alternative treatment options on a molecular level, and finally contribute to a better clinical outcome.

2 groups of different inclusion criteria (healthy or depressive disorder) each group undergoing one out of two conditions (spermidine vs. placebo Supplementation) (randomized)

double-blind study (participants nor experimenters know who receives placebo or spermidine supplementation)

Dietary Supplement: Polyamine 21 days of spermidine supplementation (3 sachets/day = 6mg spermidine/day)

Change in protein levels of autophagy biomarkers (LC3II & p62) of isolated PBMCs (peripheral blood mononuclear cells) by Western Blotting.

Change in protein levels and protein phosphorylation by untargeted mass spectrometry-based proteomics and phosphoproteomics of isolated PBMCs (peripheral blood mononuclear cells).

Absolute number (per Liter) and relative amounts of neutrophils, lymphocytes, monocytes, eosinophils, basophils, and immature granulocytes (in %)

Comparison of Saliva Cortisol Levels in nmol per Liter (nmol/L) after Dexamethason intake between spermidine and Placebo group

Sleep diary to assess overall sleep quality assessed as ratio of the total time spent asleep (in hours) to the total amount of time spent in bed (in hours) per night

Pittsburgh Sleep Quality Index (PSQI): self-report questionnaire to assess sleep quality over a 1-month time interval consisting of 19 individual items.

Warwick-Edinburgh Mental Well-being Scale (WEMWBS): self-reported 14-item scale to assess Overall mental well-being

Inclusion Criteria: Present written declaration of consent Healty or diagnosed with depression BMI between 17 and 40 Exclusion Criteria: Insufficient linguistic communication Pregnancy or lactation Gluten, histamine or wheat seedling intolerance Drug abuse or alcohol dependency Current spermidine substitution

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