Causes of astrogliosis

Astrocytes are exquisitely sensitive to very small fluctuations in the CNS intracellular environment and readily attain a reactive phenotype in the face of such changes. One acknowledged cause of increased astrocyte reactivity is increased intracellular levels of PICs, NO, and ROS secreted by activated microglia [163–165]. Other triggers include glucose deprivation, increased levels of ATP and other gliotransmitters, and activation of surface toll-like receptors by commensal lipopolysaccharide (LPS) originally translocated from the intestine. It is important to note that the development of reactive astrogliosis provoked by these stimuli, particularly the inflammatory mediators such as the PICs, ROS, and NO, is accomplished via major changes in astrocyte gene transcription patterns, which drive morphological, physiological, and biochemical changes ([Reference Romano, Koczwara, Gallelli, Vergara, Micioni Di Bonaventura and Gaetani142]; reviewed by [Reference Sofroniew and Vinters166]).

Although there are a myriad of changes in signaling pathways in activated astrocytes compared with those existing in these glial cells in their physiological state, from the perspective of this article, the most noteworthy change is the chronic activation of the NF-κB, MAP kinase, and Jak/Stat pathways, which result in high intracellular levels of ROS, RNS, and PICs (reviewed by [Reference Sofroniew167]). It is noteworthy that STAT-3 is of paramount importance as the weight of evidence suggests that activity of this transcription factor is an indispensable element in the development and maintenance of reactive astrogliosis [168–170].

Readers interested in the details of mechanisms underpinning the advent and persistence of a reactive or dysfunctional state in astrocytes are invited to consult the work of [Reference Johnstone, Billaud, Lohman, Taddeo and Isakson171] and [Reference Lutas, Birnbaumer and Yellen60]. However, the key points to bear in mind during a perusal of the following sections of this article are that many of the factors underpinning the loss of homeostatic functions normally exerted by astrocytes in their physiological state stem from the changes in transcription orchestrated by the chronic activation of the pathways and transcription factors discussed above and/or the ensuing increases in levels of PICs, ROS, and RNS. These adversely affect the transcription and/or function of crucial membrane receptors and impair mitochondrial respiration and dynamics [Reference Bough and Rho59,Reference Stout, Costantin, Naus and Charles172,Reference Pehar, Harlan, Killoy and Vargas173]. The importance of the latter is difficult to overemphasize as the homeostatic roles of astrocytes depend on adequate performance of mitochondria [Reference Tay, Béchade, D'Andrea, St-Pierre, Henry and Roumier55,Reference Du, Zhang, Chen, Zhu, Yang and Zhang56,Reference Bataveljic, Nikolic, Milosevic, Todorovic and Andjus174], and on a wider note, a host of neural–glial interactions also depend on optimal mitochondrial function [Reference Brambilla, Martorana and Rossi175]. Hence, a therapeutic approach, such as the KD, capable of reducing oxidative stress and inflammation in the brain in vivo while improving mitochondrial function has the potential to mitigate against the severity of astrogliosis and improve CNS homeostasis.

Consequences of astrogliosis

The normal role of astrocytes in regulating other dimensions of CNS homeostasis such as neurotransmitter levels, water transport, waste product clearance, ion homeostasis, and glucose and oxygen delivery to neurones (reviewed by [Reference Guillamón-Vivancos, Gómez-Pinedo and Matías-Guiu176] and summarized in Figure 2) are impaired when astrocytes are in a reactive state [Reference Koyama177,Reference Elsayed and Magistretti178]. These observations are underpinned by the fact that astrogliosis results in adverse changes in astrocyte phenotype, signaling pathways, and surface receptor expression, which normally enable these cells to perform their essential role in the regulation of various dimensions involved in the maintenance of CNS homeostasis as described above. Disruption of the neurovascular unit owing to a loss of astrocyte end-feet and other cellular protrusions is perhaps the most damaging physical change as far as CNS homeostasis is concerned. The physical connection between BBB epithelial cells and neurones is needed to deliver nutrients and oxygen to the latter (reviewed by [Reference Yudkoff, Daikhin, Nissim, Horyn, Lazarow and Luhovyy179]). Examples of changes in receptor levels and function [Reference Yudkoff, Daikhin, Nissim, Lazarow and Nissim180] include oxidative modification of glutamate receptors leading to inhibition of astrocyte-mediated glutamate uptake, nitrosylation of the gap junction channel connexin 43 (Cx43), and several gap junction pannexins (reviewed by [Reference McKenna181]) leading to dysregulated calcium signaling and ATP transfer between astrocytes and neurones and other astrocytes [Reference Valdebenito, Ruminot, Garrido-Gerter, Fernandez-Moncada, Forero-Quintero and Alegria182]. Astrogliosis is also associated with profound disturbances in potassium homeostasis as a result of oxidative modification and downregulation of Na⁺, K⁺-ATPase (NKA) [Reference Yudkoff, Daikhin, Nissim, Lazarow and Nissim180], and the weak inwardly rectifying K_ir family potassium channel K_ir4.1 [Reference Puchalska and Crawford183,Reference Dahlin, Elfving, Ungerstedt and Amark184]. The effects of astrogliosis in disrupting CNS homeostasis are summarized in Figure 3.

Figure 2. The multiple roles of astrocytes in central nervous system homeostasis.

Figure 3. Disruption of central nervous system homeostasis resulting from astrogliosis.

Given this information, it probably comes as no surprise to learn that astrocyte dysfunction, astrogliosis, and the accompanying global loss of the physiological functions of astrocytes in the regulation of CNS homeostasis play a major role in the development and acceleration of most if not all neurodegenerative [Reference Calderon, Betancourt, Hernandez and Rada185,Reference Hertz, Chen and Waagepetersen186] and neuroprogressive disorders [Reference Anlauf and Derouiche187,Reference Schousboe, Bak and Waagepetersen188]. In addition, treating the causes and/or consequences of astrogliosis is a major therapeutic objective [Reference Woolf, Curley, Liu, Turner, Charlton and Preul61,Reference Paoli, Bosco, Camporesi and Mangar63,Reference Sofroniew and Vinters166]. How that might be achieved, at least in part, via dietary induced ketosis will be discussed in the next section of this article. In order to facilitate this endeavor, we will divide the following section into effects on the glutamate–glutamine cycle, glutamine synthetase, glutamate secretion, glutamate uptake via EAATs, NKA, K_ir4.1 receptors, aquaporin-4 (AQP4), gap junctions, and K_ATP channels.

Effect of nutritional ketosis on the astrocyte glutamate–glutamine cycle

There is evidence that the entry of KBs into astrocytes stimulates mitochondrial metabolism and accelerates flux through the TCA cycle resulting in a number of consequences including the upregulation of the glutamate–glutamine cycle ([Reference Balietti, Giorgetti, Di Stefano, Casoli, Platano and Solazzi71,189–191]; reviewed by [Reference Dienel and Cruz144]). The mechanisms underpinning these observations are a matter of debate but stimulation of glycolysis, enhanced efficiency of pyruvate utilization, and increased levels of succinate to overcome a shortfall of oxaloacetate in an environment of glucose restriction all appear to be involved ([Reference Hyatt, Kephart, Holland, Mumford, Mobley and Lowery74,Reference Ben Haim, Carrillo-de Sauvage, Ceyzériat and Escartin157,Reference Marsman, van den Heuvel, Klomp, Kahn, Luijten and Hulshoff Pol192]; reviewed by [Reference Hashimoto, Bruno, Nierenberg, Marmar, Zetterberg and Blennow193]).

The weight of evidence suggests that this phenomenon results in increased synthesis of glutamine and upregulated levels of GABA coupled with decreased synthesis of glutamate [Reference Balietti, Giorgetti, Di Stefano, Casoli, Platano and Solazzi71, 189–191, Reference Chen, Henter and Manji194, Reference Madeira, Vargas-Lopes, Brandão, Reis, Laks and Panizzutti195], although the latter appears to be highly dependent on astrocyte KB concentration [Reference Woodman, Landwehrmeyer, Lindenberg, Franz and Behrens196]. The existence of the glutamate–glutamine cycle is limited to astrocytes as these glial cells uniquely possess the two enzymes needed for its operation, namely pyruvate carboxylase, which enables the synthesis of new TCA cycle intermediates via the replenishment of oxaloacetate, and glutamine synthetase, which enables the synthesis of glutamine [Reference Pioro, Majors, Mitsumoto, Nelson and Ng197,Reference O'Gorman Tuura, Baumann and Baumann-Vogel198].

These neurotransmitters must be continually re-synthesized as uptake of GABA by astrocytes is limited and glutamate is used as a substrate for oxidation to compensate for the huge energetic costs of glutamate uptake (reviewed by [Reference Clark and Vissel199]). Readers interested in the biochemistry underpinning the glutamate–glutamine cycle and the re-syntheses of glutamate and glutamine are invited to consult a comprehensive review on these matters by O'Gorman Tuura et al. [Reference O'Gorman Tuura, Baumann and Baumann-Vogel198].

However, there are some key points germane to the discussion below. First, the cycle is highly dependent on energy supplied by glucose oxidation and optimal mitochondrial function [Reference Kim and Yoon200]. Second, KBs are the preferred oxidative substrate for “powering” the glutamate–glutamine cycle in the environment of cerebral glucose hypometabolism seen in neurodegenerative and neuroprogressive disorders [Reference Robel, Buckingham, Boni, Campbell, Danbolt and Riedemann201]. Third, and perhaps expectedly, evidence of dysfunction or dysregulation of this cycle is present in at least some regions of the brain in patients with SZ [Reference Robel and Sontheimer202], MDD [Reference Ortinski, Dong, Mungenast, Yue, Takano and Watson203], BPD [Reference Jackman, Uliasz, Hewett and Hewett204], AD [Reference Shi, He, Hewett and Hewett205], HD [Reference Mei, Wu and Zhou206], ALS [Reference Eid, Thomas, Spencer, Runden-Pran, Lai and Malthankar207], and PD [Reference van der Hel, Notenboom, Bos, van Rijen, van Veelen and de Graan208]. Moreover, impairment of this cycle is a major element in the development of disturbed glutamate homeostasis, with glutamate excitotoxicity, and reduced GABAergic signaling, which play a causative role in the pathogenesis of most if not all neurodegenerative and neuroprogressive conditions [Reference Swamy, Norlina, Azman, Suhaili, Sirajudeen and Mustapha209,Reference Castegna, Palmieri, Spera, Porcelli, Palmieri and Fabis-Pedrini210]. Finally, the weight of evidence suggests that the ultimate source of glutamate and GABA dyshomeostasis seen in these illnesses is the presence of chronic astrogliosis [Reference Kosenko, Llansola, Montoliu, Monfort, Rodrigo and Hernandez-Viadel211,Reference Rodrigo and Felipo212].

Nutritional ketosis, astrogliosis, and glutamate synthetase

Astrogliosis is associated with reduced activity of glutamine synthase and GABA synthesis and relative failure of astrocyte-mediated glutamate reuptake as well as disruption of the glutamate–glutamine cycle [211–213]. Astrogliosis is also associated with increased expression of the cysteine/glutamate antiporter channel (X_c⁻) resulting in increased glutamate signaling and oxidative downregulation of the astrocyte glutamate uptake receptors EAAT1 and EAAT2, which in turn leads to the development of glutamate-mediated N-methyl-d-aspartate (NMDA) receptor excitotoxicity [Reference Sofroniew and Vinters166,Reference Ma, Wang, Parikh, Green, Hoffman and Chlipala214,Reference Tae-Woo Kim, Park, Jung and Kim215]. Impaired glutamate synthetase (GS) activity results in the accumulation of glutamate and impaired glutamate uptake thus making a contribution to the development of excitotoxicity [Reference Chang, Augustin, Boddum, Williams, Sun and Terschak216].

Several research teams have reported that GS activity in humans and animals is downregulated in a cerebral environment of chronic oxidative and nitrosative stress and that such downregulation results in elevated intracellular glutamate and reduced glutamine levels [217–220]. Moreover, there are replicated in vivo data demonstrating that a major cause of GS inhibition in vivo is nitrosylation or nitration of functional residues secondary to excessive NMDA receptor activity and perhaps even more importantly that in vivo inhibition of extrasynaptic NMDA results in the upregulation of GS activity and increased levels of glutamine [Reference Olson, Vuong, Yano, Liang, Nusbaum and Hsiao221,Reference O'Donovan, Sullivan and McCullumsmith222].

This is of interest as a mechanism known to downregulate NMDA activity in vivo is NO-mediated S-nitrosylation of functional thiol groups in NMDA receptor subunits and animal studies have reported raised NO levels in the CNS following consumption of a diet aimed at inducing ketosis [223–225].

It is also noteworthy that ex vivo studies have demonstrated a direct inhibitory effect of MCFAs on NMDA receptor excitotoxicity via the inhibition of NMDA AMPA subunits [226–228] which is of interest as MCT supplemented versions of the KD produced significant benefits in the treatment of patients with AD. Ex vivo data also suggest a positive effect of MCFAs on astrocyte mitochondrial biogenesis (reviewed by [Reference Krzyzanowska, Pomierny, Bystrowska, Pomierny-Chamiolo, Filip and Budziszewska229]).

Nutritional ketosis, astrogliosis, glutamate release, and synthesis

There are some ex vivo and in vivo data suggesting that KBs suppress the release of glutamate from astrocytes and neurones by inhibiting the actions of vesicular glutamate transporters (VGLUTs) and via an as yet undelineated mechanism [Reference Mulrooney, Marsh, Urits, Seyfried and Mukherjee230]. The reduction of glutamate synthesis by BHB in vitro in a dose-dependent manner has also been reported [Reference Woodman, Landwehrmeyer, Lindenberg, Franz and Behrens196]. An in vivo study involving rodents conducted by Olson et al. also appears to be worthy of consideration in the context of the potential beneficial effects of the KD on glutamate and GABA homeostasis. These authors reported that the reduction in glutamate and increase in GABA levels seen in the hippocampus of their study animals was effected by KD-induced changes to the microbiota [Reference Romera, Hurtado, Mallolas, Pereira, Morales and Romera231].

Astrogliosis and EAAT function

However, despite the presence of data suggesting that diet-induced ketosis may have beneficial effects on some drivers of glutamate and GABA dyshomeostasis, it must be noted that there appears to be no direct evidence that a KD exerts positive effects on the activity or levels of astrocyte glutamate transporters (EAAT1 and EAAT2). This is an important point as the weight of evidence suggests that dysfunction and/or downregulated expression of EAAT2, which is responsible for approximately 90% of glutamate reuptake by humans astrocytes, is a major cause, if not the major cause, of glutamate-mediated NMDA receptor excitotoxicity, which appears to be causatively implicated in the pathogenesis and pathophysiology of all neurodegenerative and neuroprogressive disorders [Reference Yang, Gozen, Watkins, Lorenzini, Lepore and Gao232,Reference Walls, Waagepetersen, Bak, Schousboe and Sonnewald233].

The results of human and animal studies point to a major cause of such downregulated expression or dysfunction of these receptors seen in all these illnesses as being the upregulated canonical NF-κB signaling and elevated levels of PICs such as tumor necrosis factor alpha (TNF-α) and interleukin (IL)-1β, ROS and RNS characteristic of the intracellular environment of reactive astrocytes ([Reference Waagepetersen, Sonnewald and Schousboe234]; reviewed by [Reference Walls, Waagepetersen, Bak, Schousboe and Sonnewald233]). TNF-α appears to downregulate the transcription of the EAAT2 gene [Reference Swamy, Norlina, Azman, Suhaili, Sirajudeen and Mustapha209,Reference Hertz, Gerkau, Xu, Durry, Song and Rose235] while IL-1β primarily has a negative effect on the membrane density of the receptor by enhancing its endocytosis and sequestration in the cytoplasm [Reference Kaur and Kaur236,Reference Vasconcelos, Kinoshita, Yshii, Marques Orellana, Bohmer and de Sa Lima237]. There is also evidence suggesting that the function of EAAT2 (and indeed EAAT1) is compromised in neurodegenerative and neuroprogressive illnesses as a result of S-nitrosylation of crucial thiol residues, which play an indispensable role in their function (reviewed by [Reference Larsen, Holm, Vilsen and MacAulay238]).

There is robust in vivo evidence that increasing ROS scavenging and GSH production, using N-acetylcysteine, can increase EAAT and decrease X_c⁻ expression in reactive astrocytes [Reference Dinuzzo, Mangia, Maraviglia and Giove239]. Hence, the consumption of a KD, which also results in increased GSH production in the brain via the upregulation of Nrf2 [Reference Nakamura and Lipton43,Reference Rose, Brian, Woods, Pappa, Panayiotidis and Powers46] would be expected to have a similar beneficial effect. There are also an increasing number of publications reporting reduced levels of NF-κB and PIC levels in astrocytes and indeed other regions of the brain following ingestion of various manifestations of the KD [Reference Pinto, Bonucci, Maggi, Corsi and Businaro1,Reference Hertz, Song, Xu, Peng and Gibbs240] and hence the diet seems to have the capacity to exert a corrective influence on several elements driving the EAAT2 downregulation seen in neurodegenerative and neuroprogressive disorders.

In addition, EAAT transcription is a downstream target of PPAR whose upregulation has a range of neuroprotective effects in neuropathological conditions in vivo [Reference Xu, Song, Xue, Gu, Hertz and Peng241,Reference Larsen, Stoica and MacAulay242]. This is of particular importance as there are several studies reporting upregulation of PPAR activity following the prolonged ingestion of a KD and this provides another mechanism by which diet-induced ketosis could upregulate the transcription of EAATs in reactive astrocytes [Reference Crunkhorn127,Reference Chistyakov, Aleshin, Astakhova, Sergeeva and Reiser131]. In addition, there is an accumulating body of data suggesting that the upregulation of PPAR reduces neuroinflammation, which is of interest because the presence of this phenomenon is a major trigger of increased astrocyte reactivity as discussed above [Reference Le Foll, Dunn-Meynell, Miziorko and Levin136]. Furthermore, PPAR activation may rescue the compromised mitochondrial bioenergetics and dynamics seen in diseases such as AD and SZ [Reference Takahashi, Iizumi, Mashima, Abe and Suzuki137,Reference Thevenet, De Marchi, Domingo, Christinat, Bultot and Lefebvre138].

Nutritional ketosis, astrogliosis, and NKA function

This is of paramount importance from the perspective of this article. Glutamate and GABA uptake are energy consuming processes as previously discussed [Reference Clark and Vissel199,Reference Franzon, Lamers, Stefanello, Wannmacher, Wajner and Wyse243] due in part to the reliance of EAATs on the option function of NKA receptors which coexist in the same molecular complex [Reference Novaes, Dos Santos, Dragunas, Perfetto, Leza and Scavone244] (reviewed by [Reference Franzon, Lamers, Stefanello, Wannmacher, Wajner and Wyse243]). This enzyme, as the name suggests, is in turn dependent on adequate supplies of ATP [Reference Roy, Dasgupta, Banerjee, Chowdhury, Mukhopadhyay and Saha245] and hence its function is likely to be compromised in an environment of impaired bioenergetics characteristic of astrocytes in their reactive state [Reference Tay, Béchade, D'Andrea, St-Pierre, Henry and Roumier55].

Improving the function of NKA is clearly a desirable therapeutic target and in that context, it is important to note that several research teams have reported that protracted, acute, or intermittent ketosis activates or increases the expression NKA pumps in the brain [Reference Veech, Chance, Kashiwaya, Lardy and Cahill68,Reference Banerjee, Dasgupta, Rout and Singh246,Reference de Lores Arnaiz and Ordieres247]. It should be stressed that this finding is not only important from the perspective of glutamate homeostasis as the interplay between NKA and EAATs, but also plays an important role in regulating levels of K⁺ ions throughout the CNS [Reference Seidel, Faideau, Aiba, Pannasch, Escartin and Rouach248]. From the perspective of K⁺ homeostasis, however, the weight of evidence suggests NKA is the most important player in this molecular partnership and plays the dominant role in K⁺ uptake into astrocytes, which in turn regulates neural function and excitability [Reference Sofroniew167]. The dependence of K⁺ uptake into astrocytes on NKA activity goes some way to explaining evidence supplied by several authors confirming that the maintenance of K⁺ homeostasis is the most energy intensive role of astrocytes [Reference Thakurta, Banerjee, Bagh, Ghosh, Sahoo and Chattopadhyay249] and thus dependent on adequate supplies of ATP [Reference Rodrigo, Miranda-Merchak, Valenzuela Grau, Bachler and Vergara250,Reference Namazi, Jamshidi Rad, Attar, Sarrafzadegan, Sadeghi and Naderi251]. The function of NKA and its indispensable role in astrocyte is well documented and hence will not be considered here but any readers interested in a detailed consideration of the biochemistry underpinning its structure and functions are referred to excellent reviews by Roy et al. [Reference Roy, Dasgupta, Banerjee, Chowdhury, Mukhopadhyay and Saha245] and Rodrigo et al. [Reference Rodrigo, Bachler, Araya, Prat and Passalacqua252].

However, from the perspective of this article, it should be emphasized that the activity of NKA is not just dependent on adequate supplies of ATP but is heavily influenced by the redox state of the intracellular and extracellular environment. Unsurprisingly, animal and human studies confirm that oxidative and nitrosative stress inhibits NKA activity in rat hippocampus and prefrontal cortex [Reference Gerbi, Barbey, Raccah, Coste, Jamme and Nouvelot253,Reference Gerbi and Maixent254] and in patients with SZ and BPD [Reference Gerbi, Maixent, Barbey, Jamme, Pierlovisi and Coste255,Reference Morris, Walder, Puri, Berk and Maes256]. A combination of oxidative stress would also appear to explain the downregulation of NKA activity seen in MDD, AD, and other neurodegenerative diseases (reviewed by [Reference Fraser, Whiting, Andrew, Macdonald, Musa-Veloso and Cunnane257]). These are important observations as accumulating evidence suggests that downregulation of NKA is partly responsible for the impaired ability of reactive astrocytes to regulate K⁺ homeostasis [Reference Kosenko, Llansola, Montoliu, Monfort, Rodrigo and Hernandez-Viadel211,Reference Taha, Ryan and Cunnane258].

The causative role played by oxidative and nitrosative stress in NKA downregulation is further emphasized by studies reporting that prolonged use of antioxidant combinations such as vitamin C and E or N-acetylcysteine, α-tocopherol, and α-lipoic acid can produce clinically significant increases in NKA activity in the brain and periphery [Reference Liu, Kobayashi, Jin, Wada and Nao-I259,Reference Hassan, Luo, Majumdar, Dominguez, Busik and Bhatwadekar260]. Thus, there is a prospect that the well-documented antioxidant properties of the KD may exert a similar effect assuming a similar effect size and may well underpin the observations reporting a positive effect of the KD on improving NKA function discussed above. However, there may be another mechanism by which nutritional ketosis may improve NKA function, which would appear to be under-discussed.

Briefly, several authors have also reported strong negative correlations between the extent of membrane lipid peroxidation and NKA activity in vivo in illnesses as diverse as cardiovascular disease, SZ, and BPD [Reference Gerbi, Maixent, Barbey, Jamme, Pierlovisi and Coste255,Reference Morris, Walder, Puri, Berk and Maes256,Reference Thompson, Chen, Luo, Xiao, Cummins and Bhatwadekar261]. The relationship between increased membrane lipid peroxidation and increasing NKA dysfunction appears to be mediated by decreased membrane fluidity and PUFA content [Reference Hassan, Luo, Majumdar, Dominguez, Busik and Bhatwadekar260,Reference Sibille, Dao Duc, Holcman and Rouach262]. This is of importance as several studies have reported a significant increase in NKA activity following dietary supplementation with PUFAs [Reference Hassan, Luo, Majumdar, Dominguez, Busik and Bhatwadekar260,263–265]. These observations may be explained by reference to data confirming that dietary PUFAs can integrate into lipid membranes in the periphery and the brain combatting the drivers of lipid peroxidation and increasing membrane fluidity via mechanism (reviewed by [Reference Larsen and MacAulay266]). In this context, it is noteworthy that the KD can elevate levels of PUFAs in the circulation, cerebrospinal fluid, and brain [Reference Lee, Ghetti, Pinto-Duarte, Wang, Dziewczapolski and Galimi267,Reference Olsen, Khakh, Skatchkov, Zhou, Lee and Rouach268] and hence this property may afford yet another route by which diet-induced ketosis might upregulate NKA levels and function. There is also some evidence to suggest that reducing lipid membrane peroxidation and improving membrane stability in astrocytes may help to increase the expression of another receptor, which also plays an indispensable role in K⁺ homeostasis mediated by these glial cells, namely the aforementioned weak inwardly rectifying K_ir family containing K_ir4.1 [269–271]. This is important as the downregulation of this receptor in reactive astrocytes is the other major cause of impaired K⁺ homeostasis in the brain [Reference Puchalska and Crawford183,Reference Dahlin, Elfving, Ungerstedt and Amark184].

Nutritional ketosis, astrogliosis, and K_ir4.1 function

These findings are a reflection of the fact that astrocyte-mediated K⁺ buffering is mainly enabled by the presence of K_ir family potassium channels containing K_ir4.1 and K_ir4.1/5.1 subunits [Reference Zurolo, De Groot, Iyer, Anink, Van Vliet and Heimans272,Reference Schirmer, Srivastava, Kalluri, Bottinger, Herwerth and Carassiti273]. Much evidence suggests that the K_ir4.1 is the most important channel in astrocyte-mediated spatial buffering and may be responsible for up to 45% of potassium buffering in the hippocampus [Reference Marnetto, Valentino, Caldano and Bertolotto274,Reference Srivastava, Aslam, Kalluri, Schirmer, Buck and Tackenberg275]. Readers interested in regarding the structure and mechanisms underpinning the operation of K_ir4.1 and other astrocyte K_ir family channels are invited to consult the work of Brill et al. [Reference Brill, Goldberg, Karni, Petrou, Abramsky and Ovadia276].

K_ir4.1 activity has been associated with several other elements involved in astrocyte structure and function such as the regulation of astrocyte cell volume, astrocyte K⁺ conductance, resting membrane potential, and glutamate uptake [Reference Wilcock, Vitek and Colton277,Reference Medina, Watson, Bunney, Myers, Schatzberg and Barchas278]. Importantly, several research teams have reported that inhibition of this channel leads to increased K⁺ concentrations in the extracellular space and impaired glutamate uptake [Reference Imbrici, Camerino and Tricarico279,Reference Sun, Yan, Zou, Wang, Li and Wang280]. The resultant increase of glutamate in the synaptic cleft resulting from K_ir4.1 inhibition results in abnormal modulation of synaptic transmission and network level communication [Reference Wilcock, Vitek and Colton277,Reference Jin, Yu, Wu, Zhang, Shi and Chen281] and is associated with the development and maintenance of neuroinflammation [Reference Skowronska, Obara-Michlewska, Czarnecka, Dabrowska, Zielinska and Albrecht282,Reference Skowronska, Obara-Michlewska, Zielinska and Albrecht283]. The pathological significance of K_ir4.1 downregulation in a neuroinflammatory environment is further emphasized by the results of several in vivo studies reporting reduced expression and/or function of this receptor in several neurodegenerative diseases, most notably multiple sclerosis and AD [284–287].

Downregulation of K_ir4.1 is also seen in patients with MDD [Reference Jiang, Wang, Zhao, Feng, Shen and Yu288] and there is some evidence to suggest that reduced K_ir4.1 expression plays a causative role in the development of SZ and ASD [Reference Wang, Li, Feng, Ren, Shen and Zhao289].

Given the potential therapeutic importance of improving K_ir4.1 expression and/or function, it is encouraging to note that there is evidence that some of the elements responsible for the downregulation of astrocytic K_ir4.1 receptors in an environment of chronic neuroinflammation are very similar if not identical to the drivers of impaired EAAT expression and activity discussed above with increased IL-1 [Reference Djukic, Casper, Philpot, Chin and McCarthy270,Reference Skowronska, Obara-Michlewska, Czarnecka, Dabrowska, Zielinska and Albrecht282,Reference Ho, Chan, Yeh, Kuo, Liu and Wang290] and glutathionylation [Reference Nagelhus and Ottersen291] playing important inhibitory roles. Hence, the proven capacity of nutritional ketosis to reduce levels of oxidative stress and inflammation in the brain discussed on several occasions above might be expected to improve the expression and function of this receptor assuming that such reductions are of sufficient magnitude needed to produce such an effect. Other factors known to reduce K_ir4.1 expression in vivo seen in neurodegenerative and neuroprogressive conditions include high levels of extracellular glutamate, which stimulate NMDA receptors located on astrocytes leading to K_ir4.1 downregulation [292–294].

Hence, improvements in glutamate reuptake by astrocytes coupled with amelioration of glutamate dyshomeostasis, which may stem from nutritional ketosis would be expected to produce concomitant improvements in K_ir4.1-mediated astrocyte K⁺ buffering. Increased levels of ATP fostered by the bioenergetic and metabolic consequences of induced ketosis may also be of therapeutic benefit as far as improving K⁺ homeostasis mediated by K_ir4.1 is concerned as the optimum function of this receptor is also dependent on adequate levels of ATP [Reference Satoh, Tabunoki, Ishida, Saito, Konno and Arima285,Reference Milton and Smith286,Reference Foglio and Rodella295,Reference Xu, Chen and Li296]. Consequently, the documented improvements in oxidative stress, neuroinflammation, glutamate homeostasis, and ATP production in the CNS following prolonged ingestion of various KDs would be expected to result in beneficial effects on K_ir4.1 levels and function.

Nutritional ketosis, astrogliosis, AQP4, and gap junction function

There are replicated in vivo data confirming that nutritional ketosis and/or BHB administration may upregulate AQP4 activity [Reference Sahni, Zhang, Sosunov, Galkin, Niatsetskaya and Starkov70,Reference Retamal297] and normalize Cx43 gap junction function [298–300]. This is also important from the perspective of K⁺ homeostasis as the activity of K_ir4.1 channels in astrocyte-mediated K⁺ buffering is aided by the activity of AQP4 (reviewed by [Reference Zhao, Xin, He and Hu301]) and astrocyte gap junctions [Reference Srivastava, Aslam, Kalluri, Schirmer, Buck and Tackenberg275].

The expression, structure, and activity of AQP4 is significantly compromised in the environment of chronic neuroinflammation seen in neurodegenerative and neuroprogressive disorders [Reference Eugenin, Basilio, Saez, Orellana, Raine and Bukauskas302,Reference Tanner, Lutas, Martinez-Francois and Yellen303]. These ROS- and NO-mediated alterations lead to compromised performance of the receptor in regulating K⁺ homeostasis and several other dimensions of CNS homeostasis such as water balance, glutamate uptake, adult neurogenesis, and astrocyte migration (reviewed by [Reference Ma, Berg and Yellen304]). Impaired expression of AQP4 perpetuates and exacerbates neuroinflammation and astrogliosis and this phenomenon also underpins the detrimental role played by this receptor in the pathophysiology of AD, PD, MDD, and ASD [Reference Kawamura, Ruskin, Geiger, Boison and Masino305] (reviewed by [Reference Tanner, Lutas, Martinez-Francois and Yellen303]).

Astrocytic Cx43 gap junctions and pannexin hemichannels are held in an open configuration in a state of chronic neuroinflammation and oxidative stress as a result of the S-nitrosylation and oxidative modification of regulatory cysteine thiol motifs leading to conformational changes and loss of functional plasticity (reviewed by [Reference Sun and Feng306,Reference Szeto, Chen, Sun and Feng307]). This compromised function not only impairs their role in K⁺ homeostasis but also negatively impairs astrocyte-mediated glutamate uptake and dispersal [Reference Yang, Jin and Jiang308,Reference Yang, Shi, Cui, Wu and Jiang309]. There is also evidence that a state of chronically open gap junctions induces and exacerbates neuroinflammation [Reference Yang, Jin and Jiang308,Reference Yang, Shi, Cui, Wu and Jiang309] (reviewed by [Reference Yang, Shi, Chen, Cui, Konduru and Shi310]).

These consequences would appear to underpin at least in part evidence implicating gap junction and pannexin hemichannel dysfunction as another causative factor in the development of neurodegenerative and neuroprogressive disorders [Reference Yang, Shi, Cui, Wu and Jiang309,Reference Yan, Ma and Zhang311] (reviewed by [Reference Kozoriz, Church, Ozog, Naus and Krebs312]).

Nutritional ketogenesis, astrogliosis, and K_ATP channel function

Several animal studies have reported that prolonged ingestion of the KD or administration of the KD results in the opening of K_ir6.1 family K_ATP channels located in cell plasma membranes (sK_ATP) and in the outer membranes of mitochondria (mtK_ATP) [Reference Morris, Walder, Carvalho, Tye, Lucas and Berk44, 313–315]. It could be argued that this effect results from increasing ATP levels, which are increased following ingestion of various KDs as discussed above. However, given the fact that these channels are opened in an environment of low ATP and high ATP [Reference Liu, Lu, Wan, Auyeung and Mattson316,Reference Busija, Lacza, Rajapakse, Shimizu, Kis and Bari317] and the significant increase in ATP production in the brain induced by the KD, it is possible that K_ATP opening is mediated by decreasing oxidative stress and neuroinflammation. This argument is strengthened by evidence demonstrating inhibition of K_ATP channels by glutathionylation in an environment of increased oxidative stress [Reference Yang, Shi, Cui, Wu and Jiang309–Reference Yan, Ma and Zhang311, Reference Cao, Dai, Zhang, Chen, Yang and Hu318].

Irrespective of the mechanisms underpinning such upregulation, however, there is evidence to suggest that increased activity of these channels also has positive consequences for astrocyte function. For example, one such consequence is improved sequestration of K⁺ into mitochondria via a mechanism, which is similar in many respects to the mechanism enabling the sequestration of iron [Reference Kozoriz, Church, Ozog, Naus and Krebs312,Reference Thomzig, Wenzel, Karschin, Eaton, Skatchkov and Karschin313]. There is also evidence, albeit in vitro, of a positive association between the activation of astrocytic mtK_ATP channels and upregulation of electrical coupling between astrocytes in the hippocampus which is an effect mediated via increased efficiency of Cx43 gap junction function secondary to upregulated ERK signaling in astrocytic mitochondria (reviewed by [Reference Takeuchi and Suzumura299]). It is also noteworthy that opening of astrocyte mtK_ATP channels may also be important from the perspective of CNS homeostasis as its upregulation appears to be an important element in maintaining the stability of the wider astrocyte neurovascular unit [Reference Sun and Hu314].

There are also replicated data suggesting that opening mtK_ATP channels may make a significant contribution to astrocyte survival in an environment of chronic inflammation and oxidative stress by inhibiting the translocation of Bax and the release of cytochrome c oxidase from mitochondria into the cytosol thereby inhibiting ROS- or TNF-mediated apoptosis [Reference Zhang, Zhou, Ding, Zhou, Sun and Hu315–Reference Busija, Lacza, Rajapakse, Shimizu, Kis and Bari317]. Finally, one team of researchers has produced tantalizing evidence of an association between the opening of K_ATP channels and the inhibition of astrocyte activation and the prevention of astrogliosis [Reference Cao, Dai, Zhang, Chen, Yang and Hu318].