Vitamin D, Cognition and Alzheimer’s Disease: The Therapeutic Benefit is in the D-Tails

An unambiguous action on cognitive decline in aging or AD-like animals

Data obtained with vitamin D supplementation of animal models unambiguously show an improvement in memory and cognitive function along with a decrease in several markers of AD pathology. Interestingly, available experimental data points to a more pronounced effect of vitamin D when delivered to aging or AD-like animals. In a recent study, Latimer et al. showed that a high vitamin D3 diet (10,000 IU/Kg/day) lasting 5 to 6 months prevented cognitive decline in aging rats. More specifically, the authors described the superiority of a high diet compared to a low and medium one (100 or 1,000 IU/Kg, respectively) in significantly improving performance in the Morris water maze (MWM), a hippocampal-dependent learning and memory task [59]. At approximately the same time, Briones and Darwish performed subcutaneous injections of 1,25(OH)2D3, for 21 days, to both young and aged rats (6 months and 20 months, respectively) and observed an attenuation of cognitive impairment only in older animals [60, 61]. Both these studies underlie a role for vitamin D supplementation in the preservation of cognitive function in aging rats. Concerning AD, an important number of animal models now allow for understanding and compound testing in order to better apprehend and possibly treat this debilitating disease. Several groups have used these models to test the hypothesis that vitamin D has protective effects in AD. Taghizadeh et al. demonstrated that the absence of vitamin D in the diet of an adult AD rat model intensifies learning and memory deficits measured by the MWM [62]. A second study by Yu and colleagues used young AβPP transgenic (Tg) mice, under a 12 IU/g vitamin D diet and compared their behavior to those under a control (2.4 IU/g) or deficient (0 IU/g) diet from weaning to 6 months of age. They found an amelioration of performance in the MWM for the animals under the higher vitamin D diet compared to the other groups. However, the group does not present data on the effect of a vitamin D enriched diet in non-AD animals [63]. Our group has observed that a 4-month vitamin D3 supplementation in an AD mouse model (5xFAD mice) does not impact memory function at a young age (5 months) when no cognitive deficits are present (unpublished data). Conversely, vitamin D3 rescues working memory deficits at 8 months of age but this effect is only observed in Tg animals and not in aging wild-type (Wt) mice [64]. Similarly, intraperitoneal injections of 1,25(OH)2D3 for 8 weeks in 8-week-old animals restores learning and memory deficits in an AD mouse model but does not affect cognitive function in their Wt counterparts [65]. To our knowledge, the only study demonstrating an effect of vitamin D treatment preferentially in Wt animals is that of Bennett and colleagues. The authors conducted a study based on a vitamin D2-enriched button mushroom diet in Wt and AD Tg mice. Behavioral analysis was carried out after 7 months of supplementation and revealed subtle changes in cognitive function. Vitamin D2 improved cognitive performance more reliably in Wt mice rather than Tg [66]. The main difference with other studies is the use of vitamin D2 rather than D3. Animal studies therefore support the hypothesis that vitamin D action is likely to be age-dependent and show a clear effect of vitamin D supplementation in restoring cognitive deficits in an AD-like context. The mechanisms by which vitamin D can impact AD pathology are yet to be fully elucidated although a number of processes at play during the development of the pathology have been shown to be targeted by the vitamin D signaling system. Considering the mode of action of this steroid hormone, it is likely that its biological function will be multiple, ranging from direct action on amyloidogenesis to restoration of neurotransmission, modulation of inflammation, regulation of apoptosis, oxidative stress, and vascular processes (Fig. 2). Several studies have made important contributions to the understanding of vitamin D mechanism of action in an AD-like brain.

Vitamin D and amyloidogenesis

On top of the observation that vitamin D supplementation leads to improved cognitive function, all the studies in an AD-like context have also shown that vitamin D treatment, regardless of the model tested, the dosage, the molecule chosen, and the time of treatment decreases the amyloid burden, suggesting a link between vitamin D function and amyloidogenesis [60–66]. The behavioral changes reported in the study by Yu and colleagues were accompanied by a diminished Aβ load, along with an increase in astrocytic reactivity, NGF levels, and decreased TNFα in the brain of treated mice. Interestingly, the authors show a change in AβPP processing, with a variation in AβPP end products depending on the vitamin D regimen [63]. Grimm and collaborators recently studied the impact of a relatively low hypovitaminosis D on AβPP processing in mice and cell cultures. The data reveal an increase in Aβ caused by an increase in β-secretase level and activity, concomitant with a decrease in neprilysin levels [67]. Our team has recently demonstrated that a 5-month vitamin D3 enriched diet decreases the number of amyloid plaques in the frontal cortex, neocortex, and hippocampus of female 5xFAD mice. This reduction is accompanied by decreased astrocytic reactivity and increased microglial activation in the frontal cortex. Moreover, transcriptomic profiling of these brain regions reveals that vitamin D3 supplementation regulates the expression of a large number of genes associated to AD in both Wt and Tg animals [64]. Other clues indicating direct interactions between vitamin D and the amyloid pathway arise from several studies. For instance, overexpression of the VDR or treatment with vitamin D was shown to suppress AβPP transcription in neuroblastoma cells [35]. VDBP, which is increased in thecerebrospinal fluid of AD patients, was also shown to interact directly with Aβ in the brain of both AD patients and animal models. Binding of VDBP with Aβ was associated with reduced aggregation of the amyloid peptide and cellular death in vitro. Overall, the presence of VDBP rescues animal models from synaptic loss and memory deficits induced by Aβ [68]. A team provided extensive work on the crosstalk operated between vitamin D and Aβ signaling pathways. After demonstrating that Aβ treatment of hippocampal and cortical neurons induces VDR repression, they showed that VDR silencing in vitro leads to similar effects to Aβ treatment. Their studies further indicate that vitamin D treatment of cortical neurons reverses the cytotoxic effects induced by Aβ mentioned above as well as prevents upregulation of iNOS [48, 49, 69, 70]. Further in vitro studies examined the effect of vitamin D in combination with memantine on cortical axon degeneration. These two compounds act in a synergistic manner to protect against glutamate or Aβ-induced toxicity in cortical neuronal cultures [71]. Altogether, these findings strongly support a role for vitamin D signaling in reducing amyloidogenesis, one of the hallmarks of AD pathology. Moreover, the putative crosstalk between Aβ pathology and VDR signaling appears to provide neuroprotective mechanisms againstAβ-induced toxicity potentially limiting the decline in cognitive function. This underlies a specific action of vitamin D in preventing or halting cognitive impairment in AD, but what other mechanisms are at play in a “healthy” aging brain?

Synaptic transmission and calcium homeostasis

Brain aging is partly characterized at the cellular level by neuronal death, decreased neuronal excitability, impaired synaptic transmission and altered calcium homeostasis. Along with regulating amyloid processing and deposition, ultimately modulating neurotransmission, vitamin D has also been shown to act on calcium channels and neurotransmitter release in aging brains. Analysis of electrophysiological recordings after vitamin D treatment reveals a modulation of L-type calcium channel (L-VGCC) current and density in CA1 hippocampal neurons of aged rats [72] but no change in long term potentiation (LTP) in CA1 neurons of normal rats [59, 73]. However, a significant increase in neuronal excitability in aging rats [59] and synaptic potentiation in the CA3-CA1 pathway of an AD rat model has been described [74]. Vitamin D-VDRpathway disruption or activation of calcium channels in hippocampal neurons has been reported in several studies. VDR silencing in vitro leads to upregulation of L-type voltage sensitive calcium channels (LVSCC) and NGF, a process typically observed in aging neurons while vitamin D treatment of cultured neurons decreases LVSCC expression, conferring neuroprotection in an aging nervous system [47–49, 69, 72, 75]. Conversely, absence of 1,25(OH)2D3 in mice induces an upregulation of LVGCCs in the dentate gyrus of 8-week-old mice, which results in increased cell proliferation and neurogenesis [76]. It appears that depending on the time frame for treatment, vitamin D may modulate both neuroprotection and neurogenesis by finely regulating the expression of calcium channels on a timely basis. The study by Latimer and colleagues further reveals that vitamin D3 treatment directly modulates the expression of genes and proteins involved in synaptic vesicle trafficking and neurotransmitter release in aging rats [59]. A result confirmed by the transcriptomic study performed in AD female mice after 5 months of vitamin D3 supplementation [64]. Interestingly, earlier work on the effects of vitamin D deficiency during development had already demonstrated that the adult brain later displayed altered expression of a number of genes and proteins involved in calcium regulation, synaptic plasticity and neurotransmission [77, 78]. This consolidates the hypothesis that vitamin D deficiency at earlier ages has repercussions on nervous system processes such as neurotransmission, implicated in aging and AD-like pathology.

Regulation of oxidative stress, brain energy metabolism, and vascular processes

Another possible mechanism for Aβ clearance operates through passage of the blood-brain barrier (BBB). This physical, relatively impermeable, barrier is composed mainly of endothelial cells, astrocytes, and pericytes. In 2011, Ito et al. demonstrated that, 24 hours following an intraperitoneal injection of 1,25(OH)D in mice, elimination of radioactive human Aβ was increased 1.3 times and that the total level of brain Aβ was reduced. This brain-to-blood efflux likely involves internalization of Aβ peptides within brain capillaries [53]. This hypothesis is further confirmed by the work of Durk and colleagues who report increased expression and transport activity of P-glycoprotein in brain capillaries and endothelial cells after incubation with 1,25(OH)D3, resulting in reduced accumulation of human Aβ within these cells [52]. These vitamin D-related activities likely occur via activation of the nuclear receptor VDR present on the cells comprising the BBB [52, 53, 79, 80]. Moreover, action of 1,25(OH)D on brain pericytes elicits anti-inflammatory responses at the transcriptomic level, likely adding to the direct protective role of vitamin D within brain capillaries under chronic inflammation constraints such as those present in AD [80]. In line with these results, the transcriptomic response of cultured mixed neuron-glial cells or whole brain regions such as the neocortex and hippocampus to 1,25(OH)D in normal conditions or in AD mice, reveals dysregulation of genes potentially capable of limiting the progression of neurodegenerative diseases with particular emphasis on AD [64, 79]. Vitamin D can also inhibit the production of inducible nitric oxide synthase (iNOS) an enzyme necessary for synthesis of nitric oxide (NO). During aging or neurodegenerative diseases such as AD, reactive nitrogen and oxygen species produced by damaged neurons, microglia and astrocytes can increase the expression of iNOS. As a result, higher levels of NO can cause cell death through inhibition of mitochondrial and neuronal respiration, further leading to neuronal excitotoxicity [81–83]. Low vitamin D diet has been shown to enhance oxidative stress through transcription of iNOS. More specifically, low levels of vitamin D result in increased tyrosine nitration of brain proteins, alterations in glucose metabolism and mitochondrial changes in the brain of rats aged 16-17 months [84]. Conversely, 1,25(OH)2D3 injections of elderly rats increases brain energy metabolism and modulates levels of reactive oxygen species in hippocampal tissues [61]. Vitamin D treatment of cortical neurons reverses the cytotoxic effects induced by Aβ and prevents upregulation of iNOS [49, 69, 70]. In activated microglia, 1,25(OH)2D3 suppresses the production of NO and the inflammatory mediators, TNFα and interleukin-6 (IL-6), in a dose-dependent manner suggesting direct anti-inflammatory roles for vitamin D in the brain [85]. Local conversion of vitamin D to the active 1,25(OH)2D3 in the brain may be a direct neuroprotective response to CNS inflammation followed by inhibition of NF-κB related iNOS induction [86].

Modulation of inflammatory processes

Another very important component of neurodegenerative diseases such as AD is the establishment of chronic inflammatory processes. Vitamin D is now known as an effective immune modulator and as such has the potential to regulate the inflammatory state in AD pathology. Vitamin D can i) upregulate expression of several neurotrophins, ii) increase secretion of the anti-inflammatory cytokine IL-4, iii) reduce secretion of pro-inflammatory cytokines TNF-α and interleukin-1 beta (IL-1β), and iv) inhibit differentiation of dendritic cells [44–46, 87]. The VDR has been observed on human and rodent microglia, the brain macrophages responsible for immune surveillance and phagocytosis [88]. Supplementation with vitamin D3 for 5 months largely impacts inflammatory and immune gene expression profiles translating into improved functional outcomes in an AD mouse model [64]. Administration of 1,25(OH)D for 21 days induced a change in the inflammatory state and amyloid burden of aged rats (20 months) but had no significant effect in younger ones (6 months). More specifically, the increase in pro-inflammatory cytokine Il-1β and the decrease of anti-inflammatory cytokine Il-10, observed in aged compared to young rats, is reversed after subcutaneous injections of vitamin D [89]. Lee at al. showed that 12-month-old mice injected with vitamin D displayed a change in retinal macrophage morphology and number, indicative of increased phagocytic activity. This process was accompanied by a reduction in complement component C3, a chronic inflammation marker. Overall, this resulted in decreased Aβ load in the retina of these mice, improving visual function [90]. In AD, macrophage phagocytosis of Aβ is defective resulting in decreased Aβ clearance and increased Aβ deposition and inflammation in the brain. Mizwicki et al. demonstrated that vitamin D3 is capable of promoting recovery of Aβ phagocytosis in cultured human macrophages from AD patients possibly by rebalancing the inflammatory state through the modulated transcription of TLRs, IL-1, IL1R1, and chemokines [89, 91].

On top of the mechanisms discussed above concerning the neuroprotective activities of vitamin D during aging or in an AD-like context (Fig. 2), a certain number of hypotheses need to be tested. These are based on the common pathways altered in AD and known to be targets of 1,25(OH)D in the brain or elsewhere. These include action on miRNAs, TLRs, angiogenesis, glycation, and oxidative stress, all these processes being altered in aging brains and particularly in AD [92]. The road to unraveling the mechanisms of action of vitamin D in an aging brain is a long one, as it is now clear that this misnamed vitamin is a potent neurosteroid with pleiotropic actions that are tissue-, time-, individual-, dose-, and pathological context-specific. Due to its capacity to interact with both nuclear and membrane receptors, the downstream effects of vitamin D supplementation are multiple and crosstalks between various signaling pathways render its study rather complex. It is therefore not surprising that clinical observations lead to contradictory results as we are only starting to discover the full potential of this steroid hormone in the nervous system.

A potential gender effect

Interestingly, studies performed with female, male, or mixed populations show contrasting results. All studies based on women cohorts report a clear association between hypovitaminosis D and cognitive impairment, while the unique study in men reports no association and reports from both male and female populations are controversial (Table 2). In one study, Annweiler et al. found that low dietary intake of vitamin D (defined as <35 μ/week, or 400 IU/year) in a large cohort of women of mean age 80.5, was significantly associated with global cognitive performance as measured by the Pfeiffer Short Portable Mental State Questionnaire (SPMSQ) [96]. In a second cross-sectional study, they found that vitamin D deficiency, defined as <10 ng/mL (<25 nmol/L), was associated with lower SPMSQ scores in elderly women aged 75 years and older [95]. Conversely, a study among healthy, older Europeans aged 55 to 87 years, demonstrated that seric 25(OH)D concentrations of >32.1 ng/mL (>80 nmol/L), considered optimal, were associated with significantly fewer errors in tests of spatial working memory, particularly in women [111]. One 4-year longitudinal study of 6,257 elderly women reports that lower 25(OH)D blood concentrations at baseline were associated with increased incidence of global cognitive decline measured by the Mini-Mental State Examination (MMSE) test in elderly women [97]. Importantly, this was not observed in a similar study performed in men exclusively [99]. Association of baseline serum 25(OH)D deficiency with the onset of dementia or AD was also observed in two cohorts of women over 75 years old [93, 94]. However, a post-hoc analysis of the largest clinical trial on this topic, the Women’s Health Initiative Calcium and Vitamin D trial, which included over 4,000 women aged 65 and older, found no effect of a 7.8 year supplementation with 100 mg calcium carbonate and 400 IU of vitamin D3 on the incidence of dementia, MCI, or other cognitive measures [98]. Of important note, supplementation with vitamin D during this clinical trial was associated with calcium supplementation. This renders interpretation of results more difficult as it is known that high calcium levels are associated with increased risk of cognitive decline in humans and animals [128, 129]. The dual supplementation could therefore be counterproductive [130]. No interventional study has been performed in men exclusively and 8 pre-post or randomized control trials have been performed in a mixed population (Table 2). Among them, cognitive improvements were found after 8 weeks of supplementation for global cognitive performance [121] and after 4 weeks [120] to 16 months of supplementation [115] for executive functioning. However, these studies were pilot nonrandomized pre-post studies performed in small cohorts of patients. Despite this limitation, the latter findings were consistent with the result of one placebo-controlled randomized clinical trial that reported after 6 months an improvement of information processing speed in the supplementation arm compared to the placebo arm [119]. Nevertheless, a meta-analysis concluded that, although cognition favorably evolves in patients receiving vitamin D supplementation (“before and after” approach), the between-group comparison of the cognitive scores at the end of the follow-up (“comparative” approach) do not show better cognitive scores in the supplementation arm compared to the comparative arm [131]. Thus, although, there appears to be a clear link between vitamin D status reflected either by 25(OH)D serum levels or nutritional intake of vitamin D and cognitive function in women, these studies do not allow for comparison of both genders and might not be representative of a general population. Moreover, some studies have shown such associations in mixed population while others report no link (Table 2), forcing us to consider other possible confounders.

A possible age threshold

The latest study on the topic of vitamin D and cognitive decline was performed in individuals of different ages obtained from The Tromso study. The report highlights a positive association between serum 25(OH)D and cognitive function in individuals aged 65 years and over [103]. The interesting result from this study is that levels of 25(OH)D appear to be predictive of cognitive outcome only in older individuals. Memory and cognitive performance tests were performed at different time points, over a period of 7–13 years, and only the latest time points were significantly correlated with baseline vitamin D levels [103]. We therefore decided to compare studies that allowed for a clear distinction between ages, i.e., studies performed on individuals strictly under 65 or over 65 years of age. As reported in Table 2, this criterion allowed us to discriminate 6 studies conducted in individuals less than 65 years of age and 14 studies comprising of individuals aged over 65 exclusively. The results are quite pronounced as all studies, except one, investigating individuals under 65 years of age report no association between 25(OH)D serum levels and cognitive performance, and conversely nearly all studies in older individuals report a link between vitamin D status and cognitive outcome (Table 2). Cross-sectional studies in adolescents (age 12–16.9 years), based on the data from the NHANES III cohort, found no association of 25(OH)D seric levels and performance in Intelligence Scale and Wide-range Achievement Test [110, 113]. Similar reports, carried out in young adults (20–59 years), showed once again no significant association between 25(OH)D concentrations and measures of cognitive function [110, 113]. Another study examined the link between cognition and vitamin D in earlier stages of life. It demonstrated no correlation between seric levels of both 25(OH)D2 and 25(OH)D3 at mean age 9.8 years, with academic performance outcomes at ages 13-14 and 15-16. This study included a cohort of 3,171 individuals [112]. The only study, to our knowledge, describing an association between cognitive performance and seric 25(OH)D levels in non-aged, non-demented individuals was carried out in a lifespan approach by Maddock and colleagues in 2014 [109]. This study included 6,496 mid-life individuals from a British cohort whose information on childhood cognitive ability, educational attainment, and vitamin D-related behaviors was collected at several points from birth to age 50. At the age of 45, seric vitamin D concentration was measured and, at age 50, verbal memory, verbal fluency, and speed of processing were tested. The results showed a non-linear U-shaped association between 25(OH)D concentrations at age 45 years and immediate word recall at age 50 years, i.e., people with both lower and higher 25(OH)D concentrations performed worse on the cognitive task [109], but this result was likely due to the recent supplementation of those with prior hypovitaminosis D. The controversial link between low vitamin D status and cognitive performance in non-aged individuals has led to only one randomized control trial during which 5,000 IU of vitamin D3 was given daily to young adults for 6 weeks. Thecontrol trial concluded that vitamin D supplementation had no effect on cognitive and emotional measures, such as working memory and depression although enhanced working memory performance over time was observed in both the supplemented and comparative group [118]. The short period of supplementation and the relatively high serum 25(OH)D concentrations at baseline, possibly above the cerebral needs for vitamin D (if any), could have masked the cognitive effect of vitamin D supplementation. However, the null findings concerning vitamin D status and cognitive outcome in young individuals indicates that the time-frame considered for associating 25(OH)D to cognitive performance is of prime concern and that longer follow-up times from longitudinal studies might help decipher a link between hypovitaminosis D at younger ages and cognitive decline in elderly populations. Among the studies investigating the link between vitamin D status and cognitive outcome in elderly populations, two large population-based reports indicate no association between 25(OH)D levels and cognitive performance [110, 114]. Interestingly, this contrasts largely with a study by Llewellyn and colleagues, which performed analysis on the same NHANES III cohort than the above-mentioned studies [106]. The two major differences are i) Llewellyn et al. focused on individuals aged strictly 65 years and older whereas McGrath et al. and Tolppannen et al. included a larger age group, from 60 to 90 years old, and ii) the cognitive measures considered for assessment of performance differed. It appears that the effects of hypovitaminosis D on brain function and cognition do not emerge until later in life. Most importantly, it is possible that vitamin D status does not impact cognitive measures in healthy individuals but is rather linked to the onset of cognitive decline during aging. Finally, the type of cognitive test used for analysis might impact results as vitamin D status could potentially be associated to domain-specific functions.

AD versus non-pathological aging

A review of 37 studies from Balion et al. allowed the authors to compare MMSE scores with levels of 25(OH)D. They found that higher average MMSE scores were associated with 25(OH)D concentrations >50 nmol/L and ultimately that lower vitamin D levels increased the risk for AD [124]. Another meta-analysis concluded that an individual without AD shows a 40% increased chance of having higher vitamin D levels than matched controls [125]. Despite a growing amount of studies linking lower vitamin D to cognitive decline in an associative manner, data specific to hypovitaminosis D and incidence of AD is scarce. However, in every study, a link between vitamin D status and dementia has been observed while studies investigating vitamin D deficiency in the case of non-pathological aging is more controversial (Table 2). Two 7-year follow-ups, the first in 40 women of mean age 78.4 years [93] and the second in 498 women aged 75 years and older [94] aimed at studying the association between levels of vitamin D and the risk of AD. The first report, performed on a relatively small, restricted cohort showed that baseline serum 25(OH)D deficiency associates with the onset of non-AD dementias but not with AD [93]. However, the second study, based on vitamin D dietary intake and including a larger population found that baseline dietary intake of vitamin D was inversely associated with the onset of AD but not with non AD-dementia [94]. These apparent contradictory results could be due to the difference in cohort size for both studies or to the measure used for assessment of vitamin D status. In line with the second observation, two recent longitudinal studies have put forward an association between low levels of seric 25(OH)D and the risk of AD [100, 105]. One study followed 1,658 men and women of mean age 73.6 years for 5-6 years [105], while the second was performed on 10,168 individuals over a period of 30 years [100] and both report an increased risk for AD with decreased levels of 25(OH)D. The latter study, due to the long period of time considered and the size of the cohort, provides a solid basis to the hypothesis that vitamin D deficiency at younger ages is associated to increased risk of cognitive decline, dementia, or AD later on.

To our knowledge, only three prospective studies have investigated the effects of vitamin D supplementation in AD patients. A first study by Stein and colleagues consisted, for phase 1, in the administration of 3,000 IU per day of vitamin D2, for 8 weeks, in 13 older patients. The group found that the increased seric levels of 25(OH)D were accompanied by a significant improvement in the Alzheimer’s Disease Assessment Scale (ADAS-cog) score. However, the second phase of Stein et al.’s study, consisting of a randomized control study in which participants received either physiological doses (1,000 IU per day) or supra-physiological doses (6,000 IU per day) of vitamin D2 for 8 weeks, showed no difference between groups in terms of the ADAS-cog score or the MMSE [121]. However, the very small number of participants in this study along with the shortduration of treatment and possibly the use of vitamin D2 impedes general conclusions to be drawn. The second pre-post study proposed the combination of memantine, a modulator of NMDA receptors, and vitamin D as an effective treatment for improving cognition in AD patients. This pilot study demonstrated the efficacy of combining both treatments for 6 months measured by an increase of MMSE scores compared to the administration of vitamin D or memantine alone [116]. Other clinical trials, based on vitamin D supplementation and assessing cognition decline, as a primary or secondary outcome, are currently performed in aging patients (VITAL-cog, DO HEALTH) or with pathologies such as amnesia(D-cog), Alzheimer’s disease (AD-IDEA), Parkinson’s disease, and type II diabetes (THINK-D). Details can be found on the website clinicaltrials.gov.

A positive effect on executive functions and not episodic memory

As described above, there is robust evidence that vitamin D insufficiency is associated with cognitive impairment as a whole among older adults [122]. However, it is not fully elucidated which domain-specific cognitive functions are disturbed in the case of vitamin D insufficiency and explain the association of serum 25(OH)D concentration with the composite cognitive scores. For instance, as mentioned previously, the contradictory studies by McGrathet al., Tolppanen et al., and Llewellyn et al. based on the cohort from the NHANES III survey might be representative of the different cognitive measures incorporated for analysis. In the work by Llewellyn et al., the authors found an association of 25(OH)D with cognitive impairment calculated as a composite score, i.e., a global measure of cognitive performance assessing both memory, learning, orientation, and attention [106]. In contrast, in the initial observations by McGrath et al. and Tolppanen et al., the authors used only a “memory and learning score”, and found inconclusive results [110, 114], making it unlikely that episodic memory is the domain-specific cognitive function primarily altered in the case of vitamin D insufficiency. A meta-analysis was consistent with this assumption, and showed that the association between vitamin D insufficiency and episodic memory remains uncertain due to modest and clinically non-relevant results [131]. In contrast, this meta-analysis provided compelling evidence that lower serum 25(OH)D concentrations predict executive dysfunction [131]. This hypothesis is corroborated by the findings of Buell and colleagues investigating elders (aged >65) receiving home health services. The authors found that cognitive performance, particularly executive function and not memory, was associated with circulating vitamin D of 50 to 75 nmol/L [102]. An interventional study by Annweiler and colleagues also demonstrated that supplementing elderly individuals with vitamin D3 during 16 months improved executive function [115]. Executive functions refer to a heterogeneous set of high-level processes that control and regulate other abilities and behaviors [132]. To override this heterogeneity, a more recent study aimed to determine which executive subfunction was disturbed in the case of vitamin D insufficiency and found a decrease in mental shifting, but neither in cognitive inhibition nor in information updating, among community-dwelling older adults with vitamin D insufficiency [133]. Mental shifting is the ability to adjust thinking or attention in response to changing goals and/or environmental stimuli in order to behave appropriately [132]. This may thus help to better understand the involvement of vitamin D in the cognitive decline and in the course of dementia. However, to make firm conclusions about the domain-specific cognitive functions affected by vitamin D insufficiency, further studies using a universal battery of cognitive measures will help decipher the subtle specificities by which vitamin D levels are associated to cognitive outcome.

The genetic background

One major difference between animal models and humans is the controlled genetic and environment that offers laboratory work. The possibility that individual differences in vitamin D metabolism exist is not to be undermined as polymorphisms in VDR have been reported as potential risk factors for AD [30, 32, 33]. VDR is not the only metabolite required for proper vitamin D action, as the several enzymes involved in metabolism are key to drive functional vitamin D outcome. If polymorphisms or simply decreased activity of these enzymes is present in certain individuals, vitamin D treatment might not be effective. Moreover, it still remains unknown how vitamin D metabolites enter the nervous system, whether it be by passive diffusion or the use of energy-dependent transporters such as megalin. A recent report suggests that sex-specific gene variations in both the VDR and megalin receptor associate with age-related cognitive decline in a relatively large cohort of US citizens [36]. Thus, defects in transport, metabolism, or action of vitamin D metabolites in an individual-dependent manner need to be taken into considerations when implementing adaptive clinical trials possibly based on Bayesian models rather than classical randomized control trials.

Assessing a possible gender effect

Inter-individual differences, especially inter-gender dissimilarities, should also be taken into account. Sex differences in observed associations of megalin SNPs may be related to interaction of megalin with both estrogen and vitamin D and competitive binding of both steroid hormones to this shared endocytic receptor. Our team has recently observed in a mouse model of AD that vitamin D3 treatment impacts male and female mouse differently, at the behavioral level, according to the time frame for supplementation (unpublished data). Moreover, we have shown that vitamin D likely interacts with the estrogen receptor, Esr1, to regulate molecular pathways relevant to AD pathogenesis [64]. A number of studies suggest a strong link between the drop in estrogen levels occurring during menopause and the development of AD [135–137]. Animal studies could help clarify this matter; however, future clinical trials might consider stratifying results in a gender-specific manner.

Assessing vitamin D status and responsiveness for nervous system related functions

Whether 25(OH)D serum levels appropriately reflect an individual’s capacity to use vitamin D metabolites, particularly in relation to nervous system functions, is of great importance. For instance, conditions such as liver disease and nephrotic syndrome result in reduced VDBP and albumin levels that lead to a reduction in total 25(OH)D and 1,25(OH)D levels without necessarily affecting the free concentrations of these metabolites [138]. As an example, ethnic differences in 25(OH)D levels have been reported. African Americans present lower VDBP levels, hence lower total 25(OH)D levels but without evidence of vitamin D deficiency, potentially due to the action of stable levels of free fractions of 25(OH)D or 1,25(OH)D [18]. If the free fractions are the ones to enter the nervous system, total 25(OH)D serum levels might not reflect the levels of vitamin Dmetabolites entering the brain. Moreover, megalin has been shown to be present at the BBB, in choroid plexus, suggesting a possible role for endocytic uptake of vitamin D or 25(OH)D linked to VDBP into nervous system tissues. The presence of CYP27B1 in the brain allows for local synthesis of 1,25(OH)2D3 in this tissue creating an autocrine/paracrine system for metabolism of active vitamin D compounds. In such systems, metabolites are produced, act and are degraded locally without affecting serum 25(OH)D levels. Maybe alternative approaches to complement the measurement of circulating 25(OH)D are now required to evaluate more precisely an individual’s response and needs for vitamin D in the context of brain function. One approach might be to consider CSF levels of vitamin D metabolites rather than serum levels as a reflector of vitamin D status in the nervous system. 25(OH)D, 24,25(OH)D and 1,25(OH)D have been shown to be present in human CSF, although in lower concentrations than in sera [43] and are reduced in AD patients [139]. A complementary approach to measuring either serum or CSF levels of vitamin D metabolites could be to measure genomic effects of vitamin D on its target genes for profiling an individual’s vitamin D status and capacity to respond to supplementation [140].

An appropriate supplementation according to baseline levels

Since recommendations regarding the use of cholecalciferol supplementation to reach optimal levels of 25(OH)D in humans are under debate, possible conversion of doses from animals to humans might be considered in the context of neurological diseases. Indeed, animal studies show improvement of cognitive function in animal models of aging or AD with specific doses of vitamin D. However, the method for translating doses from animal models to human clinical trials should be carefully considered. Deriving interspecies equivalents for therapeutic interventions has usually been done using Body Surface Area scaling; however, it appears that this might not be as adequate as advanced allometric and physiologically based pharmacokinetic modeling for animal-to-human dosage conversion [141]. This is particularly relevant in the case of vitamin D supplementation, as assessing the “pharmacologically active dose” required in regards to brain function is of great need.

Daily intake of 800 IU is the current recommendation of the IOM for people aged 70 years and older based on estimates that this dose will produce or exceed a threshold of 25(OH)D of 20 ng/mL in 97.5% of people [14]. A randomized double blind study of elderly nursing home residents showed that after 16 weeks of 800 IU/day of vitamin D3, 25(OH)D levels were 33 ng/mL. There was little difference between an 800 IU/day and 2,000 IU/day supplementation except for one participant who did not reach 20 ng/mL under the 800 IU/day regimen indicating that some individuals need higher doses. With 2,000 IU/day, no one reached 50 ng/ml or higher. This study also underlined the complex relationship between baseline 25(OH)D status and responses to supplemental vitamin D. When using doses of 800 IU, 2,000 IU, or 4,000 IU/day, increases in 25(OH)D were consistently higher in those with the lowest baseline [142].

It is noticeable that most trials, showing no cognitive difference in the supplementation arms compared to the comparative arms, have actually included participants with relatively high serum 25(OH)D concentrations at baseline, possibly above the cerebral needs for vitamin D (if any). Moreover, the comparative arm of several trials continued to receive vitamin D supplements, either as part of the study [121] or as part of routine care [120]. So it will be important in the future to examine the cognitive effect of vitamin D supplementation in populations exhibiting vitamin D insufficiency at baseline. The definition of vitamin D insufficiency is consensually not based on what is called a “population-based reference value” (i.e., normality is the reference range involving 95% of the population) but rather on “health-based reference values” (i.e., normality is the 25(OH)D concentration range that prevents adverse health effects) [143]. Defining a “cognition-based reference value” is still the matter of debate.

The nature of the delivered molecule also needs to be considered. The difference in binding affinity of vitamin D2 metabolites to VDBP being weaker than that of vitamin D3, this would lead to a shorter half-life and an increased rate of clearance from circulation. The apparent inefficiency of 25(OH)D2 binding to VDBP may be advantageous in the setting of extra-renal metabolism of 25(OH)D where decreased binding to VDBP may increase the uptake of 25(OH)D2 by target cells. Future studies in animal models may aid in evaluating the relative efficacy of each metabolite in reference to cognitive outcomes. In parallel, human studies should incorporate separate measures for 25(OH)D2 and 25(OH)D3 in serum and CSF along with total versus free concentrations of each molecule after supplementation with either metabolite.

It will also be important to determine whether vitamin D insufficiency triggers AD (i.e., results in a pathological dysfunction of the brain leading to AD) or whether it removes protection of the CNS against AD. Just enough supplements to correct vitamin D insufficiency should be sufficient if vitamin D is a neuroprotectant, although high to very high dose of supplements should be used if vitamin D controls the CNS, with the aim of boosting mental faculties. Also, if vitamin D insufficiency explains partially the pathological process of AD, it may also enhance the efficacy of standard anti-dementia treatments or account for the resistance to these treatments. In line with this, a 6-month controlled trial showed that the combination of memantine+vitamin D was superior to memantine alone and vitamin D alone in preventing cognitive decline among AD patients [116]. Even preliminary, this result certainly engages clinicians to replenish vitamin D before starting anti-dementia treatments or at least to use vitamin D as an adjunct to these treatments.