Fifty million people worldwide have dementia [1
]. In individuals aged more than 70 years, dementia is the second largest cause of death [3
]. Alzheimer’s disease (AD) is the most commonly observed form of dementia that composes nearly 70% of cases [4
]. The most influential genetic risk factor known for AD is the inheritance of the ε4 allele of apolipoprotein E (APOE4) [5
]. The sign of AD typically starts with memory decline. On the other hand, Mild Cognitive Impairment (MCI) is diagnosed when there is evidence of memory impairment, but the patient retains general cognitive function and activities of daily living and is not demented [9
]. MCI is sometimes a transition between aging and dementia [10
]. It is reported that more than 10% of MCI individuals develop dementia in a year [11
]. Therefore, beneficial intervention on MCI should be sought in order to decrease the number of patients who suffer from AD in the future. Current evidence does not support the use of pharmacologic treatments for cognitive protection in persons with MCI [12
]. Thus, intervention to MCI aiming to reduce risk from AD dementia onset could target lifestyle improvements including augmentation of intellectual or physical activities, and nutritional adjustment [13
In randomized controlled trials from our laboratory and from others, anserine and carnosine supplementation (ACS) in seniors helped to preserve cognitive function, primarily verbal episodic memory, and brain perfusion [16
]. Carnosine is an anti-inflammatory dipeptide, consisting of beta-alanine and histidine, and is present in the millimolar range in skeletal muscle, and in the hundred-micromolar range in the vertebrate brain [20
]. Anserine is a natural derivative of carnosine and is present at high levels in the breast skeletal muscle in chicken. Anserine and carnosine are called as imidazole dipeptides, which have physiological functions in common such as anti-oxidation, pH buffering, metallic chelation, but anserine is not cleaved by human carnosinase, which is abundant in human serum [21
]. Beneficial effects of ACS, which was observed in the cognition of healthy elderly subjects, led us to investigate the effects of ACS in MCI individuals with hope for finding any benefit for dementia prevention. Moreover, the results of our previous trial showed that ACS preserved brain perfusion in individuals carrying APOE4 [19
]. However, efforts for almost 25 years to elucidate the mechanism of how this allele increases the risk for AD onset have not yielded enough results.
In the present study, we conducted a randomized, double-blind, placebo-controlled trial of oral ACS for 12 weeks with subjects of MCI patients referring to a memory clinic in the Tokyo Metro Area. APOE4 genotyping of participants was performed, then the results were utilized in the subanalysis conducted separately for the subjects with or without APOE4. MCI patients were enrolled in the trial and randomized to enter the active group (Active group) or the placebo group (Placebo group). They consumed 750 mg of anserine and 250 mg of carnosine per day or a placebo. The dose of anserine and carnosine for supplementation was equivalent to that in previous studies. [16
]. At the baseline and follow-up, psychometric examinations including MMSE and Clinical Dementia Rating (CDR) were performed. Participants also underwent electroencephalograms (EEG) recording, and the data were used to estimate cortical neural activity impairment that is sensitive to the early stage of AD. [23
]. This study aimed to evaluate the effects of ACS on cognitive decline in MCI individuals, and as secondary, to consider the capability of ACS to alleviate cognitive deterioration by APOE4.
In this communication, we conducted a randomized, double-blind, placebo-controlled trial to investigate the effect of anserine and carnosine supplementation on MCI subjects. In the previous studies from our laboratory and others [16
], it was shown that ACS helps preserve cognitive function in older adults with normal cognition and preserves brain blood flow especially in individuals carrying APOE4 [16
]. Therefore, in the present study with subjects of MCI patients, we went on to an investigation of the cognitive function of APOE4 positive subjects, especially. Though statistically significant effects by ACS on the changes of cognitive tests during the trial were not obviously observed in the total subjects, the effect for the preservation of the cognitive function in APOE4 (+) MCI subjects may exist. It was shown in the difference of MMSE score changes from the baseline and to the follow-up between the two groups (from 27.1 to 26.9 in the Active and from 26.7 to 25.1 in the Placebo). In the CDR test, we detected a significant difference in the change of the gloCDR score between the two groups (p
= 0.0261; −0.25 in the Active group and 0 in the Placebo) in APOE4 (+) subjects. EEG data, as well as Aβ1-42
in the plasma data, supported the notion that ACS may help to maintain the cognitive function, especially in APOE4 (+) subjects.
In the total subjects, we detected a difference only in the change of the gloCDR score in total subjects between the two groups (Table 2
). The transition from MCI to the normal cognitive status is called reversion, which is not rare without any specific medical intervention. The rate reported being about 24% in a meta-analysis [33
]. CDR is a dementia rating instrument which requires information from a person closely observing the subject, and gloCDR has been often utilized to find reverters in patients, as a supplementary to clinical interview [26
]. The score of gloCDR that corresponds to reversion is the change from 0.5 to 0. In the active group, the gloCDR score improved from 0.5 to 0 in half of the eight APOE4 (+) subjects, but in the placebo group only two of the 17 APOE4 (-) subjects did. The risk for dementia onset in reverters drops to about 40% of that in MCI individuals without reversion [34
]. These observations might imply a possibility that ACS has a promotive effect for reversion, and, as a secondary effect, for the risk reduction of the AD onset, in APOE4 (+) MCI subjects.
The overall tendency in the number of diagnoses by clinical specialists in the study might be consistent with the potential of ACS for prevention from dementia onset. Of the 25 subjects, four in Placebo group developed dementia due to AD in the DSM-5, while all subjects in the Active group remained MCI at follow-up.
In this communication, the number of subjects suggested to become demented was different for each test since we reported independent test scores, though the efficacy of ACS might be shown in MMSE as well as in CDR. MMSE is a screening tool with good specificity for mild AD [36
]. The fact that seven subjects in the Placebo group and only two subjects in the Active group went below the cut-point on MMSE might also reflect the efficacy of ACS for prevention from dementia. In CDR, a direct effect against developing dementia in MCI was not revealed clearly, compared to the assumable potential through promoting reversion from MCI to cognitive normal. One subject reached the gloCDR score of 1.0 or above (one ApoE4/E3 subject in the Placebo group with gloCDR 1.0 at follow-up, shown in Figure 3
), and none in the Active group did, which was not significantly different. WMS-1 and WMS-2, and ADAS did not show cognitive amelioration by ACS in our study.
In our previous publication, we demonstrated cognitive amelioration by ACS on the delayed recall test WMS-2 with cognitive normal healthy elderly subjects [16
]. The average WMS-2 score in the previous study was over 10 out of total 25, while that in the present study was around 5. ADAS is pointed out to be suited for use in mild-to-moderate AD dementia but lacks appropriate sensitivity of some items for MCI or mild dementia due to AD [34
]. It may be supposed that the mismatching of test instruments and the subject population explain the failure to detect the effects of ACS in some tests in the present study. We experienced challenges in the selection of cognitive tests for assessment of cognitive function in the MCI stage and early dementia.
ACS might protect against the development of AD dementia and promote amelioration of cognitive decline in APOE4 (+) MCI individuals. We want to mention how ACS could help APOE4 (+) subjects. The EEG sNAT analysis showed a tendency toward improvement in the APOE4 (+) subgroup (Figure 5
), in this EEG-based evaluation, locations with neuronal hypoactivity and undersynchrony in AD subjects are statistically correlated with the locations of regional cerebral blood flow (rCBF) reduction measured by SPECT [23
]. In the previous study with cognitively healthy older adults [19
], brain blood flow in the prefrontal area was preferentially preserved in APOE4 (+) individuals. In the present study, plasma amyloid beta 42 of APOE4 (+) MCI subjects showed a trend for decrease by ACS. It is reported that the plasma amyloid beta 42 is just within a moderate decrease during the preclinical or prodromal AD stages and in mild-to-moderate AD [38
], and increased levels of amyloid beta is associated with vascular disease [38
]. The observed trend in amyloid beta 42 concentration in plasma might reflect the beneficial effect of ACS on blood vessels when ACS ameliorated cognitive decline in APOE4 (+) MCI subjects.
In a transgenic AD mouse model, we have demonstrated that anserine supplementation, as well as carnosine supplementation, prevented memory deficits [40
]; it also reversed the blood-vessel abnormality and elevated RAGE expression in these mice [40
]. We have reported that anserine supplementation in AD model mice significantly prevented damage to brain microvascular pericytes, triggered by the accumulation of amyloid-beta peptides, toward the normal level [41
]. In a series of elegantly coordinated preclinical and clinical studies, Zlokovic et al. demonstrated the role of brain blood-vessel pericytes in enabling brain microcapillaries to maintain the flow of brain blood and suggested that pericyte degeneration contributes to the etiology of AD carrying APOE4 [42
]. In addition to the effect on pericytes, we have also reported that ACS suppressed the gene expression level of a blood chemokine, CCL24, in cognitively normal older adults [46
]. It has been reported that APOE4 also has a considerable influence on peripheral blood macrophage, as well as brain microglia when these cells were treated with lipopolysaccharide (LPS). In these experiments, following LPS stimulation, apoE4-macrophage showed higher inflammatory reaction than apoE3-macrophage [47
]. Therefore, it can be assumed that once anserine/carnosine supplements are ingested, these peptides directly and/or indirectly resulted in the protection of the degenerative cellular changes of brain microvascular pericytes which can be triggered by the accumulation of amyloid-beta peptides [8
]; however, further studies are required to determine the precise mechanisms by which ACS affects brain function in APOE4 (+) individuals.
Our study has some limitations. First of all, the sample size adopted in the present study may not be sufficient to detect the score differences, if they were, in all the cognitive tests performed. Second, ACS was significantly affected in APOE4 (+) subjects, but not in subjects without the allele significantly. It can be possible that blood-vessel alterations occur more rapidly in ApoE4 (+) than in ApoE4 (-) individuals, as is implied in a previous study [19
], but further study is necessary to verify the effect of anserine and carnosine in MCI subjects. We are underway to prepare and start the next RCT study to evaluate the effect of ACS. Third, changes in EEG did not differ between the two groups statistically. It may be due to the small sample size. Further study with appropriate sample size and follow-up duration would help us understand the significance of these results for various populations. Authors should discuss the results and how they can be interpreted in perspective of previous studies and the working hypotheses. The findings and their implications should be discussed in the broadest context possible. Future research directions may also be highlighted.