**2. Ascorbic Acid Biochemistry and Transport**

AA concentration is higher in the brain than almost all other organs, and in fact may only be equaled in concentration in the adrenal glands. Scurvy, the classical clinical syndrome of AA deficiency, is rare, although not unheard of, in developed populations, but sub-clinical deficiency is still widespread, particularly in at-risk populations such as the elderly, hospitalized and those with poor access to good nutrition. One or more symptoms of scurvy were observed in 18 out of 145 (12%) of elderly patients on admission to hospital, and all but one of this group were classed as being AA deficient as confirmed from plasma levels [10]. Interestingly, 7 of the 23 (30%) controls (no signs of clinical scurvy) also had AA deficiency according to plasma levels, and all of the patients included in the study were classed as having depleted serum AA. Animal studies have shown that brain preferentially retains AA at the expense of other organs with cerebellar, hippocampal and cortical areas appearing to retain AA the most effectively under conditions of depletion which may have a bearing on diseases that target specific areas of the brain [11]. However, the brain is unable to hold maximal or optimal or even sufficient levels in the face of chronic insufficiency, which may have important effects on pathological aging and neurodegenerative diseases. Some populations, such as the elderly and smokers likely require even higher intakes of AA [12]. Roles for AA are widespread and include its vital role as an antioxidant as well as action as a cofactor for a number of dioxygenase enzymes involved in the synthesis of carnitine, collagen, and neurotransmitters including dopamine, norepinephrine and serotonin (reviewed [13]). AA can inhibit LDL oxidation and increase resistance of LDL to oxidation (for review, see [4,14]). AA also plays a role in the function of endothelial nitric oxide synthase (eNOS) by recycling the eNOS cofactor, tetrahydrobiopterin, which is relevant for arterial elasticity and blood pressure regulation [4,15]. Each of these roles plus atherogenic factors may contribute to the preventative role of AA in the development of cognitive impairment.

Evidence of altered glutamate transport (e.g., changes in EAAT2 and EAAT3 transporters) is seen in human AD postmortem samples, particularly in patients with hippocampal sclerosis [16]. AA is released from astrocytes as glutamate is taken up, and this relationship is termed a hetero-exchange although this does not fully represent how the two processes are tethered [17,18]. It is presumed that AA moderates the oxidative stress induced by glutamate [19] and so is protective against overstimulation and cell death. This relationship has been more closely investigated in relation to Huntington's disease [20–22], which also involves cell death. GLT-1 is a high affinity transporter that relies on Na-dependent transport across an electrochemical gradient for rapid removal of glutamate from the synapse. It is sensitive to oxidative stress, and disruption of the transporter leads to glutamate accumulation and hyperstimulation of receptors. Memantine is the first of a new class of drugs for AD that blocks NMDA receptors and its efficacy suggests that further research into glutamatergic signaling and AD is warranted. Therefore, as the role of glutamate transport and NMDA receptors in AD becomes clearer, this may be revealed as another key area where high intracellular AA levels are critical for brain health.

Further excitement has recently been generated in the field of epigenetics with a potential new answer for why AA is concentrated so strongly in CSF and brain parenchyma (reviewed in [23]). Nutrition is perhaps the quintessential example of environmental modification of the genome, and recent work has highlighted a direct role for AA that cannot be replicated with other antioxidants. 5-mc (5-methylcytosine) is oxidized to 5-hmc (5-hydroxymethylcytosine) as part of dynamic DNA demethylation. This reaction, and further oxidation of 5-hmc, are both catalyzed by the activity of TET (ten-eleven translocation) dioxygenase enzymes, for which AA is a critical co-factor (needed for the reduction of iron Fe3+ to its active form Fe2+) [24–26]. Thus AA is vital for neuronal repair as

well as new cell generation and here may play a direct role in the transcription and expression of hundreds of different genes. 5-hmc and Tet proteins are abundant in brain and knockout of Tet1 in mice indicated that it may be involved in synaptic plasticity and memory extinction in addition to DNA methylation [27]. The specific relation to AD and other degenerative disorders is not yet clear, but this exciting field may provide new clues.

AA is a one-electron donor that readily reacts with a range of reactive oxygen species (ROS) to neutralize or decrease their reactivity. Loss of the electron leads to formation of the ascorbate free radical, which can be efficiently recycled to ascorbic acid through enzymatic means (summarized in Figure 1). Ascorbate radicals react preferentially with themselves forming ascorbic acid and dehydroascorbate. This oxidized form of AA can also be recycled back to ascorbic acid, although in some cases it undergoes irreversible ring opening and may be lost. AA also supports the regeneration of other antioxidants, such as vitamin E and glutathione (GSH), in biological tissues, thus combatting oxidative stress through various pathways. This close relationship between GSH and AA is such that AA has often been observed to "take the first hit" for GSH in response to oxidative stressors, and GSH is involved in reduction of dehydroascorbate to ascorbate [28].

**Figure 1.** Summary of ascorbic acid oxidation and recycling. Adapted from [13].

It should also be considered that AA and some of its degradation products may be involved in some potentially damaging functions [29]. For example, the Maillard reaction, most commonly studied in relation to food, is also a step in the formation of advanced glycation end products (AGEs). Evidence from a mouse model that selectively over-expressed the vitamin C transporter SVCT2 in the eye [30] implicated AA in age-related damage to crystalline proteins in the lens. There is also some debate as to the potential pro-oxidant role of AA via the Fenton reaction. AA reacts with metal ions in enzymes (e.g., hydroxylases, oxygenases) that require them to be in a reduced state for optimal enzyme function. *In vitro*, reactions with these ions can lead to the production of hydroxyl radicals and other reactive molecules. *In vivo*, catalytic metal ions are less available as their levels are typically kept in check in healthy individuals by metal binding proteins (e.g., ferritin, transferrin). Thus, *in vivo*, the evidence typically supports the antioxidant roles of AA (reviewed in detail [31,32]). One exception may be in the case of iron overload. A limited pro-oxidant effect of increased dietary AA, seen as elevated liver malondialdehyde in combination with decreased glutathione peroxidase, was found in mice fed supplemental dietary iron (although only in the lower and not the high iron diet group) [33]. It should be noted though that this study was performed under conditions of AA sufficiency, in wild-type mice that synthesize their own AA. Such an effect of pro-oxidation reactions was not found in plasma from adults or pre-term infants [3], or in AA- and iron-supplemented guinea pigs [34] and in general AA is not considered a risk factor even in patients with hereditary hemochromatosis [35]. High iron is thought to be a risk factor for AD [36] although the data are equivocal. In a study of 116 AD patients compared to 89 healthy controls, dysregulated iron homeostasis (lower serum iron, ferritin and transferrin, combined with several genetic markers for altered iron metabolism) was associated with AD [37]. The full relationship between AA, iron and AD may warrant further investigation. A further indication of the complexity of the relationships between AA, amyloid and oxidative damage is that in isolated rat brain mitochondria, and in the presence of AA and iron, amyloid-β1–42 was actually found to have an antioxidant effect and prevent formation of hydrogen peroxide, presumably through metal chelation [38]. Although aggregated amyloid-β1–42 cannot be argued to be healthy in brain tissue, this finding certainly raises interest for researchers in this field who have typically considered amyloid-β to be solely detrimental and an inducer of ROS.

Details of the complexity of the transport of AA within the central nervous system have been understood for almost half a century. Seminal work by Hammarström [39] using radio-labeled AA in guinea pigs first showed that peripherally-administered AA did indeed reach the brain, but that it appeared to travel via the choroid plexus and not traverse the blood brain barrier directly. Reports that AA levels in the CSF exceeded those found in blood, and were less affected by variations in plasma AA, suggested the existence of an active and saturable transporter for AA in the choroid plexus, which was then determined to be the case [40]. The sodium dependent vitamin C transporter 2 (SVCT2) itself was not described until much later but is responsible for transport both at the choroid plexus and in the neurons. There are two sodium-dependent vitamin C transporters, SVCT1 and SVCT2, which are responsible for transport of AA. Distribution of the two transporters varies across organs [41–43], but SVCT2 is the only transporter expressed in the brain. Regulation of the active transport of AA by these transporters may also vary across organs and in particular disease states (reviewed [44]). It allows for accumulation of AA in cells against the concentration gradient in SVCT2-dependent tissues. Following characterization of SVCT1 [43,45] single nucleotide polymorphisms (SNPs) were identified in both SVCT1 and SVCT2 and some studies are now being performed to ascertain how they alter AA status and whether they confer additional risk for diseases [46,47]. Several SNPs have been identified in the *SLC23A1* gene coding for SVCT1, and it is argued that this may be less constrained than the *SLC23A2* gene [45,48]. Altered function of the SVCT1 would have important effects on AA absorption and excretion and so would impact

nutritional requirements. One synonymous and three nonsynonymous SNPs resulted in diminished vitamin C uptake in *Xenopus laevis* oocytes [49]. One of these SNPs led to an 80% decrease in uptake, and was found to have a relative occurrence of 6%–17% in African-Americans [49]. These studies are of interest to brain research because it is presumed that such SNPs could cause chronically lower circulating AA levels, which would also potentially affect brain levels. Although fewer SVCT2 SNPs have been identified, these could have an even greater effect on brain AA levels and impact disease progression.

Expression of SVCT2 mRNA and protein varies in brain and other organs during development, presumably directly linked to very high, and changing AA levels during this critical period Nevertheless developmental regulation appears to be particularly specialized and AA regulation of SVCT2 is not common to all cell types, nor to all ages. Thus, SVCT1 and SVCT2 expression, including in brain, is regulated by several disease states associated with oxidative stress (e.g., streptozotocin-induced diabetes and middle cerebral artery occlusion) [50,51] supporting the idea of potential for change in neurodegenerative disorders such as AD. Brain levels of AA in humans are dependent on the SVCT2 (see Figure 2) but also on dietary intake and intestinal absorption via SVCT1. Given that AA can also mediate the permeability of endothelial cell layers in culture [52,53], it can be seen that individual variability in either SVCT1 or SVCT2 transporter function could directly impact brain levels and vasculature in a similar manner to dietary deficiency. One particularly interesting question that may yet be answered with animal studies is the question of how SVCT2 transporter function may change with age and disease status and what impact this would have on AA levels. SVCT2 mRNA and protein both vary during development showing an inverse correlation with brain AA levels, whereas no such changes were seen in brain during AA deficiency in gulo-/- mice indicating that adult brain is unable to respond to long periods of deficiency by altering transport [54–56]. Changes in transporter function have not, to our knowledge, been studied in normally aging or AD mouse models, but if transporter function varied among groups, this could lower both CSF and intraneuronal AA levels independently of dietary intake and circulating AA levels.

There are several animals that, like humans, do not synthesize their own AA including guinea pigs, primates and some fish [57]. Nevertheless, transport is conserved across species and study of AA transporters *in situ* can be conducted in most animal models. Cell lines and primary culture techniques are available to study different organs and tissues, which have yielded useful information on expression, membrane location and transport kinetics of SVCT1 and SVCT2 in, for example, intestinal CaCo-2 cells, epithelial cells, endothelial cells, hepatocytes, muscle, intervertebral discs, Schwann cells and others [58–65]. An important consideration is that SVCT2 is regulated to some degree both by AA levels and by oxidative stress and can therefore develop in culture, as seen in astrocytes [66,67], which renders this approach potentially problematic. Other weaknesses of culture systems include the difficulties of maintaining a constant AA level in the media because it is so readily oxidized to dehydroascorbate. Dehydroascorbate can be lost reasonably quickly and can be transported into cells via glucose transporters and then recycled back to ascorbate within the cell. HPLC methods for measuring AA are very accurate if care is taken with sample preparation to minimize loss, and radiolabeled AA is available for assays for quantification in culture. Nevertheless to accurately model human *in vivo* situations, particularly in reference to specific diseases such as AD, animal models are also needed.

Different AA levels across brain regions have been reported in human and rodent brains [11,68,69]. Figure 2 shows a schematic representation of the distribution of SVCT2 (red, solid line) in the brain, and SVCT1 in intestines (green, dotted line). SVCT2 is highly, but not necessarily regularly, distributed throughout the brain. Figure components are not drawn to scale.

**Figure 2.** Location and distribution of SVCT1 and SVCT2 transporters and their importance in maintaining optimal brain ascorbic acid levels.

**Inset 1**. **Cortex and lining of lateral ventricle.** It was originally proposed that AA might follow a pattern of decreasing concentration with increasing distance from the ventricles following a pattern of diffusion [68]. It has since been shown that SVCT2 and AA distribution (indicated by red coloring) depend on cell type and correlate with neuronal density [70,71]. *In situ* hybridization in adult rat showed SVCT2 distribution throughout the brain, with highest expression in olfactory bulbs, hippocampus, cerebellum, cortex and choroid plexus [42]. High SVCT2 expression is seen in the ependymal cells lining the ventricular walls, with decreasing concentration in more external areas of the cortex by *in situ* hybridization and immunohistochemistry techniques [71]; **Neurons.** Primary culture of embryonic (E17) neurons shows SVCT2 in soma and cell processes [71]. Primary culture of mouse hippocampal cultures (E16–17) showed punctate staining of SVCT2 in axons [72]. Using immunohistochemical techniques in hippocampus, SVCT2 was observed in cell bodies but not processes by immunohistochemistry [73]; **Astrocytes** take up

dehydroascorbate via glucose transporters (GLUTS) and recycle it to ascorbic acid, which can be taken up by neurons. SVCT2 was not observed in astrocytes by *in situ* hybridization in adult rat brain, even following excitotoxic stimulation with quinolinic acid, although SVCT2 was observed through Northern blot technique in cultured astrocytes from rat embryo [66,74]; **Inset 2. Cortex and lining of lateral ventricle.** Panels A–C show cortex and ventricle. In panel A, AA is stained with silver stain [75] and can be seen at its most concentrated closest to the ventricle lining. In panel B, fluorescent stain for the S19 antibody staining for SVCT2 shows high expression in cells along the ventricle lining. Panel C shows DAPI stain for cell bodies. (Images courtesy of J.M. May); **Inset 3. Cross-section of Cerebral blood vessel.** There is no SVCT2 present in blood vessels in the brain. Glucose transporters (GLUTS) are found in endothelial cells and may transport dehydroascorbate. This contributes a negligible amount to brain AA stores under normal circumstances because there is typically competitive inhibition by glucose. An exception to this state may be after transient ischemia in which case SVCT2 has been shown to develop in mice [76]; **Inset 4. Choroid plexus.** SVCT2 is located throughout the choroid plexus for transfer of AA from blood to CSF. High expression of SVCT2 in choroid plexus cells has been shown by *in situ* hybridization in several studies [42,71,74]; **Inset 5. Tanycytes associated with the hypothalamus in the wall of the third ventricle.** Tanycytes are highly specialized hypothalamic glial cells that are capable of forming tight junctions and participate in the formation of the barrier between CSF and brain tissue. SVCT2 has been shown by *in situ* hybridization and immunohistochemistry in different subtypes of tanycytes in mouse brain [75]; **Inset 6. SVCT1 and SVCT2 expression in the intestine**. Expression of both transporters allows for uptake of AA from food and distribution around the body (and to brain). SVCT1 is located on the apical side of the enterocytes and is responsible for taking up AA as it passes through the intestinal lumen. SVCT2 is located on the basolateral side of cells. This is thought to be more important in animals that can synthesize their own AA, but do not consume an AA rich diet, and must still supply enterocytes with AA from blood [60]. Similar localization of SVCT1 on the apical membrane with SVCT2 found on the basolateral membrane is also seen in kidney cells. How AA passes out of the cells into the interstitial fluid and blood supply is still not confirmed, and is a conundrum given the typical direction of the SVCT2 for uptake into cells.
