Phospholipid, arachidonate and eicosanoid signaling in schizophrenia

This paper reviews the potential role of arachidonic acid in the pathophysiology of schizophrenia. We discuss how abnormal levels of arachidonic acid may arise, and how dysregulation of signaling molecules derived from it have the potential to disrupt not only dopamine signaling, but numerous other physiological processes associated with the illness. Pharmacological doses of niacin stimulate the release of arachidonic acid; and arachidonic acid-derived molecules in turn dilate blood vessels in the skin. A blunted skin flush response to niacin is reliably observed among patients with schizophrenia. The niacin response abnormality may thus serve as a biomarker to identify a physiological subtype of schizophrenia associated with defective arachidonic acid-derived signaling.


Introduction
Schizophrenia (SZ) is a complex behavioral syndrome associated with diverse biochemical and physiological abnormalities.This paper will describe how arachidonic acid (AA) and abnormalities related to its metabolism may to some extent unify some seemingly unrelated biochemical findings in SZ (Horobin, 1998;Skosnik and Yao, 2003).We will also describe how a blunted skin flush response to niacin may serve as a biomarker for AA-related signaling defects, and suggest that AA-related abnormalities may represent a distinct physiological subtype within the SZ syndrome (Messamore, 2003;Messamore et al., 2010;Yao et al., 2015).

Heterogeneity of the Schizophrenias
Despite early acknowledgement of the likelihood that SZ is not a unitary illness (Bleuler, 1920), for most of the 20th century it was usually studied as if it were a single disease entity.The dominant view was that of a unitary illness with differing modes of expression.This view of SZ, however, is no longer tenable.The variable therapeutic response to antipsychotic medication provides strong evidence against a unitary cause of SZ (Garver et al., 2000).In fact, the preponderance of evidence is consistent with SZ having numerous, etiologically distinct causes (Jablensky, 2006).Efforts toward understanding its various causes, and toward developing improved, etiologically-focused treatments, may be more fruitful if the schizophrenias were deconstructed into physiologically-based Fig. 1.An overview of phospholipids turnover, arachidonic acid, and eicosanoid signaling in schizophrenia symptomatology (adapted from Skosnik and Yao, 2003).Abbreviations: AODS, antioxidant defense system; NT, neurotransmitters; RO, reactive oxygen species; apoD, apolipoprotein D; PLA 2 , phospholipase A 2 ; PLC, phospholipase C; LOX, lipooxygenase; AA, arachidonic acid; DAG, diacylglycerol; COX, cyclooxygenase; 2-AG, 2-arachidonoyl glycerol; GAP, growthassociated protein; PKC, phosphokinase C; PGG 2 .prostaglandin G 2 ; PGH 2 , prostaglandin H 2 ; PGD 2 , prostaglandin D 2 ; CB, cannabinoid.
intermediate phenotypes (Braff et al., 2007;Keshavan et al., 2008).We and others have observed evidence suggesting that one of these intermediate phenotypes may involve phospholipid signaling abnormalities that prominently involve dysregulation of AA levels or the actions of its physiologically active metabolites.

Phospholipid, arachidonate and eicosanoid (PAE) signaling in SZ
A phospholipid turnover abnormality is present in many cases of SZ.Disordered phospholipid turnover with resulting changes in the levels and metabolic destinations of AA can unite seemingly unrelated neurochemical and clinical observations in SZ.
A model relating phospholipid signaling to SZ-relevant neurochemical abnormalities can be found in Figure 1.As shown, several factors -including oxidative stress and cytokine release -have the potential to lyse membrane phospholipids, resulting in the release of AA and, over time, reduce its cell membrane levels.Overactivity of phospholipase A 2 (PLA 2 ) or phospholipase C (PLC) enzymes would deplete the membrane-lysable pool of AA.In turn, changes in the availability of AA and AA-derived signaling molecules affect the release or circulating levels of several neuroactive molecules, including: GAP-43; the neurotransmitters dopamine and glutamate, and the endocannabinoids anandamide and 2-arachidonoylglycerol (2-AG).Alterations in AA may also affect the inflammatory response, which can then affect PLA 2 release via cytokines, further exacerbating phospholipid turnover and AA release.These diverse disruptions have the potential to impact many neuronal signaling pathways relevant to psychosis (Horrobin, 1998;Peet et al., 1994;Skosnik & Yao, 2003).
Low levels of AA have been observed postmortem in the cerebral cortex of SZ patients (Horrobin et al., 1991;Yao et al., 2000).Low AA levels have been identified in red blood cell (RBC) membranes from patients with chronic SZ (Glen et al., 1994;Peet et al., 1994;Vaddadi et al., 1986;Yao et al., 1994a), as well as first-episode neuroleptic-naïve SZ (Avrindakshan et al., 2003;Reddy et al., 2004).Thus, abnormally low AA levels have been observed in brain as well as RBC membrane phospholipids from patients with SZ (further reviewed by Conklin et al., 2007;Peet, 2007;Skosnik and Yao, 2003;Yao, 2003).These changes could conceivably lead to decreased synthesis of eicosanoids.Collectively, these changes can account for numerous physiological and clinical observations in SZ.
In addition to the formation of second messengers, AA released from membrane phospholipids can be converted to a variety of biologically active metabolites, collectively termed eicosanoids, through the concerted reactions of cyclooxygenase (COX), lipoxygenase (LOX) and cytochrome P-450 (CYP).Eicosanoids can modulate neural cell function, and can mediate several pathophysiological processes (Bazan, 2006).Since AA is the major C20 polyunsaturated fatty acid (PUFA) in mammalian tissues, the prostaglandin-2 (PG2) and thromboxanes-2 (TX2) series are the predominant classes of eicosanoids (Fig. 2).Inhibition of COX by nonsteroidal antiinflammatory drugs has revealed the significance of PG2 in the regulation of nerve conduction, neurotransmitter release, inflammation, pain, fever, immune responses, apoptosis and psychosis (Akhondzadeh et al., 2007).

Clinical and physiological effects of AA and eicosanoids
AA deficiency, or abnormal regulation of AA-derived signaling could explain several clinical and physiological findings in SZ.Association between low RBC membrane AA levels and the expression of the negative symptom syndrome (apathy, social withdrawal, affective flattening, etc.) has been reported (Glen et al., 1994).In other studies, low RBC membrane levels of dihomo-γ-linolenic acid, the immediate precursor of AA, were associated with increased psychosis ratings (Yao et al., 1994b) and disorganized symptoms (Doris et al., 1998).Among SZ patients receiving fatty acid supplementation therapy, increased RBC AA contents were associated with a reduction in the severity of symptoms (Peet et al., 2002).
Aspirin inhibits vascular dilation during electrical stimulation of cerebral cortex slices in vitro (Zonta et al., 2003); and pharmacological inhibition of COX-2, or COX-2 gene knockout, reduces by 40 to 50% the cerebral blood flow response of the sensory cortex to whisker stimulation in mice (Niwa et al., 2000).Thus, an eicosanoid signaling abnormality could potentially contribute to the cerebral neurovascular coupling defects that have been repeatedly observed in SZ (Berman et al., 1992;Liddle et al., 1992;Weinberger et al., 1992).
Many signs and symptoms of SZ are suggestive of excess dopamine activity (Snyder, 1981).Prostaglandin D 2 (PGD 2 ) and other PGs stimulate the production of cAMP and thereby exert functional antagonism of dopamine-D 2 receptors, which modulate inhibition of cAMP synthesis (Ono et al., 1992;Wanibuchi and Usuda, 1990).PGD 2 therefore counteracts the biochemical and behavioral effects of dopamine in vitro and in vivo.PGD 2 administration blocks the behavioral effects of dopaminergic agents such as apomorphine, L-DOPA, and amphetamine; and induces catalepsy in a manner identical to dopamine-D 2 receptor antagonists such as haloperidol (Ono et al., 1992;Wanibuchi and Usuda, 1990).Deficient PGD 2 signaling would thus be expected to produce the signs of excessive dopamine activity that are seen in SZ.A role for PGs as endogenous anti-dopaminergic agents is supported by the findings that inhibitors of PG synthesis can cause psychotic symptoms in some people and can worsen psychotic symptoms in SZ patients (Hoppman et al., 1991;Sussman and Magid, 2000).The relationship between SZ and COX products is complex, however, as there are also reports suggesting that COX inhibitors may augment the efficacy of antipsychotic medications (Akhondzadeh et al., 2007;Laan et al., 2010;Müller et al., 2002).These seemingly contradictory findings nonetheless speak to the relevance of prostaglandins to the pathophys-iology of the schizophrenias while also pointing toward their etiologic diversity.
A deficiency of prostaglandins has previously been related to SZ (Horrobin, 1977).It is likely that reduced AA availability may in part explain a variety of clinical observations that are usually ignored by the receptor-based etiological hypotheses (Horrobin, 1998).For example, in SZ, there appears to be a lower risk of arthritis and other inflammatory diseases (Eaton et al., 1992), greater resistance to pain (Davis et al., 1979, Horrobin, 1977), and remission of psychosis during fever has been observed (Horrobin, 1977).These effects could be secondary to reduced eicosanoid signaling.

Niacin Flush response as a test for eicosanoid abnormalities
Niacin (nicotinic acid), at sufficient doses, is well-known to dilate blood vessels in the skin resulting in a visible skin flush.The biochemical mechanism (Fig. 3) of this response has been studied over the course of several decades so many of the key steps have been well described.Niacin-induced skin flushing is prominently mediated by metabolites of AA.Thus, it may serve as a physiological marker of the integrity of phospholipid-arachidonate-eicosanoid (PAE) signaling.We will now describe how the niacin response abnormality might define a physiologically distinct subtype of SZ.

Mediation of niacin flush by AA metabolites
The levels of PGD 2 and 9α,11β-PGF 2 (a stable metabolite of PGD 2 ) rise markedly in skin exudate or venous blood following oral or topical administration of niacin in humans (Eklund et al., 1979;Kobza Black et al., 1982;Morrow et al., 1992).Evidence favors PGD 2 as the predominant mediator of the flush response (Morrow et al., 1989).However PGE 2 levels also increase in response to niacin (Eklund et al., 1979;Kobza Black et al., 1982).In man, the flush response to niacin can be completely abolished by COX inhibitors such as aspirin or indomethacin (Svedmyr et al., 1977;Wilkin et al., 1982).In mice, the flush response to niacin can be eliminated by knocking out genes for either the niacin receptor or COX-1, consistent with an exclusive and obligatory role for this pathway in niacin-evoked skin flushing (Benyó et al. 2005).Classical vasodilatory mediators, such as histamine, bradykinin, serotonin, and acetylcholine do undergo changes in their skin transudate levels after niacin challenge (Morrow et al., 1992;Plummer et al., 1977).However, Papaliodis et al. (2008) showed that niacin induced the release of serotonin from human platelets in vitro and elevated blood levels of serotonin after intraperitoneal injection of niacin in rats.Although further study will be needed to clarify the possible role of serotonin, the vast majority of evidence points to a strongly predominant role of AA metabolites as primary mediators of the flush response to niacin.Other eicosanoid vasodilators, however, have not been well studied in SZ.

Mechanism of the niacin flush response
A model of our current understanding of the mechanism of niacin-induced skin flushing is illustrated in Figure 3. Niacin interacts with a specific G-protein coupled receptor, HM74a, located on dermal macrophages and adipocytes (Benyó et al., 2006;Urade et al., 1989); its activation by niacin stimulates PLA 2 mediated formation of AA (Tang et al., 2006).Formation of AA is the rate-limiting step in the biosynthesis of the vasodilatory PGD 2 and E2 (PGE 2 ) (Murakami and Kudo 2004) These prostaglandins bind to specific prostanoid receptors on vascular smooth muscle within the skin.Activation of prostanoid receptors dilates cutaneous blood vessels (Lai et al., 2007), and a visible skin flush arises from the ensuing increased blood flow (Benyó et al., 2005;Maciejewski-Lenoir et al., 2006;Morrow et al., 1989Morrow et al., , 1992)).

The abnormal niacin response in SZ
Abrams Hoffer first reported observations that a substantial portion of patients with SZ are unusually resistant to the skin flush effect of pharmacologically-dosed niacin (Hoffer, 1962).Citing this work, and supporting these observations with clinical evidence of prostaglandin defects in the illness, Horrobin (1980aHorrobin ( , 1980b) ) proposed that the niacin response abnormality could be used to demonstrate a prostaglandin signaling abnormality in SZ Abnormally attenuated skin flushing in response to niacin has been very widely replicated in samples of patients with SZ (Messamore, 2003;Smesny et al., 2003;Yao et al., 2015).The niacin response abnormality is consistently overrepresented in SZ compared to both healthy control groups (Messamore, 2003), or psychiatrically ill comparison groups with major depression (Bosveld-van Haandel et al., 2006) or bipolar disorder (Liu et al., 2007;Ross et al., 2004;Yao et al., 2015).
Consistent with the view that SZ is an etiologically heterogenous disorder, the niacin response abnormality is not present in all patients with SZ.The prevalence of the niacin abnormality was pegged at 80 to 90% in some studies (Puri et al., 2001;Ward et al., 1998).However, we have observed a prevalence of about 30% in SZ patients in two separate experiments, and 20% in first-degree relatives of patients with SZ (unpublished data).There has been very little consistency across reports with respect to the methods to stimulate or measure blood flow.This methodological variability accounts for the disparity of estimates of the prevalence of the niacin response abnormality in SZ.If, however, the same methods are used, there is remarkable consistency in the ability to find higher rates of abnormality among SZ patients versus other clinical or healthy comparison groups.

Biochemical correlates of abnormal niacin response
Lower RBC membrane AA levels are associated with decreased niacin sensitivity, but this expected correlation has only been described in a healthy control group (Messamore et al., 2010).Although lower AA levels were found in patients who did not flush in response to oral niacin (Glen et al., 1996), no correlation between AA levels and niacin response was observed in two different groups of patients with SZ (Maclean et al., 2003;Messamore et al., 2010).On the other hand, an unexpected correlation was observed between maximal niacininduced blood flow and red blood cell membrane levels of adrenic acid (22:4 n-6), the elongation product of AA.Adrenic acid has vasodilatory actions in bovine coronary arteries and in arteries of the cortical layer of the adrenal gland (Kop et al., 2010).Lack of correlation between AA levels and niacin response may result from a homeostatic imbalance within the n-6 PUFAs pathway in SZ.The significance of adrenic acid levels as they relate to niacin-induced blood flow response deserves further attention.
RBC membrane levels AA are normally tightly correlated with its immediate elongation product adrenic acid (22:4 n-6).However, this expected correlation is abolished in SZ (Messamore et al., 2010).Similar precursor-product dysregulations have also shown in tryptophan (Yao et al., 2010b) and purine (Yao et al., 2010a) pathways.Although many of these correlated relationships persist across disease or medication status, others are lost among patients with SZ.

Clinical correlates of abnormal niacin response
The extent to which abnormal niacin-induced skin flushing associates with clinical manifestations of SZ has recently been reviewed (Messamore, 2012).Impaired niacin-induced flushing has been associated with greater severity of both positive and negative symptoms of SZ (Berger et al., 2002;Glen et al., 1996;Hudson et al., 1997;Smesny et al., 2003).Niacin response impairment was significantly linked to inorganic phosphate levels revealed by 31 P magnetic resonance spectroscopy (Puri et al., 2007).This suggests that niacin-abnormal SZ patients may have higher levels of cerebral energy metabolism.Niacin response, as reflected in the 'volumetric niacin response D112, page 4 of 8 E. Messamore and J.K. Yao: OCL 2016, 23(1) D112 score' appears much more impaired among SZ patients with a history of violence (Puri et al., 2007) compared to ordinary SZ patients (Puri et al., 2002).Niacin response impairment is associated with worsened global functioning among patients with SZ (Messamore, 2012).Additionally, the niacin response abnormality is associated with significant cognitive impairment in SZ (Nilsson et al., 2015).

The niacin response abnormality is not an artifact of medication or smoking status
Evidence suggests that neither antipsychotic drugs nor smoking significantly affect the niacin skin flush response (Mills et al. 1997;Shah et al. 2000;Smesny et al. 2001;Turenne et al. 2001).The niacin skin flush is not affected by local anesthetics, suggesting that local neurotransmitter release is not involved in niacin-induced vasodilatation (Winklemann et al., 1965).There is no correlation between antipsychotic drug dose and niacin sensitivity in patients with SZ (Hudson et al., 1997;Messamore, 2003).Neither has a significant difference in niacin sensitivity been found between medicated versus unmedicated patients (Shah et al., 2000).In contrast, bipolar disorder patients who take antipsychotic medications have a normal or even enhanced flush response (Hudson et al., 1997) Moreover, niacin subsensitivity occurs in first-degree relatives of schizophrenics, which suggests that this is a heritable trait, independent of medication status (Shah et al., 1999;Waldo, 1999).On the other hand, Tavares et al. (2003) reported that 4 out of 13 SZ patients with niacin subsensitivity became sensitive to niacin after 8 weeks of atypical antipsychotic drug treatment.If such drugs do tend to normalize niacin sensitivity, then the potential bias in patient sample would be to underestimate the prevalence or magnitude of the niacin response abnormality in SZ.Taken together, comparing the niacin-induced flushing between medicated and unmedicated SZ patients, or unmedicated, non-psychotic relatives, suggests that the niacin response abnormality in SZ is not an artifact of antipsychotic medications (Chang et al., 2009;Lin et al., 2007;Maclean et al., 2003;Shah et al., 2000).

Possible mechanisms for the niacin response abnormality in SZ
As depicted in Figure 3, there are several possible explanations for the finding of niacin subsensitivity in SZ.These include: abnormal signaling at the niacin receptor; abnormal presentation of free AA to COX; abnormal COX activity; abnormal conversion of initial COX products to vasodilatory end products; or abnormal signaling at prostanoid receptors in vascular smooth muscle.Support for abnormal niacin receptor signaling is provided by a report of decreased niacin receptor expression in postmortem brain from SZ patients (Miller and Dulay, 2008).
Presentation of free AA to COX is accomplished by the action of PLA 2 , which may be abnormally active in niacinsubsensitive SZ (Hudson et al., 1996;Tavares et al., 2003).Any process leading to diminished output of prostaglandins could potentially account for niacin subsensitivity.Abnormal COX activity in SZ was indirectly demonstrated by Das and Khan (1998).
Cyclooxygenase action on AA initially produces prostaglandin H 2 , which is subsequently converted by tissue-specific isomerases to vasodilatory end-products.Isomerase abnormalities have been detected in discrete brain regions from patients with a variety of mental illnesses (Maida et al., 2006).
It is intriguing to note that none of these pathways is specifically targeted by existing antipsychotic medications.Discovering the mechanisms responsible for the niacin response abnormality may inform the development of novel treatment strategies.Such treatments would likely involve targets outside the traditional monoamine receptors and thus would be expected to augment the efficacy of current medications.Elucidating the mechanism of the niacin response abnormality may also lead to physiologically-informed categorization of mental illness.

Conclusions
There is a compelling need to categorize the schizophrenias according to physiological criteria.Ideally such physiologically-defined categories would lead more easily than the older, traditional schema to insights about biochemical or genetic causes − and would suggest more tailored and effective treatments.Although this goal is desirable, there have been relatively few leads as to which physiological abnormalities may be more promising to follow.
We suggest that the niacin skin flush response abnormality is a viable candidate for the physiological subtyping of SZ In contrast to the incompletely-understood etiology of SZ, the mechanism of niacin-induced skin flushing in man is relatively well-characterized.This detailed knowledge, coupled with the accessibility of skin for scientific study, presents a technically feasible, straightforward, and economically attractive opportunity to broaden our understanding of the biochemical changes that accompany SZ.
It is intriguing to note that none of the biochemical pathways mediating the niacin response is specifically targeted by existing antipsychotic medications.Discovering the mechanisms responsible for the niacin response abnormality may inform the development of novel treatment strategies.Such treatments would likely involve targets outside the traditional monoamine receptors and thus would be expected to augment the efficacy of current medications.As the metabolic and genetic underpinnings of this biomarker are elucidated, it should yield valuable insights into the complex pathophysiology of schizophrenic illness.