PREVENTING NEURGENESIS DEFICITS AND ENHANCING NEUROGENESIS

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a nonprovisional application of and claims priority to U.S. Provisional Patent Application 62/073,339, entitled "Use of Cotinine in Preventing Neurogenesis Deficits, Oxidative Stress and Cardiovascular Events by Inhibiting Neuroinflammation", filed on October 31 , 2014, the contents of which are herein incorporated by reference.

FIELD OF INVENTIO N

This invention relates to treatment of chemotherapy or stress-related side effects. More specifically, the present invention provides therapeutic methods and compositions for treating chemotherapy treatment or reducing stress-related neuroinflammation.

BACKGROUND OF THE INVENTION

The stimulation of the hypothalamic-pituitary-adrenal (HPA) axis by chronic stress leads to increased inflammation and oxidative stress (Zunszain, et al., (2013) Inflammation and depression. Current topics in behavioral neurosciences, 14, 135- 151 ). H PA activation also inhibits the expression of factors promoting synaptic plasticity, neurogenesis, and neuronal survival (Dwivedi, (2009) Brain-derived neurotrophic factor: role in depression and suicide. Neuropsychiatry disease and treatment, 5, 433-449). Increasing evidence suggests that this cascade of events participates in both the development and maintenance of depression (Kawahara, et al., (1997) Cortical spreading depression induces long-term alterations of BDNF levels in cortex and hippocampus distinct from lesion effects: implications for ischemic tolerance. Neurosci Res, 29, 37-47; Kim, et al., (2007) Low plasma BDNF is associated with suicidal behavior in major depression. Prog Neuropsychopharmacol Biol Psychiatry, 31 , 78-85; Lee, et al., (2009) Reduced platelet BDNF level in patients with major depression. Prog Neuropsychopharmacol Biol Psychiatry, 33, 849-853; Shi, et al., (201 0) Intranasal administration of nerve growth factor produces antidepressant-like effects in animals. Neurochemical research, 35, 1302-1314).

Inflammation is an inherently beneficial mechanism of the immune system by which an area which has become damaged or contains unfamiliar agents, such as protein aggregates, viruses or, bacteria, can be signaled to by other cells so that it may be repaired and returned to a normal health state (Guy, et al., Inflammatory Neurodegeneration and Mechanisms of Microglial Killing of Neurons. Mol Neurobiol. 201 0; Purcell & Gorman, Immunoproteomics. Molecular and Cellular Proteomics. 2004. Vol 3, 1 93-208). The innate immune system responds to foreign pathogens based on conserved patterns, but it can mount a response to a novel threat not previously encountered (Purcell & Gorman, Immunoproteomics. Molecular and Cellular Proteomics. 2004. Vol 3, 1 93-208). Both systems can lead to the development of inflammation. Cytokines and chemokines are factors which allow for immune cells to communicate with each other and play an important role in neuroinflammation (Whitney, et al., Inflammation mediates varying effects in neurogenesis: relevance to the pathogenesis of brain injury and neurodegenerative disorders. J Neurochem. 2009 Mar;1 08(6):1343-59). All these factors can be activated by stress (Lucassen, et al., "Regulation of adult neurogenesis by stress, sleep disruption, exercise and inflammation: I mplications for depression and antidepressant action." European Neuropsychopharmacology 20.1 (201 0): 1 -1 7; Song & Wang, Cytokines mediated inflammation and decreased neurogenesis in animal models of depression. Prog Neuropsychopharmacol Biol Psychiatry. 201 1 Apr 29;35(3):760-8). The brain has specialized immune cells called glia which consists of microglia and astrocytes, which protect the blood-brain barrier which tightly controls which substances can enter the brain (Brown & Neher, Inflammatory Neurodegeneration and Mechanisms of Microglial Killing of Neurons. Mol Neurobiol. 201 0 Jun;41 (2-3):242-7). Inflammation causes damage to the blood-brain barrier allowing previously restricted substances in, such as leukocytes and cytokines (Brown & Neher, Inflammatory Neurodegeneration and Mechanisms of Microglial Killing of Neurons. Mol Neurobiol. 201 0 Jun;41 (2-3) :242-7). Depressed patients show increased levels of the pro inflammatory cytokines I L1 , IL6, and TNF-alpha in the brains, people with many different types of diseases experiences depression at a 5-10 times greater rate than healthy individuals, and when patients are given Interferon-gamma for the treatment of cancer, they develop symptoms that are similar to depression (Song & Wang, Cytokines mediated inflammation and decreased neurogenesis in animal models of depression. Prog Neuropsychopharmacol Biol Psychiatry. 201 1 Apr 29;35(3):760-8).

Nicotine's suppressive effects on microglia activation mediate some of the neuroprotective actions of nicotine (Barreto, et al., Beneficial effects of nicotine, cotinine and its metabolites as potential agents for Parkinson's disease. Front Aging Neurosci. 2015 Jan 9;6:340), and occur through a7nAChR activation, and subsequent activation of signal transducer and activator of transcription 3 (STAT3). Importantly, from the regulators analyzed so far, STAT3, SOCS-3, PI3K and MyD88s have also been implicated in mediating the anti-inflammatory effect of the a7nAChRs in immune cells (Paris, et al., Anti-inflammatory activity of anatabine via inhibition of STAT3 phosphorylation. Eur J Pharmacol. 2013 698:145-53; Shen, eta I., Neurotrophin-4 (Ntf4) mediates neurogenesis in mouse embryonic neural stem cells through the inhibition of the signal transducer and activator of transcription-3 (STAT3) and the modulation of the activity of protein kinase B. Cell Mol Neurobiol. 201 0 Aug;30(6):909-1 6; Bagaitkar, et al., Cotinine inhibits the pro-inflammatory response initiated by multiple cell surface Toll-like receptors in monocytic TH P cells. Tob Indue Dis. 201 0 Nov 23;1 0(1 ):18; Maldifassi, et al., A new I RAK-M-mediated mechanism implicated in the anti-inflammatory effect of nicotine via alpha7 nicotinic receptors in human macrophages. PLoS One. 2014 Sep 26; 9(9), e1 08397; artel I i, et al., The cholinergic anti-inflammatory pathway: a critical review. Auton Neurosci. 2014 May;1 82:65-9; Pavlov, et al., The cholinergic anti-inflammatory pathway: a missing link in neuroimmunomodulation. Mol Med. 2003 May-Aug;9(5-8) :1 25-34; Rehani, et al., Cotinine-induced convergence of the cholinergic and PI3 kinase- dependent anti-inflammatory pathways in innate immune cells. Biochim Biophys Acta. 2008 Mar;1 783(3):375-82; Wang, et al., Nicotinic acetylcholine receptor alpha7 subunit is an essential regulator of inflammation. Nature. 2003 Jan 23;421 (6921 ) :384-8). Several lines of evidence have shown that activation of nAChRs, such as D7nAChRs, improves attention, and learning and memory in rodents and non-human primate models of several neurological and psychiatric disorders including schizophrenia (Jones, e al. , Muscarinic and nicotinic acetylcholine receptor agonists and allosteric modulators for the treatment of schizophrenia. Neuropsychopharmacology. 2012 Jan;37(1 ):1 6-42; Leiser, et al., A cog in cognition: how the alpha 7 nicotinic acetylcholine receptor is geared towards improving cognitive deficits. Pharmacol Ther. 2009 Jun:122(3):302-1 1 ; Lieberman, et al., A randomized exploratory trial of an alpha-7 nicotinic receptor agonist (TC- 561 9) for cognitive enhancement in schizophrenia. Neuropsychopharmacology. 2013 May;38(6):968-75; Winterer, et al., Allosteric alpha-7 nicotinic receptor modulation and P50 sensory gating in schizophrenia: a proof-of-mechanism study. Neuropharmacology. 2013 Jan;64:1 97-204; Young, J. W. and Geyer, M. A. (2013) Evaluating the role of the alpha-7 nicotinic acetylcholine receptor in the pathophysiology and treatment of schizophrenia. Biochem Pharmacol. 2013 Oct 15;86(8) :1 122-32).

In the brain, once inflammation has started, uncontrolled feedback loops can lead to brain damage and decreased neurogenesis (Whitney, et al., Inflammation mediates varying effects in neurogenesis: relevance to the pathogenesis of brain injury and neurodegenerative disorders. J Neurochem. 2009 Mar;108(6) :1 343-59). Cholinergic compounds related to nicotine present in plants of the Solanacea family including green tomato, peppers and eggplant, such as anatabine, regulate cytokine production and display anti-inflammatory properties (Paris, et al., Anti-inflammatory activity of anatabine via inhibition of STAT3 phosphorylation. Eur J Pharmacol. 2013;698:145-153).

It has been found that brain inflammation induced by stress is also accompanied by a decrease in neurogenesis (Song & Wang, Cytokines mediated inflammation and decreased neurogenesis in animal models of depression. Prog Neuropsychopharmacol Biol Psychiatry. 201 1 Apr 29;35(3):760-8). Neurogenesis is a process by which new neurons are created (Whitney, et al., Inflammation mediates varying effects in neurogenesis: relevance to the pathogenesis of brain injury and neurodegenerative disorders. J Neurochem. 2009 Mar;108(6):1343-59). This process is very prominent during development, but considerably decreases in the adulthood (Whitney, et al., Inflammation mediates varying effects in neurogenesis: relevance to the pathogenesis of brain injury and neurodegenerative disorders. J Neurochem. 2009 Mar; 108(6):1343-59). Only two areas are known to undergo neurogenesis in the adulthood (Lucassen, et al., "Regulation of adult neurogenesis by stress, sleep disruption, exercise and inflammation: Implications for depression and antidepressant action." European Neuropsychopharmacology 20.1 (201 0): 1 -1 7); the subgranular zone of the dentate gyrus and the subventncular zone of the hippocampal formation (Whitney, et al., Inflammation mediates varying effects in neurogenesis: relevance to the pathogenesis of brain injury and neurodegenerative disorders. J Neurochem. 2009 Mar; 108(6):1343-59). Neurons developed in the subventncular zone are destined for the olfactory bulb (Whitney, et al., Inflammation mediates varying effects in neurogenesis: relevance to the pathogenesis of brain injury and neurodegenerative disorders. J Neurochem. 2009 Mar; 1 08(6) :1343-59).

It is thought that adult neurogenesis is a critical component of learning, memory and mood stability (Whitney, et al., Inflammation mediates varying effects in neurogenesis: relevance to the pathogenesis of brain injury and neurodegenerative disorders. J Neurochem. 2009 Mar; 108(6):1343-59). Decreased levels of neurogenesis have been reported in patients with depression and adults who are under stress (Purcell & Gorman, Immunoproteomics. Molecular and Cellular Proteomics. 2004. Vol 3, 1 93-208). These conditions produce both chemical and structural changes in the brain that can explain changes in behavior (Purcell & Gorman, Immunoproteomics. Molecular and Cellular Proteomics. 2004. Vol 3, 1 93- 208). Hippocampal volume can be reduced by both depression and reduced neurogenesis (Song & Wang, Cytokines mediated inflammation and decreased neurogenesis in animal models of depression. Prog Neuropsychopharmacol Biol Psychiatry. 201 1 Apr 29;35(3):760-8).

A deficit in the expression of the a7nAChRs has been found involved in the etiology and development of AD and many others mental health conditions including the Tourette's syndrome, PD, bipolar disorder and schizophrenia. Unfortunately, the fact that a7nAChRs, become rapidly desensitized by its agonists has limited the benefits of drugs directed to increase the levels of ACh such as the acetylcholinesterase inhibitors (AChEI) as well as agonists and partial agonists binding to the canonical ACh binding site of these receptors (Williams, et al., Positive allosteric modulators as an approach to nicotinic acetylcholine receptor- targeted therapeutics: advantages and limitations. Biochem Pharmacol. 201 1 Oct

15;82(8) :915-30). 7nAChRs are activated by conformational changes induced by ligands such as competitive antagonists, full and partial agonists that bind the receptor at the classical agonist binding site (orthosteric). However, a7nAChRs also are positively or negatively regulated by allosteric modulators that bind not to the orthosteric but allosteric sites. The study of a7 receptor responses to agonists measured from transfected cells and Xenopus laevis oocytes revealed that desensitization of these receptors is nearly immediate and the probability for a7nAChR channel to open after agonist application is only 0.002. In presence of higher concentration of the agonists this receptor is converted to a ligand-bound non-conducting state. However, this state can be destabilized by PAMs such as

PNU-120596 (Williams, et al., Intrinsically low open probability of alpha7 nicotinic acetylcholine receptors can be overcome by positive allosteric modulation and serum factors leading to the generation of excitotoxic currents at physiological temperatures. Mol Pharmacol. 2012 Oct;82(4) :746-59). Cotinine, a component of tobacco leaves and the predominant metabolite of nicotine, is also anti-inflammatory (Rehani, et al., (2008) Cotinine-induced convergence of the cholinergic and PI3 kinase-dependent anti-inflammatory pathways in innate immune cells. Biochimica et Biophysica Acta (BBA) - Molecular Cell Research, 1 783, 375-382) and has anti-depressant properties, as it facilitates serotonin (5-HT) release in the brains of rats (Fuxe, et al., (1 979) On the action of nicotine and cotinine on central 5-hydroxytryptamine neurons. Pharmacol Biochem Behav, 1 0, 671 -677). Due to its origin, it has been used as a biomarker for cigarette smoking (Echeverria-Moran, Cotinine: Beyond that Expected, More than a Biomarker of Tobacco Consumption. Front Pharmacol. 2012; 3:1 73). Cotinine crosses the blood-brain barrier and is non-addictive in humans (Echeverria & Zeitlin,

Cotinine: A Potential New Therapeutic Agent against Alzheimer's disease. CNS Neuroscience & Therapeutics 1 8 (2012) 51 7-523). Cotinine-treatment reduces anxiety and enhances the extinction of contextual fear memory in mice (Zeitlin, et al., (2012) Cotinine enhances the extinction of contextual fear memory and reduces anxiety after fear conditioning. Behav Brain Res, 228, 284-293) and also stimulates the Akt/GSK3p pathway (Echeverria, et al., (201 1 a) Cotinine Reduces Amyloid-b Aggregation and Improves Memory in Alzheimer's Disease Mice. Journal of Alzheimer's Disease, 23, 1 -1 9). The activation of this pathway stimulates neurogenesis and neuronal survival, and decreases depressive-like behavior (Wada, (2009) Lithium and neuropsychiatric therapeutics: neuroplasticity via glycogen synthase kinase-3beta, beta-catenin, and neurotrophin cascades. J Pharmacol Sci, 1 1 0, 14-28; Riadh, et al., (2012) Neuroprotective and neurotrophic effects of long term lithium treatment in mouse brain. Biometals, 24, 747-757; Hur, & Zhao, GSK3 signalling in neural development. Nat Rev Neurosci. 2010 Aug;1 1 (8) :539-51 ). In fact, most currently prescribed antidepressants, such as serotonin reuptake inhibitors (SSRIs), monoamine oxidase (MAO) inhibitors and tricyclic antidepressants, also activate this pathway activate this pathway (Beaulieu, et al., (2009) Akt/GSK3 signaling in the action of psychotropic drugs. Annu Rev Pharmacol Toxicol, 49, 327-347; Echeverria, et al. , (201 1 a) Cotinine Reduces Amyloid-b Aggregation and Improves Memory in Alzheimer's Disease Mice. Journal of Alzheimer's Disease, 23, 1 -1 9), which, in turn, stimulates the expression of neurotrophic factors such as the brain-derived neurotrophic factor (BDN F) (Id.) and the vascular endothelial growth factor (VEGF).

The pharmacodynamic effects of cotinine showed that cotinine was a very poor agonist of a 7nAChRs; However, neurochemical, pharmacological and behavioral data suggested that cotinine elicited 7nAChRs signaling. This evidence suggested that cotinine was a positive allosteric modulator (PAM) of the a7 receptor. Recently, it has been shown data, using electrophysiological techniques to analyze the activity of a7nAChRs expressed in Xenopus oocytes, suggesting that S-cotinine is a a7nAChR PAM inhibiting the receptor desensitization while enhancing its activation by Ach, which supports the hypothesis that cotinine should be beneficial as an adjunctive therapy to the AChEI for the treatment of AD (Terry et al., 2015). Previous study working with primary monocytes isolated from whole human blood, demonstrated that pre-treatment with cotinine for two hours inhibited the inflammatory response to gram negative bacteria and dramatically suppressing the production of cytokines such as TNF (EC50 =100 ng/ml, 0.3 μΜ). Cotinine blocked more than 80% of the release of TNF-a induced by Pheudomona gingivalis (MOI=1 0). This effect was suppressed by a-BTX (2 μg/ ml) suggesting that this effect was mediated by a7nAChRs. This effect was dependent on PI3K activity and was accompanied by Akt activation and the inhibition of GSK3 D Dby phosphorylation at Serine 9 (Akt phosphorylation site). This is interesting because a similar activation of Akt and inhibition of GSK3P was found in brain homogenates from cotinine-treated Tg6799 mice. Cotinine's anti-inflammatory effect was independent of N FkB but it was accompanied by the stimulation of IL-1 0 release (Eom & Jope, Blocked inhibitory serine-phosphorylation of glycogen synthase kinase 3alpha/beta impairs in vivo neural precursor cell proliferation. Biol Psychiatry. 2009 Sep 1 ;66(5):494-502; Rehani, K. , Scott, D. A., Renaud, D., Hamza, H., Williams, L. R., Wang, H. and Martin, M. (2008) Cotinine-induced convergence of the cholinergic and PI3 kinase-dependent anti-inflammatory pathways in innate immune cells. Biochim Biophys Acta. 2008 Mar;1783(3):375-82). Cotinine's anti-inflammatory effects involved the activation of TLRs. In fact, in monocytic cells, cotinine suppressed the cytokine production resultant upon agonist-specific engagement of the TLRs (TLR 2/1 ; 2/6; 4 and 5) (Bagaitkar, J., Zeller, I., Renaud, D. E. and Scott, D. A. (2012) Cotinine inhibits the proinflammatory response initiated by multiple cell surface Toll-like receptors in monocytic TH P cells. Tob Indue Dis. 2012 Nov 23; 10(1 ):18). Cotinine improved working memory in transgenic mice (Echeverria & Zeitlin, Cotinine: A Potential New Therapeutic Agent against Alzheimer's disease. CNS Neuroscience & Therapeutics 1 8 (2012) 51 7-523; Patel et al., Cotinine halts the advance of Alzheimer's diseaselike pathology and associated depressive-like behavior in Tg6799 mice. Frontiers Aging Neuroscience. 2014. In Press, available online). Cotinine offers many advantageous characteristics to other PAMs, including anti-inflammatory actions, no addictive properties, good solubility in water, long plasma half-life and nonsignificant toxicity in humans, no increase in heart rate or blood pressure (Echeverria & Zeitlin, Cotinine: A Potential New Therapeutic Agent against Alzheimer's disease. CNS Neuroscience & Therapeutics 1 8 (2012) 51 7-523; Grizzell & Echeverria, New insights into the mechanisms of action of cotinine and its distinctive effects from nicotine. Neurochem Res. 2014 Jun 27. Epub.). Few in vivo studies suggest an anti-tumorigenic effect of cotinine. One study evaluated the effect of cotinine and nicotine-N'-oxides on tumor development in F344 rats initiated with N-[4-(5-nitro-2-furyl)-2-thiazolyl]formamide (FAN FT) (LaVoie, et al., Evaluation of the effects of cotinine and nicotine-N'-oxides on the development of tumors in rats initiated with N-[4-(5-nitro-2-furyl)-2-thiazolyl]formamide. J Natl Cancer Inst. 1 985 Dec;75(6) :1 075-81 ).

Two other compounds act similarly to cotinine, i.e. as a PAM of nAChRs. Similar to cotinine, galantamine is an alkaloid extracted from plants that is neuroprotective (Kihara, et al., Galantamine modulates nicotinic receptor and blocks Abeta- enhanced glutamate toxicity. Biochem Biophys Res Commun. 2004 Dec 1 7;325(3):976-82). Galantamine acts at a7nAChR, as well as 3/β4, α4/β2, and α6/β4 nAChR (Maelicke, et al., Allosteric sensitization of nicotinic receptors by galantamine, a new treatment strategy for Alzheimer's disease. Biol Psychiatry.

2001 Feb 1 ;49(3):279-88; Samochocki, et al., Galantamine is an allosterically potentiating ligand of the human alpha4/beta2 nAChR. Acta Neurol Scand Suppl. 2000;176:68-73). However, galantamine can induce undesired side effects similar to other cholinergic drugs, such as gastrointestinal symptoms and new therapies or forms of delivery to reduce the side effects are currently been investigated

(Bhattacharya, et al., Nasal Application of the Galantamine Pro-drug Memogain Slows Down Plaque Deposition and Ameliorates Behavior in 5X Familial Alzheimer's Disease Mice. J Alzheimers Dis. 2015 May 7;46(1 )123-36; Maelicke, A., Hoeffle-Maas, A., Ludwig, J., Maus, A., Samochocki, M., Jordis, U. and Koepke, A. K. (2010) Memogain is a galantamine pro-drug having dramatically reduced adverse effects and enhanced efficacy. J Mol Neurosci. 2010 Jan;40(1 -2):135-7). Anatabine is another alkaloid present in plants of the Solanacea family, similar to cotinine. Experiments on anatabine-induced microglia activation have been investigated (Paris, et al., Anti-inflammatory activity of anatabine via inhibition of STAT3 phosphorylation. Eur J Pharmacol. 2013 Jan 5;698(1 -3):145-53). Anatabine prevented the activation of the transcription factors STAT3 and NFkB induced by LPS in neuronal cell lines and human microglia. In vivo anatabine inhibited cytokine production (IL-6, IL-1 β and TN F-a) in the plasma, kidney and spleen of animals injected with LPS and concomitantly opposed STAT3 phosphorylation induced by LPS in the spleen and kidney (Paris, et al., Anti-inflammatory activity of anatabine via inhibition of STAT3 phosphorylation. Eur J Pharmacol. 2013 Jan 5;698(1 -3):145- 53).

However, to date there are no methods in place to ameliorate neurogenesis deficits. Accordingly, there is a need in the art for therapeutic methodologies for increasing or enhancing neurogenesis in neurogenesis deficient individuals, including stress- induced neurogenesis deficits, increasing neurogenesis gene expression, and increasing neuronal cell formation.

SUMMARY OF THE INVENTION

To define whether cotinine activity correlated with changes in neuroinflammation and neurogenesis in the brain, forced swimming stress was used to induce depressive-like behavior and investigate the effect of cotinine on changes in the expression of genes involved in neuroinflammation or neurogenesis, in the hippocampus. Analysis showed that administering a therapeutically effective amount of (5S)-1 -methyl-5-(3-pyridyl) pyrrolidin-2-one to a patient experiencing stress-related neurogenesis deficiency improves neurogenesis despite the stress conditions. Useful doses of (5S)-1 -methyl-5-(3-pyridyl) pyrrolidin-2-one are between about 0.1 mg/kg to about 10 mg/kg, such as 0.1 mg/kg, 0.2 mg/kg, 0.3 mg/kg, 0.4 mg/kg, 0.5 mg/kg, 0.75 mg/kg, 0.9 mg/kg, 1 .0 mg/kg, 1 .0 mg/kg, 1 .1 mg/kg, 1 .2 mg/kg, 1 .25 mg/kg, 1 .4 mg/kg, 1 .5 mg/kg, 1 .75 mg/kg, 1 .8 mg/kg, 1 .9 mg/kg, 2.0 mg/kg, 2.25 mg/kg, 2.5 mg/kg, 2.75 mg/kg, 3.0 mg/kg, 3.25 mg/kg, 3.5 mg/kg, 3.75 mg/kg, 4.0 mg/kg, 4.25 mg/kg, 4.5 mg/kg, 4.75 mg/kg, 5.0 mg/kg, 5.25 mg/kg, 5.25 mg/kg, 5.5 mg/kg, 5.75 mg/kg, 6.0 mg/kg, 6.25 mg/kg, 6.5 mg/kg, 6.75 mg/kg, 7.0 mg/kg, 7.25 mg/kg, 7.5 mg/kg, 7.75 mg/kg, 8.0 mg/kg, 8.25 mg/kg, 8.5 mg/kg, 8.75 mg/kg,. 9.0 mg/kg, 9.25 mg/kg, 9.5 mg/kg, 9.75 mg/kg, or 1 0.0 mg/kg. Specific doses include about 5 mg/kg or exactly 5 mg/kg.

Alternatively, galantamine can be administered in place of (5S)-1 -methyl-5-(3- pyridyl) pyrrolidin-2-one. Dosages of galantamine are optionally about 1 6 mg/kg/day to about 24 mg/kg/day, and low micromolar concentrations, of 0.1 μΜ to 1 μΜ have been tested. Useful dosages are 1 6 mg/kg/day, 1 7 mg/kg/day, 1 8 mg/kg/day, 19 mg/kg/day, 20 mg/kg/day, 21 mg/kg/day, 22 mg/kg/day, 23 mg/kg/day, or 24 mg/kg/day. Similarly, anatabine can be administered in place of (5S)-1 -methyl-5-(3-pyridyl) pyrrolidin-2-one. Dosages of anatabine are from about 0.1 8 mg/kg/day to about 30 mg/kg/day. Useful examples include 0.1 8 mg/kg/day, 1 .6 mg/kg/day, 3.2 mg/kg/day, 5 mg/kg/day, 7.5 mg/kg/day, 1 0 mg/kg/day, 12.5 mg/kg/day, 15 mg/kg/day, 1 7.5 mg/kg/day, and 20 mg/kg/day.

The therapeutically effective amount of (5S)-1 -methyl-5-(3-pyridyl) pyrrolidin-2-one is optionally administered intramuscularly, intraperitoneal^ or orally. The composition can be administered daily for up to 2 months after diagnosis of stress- related neuroinflammation, such as from day 1 through day 53 after diagnosis of stress-related neuroinflammation. Alternatively, the composition can be administered starting 7 days before an anticipated stress event.

In some variations, the composition of (5S)-1 -methyl-5-(3-pyridyl) pyrrolidin-2-one is administered with a ligand of nicotinic receptor subtype a7 or α4β2. The nicotinic receptor ligand and (5S)-1 -methyl-5-(3-pyridyl) pyrrolidin-2-one can be administered concurrently or individually. Where the two compositions, i.e. the nicotinic receptor ligand and (5S)-1 -methyl-5-(3-pyridyl) pyrrolidin-2-one are administered individually, there can be a brief time lapse between the two dosings, such as 1 minute, 5 minutes, 1 0 minutes, 1 0 minutes, 15 minutes, 20 minutes, 30 minutes, 45 minutes, or 1 hour. The nicotinic receptor ligand is optionally cytisine, epibatidine, varenicline, acetylcholine, nifene, or nicotine. Alternatively, an acetylcholinesterase inhibitor can be administered with the composition of (5S)-1 -methyl-5-(3-pyridyl) pyrrolidin-2-one. As with the ligand, the acetylcholinesterase inhibitor can be administered concurrently with (5S)-1 -methyl-5-(3-pyridyl) pyrrolidin-2-one or administered individually, with a time lase as described above. I n some variations, the acetylcholinesterase inhibitor is administered at about 1 mg/kg to about 23 mg/kg, once per day. A nonlimiting example of an acetylcholinesterase inhibitor is Aricept™ (donepezil hydrochloride, Pfizer Inc., NY, NY).

A composition of (5S)-1 -methyl-5-(3-pyridyl) pyrrolidin-2-one is altneritvely used to inducing neurogenesis gene expression in a cell is exposed to stress-related neurogenesis deficiency by contacting the cell with a therapeutically effective amount of the composition. Examples of the nueorgenesis genes that are induced include the neurogenesis gene is Vegfa,, Errb2, Egf, Gdnf, Artn, or a combination thereof. Genetic analysis allows for the measurement of the relative number of RNA transcripts that an organism is producing at any given time (Kadakkuzha & Puthanveettil, Genomics and proteomics in solving brain complexity. Mol Biosyst. Apr24. Epub ahead of print). RNA transcripts are produced from the chromosomal genes in order to produce proteins through transcription and translation, respectively (Kadakkuzha & Puthanveettil, Genomics and proteomics in solving brain complexity. Mol Biosyst. Apr24. Epub ahead of print). Once mRNA is made it is translated into proteins that the organism uses to respond to the environmental changes (Kadakkuzha & Puthanveettil, Genomics and proteomics in solving brain complexity. Mol Biosyst. Apr24. Epub ahead of print).

Gene expression PCR arrays allow for the measurement of a set of mRNA transcripts in an organism at a given time using real time polymerase chain reaction (RT-PCR) (Kadakkuzha & Puthanveettil, Genomics and proteomics in solving brain complexity. Mol Biosyst. Apr24. Epub ahead of print).

In some variations, the therapeutically effective amount of (5S)-1 -methyl-5-(3- pyridyl) pyrrolidin-2-one is optionally used a dosage, as described above, or a concentrations of 4 μιη to 30 μιη. For example, (5S)-1 -methyl-5-(3-pyridyl) pyrrolidin-2-one can be used at 4 μιη, 5 μιη, 6 μιη, 7 μιη, 8 μιη, 9 μιη, 10 μιη, 1 1 μιη, 12 μιη, 14 μιη, 15 μιη, 1 6 μιη, 1 7 μιη, 18 μιη, 20 μιη, 22 μιη, 24 μιη, 25 μιη, 26 μιη, 28 μιη, or 30 μιη. Similarly, the composition of of (5S)-1 -methyl-5-(3-pyridyl) pyrrolidin-2-one is optionally contacted or administered with a nicotinic receptor ligand or acetylcholinesterase inhibitor, as described above.

Testing was designed to analyze the gene expression patterns of mice subjected to stress for both neurogenesis and inflammation and identify a pathway through which cotinine may act. The results show that cotinine reduced the expression of several neuroinflammatory factors including chemokines, cytokines and other factors including Nos2, C3, Ccl3 and Ccr2. Cotinine also decreased the negative effects of stress on the expression of various neurogenesis genes including Artn, Erbb2, Egf, Gdnf and Vegfa. This evidence suggests that cotinine has multiple beneficial effects underlying its pro-cognitives and antidepressant effects. Several neuroinflammatory and neurotrophic factors whose expression is modified by cotinine in forced swimming mice were identified, further elucidating mechanisms by which inflammation causes decreased neurogenesis and the connection between increased inflammation, decreased neurogenesis and depression.

BRIEF DESCRIPTION OF THE DRAWINGS

For a fuller understanding of the invention, reference should be made to the following detailed description, taken in connection with the accompanying drawings, in which:

Figure 1 is a graph showing a summary of the inflammation and autoimmunity genes affected by cotinine treatment.

Figure 2 is a graph showing a summary of the neurogenesis genes affected by cotinine treatment.

Figure 3 is a model showing cotinine-based effects on cellular pathways resulting in alterations in neurogenesis genes.

Figure 4 is a blot showing cotinine increased the expression of VEGF in the hippocampus of mice subjected to repetitive forced swim stress. Mice pretreated with 5 mg/kg of cotinine (Cot 5) or vehicle (Veh) were exposed to repetitive 6-min forced swim (FS) daily for 6 days (a) to under continuous treatment. Following esting, mice were euthanized and the levels of VEGF and β-tubulin in the hippocampus were analyzed by Western blot. The plots represent VEGF immunoreactivity (I R) values in the hippocampus of mice.

Figure 5 is a graph showing cotinine increased the expression of VEGF in the hippocampus of mice subjected to repetitive forced swim stress. VEGF immunoreactivity (I R) in the blots from Figure 4 were normalized to β -tubulin and expressed as percentage of the average value found in vehicle-treated mice. Western blot images are seen beneath each comparison, ns, non-significant differences.

Figure 6 is a blot showing cotinine increased the expression of VEGF in the hippocampus of mice subjected to repetitive forced swim stress compared to unstressed mice. Mice pretreated with 5 mg/kg of cotinine (Cot 5) or vehicle (Veh) were exposed to repetitive 6-min forced swim (FS) daily for 6 days (a) to under continuous treatment or controls, not subjected to the forced swim (N ES). Following resting, mice were euthanized and the levels of VEGF and β-tubulin in the hippocampus were analyzed by Western blot. The plots represent VEGF immunoreactivity (I R) values in the hippocampus of mice.

Figure 7 is a graph showing cotinine increased the expression of VEGF in the hippocampus of mice subjected to repetitive forced swim stress. VEGF immunoreactivity (I R) in the blots from Figure 6 were normalized to β -tubulin and expressed as percentage of the average value found in vehicle-treated mice. Western blot images are seen beneath each comparison, ns, non-significant differences; p < 0.001 .

Figure 8 is a plot of the gene relationships for Ccr4, Ccl3, Ccr2, Ccl2, Fos, Erbb2, Egf, Vegfa and Nos2.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT

As used herein, the singular forms "a," "an" and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "a polypeptide" includes a mixture of two or more polypeptides and the like.

As used herein, "about" means approximately or nearly and in the context of a numerical value or range set forth means ±15% of the numerical.

As used herein "animal" means a multicellular, eukaryotic organism classified in the kingdom Animalia or Metazoa. The term includes, but is not limited to, mammals. Non-limiting examples include rodents, aquatic mammals, domestic animals such as dogs and cats, farm animals such as sheep, pigs, cows and horses, and humans. Wherein the terms "animal" or "mammal" or their plurals are used, it is contemplated that it also applies to any animals.

As used herein the term "patient" is understood to include an animal, especially a mammal, and more especially a human that is receiving or intended to receive treatment.

As used herein, the term "therapeutically effective amount" refers to that amount of a therapy (e.g., a chemotherapeutic agent) sufficient to result in the amelioration of at least one stress-related neurogenesis deficiency, enhancement of neurogenesis, or induction of one or more neurogenesis genes. As used herein, enhancement of neurogenesis means a statistically-significant increase in neuronal cell formation, and induction means a statistically-significant increase in neurogenesis gene expression.

The effect of cotinine on neurogenesis and neurogenesis gene induction was analyzed using RT2 Profiler PCR arrays (SABiosciences; Qiagen N.V., Venlon NL) on the hippocampus of mice subjected to forced swimming stress. These arrays consisted of 96 well plates, with pre-dispensed sets of primers and controls, which are designed to target the gene expression of specific pathways using RT-PCR analysis. Each array contains the primers for the analysis of 84 genes in a specific pathway, 3 reverse transcription controls, 1 genomic DNA control and 3 PCR positive controls. Sample cDNA, prepared from mRNA, is loaded onto each plate and then subjected to RT-PCR. RT- PCR creates a copy of the target region of cDNA, resulting in a doubling of template in each cycle (Livak & Schmittgen, Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) Method. Methods. 2001 Dec;25(4):402-8). SYBR green is used as a labeling agent, which allows the RT-PCR instrument (DNA Engine Opticon) to measure signal levels. SYBR green intercalates between double stranded DNA molecules which are formed during PCR. The relative amount of mRNA expression can be measured because the amount of fluorescence is proportional to amount of template present (Livak & Schmittgen, Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) Method. Methods. 2001 Dec;25(4):402-8). Comparing the relative level of mRNA expression in one condition to the relative level of mRNA expression present in another condition, can indicate how an organism is regulating the levels of proteins produced in response to changes in external stimuli (Livak & Schmittgen, Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) Method. Methods. 2001 Dec;25(4):402-8).

Example 1

Two-month-old male C57BL/6J mice (The Jackson Laboratory, Bar Harbor, ME), weighing 25-30 g were maintained on a 12-hours light/dark cycle (light on at 07:00) with ad libitum access to food and water and maintained at a regulated temperature of 25 ± 1 °C. Upon arrival mice were group-housed and acclimated for 7 days before any intervention.

Mice were randomly divided into three groups as follows: group 1 controls that were not exposed to stress (NES) and treated with vehicle (phosphate buffered saline, PBS); group 2 controls subjected to forced swimming stress (FSB) and treated with vehicle; group 3 subjected to forced swimming stress and treated with cotinine (FSA).

Cotinine ((5S)-1 -methyl-5-(3-pyridyl) pyrrolidin-2-one; Sigma-Aldrich Corporation, St. Louis, MO, USA) solutions were prepared by dissolving the powdered compound in sterile phosphate buffered saline (PBS), and vehicle (PBS). Mice were treated with vehicle or cotinine (5 mg/kg) via gavage. The gavage technique was performed by well trained personnel and did not induce significant stress in the mice. All investigators were blind to treatment groups and doses were chosen based on previously studies (Zeitlin, et al., (2012) Cotinine enhances the extinction of contextual fear memory and reduces anxiety after fear conditioning. Behav Brain Res, 228, 284-293.). Treatments were administered for 7 days before the induction of stress. For all animals, treatment continued from the onset of treatment until euthanasia.

To induce chronic stress, mice were subjected to repetitive forced swimming (FS) for six consecutive days, for six minutes each day, as described previously (Furukawa-Hibi, et al., (201 1 ) The hydrophobic dipeptide Leu-lle inhibits immobility induced by repeated forced swimming via the induction of BDN F. Behav Brain Res. 201 1 Jul 7;220(2):271 -280). Each mouse was placed in an inescapable transparent plastic cylinder (40 centimeters (cm) high x 20 cm in diameter) filled with water to a depth of 30 cm, for six minutes. Water temperature was maintained at 24-25 °C and water was changed between all trials. In all cases, following exposure, animals were retrieved, dried with a hand towel and returned to their home cages. Mice assigned to NES control group were removed from the animal housing facility and taken to the behavioral testing room during the same period of time than FS mice, but remained in their home cages during the FS period. In all experiments, no mice needed assistance to avoid drowning.

Experiments were performed during the light period of the circadian cycle and in accordance with the National Institutes of Health standards.

Following behavioral experimentation, euthanasia was performed via cervical dislocation under anesthesia with isofluorane (4% induction, 2% maintenance) by investigators not assigned to prior work with the mice blinded to the treatments. Whole brains were collected and immediately stored frozen at -80 °C.

The hippocampi were dissected on ice and placed into RNAIater tubes (Life Technologies; Thermo Fisher Scientific, Waltham, MA). RNAIater is a proprietary solution which protects RNA in tissue samples from degradation to allow for analysis at a later date. RNase free conditions were created and maintained throughout the procedure by washing labware in a 0.1 % DEPC solution followed by rinsing with RNase free water. Total RNA extraction and purification was performed using RNeasy Lipid Tissue Extraction Kit (Qiagen N.V., Venlon NL). Sample homogenization was performed using Kontes Pellet Pestle Cordless Motor and/or 20 gauge needle and syringe. Samples were extracted using a Qiazol/Chloroform extraction. The samples were purified on an RNeasy spin column using multiple washes. An on-column DNase incubation was performed in order to remove contaminating DNA. The entire extraction and purification step was performed twice to remove all contaminating DNA for samples ran on the neurogenesis arrays. Samples were eluted in RNase free water.

Nucleic acid quantification was performed on the RNA, to determine how many microliters of sample to add to the cDNA reaction, as the reaction requires a specific concentration of RNA to achieve optimal results. Nucleic acid quantification was performed using a NanoDrop spectrophotometer (Thermo Fisher Scientific, Waltham, MA). The absorbance of each sample was measured by applying 1 μΙ to the platform. Concentration was calculated using the absorbance values for 260nm using Beer's Law. The spectra were examined for the presence of a single peak and the 260/280nm ratio was expected to be near 2.0 for high quality RNA. The quality and quantity of total RNA was determined spectrophotometrically with an Agilent 21 00 Bioanalyzer (Agilent Technologies, Palo Alto, CA). The samples were selected for RT- PCR experiments provided that they had a > 2.0 RNA integrity number. cDNA was synthesized using RT ² First strand kits (SABiosciences; Qiagen N.V.,

Venlon NL) to prepare cDNA from purified total RNA. First, a genomic DNA elimination step was performed. Following DNA removal, the RNA preparation was incubated at 42 ^e C for five minutes and placed on ice. First strand synthesis reaction was performed by adding 4 μΙ of 5X RT Buffer, 1 μΙ of Primer and External Control mix and 2 μΙ of RT Enzyme Mix and 3 μΙ of RNase free water to each sample. The mixture was incubated at 42 ^e C for exactly 15 minutes and then immediately heated to 95 ^e C for 5 minutes using an iCycler thermal cycler (Bio-Rad Laboratories, Inc., Hercules, CA). Finally, 91 μΙ of RNase free water was added to each cDNA synthesis reaction. Example 2

To study whether the effect of cotinine was linked to an anti-inflammatory effect, the expression of inflammation genes in the hippocampus of stressed mice was compared in treated (cotinine) versus nontreated (vehicle), using the inflammation and autoimmunity gene expression arrays. Inflammatory response and Autoimmunity PCR arrays (PAMM-077Z) (SABiosciences; Qiagen N.V., Venlon NL) were loaded and placed into a DNA Engine Opticon RT-PCR instrument (Bio-Rad Laboratories, Inc., Hercules, CA). A mix of 1 ,350 μΙ 2x RT2 SYBR Green Mastermix, 1 02 μΙ cDNA synthesis reaction, and 1 ,248 μΙ of RNase-free water was prepared for each sample. Using a multichannel pipette, samples obtained as per Example 1 were loaded into each well of the 96-well plates. Thermal cycling was performed as follows: 1 cycle of 95 °C for 1 0 minutes, followed by 40 cycles of 15 seconds at 95 ^, 1 minute at 60 °C, and data collection. A melt curve was performed to assay the reaction specificity as follows 95 °C for 1 minute, followed by an incremental increase of 0.5 °C every 2 seconds from 55 to 95 °C and data collection.

PCR array data was then analyzed. Raw data Ct values were determined and exported to an Excel spreadsheet using Opticon Monitor 3 software (Bio-Rad

Laboratories, Inc., Hercules, CA). Each plate was examined to determine the appropriate baseline and threshold values. The CtPPC for each sample was 20 Ct ± 0.5 for all arrays. Ct values ≥ 35 were considered negative. Data analysis was performed using the PCR Array Data Analysis website (SABiosciences; Qiagen N.V., Venlon NL). Data was normalized using multiple housekeeping genes-

Hprtl, Gapdh and Actb- as well as other genes on the plate with the smallest changes in raw Ct values. Fold regulation was calculated using the delta delta Ct method (Impey, et al., Stimulation of cAM P response element (CRE)-mediated transcription during contextual learning. Nat Neurosci. 1 998.1 (7):595-601 ). Delta Ct was calculated using the average Ct value for the gene of interest minus the average Ct value for the selected housekeeping genes (Livak & Schmittgen, Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) Method. Methods. 2001 Dec;25(4):402-8). These values were averaged to obtain the average delta Ct (Livak & Schmittgen, Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T))

Method. Methods. 2001 Dec;25(4):402-8). Then, delta delta Ct was calculated by subtracting the delta Ct value for the control from the delta ct value from the gene of interest (Livak & Schmittgen, Analysis of relative gene expression data using realtime quantitative PCR and the 2(-Delta Delta C(T)) Method. Methods. 2001 Dec;25(4) :402-8). Fold change was calculated using 2-Ct for each condition (Livak

& Schmittgen, Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) Method. Methods. 2001 Dec;25(4):402-8).

The results showed 61 % of the inflammation genes analyzed were down-regulated in the stressed mice treated with cotinine, as seen in Figure 1 . Using a cut off of two-fold difference in regulation, 1 0 genes were differentially expressed in the stressed mouse treated with cotinine versus the stressed mouse treated with vehicle, seen in Table 1 . The following genes were found down-regulated: Complement component 3(C3), Ccl3, Ccr2, Fos and Nos2 and up regulated Cell , Ccl2, Ccr4, TIM and Tnfsf14 in the hippocampal extracts.

Table 1 . The relative change in gene expression induced by cotinine, expressed as fold negative or positive regulation respect to control vehicle-treated mice. Gene abbreviation Gene Name Fold regulation comparing to FSB

C3 Complement component 3 -2.89

Cell Chemokine (C-C motif) ligand 1 2.21

Ccl2 Chemokine (C-C motif) ligand 2 2.46

Ccl3 Chemokine (C-C motif) ligand 3 -4.00

Ccr2 Chemokine (C-C motif) receptor 2 -2.87

Ccr4 Chemokine (C-C motif) receptor 4 2.96

Fos FBJ osteosarcoma oncogene -2.55

Nos2 Nitric oxide synthase 2, inducible -3.93

Tlr1 Toll-like receptor 1 2.32

Tnfsfl Tumor necrosis factor (ligand) 6.1 8

superfamily, member 14

FBJ osteosarcoma oncogene (Fos) expression was decreased by 2.5 fold in the brain of the mouse subjected to forced swimming treated with cotinine compared to the mouse subjected to forced swimming and treated with vehicle. Complement component 3 (C3), was decreased by 2.9 fold in the mouse subjected to forced swimming treated with cotinine compared to the stressed mouse treated with vehicle. Five members of the chemokine family were differentially expressed, with some chemokines up regulated by cotinine treatment and stress induction (Cell , Ccl2 and Ccr4), while most of the cytokines and chemokines were modestly down regulated by cotinine in the stressed mouse. More dramatic changes were observed in the chemokines Ccl3 (2.8 fold) and Ccr2 (4.0 fold).

Example 3

To study whether administration of cotinine was linked to an enhancement of neurogenesis in the hippocampus, the expression of neurogenesis genes in the brains of vehicle-treated and cotinine-treated mice were compared. Neurogenesis and a neural stem cell real-time polymerase chain reaction (RT-PCR) arrays were used to determine the effect of cotinine on the expression of neurogenesis genes in the hippocampus of mice. The RT-PCR reaction basically consists in quantifying a fluorescent dye that intercalates with the complementary DNA (cDNA) amplified in each cycle. In this array, the RT-PCR reactions allow the simultaneous detection and quantification of the expression of 84 genes coding for stem cell specific biomarkers known to have positive or negative effects on the regulation of cell proliferation, cell differentiation, synaptic function, apoptosis, cell adhesion, and cell signaling of neurogenesis. Neurogenesis RT ² Profiler PCR arrays (PAMM-404Z) (SABiosciences; Qiagen N.V.,

Venlon NL) were loaded and placed into a DNA Engine Opticon RT-PCR instrument (Bio-Rad Laboratories, Inc. , Hercules, CA). A mix of 1 ,350 μΙ 2x RT2 SYBR Green Mastermix, 1 02 μΙ cDNA synthesis reaction, and 1 ,248 μΙ of RNase-free water was prepared for each sample. Using a multichannel pipette, samples obtained as per Example 1 were loaded into each well of the 96-well plates. Thermal cycling was performed as follows: 1 cycle of 95 °C for 1 0 minutes, followed by 40 cycles of 15 seconds at 95 ^, 1 minute at 60 °C, and data collection. A melt curve was performed to assay the reaction specificity as follows 95 °C for 1 minute, followed by an incremental increase of 0.5 °C every 2 seconds from 55 to 95 °C and data collection.

PCR array data was then analyzed. Raw data Ct values were determined and exported to an Excel spreadsheet using Opticon Monitor 3 software (Bio-Rad Laboratories, Inc., Hercules, CA). Each plate was examined to determine the appropriate baseline and threshold values. The CtPPC for each sample was 20 Ct ± 0.5 for all arrays. Ct values ≥ 35 were considered negative. Data analysis was performed using the PCR Array Data Analysis website (SABiosciences; Qiagen N.V., Venlon NL). Data was normalized using multiple housekeeping genes- Hprtl, Gapdh and Actb- as well as other genes on the plate with the smallest changes in raw Ct values. Fold regulation was calculated using the delta delta Ct method (Impey, et al., Stimulation of cAM P response element (CRE)-mediated transcription during contextual learning. Nat Neurosci. 1 998.1 (7):595-601 ). Delta Ct was calculated using the average Ct value for the gene of interest minus the average Ct value for the selected housekeeping genes (Livak & Schmittgen, Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) Method. Methods. 2001 Dec;25(4):402-8). These values were averaged to obtain the average delta Ct (Livak & Schmittgen, Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) Method. Methods. 2001 Dec;25(4):402-8). Then, delta delta Ct was calculated by subtracting the delta Ct value for the control from the delta ct value from the gene of interest (Livak & Schmittgen, Analysis of relative gene expression data using realtime quantitative PCR and the 2(-Delta Delta C(T)) Method. Methods. 2001 Dec;25(4) :402-8). Fold change was calculated using 2-Ct for each condition (Livak & Schmittgen, Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) Method. Methods. 2001 Dec;25(4):402-8). Ten samples were tested using the neurogenesis gene expression arrays: three hippocampal samples from mice that were subjected forced swimming and treated with cotinine (FSA), three hippocampal samples from mice that were subjected forced swimming and treated with vehicle (FSB) and four samples from control mice that were not exposed to stress (NEB) and treated with vehicle.

It has been established that biologically significant changes in neural tissue can occur with very small changes in gene expression, not the traditional cut off of twofold change normally expected in non-brain tissues (Soverchia, et al., Microarrays - The Challenge of Preparing Brain Tissue Samples. Addiction Biology. March 2005 1 0, 5-13). For the neurogenesis arrays, a criterion of probable significant change that consists of an arbitrary cut-off of 1 .3-fold change in gene expression combined an observable opposite effect on expression induced by cotinine was established. For example, in mice subjected to forced swimming, if a gene was negatively expressed in the brain of mice treated with vehicle at -1 .3-fold, but was positively expressed in the mice treated with cotinine at 1 .2-fold, it was selected. First, to determine if forced swimming was having an impact on gene expression, we compared gene expression levels in mice of each treatment arm subjected to forced swimming to the ones found in mice that were not exposed to stress.

In the samples from mice that had been subjected to forced swimming stress and treated with cotinine (FSA), only 56% of genes were down-regulated (1 7% decrease). Next, gene expression in the hippocampus of mice that had been subjected to forced swimming and treated with cotinine were compared to the levels present in the hippocampus from stressed mice treated with vehicle. In this comparison, 67% of genes analyzed were up regulated in the hippocampi from cotinine-treated mice when compared to the hippocampal expression in vehicle- treated mice, as seen in Figure 2.

Using the results from all three comparisons, 15 genes demonstrated differential regulation. Fourteen of these genes were negatively regulated in the stressed mice treated with vehicle, but were positively regulated in stressed mice treated with cotinine. Three genes showed statistically significant differences in expression including Acetylcholinesterase gene (Ache) (p = 0.03), ErbB2 (p = 0.008) and Vascular endothelial growth factor gene (Vegfa (p = 0.03).

In the hippocampus of vehicle treated and stressed mice, the expression of the vascular endothelial growth factor gene, Vegfa, was slightly down-regulated by 1 .1 - fold compared to mice that were not exposed to stress. However, stressed mice treated with cotinine showed a 1 .3-fold up regulation of Vegfa expression when compared to stressed mice treated with vehicle. Also, cotinine induced a highly significant up regulation of the expression of V-erb-b2 erythroblastic leukemia viral oncogene homolog 2 (Errb2) by 1 .3 fold in stressed mice compared to stress control mice. By contrast, Errb2 was down-regulated by 1 .5 fold in stressed mice treated with vehicle compared to the mice that were not exposed to stress. Egf, the gene that codes for the epidermal growth factor (EGF), is up regulated 1 .5-fold in stressed mice treated with cotinine compared to the stressed mice treated with vehicle. Similarly, Egf was down-regulated by 1 .5-fold in mice exposed to forced swimming stress treated with vehicle compared to mice that were not exposed to stress. Gdnf, the gene for glial cell line derived neurotrophic factor, was down- regulated in the vehicle-treated mice exposed to forced swimming by 1 .4-fold compared to mice that were not exposed to stress, but up regulated 2.0-fold in stressed mice treated with cotinine when compared to stressed mice treated with vehicle. Similarly, Artn, the gene for Artemin, was down-regulated 1 .3-fold in the vehicle-treated stressed mice when compared to vehicle-treated mice that were not exposed to stress, but up regulated 1 .2-fold in stressed mice treated with cotinine compared to stressed mice treated with vehicle, as seen in Table 2.

Table 2. The relative change in neurogenesis gene expression induced by cotinine (Cot), for mice exposed to stress (ES) or not exposed to stress (NES), expressed as fold negative or positive regulation respect to control vehicle-treated.

a Bone morphogenetic protein 2A, ^b V-erb-b2 erythroblastic leukemia viral oncogene homolog 2, ^c Oligodendrocyte lineage, ^d RAS-related C3 botulinum substrate 1 , ^e SRY-box containing gene 2, ^f Signal transducer and activator of transcription 3, Vascular endothelial growth factor

^* p <0.05

* ^* p <0.01

In light of the results, a model was proposed for cotinine treatment as it relates to neurogenesis, as seen in Figure 3.

Example 4 VEGF, is a neurotrophin that modulates blood flow, angiogenesis (24) and is involved in neurogenesis (Jin, et al., (2002) Vascular endothelial growth factor (VEGF) stimulates neurogenesis in vitro and in vivo. Proc Natl Acad Sci U S A, 99, 1 1946-1 1 950; Fabel, et al., (2003) VEGF is necessary for exercise-induced adult hippocampal neurogenesis. Eur J Neurosci, 1 8, 2803-2812; Cao, et al., (2004) VEGF links hippocampal activity with neurogenesis, learning and memory. Nat Genet, 36, 827-835). Vegfa mRNA expression RT-PCR assay showed Vegfa up- regulated in the hippocampus of cotinine-treated FS mice when compared to vehicle-treated FS mice (p < 0.01 ). Since continine up-regulated VEGF mRNA expression in the hippocampus of FS mice, associated protein expression levels in the hippocampi of the same mice were analzed using Western blot analysis.

The Western blot analysis investigated the expression of VEGF (FS mice: n=8- 1 0/group; NES mice: n=4-5/group). Following euthanasia, mice were perfused with saline, and brain tissues were rapidly dissected and stored at -80 ^e C, as described in Example 1 . Brain tissues were then disrupted by sonication in cold lyses buffer (Cell Signaling Technology, Danver, MA, USA) containing a complete protease inhibitor cocktail (Roche Molecular Biochemicals). After sonication, brain extracts were incubated on ice for 30 min and centrifuged at 20,000 x g for 30 min at 4 ^. Protein concentrations of supernatants were measured using the Bio-Rad protein assay (Bio-Rad, Hercules, CA, USA). Equal amounts of protein were separated by gradient (4-20%), SDS-PAGE, then transferred to nitrocellulose membranes (BA83 0.2 μιη; Bio-Rad). The membranes were blocked in TBS with 0.1 % Tween 20 (TBST) containing 5% dry skim milk for 1 h. Membranes were incubated with primary antibodies in TBST overnight at 4 °C and with secondary antibodies for 1 h at room temperature (RT) in a blocking buffer. Rabbit polyclonal antibodies directed against VEGF was obtained from Abeam (Cambridge, MA, USA). A monoclonal antibody directed against β-tubulin (Promega Corporation, Madison, Wl, USA) was used to control protein sample loading and transfer efficiency. Membranes were washed with TBST and incubated with LI-COR's goat anti-mouse I RDye secondary antibodies (LI-COR Biosciences, Lincoln, NE) for 1 h at RT, washed with TBST and TBS. Images were acquired using an Odyssey Infrared Imaging System (LI-COR Biosciences) and analyzed using the NI H Image J software.

To analyze the group and treatment effects, differences between group means in the behavioral analyses were assessed using two-way analysis of variance (ANOVA) and post hoc Tukey test. Student t-test with or without Welch correction was use to compare protein expression data between two groups. Statistical analyses were conducted using statistical software packages (SPSS, Chicago, IL, USA and GraphPad Prism, San Diego, CA, USA). For all comparisons, statistical significance was considered with a = 0.05.

After normalization, VEGF staining was shown to be increased in cotinine-treated (5 mg/kg) mice versus vehcile treated mice, as seen in Figure 4. The results show that the groups differed significantly from one another (F(2,24) = 1 0.19, p = 0.0006; Figure 6 and Tukey-Kramer's post hoc analyses revealed that mice subjected to repetitive FS stress (Figure 4) had a significant decrease in the expression of VEGF in the hippocampi (p < 0.001 ; Figure 7. On the other hand, FS mice treated with cotinine showed significantly higher levels of VEGF expression in the hippocampus than vehicle-treated, FS mice, to reach levels not significantly different than those of NES mice (Figure 7 Finally, in the absence of stress, t-test revealed that cotinine induced no changes in VEGF expression (t = 0.291 0, p = 0.7795; Figure 5. VEGF is a cytokine that plays an important role modulating neurogenesis and angiogenesis (Schanzer, et al., (2004) Direct stimulation of adult neural stem cells in vitro and neurogenesis in vivo by vascular endothelial growth factor. Brain Pathol, 1 4, 237- 248; Galvan, et al., (2006) The role of vascular endothelial growth factor in neurogenesis in adult brain. Mini reviews in medicinal chemistry, 6, 667-669; Sun, et al., (2006) Vascular endothelial growth factor-B (VEGFB) stimulates neurogenesis: evidence from knockout mice and growth factor administration. Developmental biology, 289, 329-335; Antequera, et al., (2012) Encapsulated VEGF-secreting cells enhance proliferation of neuronal progenitors in the hippocampus of AbetaPP/Ps1 mice. J Alzheimers Dis, 29, 1 87-200).

It has been found that cotinine, elicits memory enhancing effects (Buccafusco, et al., (2009) Desensitization of nicotinic acetylcholine receptors as a strategy for drug development. J Pharmacol Exp Ther, 328, 364-370; Buccafusco & Terry, (2009) A reversible model of the cognitive impairment associated with schizophrenia in monkeys: potential therapeutic effects of two nicotinic acetylcholine receptor agonists. Biochem Pharmacol, 78, 852-862; Echeverria, et al., (201 1 b) Cotinine reduces amyloid-beta aggregation and improves memory in Alzheimer's disease mice. J Alzheimers Dis, 24, 81 7-835; Echeverria Moran, (2012) Cotinine: Beyond that Expected, More than a Biomarker of Tobacco Consumption. Frontiers in pharmacology, 3, 1 73) and positive effects on brain plasticity in animal models of behavior. Clinical studies investigated the effect of cotinine at doses up to 1 .5 mg cotinine base/kg and found cotinine impaired recall on the long list on a verbal recall task but did not affected the recall of a short list or altered the scores in the profile of mood state (Herzig 1998; McNeil, 1 971 ). Cotinine, in the presence of stress, increases the expression of VEGF, a growth factor important in neurogensis.

Example 5 Using the results of Example 2 and Example 3, genes that displayed differential regulation were entered into the Gene Network Central Pro database from SABiosciences to determine any established gene relationship(s) in a Gene Interaction Network. The database uses published literature to establish known gene interactions. The following nine genes were identified to have known interactions: Ccr4, Ccl3, Ccr2, Ccl2, Fos, Erbb2, Egf, Vegfa and Nos2. The results are pictured in Figure 8.

Cotinine treatment increased neurogenesis-promoting genes, including Vegfa, Egf, Erbb2, Gdnf, and Artn, while decreasing inflammation and oxidation-related genes, such as Nos2, and Fos, and most chemokine genes.

The vascular endothelial growth factor (VEGF)-encoding gene, Vegfa, provides for a neurotrophin that modulates blood flow and angiogenesis (Lee, et al., I nduction of Neuronal Vascular Endothelial Growth Factor Expression by cAM P in the Dentate Gyrus of the Hippocampus Is Required for Antidepressant-Like Behaviors. The Journal of Neuroscience, 2009. 29(26):8493- 8505). Stress has been shown to reduce the expression of Vegfa (Raison, et al., Cytokines sing the blues: inflammation and the pathogenesis of depression. Trends Immunol. 2006 Jan;27(1 ):24-31 ) and VEGF has previously been implicated in supporting the positive effects of several antidepressants by stimulating the cAMP-CREB (cAMP response element-binding protein) pathway (Lee, et al., I nduction of Neuronal Vascular Endothelial Growth Factor Expression by cAM P in the Dentate Gyrus of the Hippocampus Is Required for Antidepressant-Like Behaviors. The Journal of Neuroscience, 2009. 29(26):8493- 8505). The stimulation of this pathway has been shown to inhibit depressive behavior, decrease neurodegeneration and improve memory (Lee, et al., Induction of Neuronal Vascular Endothelial Growth Factor Expression by cAM P in the Dentate Gyrus of the Hippocampus Is Required for Antidepressant-Like Behaviors. The Journal of Neuroscience, 2009. 29(26):8493- 8505). In addition, supporting a possible role of VEGF in the actions of cotinine, experiments conducted by Lee et al. demonstrated that increases in VEGF expression stimulated neurogenesis in mice (Lee, et al., Induction of Neuronal Vascular Endothelial Growth Factor Expression by cAM P in the Dentate Gyrus of the Hippocampus Is Required for Antidepressant-Like Behaviors. The Journal of Neuroscience, 2009. 29(26):8493-8505). This evidence is consistent with the idea that an increase in VEGF induced by cAM P may underlie the effects of cotinine. All together this evidence supports the hypothesis that an increase of VEGF expression induced by cotinine mediates its effects in the promotion of neurogenesis. V-erb-b2 erythroblastic leukemia viral oncogene homolog 2 (Erbb2) is a member of the tyrosine kinase family of epidermal growth factors (Impey, et al., Stimulation of cAMP response element (CRE)-mediated transcription during contextual learning. Nat Neurosci. 1 998.1 (7):595-601 ). As a member of the epidermal growth factor family, ErbB2 participates in cell adhesion and promoting vasculature (Yang, ErbB2 Overexpression Correlates with Increased Expression of Vascular Endothelial Growth Factors A, C, and D in Human Breast Carcinoma. CANCER. 2002. 94 (1 1 ) :2855-61 ). I nterestingly, studies have shown that ErbB2 is commonly up regulated with VEGF (Yang, ErbB2 Overexpression Correlates with I ncreased Expression of Vascular Endothelial Growth Factors A, C, and D in Human Breast Carcinoma. CANCER. 2002. 94 (1 1 ) :2855-61 ). The results herein concurred, showing ErbB2 expression approximated the expression of VEGF. Up regulation of ErbB2 expression has been suggested as a mechanism of promoting neurogenesis by regulating the differentiation of radial glial cells into astrocytes (Ghashghaei, et al., Reinduction of ErbB2 in astrocytes promotes radial glial progenitor identity in adult cerebral cortex, Genes Dev. 2007. 21 (24): 3258-3271 ). Early in development, radial glial cells transform into astrocytes as ErbB2 expression is down-regulated. (Ghashghaei, et al., Reinduction of ErbB2 in astrocytes promotes radial glial progenitor identity in adult cerebral cortex, Genes Dev. 2007. 21 (24): 3258-3271 ) In animal studies, re-expression of ErbB2 allowed radial glial to support neuronal migration, a necessary mechanism in neurogenesis (Ghashghaei, et al., Reinduction of ErbB2 in astrocytes promotes radial glial progenitor identity in adult cerebral cortex, Genes Dev. 2007. 21 (24) : 3258-3271 ). ErbB2 has also been implicated in participating in regulator pathways that lead to insulin resistance after induction of cellular stress by administering cytokines in culture. (Hemi, et al., Transactivation of ErbB2 and ErbB3 by tumor necrosis factor-alpha and anisomycin leads to impaired insulin signaling through serine/threonine phosphorylation of I RS proteins. J Biol Chem. 2002 Mar 15;277(1 1 ):8961 -9) Thus, cotinine may also enhance neurogenesis and improve brain homeostasis by up regulating the expression of ErbB2.

Cotinine increased expression of Egf in stress-induced mice, whereas control treatment with vehicle resulted in decreased Egf expression, as discussed in Example 3. This suggests that cotinine can prevent the down regulation of Egf expression induced by stress and further increase its expression. Egf is known to play an important role in neurogenesis by stimulating the differentiation, proliferation and migration of neurons (Tian, et al., A study of the functional significance of epidermal growth factor in major depressive disorder. Psychiatr Genet. 2012 .22(4):1 61 -7). Tian et al demonstrated that plasma Egf levels were decreased in patients with major depressive disorder compared with control patients (Hemi, et al., Transactivation of ErbB2 and ErbB3 by tumor necrosis factor-alpha and anisomycin leads to impaired insulin signaling through serine/threonine phosphorylation of I RS proteins. J Biol Chem. 2002 Mar 15;277(1 1 ):8961 -9).

As noted in Example 3, expression of both Gdnf (Glial cell line derived neurotrophic factor) and Artn (Artemin) are up regulated in stressed mice treated with cotinine compared to stressed mice treated with vehicle. Gdnf and Artn are both neurtrophins, and members of the GDNF family (Tian, et al., A study of the functional significance of epidermal growth factor in major depressive disorder. Psychiatr Genet. 2012 .22(4):1 61 -7). These neurotrophins exert their action through the GDNF family receptor to activate the tyrosine kinase RET part of signaling pathways involved in cell survival, inflammation differentiation and apoptosis (Tian, et al., A study of the functional significance of epidermal growth factor in major depressive disorder. Psychiatr Genet. 2012 .22(4):1 61 -7). GDNF reduces oxidative stress-induced cell death in cultured neurons and supports the viability of mesencephalic dopaminergic neurons in culture by suppressing apoptosis (Tian, et al., A study of the functional significance of epidermal growth factor in major depressive disorder. Psychiatr Genet. 2012 .22(4):1 61 -7). Administration of cotinine up regulates the expression of Gdnf and Artn and may therefore positively inhibiting oxidative damage of the brain.

Nitric oxide is a free radial which can cause oxidative damage to cells (Zhou, et al., Neuronal nitric oxide synthase contributes to chronic stress-induced depression by suppressing hippocampal neurogenesis. Journal of Neurochemistry. 2007. 1 03(5):1 843-1 854), produced by nitric oxide synthase, of which there are three isozymes; neuronal NOS (nNOS), endothelial NOS, (eNOS) and inducible NOS (NOS2) (Zhou, et al., Neuronal nitric oxide synthase contributes to chronic stress- induced depression by suppressing hippocampal neurogenesis. Journal of Neurochemistry. 2007. 1 03(5):1 843-1 854). Nitric Oxide Synthase 2 (NOS2) is involved in the deleterious effects of neuroinflammation and oxidative stress (Zhou, et al., Neuronal nitric oxide synthase contributes to chronic stress-induced depression by suppressing hippocampal neurogenesis. Journal of Neurochemistry. 2007. 1 03(5):1 843-1 854). There is a correlation between increased levels of nNOS in the brain, decreased neurogenesis and depression (Lucassen, et al., "Regulation of adult neurogenesis by stress, sleep disruption, exercise and inflammation: Implications for depression and antidepressant action." European Neuropsychopharmacology 20.1 (201 0): 1 -1 7). Zhou et al showed that mice with a genetic deletion of nNOS or that were treated with nNOS inhibitors showed increased levels of neurogenesis and lower levels of depressive-like behavior (Zhou, et al., Neuronal nitric oxide synthase contributes to chronic stress-induced depression by suppressing hippocampal neurogenesis. Journal of Neurochemistry. 2007. 103(5) :1 843-1 854). These results suggest that cotinine may decrease iNOS expression in the hippocampus. Because of this effect, cotinine may have an antioxidant effect contributing to increasing neurogenesis under pathological conditions.

FBJ osteosarcoma oncogene (Fos) is an member of the activator protein 1 (AP1 -) family of transcription factors that work in concert with nuclear factor kappa beta (NFkB) to regulate the expression of genes involved in inflammation, oncogenesis and apoptosis (Nadjar, et al., Inactivation of the Cerebral NFkB Pathway Inhibits lnterleukin-1 b-lnduced Sickness Behavior and c-Fos Expression in Various Brain Nuclei. Neuropsychopharmacology. 2005. 30: 1492-1499). It has been demonstrated that c-Fos can be inactivated when the N FkB pathway is blocked (Nadjar, et al., I nactivation of the Cerebral NFkB Pathway I nhibits lnterleukin-1 fa- Induced Sickness Behavior and c-Fos Expression in Various Brain Nuclei. Neuropsychopharmacology. 2005. 30: 1492-1499). NFkB causes inflammatory damage that allows previously excluded substances to cross the blood-brain barrier when activated by inflammatory cytokines (Nadjar, et al., Inactivation of the Cerebral NFkB Pathway Inhibits lnterleukin-1 b-lnduced Sickness Behavior and c- Fos Expression in Various Brain Nuclei. Neuropsychopharmacology. 2005. 30: 1492-1499). Cotinine decreased the expression of Fos gene in mice exposed to forced swimming. Altogether this evidence indicates that decreasing c-Fos expression via cotinine can alleviate damage induced by neuroinflammation in the brain possibly by disrupting NFkB signaling.

Complement Component 3 (C3) is a component of the innate immune system, which is released by macrophages and acts as a cytokine under inflammatory conditions (Boyle, et al., Hostility, Anger and Depression Predict Increases in C3 over a 1 0-Year Period. Brain Behav Immun. 2007. 21 (6): 81 6-823), and increases under conditions of psychological stress (Boyle, et al., Hostility, Anger and Depression Predict Increases in C3 over a 1 0-Year Period. Brain Behav I mmun. 2007. 21 (6): 81 6-823). C3 has also been linked to incidence of cardiovascular diseases including myocardial infarction and stroke and his levels correlate with cardiovascular risk factors including blood pressure, BM I and lipids (Engstrom, et al., Complement C3 and C4 in plasma and incidence of myocardial infarction and stroke: a population-based cohort study. Eur J Cardiovasc Prev Rehabil. 2007. 14(3):392-7). Also, elevated C3 may contribute to dysregulation of the H PA (Zhou, et al., Neuronal nitric oxide synthase contributes to chronic stress-induced depression by suppressing hippocampal neurogenesis. Journal of Neurochemistry. 2007. 103(5):1843-1854). Five members of the chemokine family were differentially expressed, with some chemokines up regulated by cotinine treatment (Cell , Ccl2 and Ccr4) in the mouse exposed to forced swimming when compared to the stressed mouse treated with vehicle. However, most of the cytokines and chemokines were modestly down regulated by cotinine in the stressed mouse. Chemokines are small proteins responsive to basal and inflammatory conditions in the immune system and are constitutively present in glial cells and neurons in the brain (Rostene, et al., Chemokines: a new class of neuromodulator? Nat Rev Neurosci.2007. 8 (1 1 ):895- 903). They are divided into four families based on the configuration of conserved cysteine residues at their amino terminus as follows: CXC, CC, C and CX3C (Rostene, et al., Chemokines: a new class of neuromodulator? Nat Rev Neurosci.2007. 8 (1 1 ):895-903). All of the chemokines we observed to be differentially expressed belong to the CC, or beta family, which chemoattract monocytes, macrophages, basophils, T lymphocytes and eosinophils (Rostene, et al., Chemokines: a new class of neuromodulator? Nat Rev Neurosci.2007. 8 (1 1 ) :895-903).

Monocyte chemoattractant protein 1 (CCI2) is up regulated under neuroinflammatory conditions and Ccr2 is its receptor (Rostene, et al., Chemokines: a new class of neuromodulator? Nat Rev Neurosci.2007. 8 (1 1 ):895-903). Ccl2 is highly expressed in various brain tissues including the pituitary gland, glial cells and neuronal cells in rat brains and human cell lines, while Ccr2 is expressed in the spinal cord and neurons in the rat brain (Rostene, et al., Chemokines: a new class of neuromodulator? Nat Rev Neurosci.2007. 8 (1 1 ):895-903). While initial evidence demonstrated only the immune effects of chemokines, some studies have implicated chemokine as neurotransmitters or neuromodulators (Rostene, et al., Chemokines: a new class of neuromodulator? Nat Rev Neurosci.2007. 8 (1 1 ):895- 903). Ccl2 is colocalized with cholinergic neurons and dopaminergic neurons indicating that it may be involved in neurotransmission (Rostene, et al., Chemokines: a new class of neuromodulator? Nat Rev Neurosci.2007. 8 (1 1 ):895- 903). Additionally, Ccl2 reduces, in a dose dependent manner, the GABA-induced electrical responses in rat cultured neurons (Minami, et al., Brain Cytokines and Chemokines: Roles in Ischemic Injury and Pain. J Pharmacol Sci (2006).100, 461 - 470). It has been reported that the antagonism of the Ccr2 receptor reduced neuroinflammation in rodent models of multiple sclerosis and inflammatory arthritis (Minami, et al., Brain Cytokines and Chemokines: Roles in Ischemic Injury and Pain. J Pharmacol Sci (2006).1 00, 461 -470). We found a marked down regulation of Ccr2 by cotinine in the stressed mice even though we observed an increase in the ligand Ccl2. Macrophage inflammatory protein (Ccl3), is a proinflammatory chemokine involved in monocyte and microglia activation and migration (Minami, et al., Brain Cytokines and Chemokines: Roles in Ischemic Injury and Pain. J Pharmacol Sci (2006).1 00, 461 -470). In animal studies, using LPS, a known cytokine stimulant, concentrations of Ccl3 were increased (Brown, et al., Production of Proinflammatory Cytokines and Chemokines during Neuroinflammation: Novel Roles for Estrogen Receptors a and β. Endocrinology. 201 0 October; 151 (1 0)). A known anti-inflammatory, estradiol-1 7, was unable to lower the levels of Ccl3 in these studies (Brown, et al., Production of Proinflammatory Cytokines and Chemokines During Neuroinflammation: Novel Roles for Estrogen Receptors a and β. Endocrinology. 2010 October; 151 (10)). Elevated Ccl3 levels have also been linked to depression (Brown, et al., Production of Proinflammatory Cytokines and Chemokines During Neuroinflammation: Novel Roles for Estrogen Receptors a and β. Endocrinology. 201 0 October; 151 (10)). One study measured blood serum levels of Ccl3 in healthy donors and patients with moderate to severe depression and found that Ccl3 was detectable in 20% of depressed patients but absent in healthy donors (Brown, et al., Production of

Proinflammatory Cytokines and Chemokines During Neuroinflammation: Novel Roles for Estrogen Receptors a and β. Endocrinology. 2010 October; 151 (1 0)). As previously stated, inflammation can be a beneficial mechanism, but if left unchecked it can become damaging. These results suggest that cotinine may decrease the expression of these neuroinflammatory factors, especially if tested in a different animal model of stress that would induce higher levels of neuroinflammation.

The data herein suggests that cotinine may be a good candidate for neurogenesis pharmacotherapy, during highly stressful life events. Cotinine is a positive allosteric modulator (PAM) of the homomeric a7 nAChR (Moran, (2012) Cotinine: Beyond that

Expected, More than a Biomarker of Tobacco Consumption. Frontiers in pharmacology, 3, 1 73). New evidence in vivo suggests that cotinine's effects are mediated by a7 and α4β2 nAChRs (Aguiar, et al., (2013) Neuroactive effects of cotinine on the hippocampus: Behavioral and biochemical parameters. Neuropharmacology, 71 , 292-298; Wildeboer-Andrud, et al., (2014) Cotinine impacts sensory processing in DBA/2 mice through changes in the conditioning amplitude. Pharmacol Biochem Behav, 1 1 7, 144-150). Deleterious effects of stress result, at least in part, from a deregulation of the central monoamine systems. Cotinine increase the release and reduce the uptake of 5-HT in the brain of rats (Fuxe, et al., (1 979) On the action of nicotine and cotinine on central 5- hydroxytryptamine neurons. Pharmacol Biochem Behav, 1 0, 671 -677). Further, cotinine up-regulated hippocampal VEGF expression in the FS mice, as well as other neurogenesis genes. Stress reduces hippocampal neurogenesis (Gould & Tanapat, (1 999) Stress and hippocampal neurogenesis. Biol Psychiatry, 46, 1472- 1479) and the enhancement of hippocampal neurogenesis buffers the stress response as well as depressive-like behavior (Snyder, et al., (201 1 ) Adult hippocampal neurogenesis buffers stress responses and depressive behaviour. Nature, 476, 458-461 ). Furthermore, several commonly prescribed antidepressants stimulate the expression of neurogenesis genes (Newton & Duman, (2004) Regulation of neurogenesis and angiogenesis in depression. Current neurovascular research, 1 , 261 -267; Fournier & Duman, (2012) Role of vascular endothelial growth factor in adult hippocampal neurogenesis: implications for the pathophysiology and treatment of depression. Behav Brain Res, 227, 440-449).

VEGF is neuroprotective (Gora-Kupilas & Josko, (2005) The neuroprotective function of vascular endothelial growth factor (VEGF). Folia neuropathologica / Association of Polish Neuropathologists and Medical Research Centre, Polish Academy of Sciences, 43, 31 -39) and positively influences neurite outgrowth (Jin, et al., (2006) Vascular endothelial growth factor stimulates neurite outgrowth from cerebral cortical neurons via Rho kinase signaling. J Neurobiol, 66, 236-242). For example, increased VEGF levels prevent motor neuron degeneration induced by expression of a mutant form of the superoxide dismutase 1 (Lunn, et al., (2009) Vascular endothelial growth factor prevents G93A-SOD1 -induced motor neuron degeneration. Developmental neurobiology, 69, 871 -884). Furthermore, environmental enrichment, considered of therapeutic value against depression (Hannan, (2014) Environmental enrichment and brain repair: harnessing the therapeutic effects of cognitive stimulation and physical activity to enhance experience-dependent plasticity. Neuropathol Appl Neurobiol, 40, 13-25), enhances neurogenesis as well as hippocampal VEGF levels (Cao, et al., (2004) VEGF links hippocampal activity with neurogenesis, learning and memory. Nat Genet, 36, 827- 835). Moreover, increases in VEGF expression stimulate adult hippocampal neurogenesis (Fournier & Duman, (2012) Role of vascular endothelial growth factor in adult hippocampal neurogenesis: implications for the pathophysiology and treatment of depression. Behav Brain Res, 227, 440-449).

While our observation that cotinine treatment alters VEGF in the hippocampus is novel, an arterial epitheial cell study showed nicotine- and cotinine-induced cellular change in mRNA VEGF expressio (Conklin, et al., (2002) Nicotine and cotinine up- regulate vascular endothelial growth factor expression in endothelial cells. Am J Pathol, 1 60, 413-418). I n neurons, cotinine prevented the decrease in the expression of VEGF in the hippocampus of the mice exposed to repetitive FS stress. Interestingly, cotinine did not change VEGF protein levels in non-stressed mice. Therefore, it is unlikely that cotinine is influencing VEGF expression directly, but exerts its effects in the presence of stress, likely through a facilitation of molecular mechanisms of homeostasis. Altogether cotinine promotes restorative cerebral changes by stimulating signaling factors such as VEGF, which may, in turn, promote plasticity processes such as neurogenesis. Given cotinine has good pharmacokinetic properties (De Schepper, et al., (1 987) Kinetics of cotinine after oral and intravenous administration to man. European journal of clinical pharmacology, 31 , 583-588) and a positive safety profile in humans, which includes no habit-forming properties or withdrawal effects, among others (Hatsukami, et al., (1997) Safety of cotinine in humans: physiologic, subjective, and cognitive effects. Pharmacol Biochem Behav, 57, 643-650; Hatsukami, et al., (1 998a) Effects of cotinine on cigarette self-administration. Psychopharmacology (Berl), 138, 1 84-189; Hatsukami, et al., (1 998b) Cotinine: effects with and without nicotine. Psychopharmacology (Berl), 135, 141 -150; Echeverria Moran, (2012) Cotinine: Beyond that Expected, More than a Biomarker of Tobacco Consumption. Frontiers in pharmacology, 3, 1 73.), cotinine therapeutic intervention during stress is useful in increasing neurogenesis, enhancing synaptic plasticity, learning and memory, and the expression of neurogenesis factors such as VEGF during stress and neurodengenerative conditions.

In the preceding specification, all documents, acts, or information disclosed does not constitute an admission that the document, act, or information of any combination thereof was publicly available, known to the public, part of the general knowledge in the art, or was known to be relevant to solve any problem at the time of priority.

The disclosures of all publications cited above are expressly incorporated herein by reference, each in its entirety, to the same extent as if each were incorporated by reference individually.

While there has been described and illustrated specific embodiments of a method of treating chemotherapy-related side effects or stress-related neuroinflammation, it will be apparent to those skilled in the art that variations and modifications are possible without deviating from the broad spirit and principle of the present invention. It is also to be understood that the following claims are intended to cover all of the generic and specific features of the invention herein described, and all statements of the scope of the invention which, as a matter of language, might be said to fall therebetween.