Nerve Growth Factor ( s ) Mediated Hypothalamic Pituitary Adrenal Axis Dysregulation Model in Stress Induced Genesis of Psychiatric Disorders

Nerve Growth Factor(s) Mediated Hypothalamic Pituitary Adrenal Axis Dysregulation Model in Stress Induced Genesis of Psychiatric Disorders Ashutosh Kumar*1,6, Pavan Kumar*1,6, Muneeb A. Faiq3,6, Vikas Pareek2,6, Khursheed Raza1,6, Sankat Mochan1,6 , Pranav Prasoon4,6, Vivek Sharma5,6 1. Department of Anatomy, All India Institute of Medical Sciences (AIIMS). New Delhi, India 2. Computational Neuroscience and Neuroimaging Division, National Brain Research Centre (NBRC), Manesar, Haryana, India 3. Department of Ophthalmology, All India Institute of Medical Sciences (AIIMS). New Delhi, India. 4. Spinal Cord and Brain Injury Research center, Department of Physiology, University of Kentucky, Lexington, KY, USA 5. Department of Physiology, Jawaharlal Institute of Postgraduate Medical Education and Research (JIPMER), Puducherry, India 6. Etiologically Elusive Disorders Research Network (EEDRN)


INTRODUCTION
The role of Nerve Growth Factors (NGFs) in integration of physiological functioning of the nervous system is imbibing immense interest.NGFs, especially the prototype 'NGF' and Brain Derived Neurotrophic Factor (BDNF) have been reported to have important roles in maintaining neuro-endocrine homeostasis, which could have a great impact on the psychological health of individuals (1-3).Hypothalamo-pituitary-adrenal (HPA) axis is understood as the nodal point around which neuro-endocrine-homeostasis of the body is established.In this context, NGFs have been reported to have connection with genesis of psychiatric disorders; and there are some studies available on the overlap/crosstalk of NGFs-HPA interactions.We initiated our probes with extensive review of literature on NGFs' conglomeration in stress, and its causal relationship to psychiatric disorders.Our analysis revealed that a neuro-endocrine model of NGFs-mediated HPA-axis activation may be involved in stress induced genesis of conditions falling within the umbrella of psychiatric disorders.

NGFs: Descendents of Superfamily of Neurotrophin
NGFs belong to the Superfamily 'Neurotrophins' which has 'NGF' as the prototype, and it also includes BDNF, NT-3, NT-4/5 (4), NT-6 and NT-7 (5,6) (in invertebrates only) (Fig. 1a); which are more or less similar (7) in structure and function to the prototype NGF (8,9).NGFs execute their actions through binding low affinity p75 neurotrophin receptor ( p75 NTR) and high affinity Trk receptors (Trk A, B, and C) (9,10).The NGFs receptor binding has been redundant and one type of receptor can bind to more than one NGFs with differential affinity and execute common neural functions (11,12) (Fig. 1b).NGFs secretion in neurons has been found to be autocrine/paracrine and to perpetuate itself and each other's secretion (13,14).
NGFs and their receptors are regarded as a convergence point for signalling pathways related to neurocognitive functions (15) and a vast range of effects executed by NGFs in CNS, beginning from normal neuro-physiological functioning to survival and death of neurons (16)(17)(18).In adult human brain, prototypes NGF and BDNF (more specifically BDNF) have been implicated in most of the neurotrophin mediated neurocognitive functions (19,20) although NT-3 and NT-4/5 have been also known to be involved in certain functions related to synaptic plasticity in adult brain (21)(22)(23).(Intact and dashed lines are showing signaling through specific high affinity Trk receptors and a common low affinity receptor-P 75NTR respectively.)

NGFs in adult brain
NGFs and their receptors have been detected in all regions of adult brain in various animal studies; more specifically in hippocampus and neocortex (24)(25)(26).Hippocampus has been noted to have highest NGFs synthesis in brain and amongst all NGFs, BDNF is most abundant (25).The expression of the NGFs is heterogeneous and showed region specific dominance in different brain regions.In cortex, a study reported that in rat brain, expression of prototype 'NGF', BDNF, and NT-3 was different in different layers (26).
In hippocampus, NGFs were present more specifically in pyramidal and granular layer neurons and showed variations in expression in different Cornu Amonis (CA) regions, and in cerebellum were more specifically localised to Purkinje and granule cell neurons (27).
Similar to the NGFs, their receptors were also found heterogeneously distributed and showed region specific dominance indicating target regions of their ligands, for example, TrkB, receptor for BDNF, was found upregulated in ependyma and periventricular brain parenchyma and TrkA, high affinity receptor for prototype 'NGF' was more densely expressed in basal forebrain regions and striatum (28).
Uniquely, intraventricular injections of NGFs in animal brains showed that they can also diffuse to other brain regions with differential limits i.e., prototype 'NGF' reached to the cortical regions, BDNF limited to the periventricular regions, and NT-3 reached some intermediate destinations (28).
The mode of NGFs secretion in neurons has been a matter of contention.Current evidence suggests, NGFs' secretion is not only constitutional (target derived and showing retrograde axonal transport serving trophic functions in neurons) but also have prominent activity dependent, regulated secretion which move anterograde via dendrites and axons to act at the synapses with similar processes of other neurons; where they contribute extensively in synaptic transmission and plasticity (29)(30)(31)(32).
The prototype NGF was found more specifically expressed in inhibitory GABAergic neurons (co-labelled GAD65/67) than excitatory neurons (co-labelled CaMKIIa).GABAergic neurons are considered primary source of prototype 'NGF' production in CNS (33). 6 The expression of NGFs and their receptors is found in different subcortical regions (as striatum, thalamus, hypothalamus, basal forebrain nuclei, septal regions) and brainstem nuclei.However, the expression is not only restricted to neurons, but glial cells and nerve fiber bundles also display it (27,28,34).

NGFs homeostasis: as a concept
NGFs have extensive role in physiological homeostasis regulation (3,35,36) and it is reasonable to speculate for a homeostatic control system of their own (3), disruption of which may result in psychiatric disorders (37,38).Evolutionary link, therefore, has been seen amongst different NGFs by sharing common moiety, receptors, signalling cascades and mode of action (10).
Neurons co-localize more than one NGFs or their receptors (39).The NGFs have also noticed to regulate their own or other family members in autocrine/paracrine manner in CNS (14) , and their regulation also follows circadian rhythm (40,41).A change in NGFs levels reflect on levels of HPA hormones or various neurotransmitters in the brain (42,43).They also show neural activity dependent secretion, change in levels with sleep, physical activities, and responsive to the environmental changes (44)(45)(46).All this evidence suggests that NGFs maintain a homeostatic control system of their own orchestrating with other neuro-endocrine homeostatic systems in the body.

Role of NGFs in maintenance of normal behaviour
The significant role of NGFs in behavioural processes has been implicated by peer reviewed research (47,48).The role of NGFs in various neurophysiological functions which determine normal behavior is known for long; specific NGFs have been found to be involved in synaptic transmission (20,49), memory and learning (50)(51)(52), sleep (53)(54)(55), and neuronal protection (18,56).
NGFs have been reported mediating behaviour induced cerebral plasticity (19,20).A forebrain specific BDNF gene knock-out study in adult mice revealed that there was significant (30% reduction) changes in density of dendritic spines of cortical neurons that reflected in smaller brain size and compromised behaviour (difficulty in spatial learning and propensity for depression) (48).
A study on male hamsters has shown that differential action of BDNF caused dominantsubordinate relationship in a resident-intruder model in which individual hamsters were identified as winner or loser on the basis of behaviour.Losing animals had significantly more BDNF m-RNA in basolateral and medial nuclei of amygdala while winners had more BDNF m-RNA in dentate gyrus of dorsal hippocampus implicating BDNF in subsequent experience dependent behavioral plasticity.The experience dependent learning of dominant/subordinate behaviour being mediated by BDNF was further confirmed when investigators were able to block acquisition of such behaviour by K252a (a Trk receptor antagonist (57).
Another study implicated BDNF in depression related personality traits in healthy volunteers (59).
Egan et al found a polymorphic variant of BDNF (Val66Met) in subjects associated with poor episodic memory and abnormal hippocampal activation (60).Few other studies have found the BDNF polymorph (Val66Met) to be associated with anxiety trait, depression, HPA and SAM (sympathetic adrenal medullary) axis hyperactivity, and higher anticipatory cortisol response to psychological stress (61)(62)(63).
NGFs have also been implicated in various causes of psychosocial stress which may cause deviation from normal social behavior as: social displacement, disharmony, future uncertainty, bereavement, complicated upbringing and intense conflicts (64)(65)(66).
NGFs have also been implicated in regulation of reproductive behaviour through hypothalamus-pituitary-gonadal (HPG) axis, a close functionary of the HPA.An experimental study claimed that the prototype 'NGF' is the constitutive element of semen and prostatic secretions, and it can mediate influence of male on sexual behaviour of female or can induce ovulations through HPG axis (67).The prototype 'NGF' has been observed to be associated with intimate sexual behaviours; a study found serum prototype 'NGF' level, and not other NGFs higher in persons indulged in early romantic relationship (68).
NGFs have a definite role in neurogenesis, network organizations, formation and remodelling of synapses in developing brain which perpetually keeps refining under their regulation (43).
In adult brain (including humans) where neurogenesis keeps going on as a physiological event; as in hippocampal region (dentate gyrus) and subventricular zone (SVZ) of lateral ventricles (69,70); the role of NGFs in induced neurogenesis, remodelling of neuronal connections, and synaptic organizations with new experiences in these brain regions provide individuals with ability to adapt to new life situations (71,72), a disruption of which may lead to aberrant behaviour alternatively described as psychiatric disorders (73).
NGFs interact with dopaminergic neurons thereby facilitating dopamine release at synapses (74,75).Dopamine is the chief substrate for mesolimbic reward pathway.Such a function is crucial for experience dependent modulation of normal behaviour (76).A dopamine-BDNF link in reward pathway functions has been well substantiated by animal model studies of social defeat stress (77,78).
Neuronal activity dependent secretions of NGFs have been shown to involve cAMP response element binding protein (CREB) as the chief mediator.CREB is known to mediate many neurotransmitters and neuromodulators in brain.It also activates many transcription factors, and influences expression of immediate early genes involved in synaptic transmission and plasticity, and experience dependent remodelling of neuronal circuits (79)(80)(81).The extensive role of CREB in normal neuronal functions and resultant behaviour, and its NGFs mediated regulation further substantiates that NGFs may be crucial for maintenance of normal mental health and behaviour.

Plausible mechanisms for NGFs mediated HPA axis activation model
Increasing number of studies are linking NGFs, and stress to the pathophysiology of psychiatric disorders (104)(105)(106).NGFs work as a homeostatic interface between organism and environment which is too robust to get disrupted from ordinary perturbations of daily life and helps the organism to adapt to the changing conditions (107).A persistent state of mounting stress may disrupt NGFs homeostasis in the body (107,108), and in consequence may dysregulate NGFs mediated HPA axis activation (107,109).The ambiguity raised in the NGFs-HPA axis regulatory system is to negatively affect mood and cognition of the individual which, in turn, would cause more NGFs-HPA axis dysregulation, and such a vicious cycle (Fig. 2) may precipitate stable changes in individual's behaviour in an attempt to re-establish the disrupted homeostasis (110)(111)(112).If the NGFs-HPA axis homeostasis is not re-established, the deviant changes in behaviour may further progress into manifest psychiatric disorders (19,113).

Fig. 2. Stress induced HPA axis mediated disruption in NGFs homeostasis
Stress induced NGFs mediated dysregulation of HPA axis may end up as a psychiatric disorder (if stress causing factors persist) (2,114).NGFs show activity and stress based synthesis in various brain regions involved in stress response regulation (1,105,115) and the involved brain regions are known to differentially regulate HPA axis.For example, prefrontal cortex and hippocampus are known to inhibit but amygdala excites it (116).Additionally, chronic immobilization stress in male rats was observed to produce inverse neuronal BDNF secretion and hence growth changes mediated by it, in hippocampus and amygdala (117).
Differential regulation of HPA axis by various brain regions involved in stress response regulation is presumably the reason of phenotypic diversity observed in psychiatric disorders (116,118).
Experimental studies suggest that hippocampus is the most severely affected brain region (owing to prolonged glucocorticoid secretion) in chronic restraint stress (119)(120)(121).
Hippocampal inhibitory control of HPA axis activity, which is a normal phenomenon in homeostasis, is also known to be compromised in chronic restraint stress (116,122); and along with glucocorticoids, NGFs are known to be central players causing this dysregulation (109,112,123).Hippocampal pyramidal neurons secrete NGFs in response to stress which correspond to HPA axis activity (124,125), and also TrkB, the receptor for BDNF is found to be co-localized with glucocorticoid receptor (GR) and having mutual influence on each other's signalling in these neurons (123).A study has shown in primary rat cortical neurons that BDNF induces structural change at GR gene (phosphorylation at serine 155 and 287) and causes significant change in its transcriptome (126).Similar evidence for essential glucocorticoid-BDNF interaction for stress response have also been noted in hypothalamus controlling CRH synthesis at PVN (115).A schematic depiction of NGFs mediated hormones occurs in response of fresh stressful stimuli.An increase in cortisone release at adrenal as a stress response sends negative feedback through glucocorticoid receptor (GR) in hippocampus (but GR expression is maintained) which in turn inhibits HPA activity.Also the NGFs secretion response in challenge of fresh stress is maintained.In chronic restraint stress: HPA axis regulation gets epigenetically modified causing enduring increase in cortisone secretion at adrenal which in turn increases feedback inhibition at hippocampus resulting in significant reduction in GR expression, which further decreases hippocampal inhibitory control on HPA axis facilitating its persistent hyperactivation and exaggerated response in challenge of fresh stressful stimuli.The NGFs secretion response is also reduced.(Thickness of signaling lines and feedback loops are indicating their strength)) (127).

Evidence from animal studies
There are multiple animal studies supporting NGFs mediated regulation of HPA axis.Studies have also established dysregulation of particular NGFs (prototype 'NGF' and BDNF) and HPA axis in chronic stress giving way for genesis of psychiatric disorders.
Givalois et al showed that a single intra-cerebroventricular injection of BDNF in nonanaesthetized adult rats modified HPA axis activity.In paraventricular nuclei of hypothalalamus, an increase of BDNF was found to alter CRH (corticotrophin releasing hormone) and AVP (arginine vasopressin) synthesis, which in turn lead ACTH release (adreno-cortico-trophic hormone) from anterior pituitary and cortisone release from adrenal cortex.This change in HPA axis activity in response of exogenous BDNF was similar to the condition when a rat is subjected to immobilization stress which suggested role of BDNF as stress responsive intracellular messenger (128).
Naert et al examined the effect of chronic stress (restraint 3 hour/day for 3 weeks) in adult rats on behaviour and HPA axis activity in parallel with studying BDNF levels in hypothalamus, pituitary and hippocampus.Chronic stress induced anxiety, anhedonia and depression like states in these model animals.HPA axis activity was found highly modified with increase in the basal level of hypothalamic CRH and AVP synthesis and plasma levels of ACTH and cortisone.Added to that, basal BDNF levels were increased in the hypothalamus, pituitary and hippocampus.Also, the BDNF response to subsequently applied acute novel stress was found modified.All these findings indicated plausible role of BDNF in chronic stress induced genesis of psychiatric disorders (42).
Naert et al further tested the effect of continuous intra-cerebroventricular administration of BDNF on HPA axis activity in adult rats and found that it not only modified HPA activity but also biological rhythms.They opined that a change in HPA activity and biological rhythms occurr through regulatory effect of BDNF on AVP m-RNA expression (AVP m-RNA was found to be upregulated after continuous BDNF infusion) in suprachiasmatic nucleus (SCN) of hypothalamus (which is regarded as the biological clock) (129).BDNF and its cognate receptor Trk B are known to be expressed in SCN and showed circadian variations (41).
These investigators also tested the effect of partial inhibition of endogenous BDNF on HPA activity in adult rats.BDNF knockdown by small interfering RNA (siRNA) caused decreased endogenous BDNF production in different brain regions, which although didn't influence basal HPA activity, but the knockdown rats showed decreased BDNF production and concomitant altered ACTH and cortisone in response to restraint stress which stipulating essential role of BDNF in stress adaptation (130).
The acute and chronic intra-cerebroventricular administration of BDNF into brain of rat pups found to produce changes in HPA activation but only chronic administration of BDNF caused stable changes in HPA axis regulation resulting in persistently increased secretion of cortisone by adrenal cortex leading to deviant behaviour, while changes in HPA axis activation induced by acute doses of BDNF were adjusted by bodily homeostatic systems (128,129).The HPA axis changes induced by chronic administration of BDNF resembled that of chronic stress and presumably involve epigenetic mechanisms (131,132).The epigenetic mechanisms are supposed to induce structural adaptations in the chromosomal regions so as to register, signal or perpetuate altered activity states (133).The essential role of epigenetics in producing stable changes in HPA axis regulation has been effectively proved in animal model studies of chronic stress.Maternal separation of the rat pups (which is regarded as an equivalent to chronic stress) induced stable changes in HPA axis regulation leading to hypercortisone response in stress challenges.The HPA axis changes were mediated by epigenetic mechanisms in the form of hypermethylation in promoter regions of glucocorticoid receptor (GR) gene hence reducing expression of GR receptors, which in turn promoted cortisone hyper secretion by influencing negative feedback loops (134)(135)(136).Conversely, better maternal care caused hypomethylation in the promoter region of the GR gene increasing the GR expression leading to reduced cortisone secretion response in face of stress challenges (136).
In experiments blocking the promoter region methylation of GR by histone deacetylase inhibitor blocked any increase of cortisone secretion in stress challenges in rat pups who had comparatively got less maternal care (136).In contrast, addition of methyl groups (by introducing methyl donors) in promoter regions of GR gene in adult rats which had got good maternal care as pups induced hyper cortisone secretion thus reversing the set maternal programming for stress response (137).Environment enrichment could also reverse the maternal separation induced stress response modification (138), which is a good indication for plausible reversibility of HPA axis mediated persistent stress induced disorders.
Epigenetic modification of HPA axis stress response also occurred at higher levels in the axis than GR synthesis at adrenal, and were also mediated through other epigenetic methods than methylation as phosphorylation (126), histone modifications and micro-RNA mediated transcriptional regulation (139).
In an early life stress (ELS) study in male mice maternal separation induced increase in cortisone response in next stress challenges was found associated with sustained hypomethylation in POMC (a precursor for ACTH) gene promoter region.The mice also presented despair like behaviour and memory deficit which were supposedly mediated through AVP signalling and epigenetic adaptations at AVP enhancer locus (140).A persistent increase in AVP expression was found in hypothalamic paraventricular nuclei (PVN) associated with decreased DNA methylation at AVP enhancer locus in an another ELS study done in mice (141).
In a different ELS study in adult rats decreased AVP enhancer locus methylation in amygdala found associated with development of active coping mechanisms when presented with predatory stress challenge (142).Not only the BDNF expression, but also the prototype NGF has been found to be altered significantly in stress caused by various reasons in animal models (124,143); it was found raised in maternal deprivation in rats (124); and specific hypothalamic nuclei were reported having raised 'NGF' levels in aggression in mice (95).
A dopaminergic modulation of NGFs-HPA axis mediated stress response has also been shown in animal models of social defeat.Social defeat was found to raise BDNF levels in nucleus accumbens (NAc) which is a destination centre for dopaminergic neurons residing at ventral tegmental area (VTA) in mesolimbic reward pathway.A raised BDNF level was found associated with learning social avoidance behaviour in model animals and mediated through CRH (from PVN of hypothalamus) (77,78).

Translational studies in human
There are also reports translating the animal model observation of stress response modifications in persistent stress in human subjects (143)(144)(145).A single nucleotide polymorphism of BDNF (Val66Met) in human populations has also been found associated with altered stress response (63).

Role of epigenetic modifications in NGFs mediated HPA activation model: Behavioural adaptations to persistent stress manifesting as psychiatric disorders
NGFs have been extensively implicated in creating epigenetic memory of environmental stimulus (65,104).MeCP2 (methyl-CpG-binding domain 2), a methyl binding domain (MBD) and transcription repressor which is essential for accomplishing gene silencing effect of methylation needed to dissociate from the BDNF promoter region to reprogram the stress response (146).An increase in BDNF synthesis accompanying any neuronal activity has been a neurophysiological phenomenon.A rapid increase in its synthesis in specific brain regions has been a characteristic finding in face of stress challenges (125,147); Although methylation in the BDNF promoter (exon IV) in specific brain regions was found in chronic stress only (42,148).Hypermethylation of a CpG nucleotide at 5' end of the binding site of a transcription factor, nerve growth factor inducible protein A (NGFIA) in GR promoter region has also been reported in all rat pups which received reduced maternal care.Certain alteration in the DNA methylation pattern for NGFIA is considered as an essential step in the epigenetic reprogramming of a stress response (136,149).
Persistently raised basal levels of BDNF, and hypercortisone secretion along with other stable alterations in HPA axis set points may be inducing transcriptomic changes in expression of genes related to synaptic functions (150,151), and causing structural and functional changes in neurons in form of the synaptic organization and synaptic transmission leading to altered behaviour of the organism (20,152) which is perceived as a psychiatric disorder.

CONCLUSIVE REMARKS
Understanding the mechanisms of NGFs-HPA interactions and its disruption in persistent stress may help to understand stress induced pathogenesis of psychiatric disorders.
Experimental proof of emergence and alleviation of psychiatric disorders in loss and gain of NGFs-HPA axis homeostasis in cohort studies can establish the veracity of this model in general.

Fig. 1 a
Fig. 1 a.Superfamily of neurotrophins.NGFs-Nerve growth factors, *= In invertebrates only b. Nerve growth factors and their cognate receptors.