Neuroactive steroids and diabetic complications in the nervous system

Accepted Manuscript Review article Neuroactive steroids and diabetic complications in the nervous system S. Giatti, R. Mastrangelo, M. D'Antonio, M. Pesaresi, S. Romano, S. Diviccaro, D. Caruso, N. Mitro, R.C. Melcangi PII: DOI: Reference:

S0091-3022(17)30040-7 http://dx.doi.org/10.1016/j.yfrne.2017.07.006 YFRNE 673

To appear in:

Frontiers in Neuroendocrinology

Received Date: Revised Date: Accepted Date:

24 April 2017 19 July 2017 20 July 2017

Please cite this article as: S. Giatti, R. Mastrangelo, M. D'Antonio, M. Pesaresi, S. Romano, S. Diviccaro, D. Caruso, N. Mitro, R.C. Melcangi, Neuroactive steroids and diabetic complications in the nervous system, Frontiers in Neuroendocrinology (2017), doi: http://dx.doi.org/10.1016/j.yfrne.2017.07.006

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FIN-17-29-R NEUROACTIVE STEROIDS AND DIABETIC COMPLICATIONS IN THE NERVOUS SYSTEM Giatti S.1, Mastrangelo R. 2, D'Antonio M.2, Pesaresi M.1, Romano S.1, Diviccaro S.1, Caruso D.1, Mitro N.1, Melcangi R.C.1* 1Dipartimento

di Scienze Farmacologiche e Biomolecolari, Università degli Studi di Milano,

Milano, Italy; 2Division of Genetic and Cell Biology, San Raffaele Scientific Institute, DIBIT, Milano, Italy.

*Corresponding author: Roberto C. Melcangi, E-mail: [email protected], Telephone: +39 02 50318238, Fax: +39 02 50318202.

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Abstract Important complications of diabetes mellitus in the nervous system are represented by diabetic peripheral neuropathy and diabetic encephalopathy. In this context, an important link is represented by neuroactive steroids (i.e., steroids coming from peripheral glands and affecting nervous functionality as well as directly synthesized in the nervous system). Indeed, diabetes does not only affect the reproductive axis and consequently the levels of sex steroid hormones, but also those of neuroactive steroids. Indeed, as will be here summarized, the levels of these neuromodulators present in the central and peripheral nervous system are affected by the pathology in a sex-dimorphic way. In addition, some of these neuroactive steroids, such as the metabolites of progesterone or testosterone, as well as pharmacological tools able to increase their levels have been demonstrated, in experimental models, to be promising protective agents against diabetic peripheral neuropathy and diabetic encephalopathy.

Keywords: neurosteroids, sex steroids, progesterone, testosterone, streptozotocin, diabetic peripheral neuropathy, diabetic encephalopathy, myelin proteins, myelin lipids, sex.

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1.0 Introduction Diabetes mellitus is a worldwide emergency with 415 millions of people affected by the pathology; a number that will rise to 615 millions by 2040 (International Diabetes Federation, 2015). Diabetes is a pathological condition due to high blood glucose levels that are constantly above the normal range during the day. This pathological situation can be the result of the lack or reduced insulin secretion from pancreas or an improper insulin function on target tissues deputed to glucose disposal such as liver, skeletal muscle, adipose tissues and others; a phenomenon known as insulin resistance. The former diabetes mellitus is usually defined as type 1 diabetes (or insulin dependent diabetes mellitus, IDDM), usually diagnosed in children and young adults, as the result of an autoimmune destruction of the insulin producing beta cells in the pancreas. Type 1 diabetes accounts for 5% to 10% of all diabetic patients around the world. The latter kind of diabetes mellitus is also known as non-insulin dependent diabetes mellitus (NIDDM) or type 2 diabetes and represents the most common form of diabetes (85% to 90% of all cases of diabetes). People with either type 1 or type 2 diabetes have an increased risk of developing a number of severe health problems. Indeed, high blood glucose levels can lead to different complications affecting heart, blood vessels, kidneys, and the central (CNS) and peripheral (PNS) nervous system. In almost all high-income countries, diabetes is a leading cause of cardiovascular disease, blindness, kidney failure, and severe complications of nervous system leading to development of encephalopathy, retinopathy and peripheral neuropathy. Here, we review some of the main consequences of type 1 and type 2 diabetes in the human nervous system as well as in animal models with particular attention to important regulators of the nervous function, such as the neuroactive steroids.

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2.0 Effects of Diabetes Mellitus in the nervous system An important complication of diabetes mellitus is represented by the damage in the PNS (i.e., diabetic peripheral neuropathy) and CNS (i.e., diabetic encephalopathy). Diabetic peripheral neuropathy occurs in more than 50% of all diabetic patients (Zochodne, 2007) involving a spectrum of functional and structural changes in peripheral nerves. These include slowing in nerve conduction velocity (NCV) followed by axonal degeneration, paranodal demyelination and loss of myelinated fibers (Sugimoto et al., 2000; Vinik et al., 2000). Diabetic peripheral neuropathy is also reported in an animal model of type 1 diabetes, obtained by streptozotocin (STZ) injection. STZ is a chemical compound particularly toxic to the insulin-producing beta cells of the pancreas in mammals; upon administration STZ selectively kills pancreatic beta cells that will no longer respond to glucose induced insulin secretion resulting in a marked hyperglycemia. As reported in male STZ rats, alterations include myelin invaginations in the axoplasm (infoldings) and myelin evaginations in the Schwann cell cytoplasm (outfoldings) as well as alterations in myelin compaction such as abnormally wide incisures and abnormal separation of myelin lamellae (Veiga et al., 2006). In particular, the most abundant myelin abnormality is the presence of myelin infoldings in the axoplasm (Veiga et al., 2006). In agreement, two important myelin components, such as proteins and lipids, are strongly affected by diabetes (for details, see section 5.0). Decrease of Na+,K+-ATPase activity in peripheral nerves and reduced intra-epidermal nerve fiber density associated with impaired nociceptive threshold have also been well described in both sexes of humans and animal models (Bianchi et al., 2004; Biessels et al., 1999; Lauria et al., 2005; Yagihashi, 1997). Pathological mechanisms implicated in diabetic peripheral neuropathy are microvascular damage, increased non-

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enzymatic glycation/glycoxidation of proteins, enhanced activation of the polyol pathway and increased reactive oxygen species concentrations (Zychowska et al., 2013). Mitochondrial dysfunction also occurs in diabetic peripheral neuropathy. Indeed, structural (i.e., number and size) as well as functional (i.e., respiratory chain activity, biogenesis, reactive oxygen species) abnormalities of mitochondria have been reported in human as well as in several experimental models of diabetic peripheral neuropathy (Fernyhough, 2015). An important component of diabetic peripheral neuropathy is represented by neuropathic pain. Neuroinflammation, altered neurotransmission mediated by excitatory amino acids and release of neuropeptides may be involved in neuropathic pain. For instance, hyperexcitability and spontaneous hyperactivity of primary afferents (Chen and Levine, 2001, 2003) and spinal dorsal horn neurons (Chen and Pan, 2002) as well as increase of the release of glutamate associated with hyperactivity of the post-synaptic glutamate receptor (Calcutt and Chaplan, 1997; Malcangio and Tomlinson, 1998; Tomiyama et al., 2005) were observed in the experimental model of STZ-induced diabetes. Indeed, the frequency of glutamatergic excitatory post-synaptic currents (EPSCs) and the amplitude of evoked monosynaptic and polysynaptic EPSCs in the dorsal horn of the spinal cord were significantly higher in diabetic than in control rats (Li et al., 2010). On this basis, it has been proposed that hyperexcitability of the dorsal horn neurons caused by the enhanced glutamate release may contribute to the maintenance of diabetic neuropathic pain (Chen and Pan, 2002; Chen et al., 2009). Neuropeptides, like for instance substance P, have also an important role in spinal nociceptive processing (Millan, 1999; Ribeiro-da-Silva and Hokfelt, 2000). Indeed, in response to substance P, microglia and astrocytes produce and secrete proinflammatory cytokines that contribute to the development and maintenance of central sensitization and pain by amplifying the noxious neurotransmission (Milligan and Watkins, 2009; Watkins and Maier, 2003).

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The interest in the effects exerted by diabetes on the brain is growing due to its association with cognitive deficits and increased risk of dementia, stroke, cerebrovascular and Alzheimer disease, as well as psychiatric disorders (Biessels and Reijmer, 2014; Gispen and Biessels, 2000). Indeed, diabetes may induce neurophysiological and structural changes in white and gray matter of the CNS, like for instance swollen axonal synaptic boutons and axonal fragmentation of neurofilaments (Hernandez-Fonseca et al., 2009), swelling of axons and dendrites (Zhou et al., 2013), impairment of axonal transport (Baptista et al., 2013) as well as alterations of myelin membranes (Hernandez-Fonseca et al., 2009) and its components (i.e., lipids and proteins) (Kawashima et al., 2007; Pesaresi et al., 2010a). Many factors, such as decreased insulin secretion or action, dysregulation of glucose homeostasis, obesity, hyperleptinemia,

increased

of

glucocorticoids,

neuroinflammation,

impaired

neurotransmission, oxidative stress, apoptosis and mitochondrial dysfunction have been proposed to cause diabetes-associated cognitive decline (Gaspar et al., 2016). Type 1 diabetic subjects, mainly children, perform poorly in school compared to healthy classmates showing reduced performance and intelligence quotient. This decline was observed in diabetic boys diagnosed before the age of six but not in those diagnosed later and not in diabetic girls (Schoenle et al., 2002). Therefore, this study concluded that cognitive decline due to type 1 diabetes was associated with the male sex, with the degree of metabolic deterioration at diagnosis and with high long-term average of glycated hemoglobin, while it was not correlated with the occurrence of severe hypoglycemic episodes (Schoenle et al., 2002). In support of this finding, a more recent study, that involved 1144 type 1 diabetic subjects, almost equally divided between males and females, demonstrated that neither frequency of severe hypoglycemia nor previous treatment-group assignment was associated with cognitive decline (Diabetes Control and Complications Trial/Epidemiology of Diabetes, Interventions Complications Study Research Group, 2007). Furthermore, Heikkilä and

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coworkers, using brain magnetic resonance imaging on male type 1 diabetes subjects, found higher glucose and myo-inositol levels in frontal white matter and cortex (Heikkila et al., 2009). Neuronal loss in frontal cortex and white matter deficit have been reported in diabetic STZ rat model (Jakobsen et al., 1987). In addition, other studies demonstrated a neurobehavioral decline. Indeed, male STZ-treated rats mainly showed a deficit in learning the procedures of the Morris water maze rather than impairments of spatial learning and/or hippocampal longterm potentiation as a measure of synaptic plasticity. These defects were largely prevented by insulin treatment (Biessels et al., 1996; Biessels et al., 1998). Moreover, defects in male STZ rats have been proposed to be related with pre- as well as post-synaptic changes in hippocampus (Kamal et al., 2006). Also, male CD1 mice, rendered diabetic by STZ injection, showed cognitive decline mainly due to white matter abnormalities and atrophy (Toth et al., 2006). Altogether these observations indicate that the effects of diabetes occur both in central and peripheral nervous system. They are very heterogeneous, most of the time difficult to diagnose and in some cases show sex difference. Despite several studies focused on the molecular mechanisms driving peripheral or central neuropathy a predictable marker to help diagnosis or a therapeutic approach focused on the causes of this diabetic complication are currently unavailable in an area demanding for therapeutic progress.

3.0 Diabetes and reproductive axis Fertility and sexual functions are affected by type 1 and type 2 diabetes, in men as well as in women (Codner et al., 2012; Schoeller et al., 2012). Reproduction represents a crucial function that has to be harmonized in relation to the energetic status of the organism. As such, the events related to a successful reproduction (pregnancy, parturition, lactation and 7

maternal behaviour) require great energy expenditure, and the organism has to monitor its resources. Moreover, metabolic stress situations, like for instance anorexia nervosa, prolonged fasting or exercise, lead to suppression of the reproductive axis (i.e., hypothalamuspituitary-gonadal axis, HPG axis) (Crown et al., 2007). These data suggest that, alongside classical feedback mechanisms, other peptides and hormones are involved in regulating reproduction. Diabetic condition may affect the HPG axis at multiple levels. Indeed, female type 1 diabetic patients experience menstrual disturbances, increased risk to develop polycystic ovaries and subsequent hyperandrogenism, and a precocious decline of ovarian function. In men, the pathological consequences of type 1 diabetes are decreased testosterone (T) levels, poor sperm quality, associated to an impaired spermatogenesis (Codner et al., 2012; Schoeller et al., 2012). Also in type 2 diabetic patients, reduced T levels have been observed (Gibb and Strachan, 2014). Moreover, both males and females reported sexual dysfunctions (Doruk et al., 2005; Hakim and Goldstein, 1996). As proposed, insulin plays a key role in the control of reproduction. Indeed, insulin may regulate the master regulator of the HPG axis (i.e., the gonadotrophin-releasing hormone, GnRH). For instance, luteinizing hormone (LH) secretion in response to GnRH pulse is altered in diabetic men when compared to controls (Baccetti et al., 2002; López-Alvarenga et al., 2002). Infertility, hypogonadotropic hypogonadism and metabolic disruption have been observed in a neuronal-specific knock-out mouse for the insulin receptor (Brüning et al., 2000). In diabetic animal models, hyperglycemia produced a decrease in LH pulse frequency that could be reversed by insulin treatment (Burcelin et al., 2003; Dong et al., 1991; Kovacs et al., 2002; Xu et al., 2009). Finally, cell culture studies of hypothalamic neurons also suggested that insulin could stimulate directly GnRH secretory activity (Gamba and Pralong, 2006; Kim et al., 2005; Pralong, 2010; Salvi et al., 2006). On the other hand, further evidence suggests

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alternative mechanisms. For instance, in sheep, GnRH neurons do not express insulin receptor (Cernea et al., 2016). Moreover, insulin receptor knock-out mouse, specific for GnRH neurons, displays normal cyclicity during the estrous cycle and no reproductive defects (Divall et al., 2010). Overall, these results suggest that the influence of insulin on GnRH neurons may be indirect and possibly mediated by other factors. A possible candidate could be kisspeptins, which are potent stimulators of the HPG axis (Gottsch et al., 2004; Irwig et al., 2004). Indeed, mutations in their receptor (i.e., GPR54) are associated with hypogonadotropic hypogonadism, both in humans and rodents (de Roux et al., 2003; Seminara et al., 2003). In the mammalian brain, kisspeptins are produced and secreted by two groups of neurons present in the preoptic region and the arcuate nucleus of hypothalamus (ARC). Additionally, the ARC neurons produce not only kisspeptins but also other factors, such as neurokinin B (NKB) and dynorphin (Lehman et al., 2010), and for this reason they are called KNDy (kisspeptin/NKB/dynorphin) neurons (Cheng et al., 2010). NKB and dynorphin role in GnRH pulses is established, however some controversies still remain in how this could be mediated (Goodman et al., 1995; Kinsey-Jones et al., 2012; Navarro et al., 2009). Kisspeptin expression is regulated by metabolic status. Indeed, diabetes reduces the gene expression of these molecules in hypothalamus (Castellano et al., 2006; Castellano et al., 2009), and their exogenous administration is able to rescue gonadotrophin secretion, T levels and to ameliorate reproductive functions in diabetic animals (Castellano et al., 2006; Castellano et al., 2009). Also NKB and dynorphin are controlled by metabolic status. Indeed, high fat diet (45% in fat) increased NKB mRNA in female rats ARC (Li et al., 2012), while dynorphin was increased in hypothalamus, pituitary and spinal cord of STZ-treated rats (Berman et al., 1995; Jolivalt et al., 2006; Kim et al., 1999). These data suggest that altered kisspeptin expression, and more generally of KNDy neurons response, could impair

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hypothalamic control of the HPG axis, leading to the hypogonadotropic hypogonadism, observed in diabetic models (Castellano et al., 2006; Castellano et al., 2009). However, whether insulin could represent the mediating signal for kisspeptins and hypothalamic regulation of HPG axis seems to be improbable. In vitro (Luque et al., 2007) and in vivo (Castellano et al., 2006; Navarro et al., 2012; Xu et al., 2009) studies failed to demonstrate a direct insulin activity on kisspeptin neurons able to stimulate GnRH signal. Nevertheless, other factors could be involved. In the ARC, insulin action could be mediated by two additional populations of neurons, such as those expressing agouti-related peptide and neuropeptide Y, and a different population expressing pro-opiomelanocortin and cocaine amphetamine related transcript (Billington et al., 1991; Butler et al., 2001; Franks et al., 2006; Morton et al., 2006; Ollmann et al., 1997). These neurons are engaged in the control of body weight, energy metabolism, and glucose homeostasis (Myers and Olson, 2012), and could be linked also to reproduction (Hill et al., 2008). Briefly, compelling evidence support that insulin could affect electrophysiological properties (Qiu et al., 2014; Sato et al., 2005) and function (Hill et al., 2010) of these neurons that, in turn, directly affect GnRH neurons (Backholer et al., 2010; Leranth et al., 1988; Norgren and Lehman, 1989; Roa and Herbison, 2012). In addition, also leptin could have a role. This hormone, produced by adipose tissue, signals to the hypothalamus the amount of fat in the body, playing a permissive role in reproduction (Cunningham et al., 1999; Tena-Sempere, 2007). A direct control of leptin levels by insulin has been demonstrated in vitro (Cammisotto and Bukowiecki, 2002; Wabitsch et al., 1996) and in vivo models (Kolaczynski et al., 1996). In uncontrolled type 1 diabetes in rodents (Sindelar et al., 1999) and in diabetic patients decreased levels of leptin have been detected before insulin therapy (Azar et al., 2002). As reported, central infusion of this adipose hormone in diabetic animals was able to restore kisspeptin expression, improving LH and sex steroid levels (Castellano et al., 2006).

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Overall, these data support the idea that the GnRH secretion is regulated also by metabolic sensors impaired in diabetic conditions, leading to alterations in reproductive functions. Besides the effects on hypothalamus, diabetes seems not to affect pituitary functions, confirming that hypogonadism derives from a hypothalamic failure (la Marca et al., 1999; South et al., 1993). However, hyperglycemia negatively impacts the gonads. In women before therapy, type 1 diabetic patients claimed amenorrhea and delayed menarche. On the other hand, with current insulin administrations some reproductive abnormalities, mostly due to insulin excess, are present in these patients. In a physiological situation, insulin stimulates theca cells to produce androgens, which will be subsequently aromatized into estrogens (Cara and Rosenfield, 1988; Codner and Escobar-Morreale, 2007; Poretsky et al., 1999; Poretsky and Kalin, 1987). Hyperinsulinemia will enhance this situation, leading to an increased risk of polycystic ovary syndrome and subsequent hyperandrogenism (Poretsky et al., 1992). Similarly, insulin affects testes function during development (Nef et al., 2003) and in the adult life (Pitetti et al., 2013). Moreover, this hormone increases testicular cell proliferation (Bobes et al., 2001) and plays a role in sperm cell capacitation (i.e., the process leading to a sperm cell the ability to fertilize) (Schoeller et al., 2012). Furthermore, in STZ rat model, a decreased testicular insulin expression was observed (Gómez et al., 2009). As a consequence of impaired insulin levels, poor sperm quality (i.e., sperm with higher nuclear DNA damage and fragmentation, and increased number of mitochondrial DNA deletion) has been observed in type 1 diabetic men (Agbaje et al., 2007; Karimi et al., 2012; Mallidis et al., 2007). Contextually with central hypogonadism and reduced gonadal functions, also sex steroid production is affected by diabetes. Female subjects may experience hyperandrogenism, as described above, while in men with type 1 or type 2 diabetes the situation is fairly different. Many reports correlated insulin (as insulin resistance or dysglycemia) with low T levels (Allan, 2014; Grossmann, 2014; Kim and Halter, 2014). In particular, type 1 diabetic patients

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present, with respect to the normal population, low free T, similar total T and increased sex hormones binding globulin levels (van Dam et al., 2003). Similarly, type 2 diabetic patients showed low free T levels, and this condition occurs more frequently than in type 1 subjects (Chandel et al., 2008; Liu et al., 2013). Moreover, obesity and insulin resistance, that are conditions associated with low T levels (Kim, 2009), as well as prolonged androgen deprivation therapy, have been linked to increased risk to develop type 2 diabetes (Alibhai et al., 2009; Basaria et al., 2006; Keating et al., 2010; Keating et al., 2006). In conclusion, due to the pleiotropic effects of insulin in the HPG axis, its dysregulation produces multiple effects on reproduction affecting both central and peripheral mechanisms.

4.0 Neuroactive steroid levels and diabetes mellitus Nervous function is not only under the control of steroids coming from peripheral glands (i.e., steroid hormones) but also by those directly synthesized in the nervous system (i.e., neurosteroids). Indeed, synthesis of pregnenolone (PREG), dehydroepiandrosterone (DHEA), progesterone (PROG), T and 17beta-estradiol (17-E) occurs in the CNS and PNS (Giatti et al., 2015a; Melcangi et al., 2008; Tsutsui, 2012). Moreover, PROG and T are metabolized into dihydroprogesterone (DHP) and dihydrotestosterone (DHT) respectively (Figure 1). These neuroactive steroids are then further converted by the action of 3alpha- (3-HSOR) or 3betahydroxysteroid oxidoreductase (3-HSOR) into further metabolites. In particular DHP is converted into tetrahydroprogesterone, also known as allopregnanolone (3,5-THP) or into isopregnanolone

(3,5-THP),

while

DHT

is

converted

into

5alpha-androstane-

3alpha,17beta-diol (3-diol) or 5alpha-androstane-3beta,17beta-diol (3-diol) (Giatti et al., 2015a; Melcangi et al., 2008). These metabolic pathways have a deep impact in the mechanism of action of T and PROG.

Indeed, DHT, like its precursor T, interacts with

androgen receptor (AR), but the further metabolites of DHT, 3α-diol and 3β-diol, act by 12

different mechanisms. Thus, 3-diol is a GABA-A receptor agonist, while 3-diol is an agonist of estrogen receptor beta (ER) (Handa et al., 2008; Melcangi et al., 2008). Similarly, DHP, like its precursor PROG, still interacts with PROG receptor (PR), but the further metabolites, 3,5-THP and 3,5-THP, modulate the activity of GABA-A receptor. In particular, 3,5THP activates GABA-A receptor (Belelli and Lambert, 2005; Lambert et al., 2003; Lambert et al., 2009), while 3,5-THP antagonizes the effect of 3,5-THP (Melcangi et al., 2008). All these steroids are included in the family of neuroactive steroids. Important concepts have recently emerged in this field of research. For instance, the nervous system regulates neuroactive steroid levels in adaptation to modifications in peripheral steroidogenesis (Caruso et al., 2010). Moreover, the neuroactive steroid pattern in the brain does not fully reflect that in the plasma (Caruso et al., 2013b). Furthermore, synthesis and levels of neuroactive steroids are different in central and peripheral nervous system and differently modified by nervous pathologies (Melcangi et al., 2014; Melcangi et al., 2016). For instance, as reported in the STZ-model, three months of diabetes decreased the levels of PREG, PROG, DHP, T and 3-diol in plasma while in cerebral cortex, in addition to these neuroactive steroids, also 3,5-THP, 3,5-THP and DHT are decreased (Pesaresi et al., 2010b). At variance to what observed in cerebral cortex, in sciatic nerve only the levels of PREG, T and its metabolites (i.e., DHT and 3-diol) are decreased by three months of diabetes (Pesaresi et al., 2010b). As recently reported, neuroactive steroid levels in the hippocampus are already altered after short-term diabetes (i.e., one month) and differently versus the plasma levels. Indeed, while in plasma only a decrease of 3,5-THP, T and 3-diol was observed, in the hippocampus a decrease of PREG, PROG, 3,5-THP, T and its derivatives (i.e., DHT and 3diol) was associated to an increase in 3,5-THP levels (Romano et al., 2017). The finding that the levels of PREG (i.e., the first steroid formed from cholesterol) was altered in hippocampus but not in plasma, suggested that short-term diabetes could affect neurosteroidogenesis. This 13

hypothesis may be confirmed by the observation that the gene expression of steroidogenic acute regulatory protein (i.e., molecule involved in the translocation of cholesterol into mitochondria) and of cytochrome P450 side chain cleavage (i.e., enzyme converting cholesterol into PREG) was decreased in hippocampus of STZ rat. In addition, an impairment of cholesterol homeostasis (i.e., the substrate for the steroidogenesis) and mitochondrial dysfunction (i.e., where the limiting step of neuroactive steroid synthesis takes place) was reported (Romano et al., 2017). Interestingly, levels of neuroactive steroids are also affected in cerebral areas of Alzheimer's disease patients (Marx et al., 2006) and in an experimental model of this pathology, such as the 3xTg-AD mice (Caruso et al., 2013a). A close association between diabetes mellitus and Alzheimer's disease has been proposed (Takeda et al., 2011). Indeed, extensive abnormalities of insulin and insulin-like growth factor signalling pathways also occur in this neurodegenerative disorder (Steen et al., 2005). Thus, alterations in neuroactive steroid levels might be interpreted as a common feature of the cognitive impairment occurring in diabetes mellitus and Alzheimer's disease.

5.0 Myelin compartment: a target for the protective effects of neuroactive steroids Myelin is a multilamellar structure produced as a protrusion of the membrane of glial cells, namely oligodendrocytes in CNS and Schwann cells in PNS that enwraps and insulates axons, allowing fast conduction of nerve impulses. This structure is clearly evident in electron micrographs, that reveal the presence of a major dense line, composed of juxtapositions of intracellular cell membrane, and an intraperiod line, composed of appositions of the extracellular side of subsequent leaflets (Fernandez-Moran and Finean, 1957; Kirschner and Hollingshead, 1980).

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Myelin can be divided in compact and non-compact myelin, which possess peculiar function and composition. In particular compact myelin presents a unique protein/lipid ratio (30:70), which facilitates its insulating capacities (Norton and Poduslo, 1973). The protein composition of compact myelin varies between PNS and CNS. In fact, abundant proteins in the PNS include Myelin Protein Zero (P0), Myelin Basic Protein (MBP), peripheral myelin protein 22 (PMP22) (Greenfield et al., 1973), whereas in the CNS, Proteolipid Protein (PLP) and MBP are most represented (Lees et al., 1984). Amino acid composition, biochemical features and structure prediction identified P0, PMP22 and PLP as integral membrane proteins and MBP as extrinsic membrane proteins. The first three seem to mediate the adhesion and regulate the periodicity of both extra- and intracellular spaces, whereas MBP is linked to the maintenance of intracellular periodicity (Duncan et al., 1989; Giese et al., 1992; Snipes et al., 1992). Lipids represent up to 70% of the dry weight of myelin. The main lipid components of the myelin sheath are sphingolipids, glycerophospholipids and sterols, in a 28:44:28 ratio respectively (Chrast et al., 2010). In adult animals the turnover rates of myelin lipids range from

one

week

for

glycerophospholipids,

to

several

months

for

cholesterol,

galactosylceramide and sulfatide. Severe hypomyelination observed in mice with impaired production of cholesterol support the importance of myelin lipids not only in the formation of the myelin sheath, but also for the correct delivery of myelin proteins to the forming membrane (Saher et al., 2005; Saher and Stumpf, 2015). As observed in STZ model, the expression of myelin proteins, such as P0 and PMP22 in peripheral nerve (Leonelli et al., 2007) and MBP in spinal cord (Pesaresi et al., 2010a) and cerebral cortex (Cermenati et al., 2017) was affected by diabetes. Moreover, in this experimental model, diabetes produces several morphological alterations in the myelinated fibers of the peripheral nerves (Veiga et al., 2006). Furthermore, as demonstrated in STZ

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experimental model, diabetes also alters phospholipids, fatty acids and cholesterol content in the myelin of sciatic nerve leading to membrane fluidity modification (Cermenati et al., 2012). A similar effect has been also recently observed in myelin of the cerebral cortex (Cermenati et al., 2017). However, at variance to what observed in PNS (Cermenati et al., 2012), the effects observed in CNS were not depending on the levels of sterol regulatory element binding protein 1c (SREBP-1c), that was unaltered, but were most probably ascribable to the extensive oxidative stress and inflammation occurring in diabetic encephalopathy (Cermenati et al., 2017). Neuroactive steroids control the myelination process affecting both myelin proteins and the transcriptions factors involved (i.e., Krox-20, Krox-24, Egr-3, FosB and Sox-10) (Giatti et al., 2015b). In agreement, neuroactive steroids also exert protective effects on the damage induced by diabetes in the myelin compartment. For instance, in STZ experimental model, PROG and DHP, but not 3,5-THP, are able to counteract the decreased expression of P0 and PM22 (Leonelli et al., 2007) as well as the increase in the number of fibers with myelin infoldings induced by STZ treatment in the sciatic nerve (Veiga et al., 2006) suggesting a role of PR. These effects were also reproduced in an ex-vivo model of hyperglycemia. Indeed, as reported in Figure 2, dorsal root ganglia cultures exposed to high levels of glucose show a reduced myelination (as evaluated by immunostaining for MBP), and DHP, but not 3,5-THP treatment, was able to counteract this effect. As reported in STZ experimental model, amongst all the androgens considered, only DHT was able to stimulate the low expression of P0 observed in the sciatic nerve (Roglio et al., 2007). Neuroactive steroids are also able to counteract the effects of diabetes on lipid components of peripheral myelin (Mitro et al., 2014). Specifically, DHP, by promoting fatty acid desaturation altered by diabetes, reduces myelin structural alterations revealing a novel pathway of neuroprotection (Figure 3). These effects were due to the restored levels of SREBP-1c, the

16

main lipogenic transcription factor controlling fatty acid biosynthesis that is negatively affected by diabetes (Mitro et al., 2014). As reported in Figure 4, DHP treatment was also able to restore the lipid myelin profile in cerebral cortex of STZ experimental model (Cermenati et al., 2017). A similar DHP effect was also observed on MBP expression in spinal cord (Pesaresi et al., 2010a) and in cerebral cortex (Cermenati et al., 2017) of STZ experimental model.

6.0 Neuroprotective effects of neuroactive steroids and related pharmacological tools Neuroactive steroids are not only able to target alterations in myelin compartment as mentioned above, but also to exert a variety of other protective effects. Indeed, as demonstrated in STZ-treated rats, 3,5-THP improves NCV, thermal threshold and skin innervation density; its precursor, DHP, in addition to these parameters, also improves alterations in Na+,K+-ATPase activity (Leonelli et al., 2007). T derivatives exert similar effects (Roglio et al., 2007). In particular, 3-diol, counteracts impairment of NCV, thermal sensitivity and skin innervation density while, its precursor DHT, in addition to these parameters, also improves Na+,K+-ATPase activity (Roglio et al., 2007). Moreover, another androgen molecule, such as DHEA, prevents not only neuronal but also vascular dysfunction (Yorek et al., 2002). T metabolites are also potential agents for the treatment of diabetic neuropathic pain. Indeed, as demonstrated in STZ experimental model, DHT counteracts the effect of diabetes on mechanical nociceptive threshold while 3-diol was effective on tactile allodynia threshold (Calabrese et al., 2014). Both neuroactive steroids were able to counteract the increase of astrocyte immunoreactivity and glutamate release in the dorsal horn of spinal cord. However, only DHT was able to counteract the increase of pre- (i.e., synapsin-1 and syntaxin-1) and post- (i.e., phosphorylation of GluN2B) synaptic components, while only 3-diol counteracts the increase of expression of substance P and neuroinflammatory parameters, such as toll-like

17

receptor 4, tumor necrosis factor-alpha, transforming growth factor beta-1, interleukin-1beta and translocator protein of 18 kDa (TSPO) (Calabrese et al., 2014). As reported, type 2 diabetes increases the risk of dementia and brain atrophy in older women (Mehlig et al., 2014; Moran et al., 2013). Recently, two independent studies reported that elevated estrogen levels increase these risks (Carcaillon et al., 2014; Espeland et al., 2015b). In addition, a further recent study corroborates prior reports that higher levels of estrogens in elderly women may exacerbate the cognitive functions already altered by type 2 diabetes (Espeland et al., 2015a). Despite these studies assessing the role of neuroactive steroids in type 2 diabetes and their effects on the human nervous system, only few studies are available on this topic in animal models. For instance, Abadie and coworkers reported that, in the obese Zucker rats, DHEA severely reduced daily food intake (Abadie et al., 1993). This effect was ascribed to increased release of serotonin from hypothalamus. Indeed, serotonin is one of the feeding-inhibitory transmitter (Leibowitz, 1987) and DHEA reduced hyperphagia by modulating hypothalamic serotonin levels (Abadie et al., 1993). Altogether these results suggest neuroactive steroids as possible protective agents on the damage induced by diabetes in the nervous system. However, systemic treatment with these molecules could cause endocrine side effects. A therapeutic alternative might be the use of pharmacological tools able to increase steroidogenesis. Neuroactive steroid synthesis relies on cholesterol as precursor molecule. Therefore, the modulation of cholesterol homeostasis may represent a tantalizing strategy to control neuroactive steroid production. With this idea in mind, we demonstrated that activation of liver X receptors (LXR), a nuclear receptor controlling cholesterol homeostasis (Cummins and Mangelsdorf, 2006), increasing the levels of neuroactive steroids in the PNS of STZ rat it is able to exert neuroprotective effects (Cermenati et al., 2010). Another interesting strategy is to promote cholesterol shuttling into mitochondria. In this respect, several ligands of the TSPO have been developed (Kim and Pae,

18

2016a, b). Specifically, TSPO, previously known as peripheral benzodiazepine receptor, is a key protein favoring cholesterol entrance into the mitochondria that can be converted by the first steroidogenic enzyme cytochrome P450 side chain cleavage into PREG, the precursor of all neuroactive steroids. TSPO ligands increase neurosteroidogenesis producing anxiolytic effects without

causing

the side effects

normally associated

with

conventional

benzodiazepines such as sedation or tolerance (Schule et al., 2011). In the diabetic model of STZ rat, we found that treatment with Ro5-4864 (i.e., a ligand of TSPO) increases the low levels of neuroactive steroids in sciatic nerve and consequently resulted protective in this experimental model (Giatti et al., 2009). In addition, the same TSPO ligand as well as the LXR agonist GW3965 restores to control levels, the neuroactive steroids in the spinal cord, in the cerebellum and in the cerebral cortex of rat raised diabetic by STZ injection (Mitro et al., 2012). However, the pattern of induction was different among the three central nervous system (CNS) areas analyzed and between the two pharmacological tools (Mitro et al., 2012). In particular, the activation of LXR might represent a promising neuroprotective strategy, because the treatment with GW3965, differently from Ro5-4864 treatment, did not induce significant changes in the plasma levels of neuroactive steroids (Mitro et al., 2012). This suggests that activation of LXR may selectively increase the CNS levels of neuroactive steroids avoiding possible endocrine side effects due to increased steroid levels in the bloodstream.

7.0 Influence of the sex Diabetic peripheral neuropathy and diabetic encephalopathy, like other disorders of the nervous system (e.g., Alzheimer's disease, Parkinson's disease, Huntington's disease, multiple sclerosis, traumatic brain injury, stroke, autism, schizophrenia, anxiety and depression) show sex dimorphic features (Melcangi et al., 2016). Indeed, diabetic peripheral neuropathy is more frequent in men than in women (ratio male/female 2.9) (Basit et al., 2004; Booya et al., 2005).

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Males develop neuropathy earlier than females (Aaberg et al., 2008) and muscle weakness and atrophy is more frequent in male patients (Kiziltan and Benbir, 2008). In addition, motor nerve conduction abnormalities and ulnar nerve involvement are also more frequent and severe in males (Kiziltan and Benbir, 2008; Kiziltan et al., 2007) with men expressing lower amplitudes and conduction velocities and longer latencies than female patients (Albers et al., 1996). On the contrary, neuropathic pain and negative sensory symptoms are more frequent in females (Kiziltan and Benbir, 2008). In agreement, in STZ-treated rats, nociceptive threshold is differently affected in the two sexes (Joseph and Levine, 2003). In addition, diabetic encephalopathy is associated with cognitive deficits and increased risk of dementia, stroke, cerebrovascular and Alzheimer’s disease and psychiatric disorders, such as depression and eating disorders (Biessels et al., 2008; Biessels et al., 2002; Gispen and Biessels, 2000; Jacobson et al., 2002; Kodl and Seaquist, 2008). These alterations show sex differences in incidence, progression and severity (Andersen et al., 1999; Farace and Alves, 2000; Fratiglioni et al., 1997; Kaye, 2008; Marcus et al., 2008; Niemeier et al., 2007; Simonds and Whiffen, 2003) and these features could be related to sex difference in neuroactive steroid pattern. Indeed, the levels of neuroactive steroids are not only sex dimorphic in CNS and PNS of control animals, but the nervous pathology also affects these levels in a sex dimorphic way (Melcangi et al., 2016). For instance, in diabetic peripheral neuropathy (Figure 5), the levels of PREG, T, DHT and 3-diol present in the sciatic nerve are decreased in males but not in females, while the levels of PROG, 3,5-THP and 3,5-THP are decreased only in female animals (Pesaresi et al., 2010b). In diabetic encephalopathy (Figure 6), PROG levels show a sex difference in the cerebellum, decreasing versus control values in females, but not in males. The levels of PROG metabolites are impaired in a different way. Indeed, STZ causes a decrease in the levels of DHP and 3,5-THP in the cerebellum and spinal cord, respectively. However, this decrease was detected in males but not in females. In contrast, 3,5-THP 20

levels were decreased by STZ in the cerebellum of females, but not in the cerebellum of males (Pesaresi et al., 2010b). In the cerebral cortex the levels of these neuroactive steroids are not impacted by diabetes in a sex dimorphic way. T, DHT and 3-diol levels are decreased in spinal cord, cerebellum and cerebral cortex of male but not in female STZ animals. Taken together, these data indicate that diabetes induces, in CNS, a decrease in the levels of many neuroactive steroids in a sex dimorphic and regionally specific manner (Pesaresi et al., 2010b). These findings may have strong implications for the development of new sex-oriented therapies for the treatment of diabetic neuropathy and diabetic encephalopathy, based on the use of neuroactive steroids (Melcangi and Garcia-Segura, 2010). Indeed, we reported that gonadectomy in female, but not in male STZ animals, was able to significantly counteract the molecular and functional alteration observed in peripheral nerves (Pesaresi et al., 2011a). Interestingly, these effects were due to an increase in the levels of DHEA and its metabolites (i.e., T and DHT) directly in the sciatic nerve of diabetic rats (Pesaresi et al., 2011a). In agreement, we reported that treatment of intact diabetic animals with DHEA against peripheral nerve damage is more effective in females than in males (Pesaresi et al., 2011b).

Conclusions Data here summarized indicate that type 1 and type 2 diabetes induce damage in the PNS (i.e., diabetic peripheral neuropathy) as well as in the CNS (i.e., diabetic encephalopathy). As here reported, diabetes does not only affect sex steroid levels, coming from peripheral glands but also the neuroactive steroid content in nervous system. In agreement, as demonstrated in STZ experimental model, neuroactive steroids may act as protective agents against alterations induced by diabetes on protein and lipid components of myelin compartment as well as on morphological aspect of these membranes, functional markers of peripheral nerves and

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neuropathic pain. Interestingly, these protective effects may be mimicked by pharmacological tools that, acting on homeostasis (e.g., by activation of LXR) or shuttling into mitochondria (e.g., by activation of TSPO) of cholesterol (i.e., the substrate of steroidogenesis), are able to increase the levels of neuroactive steroids. Finally, another interesting aspect may be the influence of sex. Indeed, diabetic peripheral neuropathy and diabetic encephalopathy show sex dimorphic features, in term of incidence, functional outcomes and neuroactive steroid levels. This may be very important, because these features may represent a possible background for the development of sex-oriented therapies based on neuroactive steroids to counteract the damage induced by diabetes in the nervous system.

Acknowledgements We acknowledge support from the Fondazione Cariplo to R.C.M. (grant number 2012-0547) and from Università degli Studi di Milano to N.M. (intramural grant line-B).

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Legends to Figures

Figure 1- Schematic representation of synthesis and metabolism of progesterone and testosterone and their mechanism of action. Enzymatic conversions are represented by blue lines: solid lines indicate that the metabolic pathway is mediated by the reported enzyme, while dotted lines indicate that several metabolic conversions are necessary to lead to the steroid. Mechanism of action of neuroactive steroids is represented by black lines. StAR: steroidogenic acute regulatory protein; TSPO: translocator protein of 18 kDa; P450scc: cytochrome P450 side chain cleavage; 3β-HSD: 3β-hydroxisteroid dehydrogenase; 5α-R: 5α reductase; 3α-HSOR: 3α-hydroxisteroid oxidoreductase; 3β-HSOR: 3β-hydroxisteroid oxidoreductase; PR: progesterone receptor; AR: androgen receptor; ERβ: estrogen receptor β; HRE: hormone responsive element.

Figure 2- Neuroactive steroid treatment preserves myelin in an ex-vivo model of hyperglycemia. Dorsal root ganglia (DRGs) were dissected from embryonic day 13.5 mouse embryos, and placed in culture in neurobasal medium (containing 25 mM glucose). After 5 days in culture, myelination was induced by the addition of 50 g/ml ascorbic acid. Myelination was allowed to proceed for 10 days, after which glucose was elevated to 100 mM, with or without the addition of DHP or 3,5-THP (10nM). As shown in the figure, glucose elevation strongly reduces myelination, as measured by the number of myelin basic protein (MBP) internodes (quantified in the graph). The addition of DHP, but not 3,5-THP, completely preserves myelin in these cultures. Neurofilament (NF) and DAPI staining were used to show that neurons and cell numbers were not grossly affected by the treatment. Scale bar: 200 m. *p < 0.05 vs control; ## p < 0.01 vs glucose by one-way ANOVA followed by

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Tukey post-hoc test. 8-10 fields per DRG were counted from 6-8 DRG per condition in each experiment, from 3 different experiments.

Figure 3- Effects of diabetes and DHP treatment on sciatic nerve desaturation index. Desaturation index calculated as ratio between stearic acid (C18:0) and oleic acid (C18:1) as indicator of myelin infoldings in non-diabetic (Control), diabetic (induced by STZ) and diabetic treated with DHP (Mitro et al., 2014).

Figure 4- Effects of diabetes and DHP on cerebral cortex myelin phospholipids. Total amount of the different phospholipid species detected in cerebral cortex myelin of non-diabetic (Control), diabetic (induced by STZ) and diabetic treated with DHP (Cermenati et al., 2017).

Figure 5- Effects of diabetes on neuroactive steroid levels in sciatic nerve of male and female rats. Neuroactive steroids reported were significantly decreased in their levels in STZ rats in comparison to those observed in non-diabetic animals (Pesaresi et al., 2010b).

Figure 6- Effects of diabetes on neuroactive steroid levels in cerebral cortex, cerebellum and spinal cord of male and female rats. Neuroactive steroids reported were significantly decreased in their levels in STZ rats in comparison to those observed in non-diabetic animals (Pesaresi et al., 2010b).

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CHOLESTEROL StAR

TSPO P450scc

PREGNENOLONE

3β- HSD

PROGESTERONE

TESTOSTERONE

5α -R PR HRE

PR

5α -R

DIHYDROPROGESTERONE 3α -HSOR

TETRAHYDROPROGESTERONE

AR

DIHYDROTESTOSTERONE

3β- HSOR

3α -HSOR

ISOPREGNANOLONE

3α-DIOL

HRE

AR

3β- HSOR

3β-DIOL

ERβ HRE

ERβ

GABA-A receptor

Figure 1

Control

Diabetes Desaturation Index (C18:0/C18:1): 3.44

Desaturation Index (C18:0/C18:1): 16.66

Few or none infoldings

Several infoldings

Diabetes + DHP Desaturation Index (C18:0/C18:1): 3.83 Few or none infoldings

Fig 3

Total phospholipids 1.52 ng/μg proteins

Total phospholipids 0.74 ng/μg proteins

Control

Diabetes

Total phospholipids 1.64 ng/μg proteins Diabetes + DHP Fig 4

PREG T DHT 3a-diol

Fig 5

PROG 3a,5a-THP 3b,5a-THP

Cerebral Cortex

Cerebellum

Spinal Cord

PREG PROG DHP 3a,5a-THP 3b,5a-THP T DHT 3a-diol

PREG PROG DHP 3a,5a-THP 3b,5a-THP

PREG DHP T DHT

PREG PROG 3b,5a-THP

PREG PROG DHP 3a,5a-THP T DHT 3a-diol

PREG PROG DHP

Fig 6

Highlights Diabetes mellitus affects the reproductive axis and levels of sex steroid hormones Diabetes mellitus affects the levels of neuroactive steroids Neuroactive steroids exert protective effects in diabetic encephalopathy Neuroactive steroids exert protective effects in diabetic peripheral neuropathy The effects of diabetes mellitus in nervous system show sex dimorphic features

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