We plan to recruit 100 cognitively normal older individuals with SCD from memory clinics, neurologists and general practitioners in private practice, and the general population. Participants will be allocated to one of the two study arms using blockwise randomization stratified by age and sex with a 1:1 allocation ratio. The primary outcome is the change in memory performance between baseline and post-intervention visits (12 months after baseline). Secondary outcomes include the change in memory performance from baseline to follow-up assessment (18 months after baseline), as well as changes in neurocognitive, behavioral, and physiological parameters (including blood and neuroimaging biomarkers), assessed at baseline and post-intervention.

Background

Longer life expectancy has led to a growth of the older

population, with individuals aged 60 and older expected

to reach about 20% of the population in high-income

countries [1]. This demographic change is associated

with increased rates of age-related diseases, such as dementia due to Alzheimer’s disease (AD). The most

prevalent form of late-onset dementia is expected to

triple by the year 2050 [2], which will pose a high social

and economic impact on patients, caregivers, and society

in general. Importantly, around one third of these cases

worldwide are attributed to potentially modifiable risk

factors [3]. The development of effective strategies that

help to prevent age- and disease-related worsening of

brain structure and function may thus provide significant benefits for society and health-care systems.

Subjective cognitive decline: a target group for early intervention

Subtle neuropathological alterations related to AD are suggested to start decades before the onset of clinical symptoms [4, 5]. One early sign of pathological brain aging is the manifestation of subjective cognitive decline (SCD). In cognitively unimpaired older individuals, the presence of SCD is associated with a higher risk for objective cognitive decline and clinical progression to symptomatic disease stages [6–9]. Moreover, individuals with SCD harbor increased β-amyloid (Aβ) deposition [10], gray matter volume reduction [11–13], and neural dysfunction [14] in brain regions typically affected in AD. Thus, SCD has been conceptualized to occur at a late preclinical stage of AD [15], where aberrant brain changes are present [16] in the absence of objective cognitive impairment. This at-risk group is recognized as an eligible target population for early intervention strategies [15, 17], aiming to protect against neuropathological alterations, to restore functional and structural brain health, and to maintain cognitive abilities as long as possible.

Spermidine and its implication in healthy aging

protect against age- and disease-related brain changes and, thereby, preserve cognitive functioning [18–22]. Caloric restriction, among others, appears to be effective to improve memory performance in the elderly [23] and induce favorable neural changes in the hippocampal network [24]. Novel candidate substances proposed to mimic such beneficial effects in aging organisms are natural polyamines, in particular, spermidine and spermine [25, 26].

 Organic compounds play an important role in the maintenance of basic cellular functions like cell growth, survival, and proliferation [27]. Beyond these “microscopic” actions, there is an indication that polyamines influence “macroscopic” systems underlying learning and memory as well as age-related changes of this cognitive function. In rodent models, polyamine levels in the hippocampus are shown to be associated with memory retrieval and formation [28] and change with age in certain brain areas, including medial-temporal memory structures [29]. Lastly, the reduction of spermidine in the brain of aged flies is paralleled by memory decline [30]. Given these observational findings, it has been hypothesized that external supply of polyamines may protect against age-related memory loss. Indeed, a first study in aging fruit flies showed that spermidine-rich diet restored endogenous spermidine levels and thereby rescued memory performance [30]. This beneficial effect of spermidine intake appears to be mediated by several protective pathways [31]. For example, spermidine may act through autophagy to regulate synaptic transmission/plasticity [32] and clear cellular “waste” including pathogenic protein aggregates [30]. Nutritional spermidine is also associated with a number of cardio-protective [33] and anti-inflammatory [34] actions, which may help to preserve higher-order brain functions. Overall, these findings in aging model organisms have suggested a promising role of spermidine in the promotion of brain and cognitive health.

Spermidine supplementation in older adults with SCD: initial evidence

Spermidine supplementation is thus proposed to open a new avenue in the protection and restoration of memory abilities in higher age.

This expected benefit is of particular need in older individuals at risk for the development of dementia. It is, however, unknown whether the memory-promoting effect of spermidine is detectable in humans and to what extent this effect may be attributed to the influence of spermidine on biomarkers of healthy aging. An initial study showed that polyamine-enriched diet over 2 months increased blood spermidine levels in healthy middle-aged men [35]. Our group has conducted a first 3-month phase IIa trial with nutritional spermidine in 30 older adults with SCD (ClinicalTrials.gov identifier NCT02755246). Trial outcomes demonstrated high compliance, tolerance, and safety profiles as well as preliminary efficacy of the administered spermidine-rich plant extract [36, 37]. Specifically, we found a moderate enhancement of memory performance, measured using the mnemonic similarity task (MST) [38], in the spermidine-treated group compared with placebo intervention. This computer-based task is a sensitive measure of subtle cognitive changes induced by targeted interventions in the memory system [39]. In the pilot trial, we did not detect intervention effects on standard neuropsychological tests of memory or executive functions [37]. At this point, longer-term intervention studies with sufficient sample size are required to validate the therapeutic potential of nutritional spermidine against memory loss in older individuals and delineate possible neurophysiological mechanisms of action.

1. Age, 60–90 years 2. Presence of SCD in accordance with research criteria, recommended by the international SCD-I working group for studies on SCD [15]: the expression of subjective cognitive complaints for at least 6 months and associated concerns (worries), affirmation to consult and/or previous consultation of a doctor due to these symptoms, normal cognitive performance, and no restrictions on activities of daily living 3. Ability to provide written informed consent 4. Health insurance coverage to clarify possible incidental findings We will exclude potential participants in case one or more of the following criteria are present: 1. Dementia, according to the Diagnostic and Statistical Manual of Mental Disorders, 4th Edition (DSM-IV) [41] 2. Mild cognitive impairment (MCI), according to clinical diagnostic criteria [42] 3. Severe or untreated medical disorders (advanced cardiac or respiratory disease, severe liver, kidney or metabolic diseases, untreated thyroid dysfunctions or untreated diabetes mellitus), psychiatric disorders (untreated depression, psychosis) or neurological disorders (epilepsy, clinically manifest stroke) 4. Malignancies currently or as indicated by medical history (exception: basalioma) 5. Drug abuse or alcohol dependency 6. Current polyamine substitution and/or participation in respective intervention studies 7. Known intolerance or allergies to wheat germs, gluten or histamine 8. Contraindications to imaging techniques: claustrophobia, metallic implants (e.g., intracranial metal clips), electronic devices (e.g., cardiac pacemakers), or permanent tattoos 

 In combination with a normal diet, the intake of the planned daily dose of 750 mg extract (even in the case of multiple overdoses) is below the calculated no observed adverse effects level (NOAEL) for humans of 29 mg/kg body weight (bw)/day for cadaverine and putrescine [44]. This is also the case for spermidine and spermine, where the NOAEL is 13.5 mg/kg bw/day or 3.1 mg/kg bw/day [44]. The NOAEL for L-ornithine in humans is above 500 mg/ kg bw/day [45], and thus remains unattainable in the planned extract administration. The safety of the spermidine-rich extract for the use in humans was evaluated prior to the SmartAge trial by a chemical analysis (unpublished data) and a translational study on safety and tolerability [36]. In the intervention group, the spermidine supplement will be administered daily in the form of six capsules, each containing 125 mg extract, resulting in a daily dose of 750 mg extract or 0.9 mg spermidine, 0.5 mg spermine, 0.2 mg putrescine, < 0.004 mg of cadaverine, and 0.12 mg of L-ornithine. This amount of daily polyamine intake accounts for an increase of approximately 10– 20% of the average spermidine intake in industrial countries [46]. The dosage is within the amount of polyamines administered in an earlier intervention study [35], using approximately 10 mg/day of dietary spermidine in humans. The amount would also be obtainable by well-targeted diets (e.g., 200 g of cooked soybeans [47]).

 During the subsequent recognition phase, images are repeated (repetition items), randomly inter-mixed with novel images (foil items), and images that are perceptually similar to those pictures seen during the encoding phase (lure items). Participants will be asked to indicate for each trial, whether the image was “old”, “new” or “similar”. From the proportion of responses provided during recognition, a response bias-corrected mnemonic discrimination index will be calculated, similar to previous reports [37, 38, 48]. Secondary endpoints: Secondary outcomes include the change in memory performance (operationalized by mnemonic discrimination performance) between baseline visit (V1) and follow-up assessment (V3, 18 months after baseline). Additional secondary endpoints (see Table 1) are changes in the following outcome measures: (a) Neuropsychological parameters on verbal and visualspatial memory, attention, executive functions, and sensorimotor speed, assessed at V1, V2, and V3 (b) Behavioral parameters of lifestyle behaviors, psycho-affective characterization and perceived quality of life, assessed at V1, V2, and V3, as well as (c) Physiological parameters including autophagy signaling (measured in muscle biopsies), peripheral vascular parameters (measured in blood), and parameters of brain structure, perfusion, and function (measured using cerebral magnetic resonance imaging [MRI]) to be assessed at V1 and V2. 

In case of a positive answer to question 1, question 2 will be asked: “Since when do you have the feeling that your cognitive performance has gotten worse?” (German: “Seit wann hat sich ihre geistige Leistungsfähigkeit verschlechtert?”). In case the answer to question 2 is at least 6 months, question 3 will be asked: “Are you concerned about this cognitive worsening?”(German: “Bereitet Ihnen diese Verschlechterung Sorgen?”, possible answers: yes/no). In case of a positive answer to question 3, question 4 will be asked: “Would you seek or have you sought medical help due to this cognitive worsening?” (German: “Würden Sie diesbezüglich einen Arzt aufsuchen bzw. haben Sie dies bereits getan?”, possible answers: yes/no). Affirmative responses to diagnostic questions 1, 3, 4, and a duration of SCD for at least 6 months are mandatory for study inclusion. Potential participants that meet inclusion criteria as evaluated during pre-screening will be invited for the on-site screening visit to the Charité – Universitätsmedizin Berlin. Each individual will receive a standardized screening assessment, including neuropsychological tests and questionnaires, to certify the absence of objective cognitive impairment and current psychiatric disorder (e.g., depression). Following measures will be used: (a) Mini-Mental State Examination (MMSE) [52] score ≥ 26, (b) performance above − 1.5 standard deviation (SD) of age-adjusted norms in the Logical Memory II subscale total delayed recall (Story A and B) [53], and the Trail Making Test (TMT) A [54], (c) no deficits on selected items of the Instrumental Activities of Daily Living Scale (IADL) [55], and (d) a Geriatric Depression Scale (GDS) [56] score ≤ 10.