**5. Therapeutic and diagnostic approaches for SCZ: Focus on astrocytes**

#### **5.1 Therapeutic targets for SCZ**

Astrocytes are involved in numerous critical physiological processes in the brain, which directly or indirectly contribute to the pathogenesis of SCZ, including receptor trafficking, development and maturation of synapses, synaptic glutamate metabolism, regulation of CNS homeostasis, maintenance of integrity of the blood-brain barrier (BBB), nutrient provision to neural tissues, and regulation of neurogenesis. Based on data showing astrocyte involvement in the pathology of SCZ, astrocytes should be considered as therapeutic targets for treating this disease. In SCZ, abnormalities in neurons and neurotransmitters mainly result from the malfunction of astrocytes. Hence, correct functioning of astrocytes is required for the processes of synaptic activity and synaptic plasticity within neural networks, which is activity necessary for normal cognitive functions [155]. Therefore, targeting pathways associated with astrocytes' abnormal function may help ameliorate SCZ complications.

As hypofunctionality of NMDA receptors is believed to be one of the leading causes of SCZ, modifying NMDA receptors' function can be considered a therapeutic strategy for SCZ treatment. Targeting astrocytic glutamate reuptake presents a viable strategy for increasing glutamate sufficient to restore NMDA functionality [156]. Clozapine decreases glutamate reuptake through downregulation of glutamate transporter (GLT1) in astrocytes resulting in ameliorating hypofunctionality of NMDA receptors [157].

In addition to glutamate, several small molecules that can affect NMDA receptor functioning are linked to astrocyte activity. In preliminary trials, the NMDA receptor co-agonists glycine and D-serine have been applied as well as D-cycloserine [158]. An encouraging effect of high-dose D-serine administration was seen in SCZ patients [159]. Unfortunately, while small studies showed promising results, more extensive

trials indicated no significant difference between intervention and placebo groups when D-serine was exogenously applied [160].

An alternative approach to enhance D-serine levels in the synaptic space is to inhibit its metabolism in astrocytes. D-serine is metabolized by D-amino acid oxidase (DAAO) in astrocytes; thus, DAAO inhibitors may enhance NMDA activity by increasing endogenous D-serine concentrations. However, none of the identified human DAAO inhibitors have been approved for use in SCZ patients. Low bioavailability, high clearance rate, and inability to cross the BBB are considered the primary restrictions of these inhibitors [161].

Strategies focused on suppressing glycine reuptake in order to increase glycine's extracellular concentration in the synaptic space where it might be able to enhance NMDA functionality have been implemented, and inhibiting glycine transporter 1 (GlyT1) in astrocytes has been one approach considered for management of some SCZ symptoms. Among various GlyT1 inhibitors that have been trialed, only bitopertin has reached phase III clinical trials [155]. However, lack of efficacy has led to the discontinuation of its development as an antipsychotic [162].

In addition to glycine and D-serine, Kynurenic acid (KYNA), a metabolite of tryptophan degradation in astrocytes, can influence the function of NMDA receptors. KYNA has a preferential affinity for the NMDA receptor and can inhibit NMDA activity. According to the direct relationship between KYNA concentration and cognitive impairments in SCZ, interventions that lower brain KYNA levels may be clinically beneficial. Regrettably, at the current time, it is not feasible to target degradative enzymes or reuptake sites to enhance the removal of excess KYNA from its effector site in the brain. Moreover, exploiting the ability of depolarization events or cellular energy scarcity to reduce cerebral KYNA production is not possible. Pharmacological kynurenine aminotransferase (KAT) inhibitors are the most effective strategy to reduce KYNA production in the brain. The practicality of this approach is supported by findings that the nonspecific aminotransferase inhibitor aminooxyacetic acid readily prevents cerebral KYNA neosynthesis *in vivo*. In this regard, KAT II is the preferential target to suppress KYNA synthesis in the brain due to its high specificity toward kynurenine [158]. According to previous preclinical studies, the administration of selective KAT II inhibitors could successfully reduce extracellular KYNA levels in various rat brain regions. When taken together, considering KYNA's inhibitory effects on several neurotransmitters with a critical role in cognitive processes, any therapeutic agent or intervention that decreases KYNA levels or otherwise hinders KYNA function in the brain may lead to cognitive enhancement in SCZ or other psychiatric disorders, and the data suggest that KAT II inhibitors or pharmacological agents that weaken the function of KYNA at its receptor(s) have a high potential to be used for cognitive deficits in SCZ [158].

Astrocytes can also be a target to repair synaptic functions by moderating their effects on glycogen/lactate metabolism, as glucose uptake into astrocytes is reduced in SCZ due to a decrease in glycolysis and decreased lactate production. The decrease in lactate could have a profound effect on reductions in neurogenesis. Therefore, modifying glycogen/lactate metabolism sufficient to compensate for reduction of lactate could facilitate lactate-mediated neurogenesis, and lead to improvement of behavioral deficits in SCZ patients [163].

Reducing inflammation resulting from astrocytes can also be considered a therapeutic approach for SCZ and this approach has been shown to improve SCZ symptoms. For instance, minocycline, an antibiotic with anti-inflammatory effects, induced improvements in some SCZ patients. COX2 inhibitors, which are non-steroid anti-inflammatory drugs, have been shown to improve SCZ symptoms.

Lending support to the effectiveness of this strategy, many antipsychotic drugs exhibit anti-inflammatory effects, which could be important in their therapeutic efficacy. Given the link between inflammation and SCZ, a clear understanding of the cytokines involved in SCZ and the role played by astrocytes in linking inflammation and SCZ could lead to therapeutic strategies [156].

#### **5.2 Diagnostic approach for SCZ**

Postmortem studies identified significant changes in astrocyte density and morphology, as well as deregulated expression of several common astrocyte markers, including glial fibrillary acidic proteins (GFAP), aquaporin 4 (AQ-4), S100, glutaminase, thrombospondin (TSB-1), and excitatory amino acid transporter 2 (EAAT2) [22, 164–166]. When taken together, while data are suggestive of a role of altered astrocytic function in SCZ, the findings do differ, with some studies indicating a drop in marker levels and the number of astroglial cells compared to controls and others a rise. Although dysregulation in developing astroglial cells may have profound effects on the formation and maturation of neuronal networks, few studies have examined the status of astroglial cells during postnatal brain development, instead focusing on the postmortem examination of adult brain tissues [36]. Due to confounding factors associated with the use of postmortem tissues, differences in the brain regions evaluated, variety in the severity of the disease, and disparities in pharmacological treatments, it remains to be determined what the contribution of these markers to the disease is and if they play a role, at which developmental stage their role is most important. In light of the profound changes in astrocytic morphology and function, monitoring of alterations in astrocytes has been considered a diagnostic approach in SCZ. However, it is difficult to know what can easily be monitored from tissue noninvasively extracted in patients, which reflects astrocytic status. At the present time, identification of peripheral biomarkers that reflect neuropathological changes in SCZ has received a great deal of interest and in this arena, exosomes have been a focus of study as they are relatively easy to detect and have been proposed to be involved in psychiatric disorders [167]. Intriguingly, it is possible to identify the parent cell from which exosomes source.

Exosomes are nano-sized extracellular vesicles containing nucleic acids, proteins, lipids, and other bioactive substances secreted by cells into the surrounding body fluids, which regulate cellular communications in addition to neuroplasticity [168], trafficking of microRNA (miRNA) [169], and neuroinflammation [170, 171]. They can cross the BBB and be assayed peripherally, Exosomes derived from astrocytes would be expected to exhibit changes across the progression of SCZ. As proof of concept that exosomes can be detected and traced back to their parent cell, a high concentration of exosomal GFAP, resulting from astrogliosis was detected in plasma obtained from SCZ patients [172]. Thus, astrocytes-derived exosomes have the potential to be used for SCZ diagnosis and assessment of disease progression. However, further studies are needed to clarify to what extent circulating exosomes can serve as novel peripheral biomarkers of SCZ.
