*5.2. Inositols as Osmolytes in Astrocytes*

Inositols are described as osmolytes in plants, protecting them from heat, high salinity, and drought stress [33]. In mammals, higher concentrations of inositols in the brain, with respect to the peripheral tissues, stand mainly for their osmoregulatory role in astrocytes, as multinuclear NMR studies have suggested [166]. SMIT gene expression is increased under osmotic stress conditions and rapidly decreased when iso-osmolarity is reestablished [167]. This mechanism allows astrocytes to adapt their size in order to reduce the impact of an ionic imbalance in extracellular media. In hypotonic conditions, the volume-sensitive organic osmolyte anion channel (VSOAC) mediates the rapid efflux of inositol along with other osmolytes, leading to cell shrinkage [168].

This property of MI fluctuation in glial cells has led to its correlation as a widely accepted marker for astrogliosis. An 1H-MRS study in brains showed reduced MI levels after a traumatic brain injury, which was later normalized over time [169]. This decrease in brain MI may be a result of astrocyte cell death or either a mechanism of osmoregulation to prevent the development of brain swelling [170]. However, the activation of microglia and astrocytes leads to the accumulation of osmolytes and increased cell size, reflecting a higher MI content [171]. MI accumulation in astrogliosis is also a common feature observed during aging in the hippocampus and cortex [172], which seems to reflect changes in astroglia cell metabolism, a chronic inflammatory state, and oxidative stress [173,174].

As observed during aging, elevated MI levels in the brain are also observed during previous stages of mild cognitive impairment (MCI), AD, and pre-AD patients with Down's syndrome (DS), with a negative correlation between MI levels and cognitive performance [154–157]. Altered MI levels are also observed with bipolar disorder [175]. Changes observed in MI levels localized in the hippocampus and frontal cortex, involved several functions, including memory and task decision, make MI a valuable biomarker of early stages of cognitive decline as it can be detected in vivo in early stages of cognitive decline and correlates time-dependently with its development.

### *5.3. Changes in Inositol Derivatives and Excitability in Neurons*

It is well established that inositols are osmoregulators in astrocytes. Since this may also be true for neuronal populations, an important role of SMIT transporters and inositol transfer within the cell has been found as intrinsic modulators of neuronal excitability.

Membrane potential is highly influenced by the efflux of K<sup>+</sup> through ion-, voltage-, or metabolic-dependent channels, which are responsible for the inhibition of cell excitability. As a substrate, the inositol derivative PIP2 acts as a "metabolic sensor" and interacts with the Kir6.2 and SUR1 subunits of ATP-sensitive K<sup>+</sup> channels (KATP), stabilizing the open state of the channel (as opposed to ATP, which stabilizes the closed state of the channel), which is an important mechanism of the pancreatic release of insulin [176–179]. In neurons, excitability is tightly controlled by G protein-gated inwardly rectifying potassium (GIRK) channels and muscarine-sensitive voltage-gated potassium (Kv) channels, where KCNQ2 and KCNQ3, forming the heteromer KCNQ2/3, are major contributors in the hippocampus for M-current, a non-inactivating K<sup>+</sup> that defines phasic versus tonic firing [159,160]. As an intracellular signaling molecule, PIP2 promotes the opening of GIRK [180] and KCNQ2/3 [181,182]. According to this, the KCNQ2/3 current is inhibited when PIP2 is depleted via the activation of Gq-coupled receptors and the subsequent PLC cleavage of PIP2 [181].

Regarding the later information, PIP2 is necessary for the modulation of KCNQ2/3 heteromer activity, but recent data point to a SMIT-mediated control of channel function. SMIT1 and SMIT2 interact physically and colocalize with KCNQ2/3 in sciatic nerve nodes of Ranvier and in axon initial segments [158,183]. In the absence of MI, SMIT1 co-expression with KCNQ2/3 modulates channel ion selectivity, gating, and pharmacology, making KCNQ2/3 less sensitive to extracellular K+ and promoting M-current [183]. However, when MI is added, KCNQ2/3 currents augment with a seemingly increased PIP2 synthesis [158]. This fact is supported by increased PIP and PIP2 levels in hypertonic medium and faster cell recovery after drug-mediated suppression of KCNQ2/3 current [141]. Thus, both SMIT and MI synergistically promote hyperpolarizing currents mediated by KCNQ2/3. Interestingly, GABA modulates KCNQ2/3 in a similar way as SMIT1. The activation of KCNQ2/3 seems to be mediated by SMIT1 through KCNQ2, whereas GABA communicates with KCNQ3. GABA binds to a S5 residue in both KCNQ2 and KCNQ3, mediating a conformational change that leads to loss of SMIT1 influence over KCNQ2/3. Since SMIT1 also binds the same S5 residues, its co-expression reduces GABA potentiation with respect to KCNQ2/3. Thereby, the presence of GABA decreases SMIT1 influence over KCNQ2/3 and vice versa as a negative feedback mechanism [160].

The finding that inositol regulates neuronal excitability is especially relevant regarding correlations of altered KCNQ conductance in several models of neurodegenerative diseases. Alzheimer's disease (AD) has been correlated to a reduced expression of the GirK2, GirK3, GirK4, KCNQ2, and KCNQ3 subunits and genes encoding for the antioxidants SOD, 8-oxoguanine DNA glycosylase (OGG1), and monoamine oxidase A (MAO-A) in rats [184]. Familial-inherited epilepsy has also been associated with mutations in KCNQ2 and KCNQ3 [185–187]. The expression and function of KCNQ2 [188] and KCNQ3 [189] are both also altered in cases of bipolar disorder. Crosstalk between SMIT/MI and KCNQ channels is an important issue, as changing intracellular levels of MI and thus PIP2 levels may modulate neuronal excitability in the hippocampus. The fact that many neuropathologies are related to either SMIT- or KCNQ2/3-altered function highlights the importance of inositol in normal neuronal function.
