Department of Biology Master’s degree in Neuroscience
COMBINING LEVOTHYROXINE WITH 3-IODOTHYRONAMINE
IMPROVES NEUROCOGNITIVE AND NEUROBIOLOGICAL
ALTERATIONS ASSOCIATED WITH ADULT-ONSET
HYPOTHYROIDISM
SUPERVISOR: Dr Grazia Rutigliano TUTOR: Dr Nicola Origlia CANDIDATE: Nicoletta Maria GrittaniA mia nonna. Avrei voluto fossi qui a ricordarmi che “Studere, studere, post mortem quid valere? Un mazzo di cime di ciquere”
Abstract:
Background and objectives. Hypothyroidism is a common endocrine disorder. Symptoms include mood alterations, such as anxiety and depression, and impairment in neurocognition, that point to an underlying disruption of hippocampal function. Guidelines from international professional societies recommend Levothyroxine (L-T4) monotherapy as the treatment of choice to cure hypothyroidism, under the assumption that L-T4 is transformed to the active hormone triiodothyronine (T3) in target organs. Although L-T4 monotherapy is effective in most cases, there is a percentage of patients that keep showing hypothyroidism-related dysfunctions, even though euthyroidism is biochemically restored. 3-iodothyronamine (T1AM) is an active thyroid hormone metabolite showing pro-learning and anti-amnestic effects. Here, we aimed at investigating whether combining L-T4 with L-T3 or T1AM may have beneficial effects in the treatment of neurocognitive and associated neurobiological alterations in a mouse model of adult-onset hypothyroidism.
Materials and Methods. Hypothyroidism was induced in (n=80) six-week old C57BL/6J male mice by Methimazole (0.2 mg/g/die) and Potassium Perchlorate (0.3 mg/g/die) administration in drinking water for 49 days, while controls (n=17) received plain water. At day 21, mice were implanted with subcutaneous ALZET® osmotic pumps delivering replacement treatments for 28 days. The experimental groups were 6, as follows: (1) hypothyroid; (2) L-T4; (3) L-T4+L-T3; (4) L-T4+T1AM; (5) T1AM; (6) euthyroid. Mice were tested with behavioural assays to evaluate hippocampus-dependent memory (i.e., novel object recognition test), locomotion and anxiety (i.e., open field test and elevated plus maze test), and depression (i.e., tail suspension test). We analysed mRNA isolated from the hippocampi by quantitative real-time PCR analysis with the mouse PrimePCRTM collection panel (BIO-RAD), a predesigned 96-well panel of gene targets involved in neurogenesis
pathway for use with SYBR® Green. To assess hippocampal neurogenesis in the subgranular zone (SGZ) of the dentate gyrus, we performed immunofluorescence for specific markers, such as Ki67 for cell proliferation and doublecortin (DCX) for new-born neuroblasts and immature neurons.
Results. L-T4 serum concentrations, determined by high performance liquid chromatography coupled with tandem mass spectroscopy (HPLC-MS/MS), confirmed the validity of our model. At the behavioural level, hypothyroid mice showed significant impairment in hippocampus-dependent memory as compared to euthyroid mice. L-T4 monotherapy substantially improved the discrimination index. However, we observed the average best performances in mice treated with L-T4+T1AM. T1AM did not induce any effect per se. Hippocampus-dependent memory remained impaired in mice treated with L-T4+ L-T3. These findings were influenced neither by locomotor activity nor by anxiety- and depression-related behaviours, which remained unchanged. The real-time PCR analysis revealed consistent significant changes in the expression of genes related to neurogenesis. The ANOVA analysis revealed an upregulation in the expression of Dll1 in L-T4 + T1AM treated mice compared to hypothyroid mice, and an upregulation in the expression of Mapk1 and Mapk3 in L-T4+T1AM treated mice compared to L-T4 treated mice. Experiments of immunofluorescence preliminarily showed enhanced neurogenesis at the level of the SGZ of the hippocampus in mice treated with L-T4+T1AM and L-T4 +L-T3 relative to the other groups.
Conclusion. In our pharmacological mouse model of adult-onset hypothyroidism, L-T4+T1AM combination had beneficial effects on hypothyroidism-related neurocognitive and neurobiological alterations. Future studies should elucidate whether L-T4+T1AM combination may prevent the neurodegenerative consequences of hypothyroidism.
Summary:
1.Introduction ……… 1
1.1 Thyroid hormones ... 1
1.1.1 Thyroid hormones in adult hippocampal neurogenesis ... 6
1.1.2 Neurogenesis in adult neurogenic niches ………... 7
1.1.3 Role of thyroid hormones in adult neurogenesis ……….. 11
1.2 Neurological aspects of adult-onset hypothyroidism ………. 13
1.2.1 Unresolved issues in hypothyroidism ………. 15
1.3 3-iodothyronamine (T1AM) ………. 18
1.4 Aims...22
2.Materials and Methods ………...23
2.1 In vivo experiments ... 23
2.1.1 Induction of hypothyroidism in adult mice ... 23
2.1.2 Hormonal determination from blood samples………...25
2.1.3 Behavioural tests ...………...26
2.1.3.1 Elevated plus maze ...………. 26
2.1.3.2 Open field test ………. 27
2.1.3.3 Novel Object Recognition Test ………... 28
2.1.3.4 Tail suspension test ………. 29
2.2 Ex vivo experiments ………...31 2.2.1 Gene expression ………...31 2.2.2 Immunofluorescence.... ………....…....36 2.3 Statistical analysis ………...……...38
3. Results ………....40
3.1 Generation of hypothyroidism ………...40 3.2 Hormonal determination ………...42 3.3 Behavioural tests ………...443.3.1 Elevated plus maze ………...44
3.3.2 Open field ………...47
3.3.3 Novel Object Recognition ………....48
3.3.4 Tail suspension test ………...50
3.4 Gene expression ………....52
3.5 Neurogenesis in the subgranular zone of the dentate gyrus ………...54
4. Discussion ………...56
List of figures:
Figure 1. Hypothalamus-Pituitary-Thyroid axis. ... 2
Figure 2. Neurogenesis in the SGZ... 10
Figure 3. T1AM synthesis by deiodination. ... 19
Figure 4. Protocol for hypothyroidism induction and substitutive therapies. ... 25
Figure 5. Graphical representation of the apparatus used for behavioural tests. ... 30
Figure 6. qPCR workflow. ... 34
Figure 7. Immunofluorescence. ... 37
Figure 8. Body weight changes in our model of adult-onset hypothyroidism. ... 41
Figure 9. Serum concentrations. ... 43
Figure 10. Elevated Plus Maze.. ... 46
Figure 11. Open field. ... 47
Figure 12. Novel Object Recognition. ... 49
Figure 13. Tail suspension test. ... 50
Figure 14. Gene expression. ... 53
List of tables:
Table 1. cDNA synthesis ... 32
Table 2. Cycling Protocol ... 33
Table 3. Number of animals used for each experimental purpose...39
Table 4. Behavioural data. ... 51
List of acronyms:
AD: Alzheimer’s Disease CNS: Central Nervous System DCX: Doublecortin
DIO: Deiodinases GCL: Granule cell layer LTD: Long-term depression LTP: Long-term potentiation NSC: Neural stem cells
ODC: Ornithine decarboxylase SGZ: Subgranular zone SVZ: Subventricular zone TH: Thyroid hormones TR: Thyroid receptors TRH: Thyrotropin hormone TSH: Thyroid-stimulating hormone
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1. Introduction
The field of neuroendocrinology comprises the multiple reciprocal interactions between the Central Nervous System (CNS) and the endocrine system in the control of homeostasis and physiological responses to environmental stimuli. This is achieved through the regulated secretion of hormones, neurotransmitters, or neuromodulators by specialized cells, a process known as neurosecretion, and mainly relies on the function of the hypothalamic-pituitary axis. Diverse sensory and hormonal inputs are integrated at the level of the hypothalamus, in charge of the control of homeostasis, and sent out as chemical outputs to key regulatory sites, such as the pituitary gland, leading to hormone secretion. As a result of the action of the hypothalamus, which arranges coordinated endocrine, autonomic, and behavioural responses, the CNS, and the endocrine system cooperate, building a bridge of complex interactions.
1.1 Thyroid hormones
One important pathway that requires the coordinated action of the HP axis regards the production and the release of thyroid hormones (TH). Thyrotropin hormone (TRH) releasing neurons of the hypothalamus synapse on thyrotrophs in the pars distalis of the anterior pituitary gland which are involved in the secretion of the thyroid-stimulating hormone (TSH). TSH is a glycoprotein hormone that induces hormone synthesis and release at the level of the thyroid gland (Figure 1). The function of the thyroid is to generate the quantity of TH necessary to meet the demands of the peripheral tissues.
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The thyroid gland is composed of closely packed spherical units termed follicles, which are covered with a rich capillary network. While the interior of the follicle is filled with the clear proteinaceous colloid, the major constituent of the total thyroid mass, the area that surrounds the lumen is composed by thyrocytes disposed in a single-layered epithelium. Formation of normal quantities of TH requires the availability of adequate quantities of exogenous iodine. Thyrocytes can concentrate the required amounts of dietary iodine due to the presence of the sodium-iodide symporter (NIS). This membrane protein takes advantage of the sodium gradient across the basal membrane of the thyrocytes to actively transport iodine inside the cell against its concentration gradient: one ion of iodine enters the cell together with two ions of sodium. The thyroid cell, for tyrosine iodination to occur, must concentrate trace quantities of iodine from the blood and oxidize it via a specific peroxidase, i.e., the thyroperoxidase. The thyroid cell synthesizes a 660,000-kDa homodimer, thyroglobulin (Tg), which is then iodinated at specific tyrosine residues, leading to the formation of monoiodotyrosine (MIT) and diiodotyrosine (DIT). The coupling of two
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molecules of DIT or one of DIT plus one of MIT, leads, respectively, to formation of thyroxine (T4) and triiodothyronine (T3), which are then stored within the colloid, still as part of the Tg molecule. This yields the total supply of daily T4, but only to the 21% of total daily T3, as the remaining quantity derives from T4 deiodination (Larsen 1975). Indeed, T4 released from the thyroid cell is a pro-hormone that must undergo 5’ deiodination to form the active 3,5,3'-triiodothyronine (T3). This reaction is catalyzed by different deiodinases (DIO) and it is the source of more than 80% of the circulating T3 in humans, while the rest is believed to be directly produced in the thyroid. Deiodination can occur in a variety of target tissues, among which there is the CNS, where cerebral T4 and T3 concentrations are in equimolar range (Campos-Barros A 1996). Deiodination in the brain is associated with a differential temporal and spatial expression of D2 and D3 isoenzymes (Kohrle 1999), in fact, different regions of the brain have specific temporal patterns of development (Bernal 2002), thus requiring different regulation of T3 bioavailability to modulate the expression of a large number of cerebral genes.
There are two main pathways through which TH can reach the brain interstitial space, and these include the transport across the blood brain barrier (BBB) and the one along the cerebral-spinal fluid (CSF), requiring the presence of specific carriers (Cheng LY 1994). TH must enter the cell to bind nuclear receptors (TR) to regulate gene expression. Although TH chemical structure could suggest this process to occur through passive diffusion, it is now clear that cellular uptake and efflux of TH are mediated by transporter proteins (Braun D 2018). Several specific TH transporters have been identified in the brain, including monocarboxylate transporter 8 (MCT8) and the organic anion transporting polypeptide 1C1 (OATP1C1). MCT8 is a highly specific transporter for T3 across the cytoplasmatic membranes (Friesema EC. 2005), expressed predominantly in the choroid plexus of the ventricles, as well as in neurons of the neo- and allo-cortex, the hypothalamus and the
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folliculostellate cells of the pituitary gland (Friesema EC. 2005). Mutations of MCT8 in humans can lead to a severe X-linked psychomotor retardation, characteristic of the Allan-Herndon-Dudley syndrome (AHDS), inhibiting the entry of T3 into neurons, thus emphasizing an important role for MCT8 and TH in the development of the CNS (Friesema E. 2004). OATP1C1 is expressed widely in the CNS. In rodents, it is responsible for a preferential transport of T4 across the blood-brain barrier, and it is thought to mediate the entry of T4 into the astrocytes (Heuer 2007). Astrocytes are the main site of conversion of T4 into T3 in the brain, reaction catalyzed by type 2 deiodinase (D2) (Guadaño-Ferraz 1999). On the other hand, Type 3 deiodinase (D3) inactivates thyroid hormone by converting T3 into T2 and T4 into rT3 (Alkemade 2005). The astrocyte-generated T3 can enter neuronal cells via MCT8 or OATP1C1, where it can interact with nuclear receptors regulating genes transcription. Alternatively, T3 has been demonstrated to modulate neuronal functions through non-canonical mechanisms involving receptors in the plasma membrane.
The canonical biological actions of TH are initiated by intracellular binding of T3 to nuclear receptors (TR). These TR (α and β) are widely distributed in the adult brain, with higher densities in the phylogenetically younger regions (amygdala and hippocampus) and lower densities in the brain stem and cerebellum (Ruel J 1985). Studies conducted in knock-out (KO) mice revealed that TR mutations can have implications on brain functions, resulting in behavioural disturbances. Indeed, TRα1-KO mice show a reduced exploratory behaviour in the open-field test and a higher freezing response during fear conditioning (Venero C 2005). It has been hypothesized that TRα1 may have a role in hippocampal structure and function due to the proof that these KO animals also have a lower number of GABAergic terminals on CA1 compared to wild-type animals (Guadaño-Ferraz A 2003). An evidence in favour of this hypothesis comes from heterozygous mice harbouring a TRα1-mutation, leading to a ten-fold reduction of TH affinity, which display extreme anxiety-like
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behaviour and a reduced recognition memory, again implying a dysfunctional hippocampal circuitry that could be restored by high-dose TH therapy (Venero C 2005). The genes that are regulated by the activation of TR in the CNS are known to encode proteins of myelin, neurotrophins and their receptors, transcription factors, splicing regulators and proteins involved in intracellular signalling pathways. Thus, TH have been found to be involved in the process of terminal brain differentiation, such as dendritic and axonal growth, synaptogenesis, neuronal migration and myelination (Eayrs 1955), together with the transcription of developmentally important plasticity-related genes, such as NMDA receptor subunits, choline acetyltransferase (critical for cholinergic function) and reelin (Leach PT 2015). As already mentioned, TH can regulate transcription by binding their nuclear receptors, but they can also be responsible of non-canonical effects (Hoefig C. 2016). The latter can be initiated by the activation of truncated forms of TRs, present in the plasma membrane, cytoplasm or mitochondria, or by activation of structurally unrelated receptors such as integrin Alpha-v Beta-3 (αvβ3), a protein best known for its interactions with the extracellular matrix (Hoefig C. 2016). These membrane receptors have been detected in proliferating basal progenitors (BP) in embryonic mouse neo-cortex (Arai Y. 2011), and found to be relatively abundant in ferret basal radial glia cells (bRGCs) of the subventricular zone (SVZ) (Fietz S. 2012). The binding of TH to this receptor leads to the activation of the mitogen-activated protein kinase (MAPK) signal transduction cascade or the integration of signals from the MAPK pathways (Davis PJ 2008), each of which has been postulated to be involved in different forms of synaptic plasticity (Peng S 2010) (Correa SA 2012). Indeed, interferences on αvβ3 expressed in ferret bRGCs, leads to inhibition of proliferation and self-renewal capacity of the latter (Fietz S. 2012). This suggests that their activation through TH might be involved in the process of neurogenesis in the SVZ, where they would be acting on proliferating progenitors. These studies demonstrate that both the canonical and
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canonical effects of TH have significant neurological implications in the brain areas involved in learning and memory.
1.1.1 Thyroid hormones in adult hippocampal neurogenesis
TH are involved in a multitude of processes characteristic of the vertebrate system (Brent 2012) such as regulation of morphogenesis, seasonal rhythms of physiological functions, metabolism, immune responses, reproduction and mood (Korf 2018). Experiments aimed at investigating the effects of TH on amphibian morphogenesis revealed that thyroidal extracts transform aquatic tadpoles into terrestrial frogs, and that metamorphosis is interrupted in thyroidectomized frogs (Brown DD 2007), pointing to a potential role for TH in the regulation of development. Indeed, deficiency of TH in mammals during the critical period of brain development is associated with profound, and often irreversible, morphological defects that contribute to severe cognitive and neurological impairment (Porterfield 1993). Observational studies performed in iodine-deficient parts of the world have shown that perinatal hypothyroidism results in various abnormalities, including delayed myelination (Prezioso G 2018), implicating that the expression of specific proteins such as myelin basic protein and myelin-associated glycoprotein is dependent on normal thyroid function (Farsetti A 1991). Also, decreased maternal TH irreversibly reduces proliferation and commitment of neuronal progenitors within the ventricular zone in the foetus (Mohan V 2012). Experiments in which hypothyroxinaemia was induced in gestating rats caused abnormal neuronal migration in the cortex and hippocampus of young post-natal rats (P40) (Ausò E 2004). Indeed, TH during the perinatal period are believed to be important for the promotion of neurogenesis, neuronal proliferation, and migration of post-mitotic neurons to the different areas of the developing cerebral cortex, hippocampus and ganglionic eminence (Bernal J 2003). As a matter of fact, iodine supplementation before pregnancy and in the first and second trimesters reduces the incidence of a condition known as endemic
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cretinism that is caused by low maternal T4 levels (Prezioso G 2018). The hypothesis is that maternal T4 is converted into intracellular T3 by fetal astrocytes and taken up by neural target cells thanks to specific transporters (Morte B 2014).Among the TH receptors found in the fetal brain, TRα1 is the earliest and most widely distributed (Forrest D 1991), and it is believed to control most of the effects exerted by T3 in the latter.
TH are not only important during the critical period of brain development, but also in adulthood (Gothie JD 2017), where the process of neurogenesis takes place in specific active proliferative areas. These areas, called neurogenic niches, are characterized by the presence of neural stem cells (NSC) that will later differentiate into neurons or glia components. Alteration of adult neurogenesis in humans has been linked to cognitive deficits, neuropsychiatric disorders, and neurodegenerative diseases (Apple DM 2017).
1.1.2 Neurogenesis in adult neurogenic niches
The evidence that TH have an influence on neuronal progenitors and hippocampal morphology during development (Porterfield 1993) led to investigate if TH could also play a role in neurogenesis in later stages of life. During the last two decades, it has been consistently demonstrated that the brain exhibits morphological plasticity well into adulthood in most animal phyla, although with decreasing ability to generate new neurons along the evolution (Lindsey BW 2006) and during aging (Enwere E 2004). In the adult brain, neurogenesis occurs in the so-called neurogenic niches, specific sites of active proliferation containing NSCs. These NSCs go through asymmetric divisions, generating a pool of proliferative progenitors which will mainly give rise to neuroblasts (Sashaina E Fanibunda 2018). On the other hand, a smaller fraction of progenitors will participate to gliogenesis by generating oligodendrocyte progenitor cells, which migrate towards the corpus callosum, where they differentiate into myelinating oligodendrocytes (Menn B 2006).
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This process, observed in rodents, also occurs in human brain but the cellular and molecular mechanisms remain unclear (Nait-Oumesmar B. 2008) (Zoltán Rusznák 2016).
There are two sites in the adult brain that house the progenitors that give rise to new neurons: the subgranular zone of the dentate gyrus (SGZ) within the hippocampus and the SVZ lining the lateral ventricles. SGZ and SVZ stem cells follow a different fate: the first will generate the granular cells of the dentate gyrus, the latter will form progenitors that will migrate to the olfactory bulb through the rostral migratory stream (RMS) and differentiate into olfactory bulb interneurons (Ewa Rojczyk-Gołe˛biewska 2014). Apart from these canonical regions, numerous evidencessuggest that a limited number of new neural cells can be formed in potential niches located in distinct structures of mature brain. Relatively documented is the presence of a stable hypothalamus neurogenic site located in the hypothalamic ventricular zone (HVZ) (Kokoeva MV 2007) (Cheng 2013), furthermore, other putative areas in which these niches have been found correspond to the amygdala (Luzzati F 2003), striatum (Be´dard A 2006), substantia nigra (Zhao M 2009), and certain neocortical areas (Vaysse L 2012), although there is not enough evidence providing that these non-classical neurogenic sites are continuously active in generating new progenitors. Given that hippocampal neurogenesis in mammals is the most conserved, we decided to focus only on neurogenesis occurring in the SGZ of the hippocampus.
Progenitors retain the capacity to divide and give rise to daughter cells that undergo structural and functional maturation, eventually integrating into mature neuronal networks (Sailor K.A. 2016), joining the pool of existing neurons in the granule cell layer (GCL). The process of maturation and commitment takes around 4-6 weeks at the end of which the progenitors will become either excitatory granule cell neurons or, in lower amount, glial cells, before they integrate in the existing neurocircuitry (Goncalves J.T. 2016). The process through which a hippocampal progenitor goes from being a neural stem cell to a mature
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granule cell neuron is characterized by the presence of specific markers (S. H. Kempermann G 2015). The quiescent neural progenitor (QNP), or Type 1 cell, expresses the intermediate filament protein nestin, the glial fibrillary acidic protein (GFAP) and the transcription factor Sox2 (Figure 2). It is a radial glial cell that undergoes asymmetric slow divisions generating Type 2a cells. Those daughter cells, which keep expressing Nestin, have the peculiar ability to rapidly amplify their number, therefore they are also identified with the name of amplifying neural progenitors (ANP). The ANP generate Type 2b neuroblasts, with a limited proliferative capacity. Apart from being immunopositive to Nestin, they also acquire expression of microtubule-associated protein Doublecortin (DCX), as well as the basic helix-loop-helix transcription factor NeuroD, that contribute to neuronal cell faith determination. These Type 2b cells migrate in the GCL of the hippocampus and undergo maturation to form DCX-positive, Nestin-negative postmitotic Type 3 cells that start to express neural cell adhesion molecule and markers such as Stanthmin and TUC-4. As these immature neurons undergo maturation, they become negative for DCX, they extend their dendritic arbours, receive synaptic inputs, and send out axonal projections to the CA3 hippocampal subfield (Sashaina E Fanibunda. 2018).
Several studies have indicated a role for adult hippocampal neurogenesis in hippocampal-dependent learning and memory (Gould E. 1999) (Schinder A.F. 2004). Indeed, different brain functions such as memory and learning (Elodie Bruel-Jungerman 2007) (Goncalves J.T. 2016), olfaction (Arenkiel 2010), social and reproductive behaviour (Migaud 2016), and other cognitive processes (Zhuo JM 2016), are believed to be dependent on neurogenesis. In humans, alterations in adult neurogenesis have been linked to cognitive deficits, neurodegenerative diseases and neuropsychiatric disorders (Kaneko N 2009).
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Figure 2. Neurogenesis in the SGZ. A sagittal section of the hippocampus has been taken as an example (A, top) to show the layered structure of the dentate gyrus (A, bottom). The process of differentiation and maturation of the hippocampal progenitor into a mature granule cell (B).
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1.1.3 Role of thyroid hormones in adult neurogenesis
Adult neurogenesis is modulated by diverse intrinsic and extrinsic factors, among which there is sensitivity to hormones, neurotransmitters and growth factors present in the neurogenic niche (Mahmoud R 2016) (Vaidya V.A. 2007). In adults and children born by hypothyroid mothers, thyroid disfunction has been associated with the occurrence of many mental disorders such as mood instability, depression or dementia (Baldini IM 1997) (Dugbartey 1998) (Smith J.W. 2002). Most recently, maternal hypothyroidism was shown to be associated with increased risk of schizophrenia (Gyllenberg D 2016). However, it is not known whether schizophrenia is a direct result of TH-disruption of neurogenesis, but this could be an aspect which deserves further investigation. In rats, adult-onset hypothyroidism significantly decreases the postmitotic survival and neuronal differentiation of hippocampal progenitors, without affecting their proliferation (Ambrogini P. 2005): this reduction in progenitor survival is likely mediated through increased apoptotic cell death (Ambrogini P. 2005). Both the decline in progenitor survival and neuronal differentiation is normalized in hypothyroid animals by restauration of euthyroid status through TH replacement therapy (Desouza L.A. 2005). Analysis performed in thyroidectomized rats show a decrease in DCX+ neurons together with a decreased dendritic complexity and progenitors proliferation correlated, at the behavioural level, with a depressive phenotype (Montero-Pedrazuela A. 2006). Furthermore, hypothyroidism induced by goitrogens delays neuronal morphological maturation and lengthens the expression of TUC-4, marker for immature neurons (Ambrogini P. 2005).
The presence of several isoforms of TR (TRα1, TRβ1 and TRβ2) in adult mouse SGZ (Kapoor R. 2015) lead to further investigate the role of TH in adult neurogenesis. Particularly, immunocytochemistry of NSCs revealed that TRα1 expression appears in DLX2+ progenitors and becomes stronger DCX+ neuroblasts (Lòpez-Juàrez A 2012), thus
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suggesting that TRα1 might lead NSCs toward a neuronal faith. In support of this hypothesis, the latter effect is caused by overexpression of TRα1 in NSCs, while loss-of-function mutations of TRα1 increase the number of NSCs leading to a decrease in migrating neuroblasts (Lemkine GF 2005). The same results were observed if the overexpression was performed in progenitors through in vivo gene transfer. The hypothesis is that T3 acts as a neurogenic switch by binding TRα1, repressing the expression of Sox2, characteristic of Type1 progenitors, and driving the cells toward a neuronal faith (Lòpez-Juàrez A 2012). Moreover, T3 also downregulates cell-cycle genes such as CyclinD1 and c-Myc, promoting cell cycle arrest and neuroblast faith in NSC and progenitors (Lemkine GF 2005).
The advent of transgenic mouse models made it possible to determine at which stage the progenitors are mostly affected by adult-onset hypothyroidism, highlighting a decrease in Type 2b neuroblasts together with a decrease in survival of Type3 progenitors (Kapoor R. 2015), thus confirming that postmitotic hippocampal neurons are the ones most sensitive to decline in TH levels (Sanchez-Huerta 2016). It follows that TH may exert their role in maintenance and differentiation of the progenitor pool once they exit the cell cycle. To investigate whether the effects exerted on neurogenesis by TH are mediated directly on the progenitors or by an influence on the neurogenic niche, in vitro studies on cultures of hippocampal progenitors were performed. The treatment of these cells with TH highlighted an improved survival of DCX+ cells, confirming the results coming from in vivo experiment, but also an enhanced proliferation, as well as a shift towards glia differentiation (Desouza L.A. 2005). The latter effects, observed only in progenitor cultures in vitro, suggests that the removal of the progenitors from their niche may alter the nature of regulation in response to TH (Desouza L.A. 2005). Finally, within the neurogenic niche, TH may exert their effects on neurons, astrocytes, as well as microglia, which in turn may then modulate hippocampal
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progenitor development and maturation (Sashaina E Fanibunda. 2018) through the release of growth factors or by influencing signalling pathways within the hippocampus.
1.2 Neurological aspects of adult-onset hypothyroidism
Hypothyroidism is a chronic disease resulting from deficient production of TH or inadequate action of TH on target tissues (Chaker L 2017) . In 99% of cases, hypothyroidism arises as a primary defect of the thyroid gland to synthesize and release TH. The remaining cases are due to defects in the HPT axis, either in the production of TSH (secondary) or of TRH (tertiary). Peripheral (extra-thyroidal) hypothyroidism refers to consumptive hypothyroidism or tissue-specific hypothyroidism due to decreased sensitivity to TH (Chaker L 2017) . Primary hypothyroidism is the most common endocrine disease, with estimated prevalence between 0.2 and 5.3% in Europe and the United States (Garmendia Madariaga A 2014) (Hollowell JG 2002) (Vanderpump 2011). An association between hypothyroidism and mental activity in adults has been acknowledged since early descriptions of what Asher called “Myxoedematous madness”, referring to neurologic and psychiatric abnormalities caused by myxoedema, such as myopathy, ataxia, psychosis and confusion (Ord WM 1888). Aside the dramatic manifestations of such severe forms, adult-onset hypothyroidism has been recognized to be associated with cognitive impairment, anxiety, and mood instability (Bathla M. 2016) (Bauer M. 2008), emphasizing the importance of TH for normal brain function. Patients with adult-onset hypothyroidism not only report significantly higher scores of depression and anxiety as compared to controls (Correia N 2009), but also present cognitive impairments of memory which can be of spatial, associative, and verbal nature (Anna Göbel 2016) (Miller KJ 2007). This points to an underlying disruption of normal hippocampal function and/or connectivity (Correia N 2009). On this regard, some evidences suggest that hypothyroidism-related memory impairment in
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adult humans may be attributed to decreased conduction velocities in central and peripheral nerves (Pollard JD 1982).
Anatomically, magnetic resonance imaging (MRI) studies have revealed that adult-onset hypothyroidism results in a significant hippocampal volume reduction (Cooke GE 2014). Also, positron emission tomography with (18F) fluorodeoxyglucose and functional magnetic resonance (FMRI), performed on hypothyroid patients show lower activity in regions of the brain involved in regulation of affect and cognition (Bauer M 2009) (Zhu DF 2006). Electrophysiological studies performed in young females affected by subclinical hypothyroidism unveiled higher levels of emotional arousal, displayed as a reduction of α power activity at rest, a tendency of β2 increase, and a significant lateralization toward the right hemisphere (Menicucci D 2013). As demonstrated by previous studies, anxiety levels correspond to a right-lateralized β2 activity (Ljubomir I. Aftanas 2005). The latter is enhanced by a decrease in TH (Wiens SC 2006) (M Romano-Torres 2002), thus suggesting that subclinical hypothyroidism may lead to a greater susceptibility to negative emotions. These evidences indicate that changes in TH levels produce effects on cognitive and emotional functions, as well as on EEG rhythms.
The proof that adult-onset hypothyroidism relates to cognitive disturbances, depression and anxiety (Bathla M. 2016) (Bauer M. 2008) has been seen also in murine models. Experiments performed in adult hypothyroid rats, aimed at evaluating depression and hippocampus-dependent spatial learning and memory, revealed an increased immobility in the Porsolt forced swim test (Montero-Pedrazuela A. 2006), and an impaired performance in the Morris water maze (Hosseini M. 2010), respectively. Moreover, studies conducted in thyroidectomized rats, resulted in a higher number of errors in the radial arm water maze, made during both the acquisition phase and the short-term and long-term memory tests (Alzoubi K. 2009). This poor performance in spatial memory tasks has been reported to be
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accompanied by impaired long-term potentiation (LTP) (Artis AS 2012) and attenuated long-term depression (LTD) in CA1 (Alzoubi H. 2007), which is conceived as the electrophysiological correlate of memory (Alzoubi K. 2009) (Alzoubi K 2005). Several animal studies have provided compelling evidence that these effects may be related to changes in transcription levels of central-specific genes or in the activity of signal transduction systems, secondary to changes in tissue hormone levels. Accordingly, in hypothyroidism, there is a reduction in the basal levels of signalling proteins required for normal LTP and memory, such as cyclic-AMP response element binding protein (CREB), calcium-calmodulin dependent isoform of adenylyl cyclase (ACI), and mitogen activated protein kinases (MAPKp44/42; ERK1/2) (Alzoubi K. 2009). Moreover, findings from several laboratories suggest that extracellular signal-regulated kinase (ERK1/2) deficiencies appear to contribute to the hypothyroidism-induced impairment in LTP (Georges NZ 2004) and that Akt activity is required to sustain long-lasting LTP (Levenga J 2017).
1.2.1. Unresolved issues in hypothyroidism
Guidelines from all professional societies (including the Endocrine Society, the European Thyroid Association and the American Thyroid Association) recommend L-T4 monotherapy as the treatment of choice for hypothyroid patients, with the goal to restore biochemical euthyroidism (normal serum TSH, fT4 and fT3 concentration), reduce symptoms and prevent long-term complications (Okosieme O 2016) (Wiersinga WM 2012) (Jonklaas J 2014). With appropriate individual dosage adjustment, treatment with L-T4 is generally considered efficacious and well tolerated, and its use is associated with relatively constant serum levels of T4, given good patience compliance. Available formulations of synthetic L-T4 have a half-life of 5-7 days and provide stable, relatively constant blood levels of T4 after ingestion of an oral once-daily dose. Even though L-T4 is one of the most commonly prescribed drugs in the world (Medicine use and shifting costs of healthcare: IMS
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Institute for Healthcare Informatics. 2014), there are still uncertainties regarding to whether its use as a single drug treatment in hypothyroid patients represents optimal therapy (Wartofsky 2005). Indeed, a not negligible portion (5 to 15%) of patients treated with L-T4 monotherapy continues to complain of symptoms suggestive of TH deficiency, such as decreased psychomotor performance, cognitive disturbances, fatigue, mood swings depression and anxiety (Biondi B 2012), resulting in worse quality of life, despite TSH levels in the normal range. Persistent symptoms may be due to various causes, aside of L-T4 dosage being suboptimal. For example, comorbidity of hypothyroidism with some symptoms of dysphoria may lead to an erroneous attribution of those symptoms to hypothyroidism, therefore being unresponsive to treatment (Beatrice A 2019). Furthermore, it could be possible that in vivo generation of T3 from T4 is not equivalent to thyroidal secretion of T3 (Gullo D 2011). Another possible explanation is that serum TSH, commonly used as a stand-alone parameter to evaluate treatment response, may not reflect intracellular TH levels in tissues, where transport across the plasma membrane and metabolic transformations might be differentially regulated (Gereben B 2015) (Biondi B 2012). This interpretation seems to be supported by experimental evidence coming from thyroidectomized rats, in which correction of tissue hypothyroidism could only be obtained combining L-T4 administration with L-T3 (Escobar-Morreale H 1995) (Escobar-Morreale H. 1996). Accordingly, in a different experiment, only L-T4+L-T3 treatment normalized tissue markers of T3 -responsiveness, such as mitochondrial content, α-glycerophosphate dehydrogenase activity, and serum cholesterol levels (Werneck de Castro JP 2015).
Being T3 the active TH, an alternative treatment could be represented by T3 monotherapy. Even though this could appear a logical solution, after administration of a dose of T3, the hormone reaches a peak level in 2-4 hours and has a half-life of only one day (Jacqueline Jonklaas 2015), in contrast to the long half-life of T4. As a consequence, replacement therapy
17
with T3 is problematic in regard to the ability of a daily dose of T3 to provide stable levels of the hormone throughout a 24-hours period, requiring a higher number of daily administrations (Celi FS 2010). Given its short half-life and the potential for wide fluctuations on serum levels, replacement therapy with T3 alone has not been recommended for long-term replacement therapy for hypothyroid patients.
In attempt to better approximate physiological thyroidal secretions of T4 and T3, several studies have evaluated the potential benefit of combination of treatment with T4 and T3, but found little, if any, difference in terms of bodily pain, fatigue, body weight, serum lipids, psychiatric symptoms of depression and anxiety, and, broadly, psychological and physical well-being and quality of life (Biondi B 2012) (Grozinsky-Glasberg S 2006) (Ma Chao 2009) (Joffe RT 2007). Despite these negative results, intriguingly, about half of the patients expressed a subjective preference for combined L-T4+L-T3 therapy versus L-T4 monotherapy (Nygaard B 2009) (Walsh JP 2003) (Bunevicius R. 1999) (Escobar-Morreale H 1995) (Bunevicius R 2002). However, at present, all clinical guidelines recommend against the routine use of combination therapy.
Finally, it has been hypothesised that hypothyroidism may be associated to decreased tissue levels of downstream derivatives of TH. Among these, the 3-iodothyronamine (T1AM), has recently emerged as an active TH metabolite with neurological effects, hence representing a possible candidate to augment L-T4 in the attempt to ameliorate persistent neuro-psychiatric symptoms of hypothyroidism (Zucchi R. 2019). As a matter of fact, mice treated with methimazole, had a decrease in T1AM tissue concentration throughout the development of hypothyroidism, which remained undetectable even after L-T4-replacement, despite an almost full recovery of fT4 and fT3 (Hackenmuller SA 2012). This indicated that the intact function of the thyroid gland is needed for T1AM biosynthesis (Hackenmuller SA 2012). According to these findings, we could speculate that reduced availability of T1AM might
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possibly result in some neuropsychiatric symptoms traditionally attributed to TH. This leaves an open window to whether there could be other combined treatments with L- T4 that could recover the persisting symptoms of L-T4 monotherapy treated patients.
1.3 3-iodothyronamine (T
1AM)
3-iodothyronamine (T1AM) is an endogenous amine that, due to the close similarity between chemical structures, has been hypothesized to be derived from TH metabolism (Figure 3). T1AM belongs to the family of thyronamines (TAM) which are decarboxylated metabolites of TH (Scanlan TS 2004) and have been found initially in the rodent brain, and then in other tissues as well as in mammalian and human blood (Frascarelli S 2011). The interest elicited by TAM comes from their proven effect at cardiac (Scanlan TS 2004), metabolic (Zucchi R 2014), cytoprotective (Kohrle J 2019) and cerebral level (Zucchi R 2014), thus leading to the possible usage of these TH-derived metabolites as pharmacological agents.
Although the biosynthetic pathway which generates T1AM is not precisely known, T1AM is believed to be generated through sequential deiodination by deiodinase selenoenzymes (DIO), and decarboxylation by ornithine decarboxylase (ODC). DIO selenoenzymes are intracellular integral membrane proteins that may require reduced intracellular cofactors for reductive deiodination of TH precursors. These precursors can be either synthesized and secreted by the thyroid gland or generated from T4 and T3 in extra-thyroidal tissues expressing one of the three DIO selenoproteins. For the generation of T1AM through deiodination there are two possible routes that have been proposed based on in vitro studies: one consisting in the sequential reductive deiodination of 3,3’-T2 , the other in which the substrate for T1AM formation is 3,5-T2AM (Piehl S 2008) (Figure 3).
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Figure 3. T1AM synthesis by deiodination.
So far, biosynthesis of iodothyronines has been only observed in the follicular structures of the thyroid (Colin IM 2013). To determine if the thyroid gland has also a role in TAM biosynthesis and excretion, hypothyroidism was induced in adult male mice, followed by L-T4 monotherapy with normal and radioactively labelled L-T4 (Hackenmuller SA 2012). Concentrations of T1AM, evaluated with mass spectrometry in the liver, revealed no trace of radioactively labelled T1AM, while the endogenous one was found in lower concentration during the induction of hypothyroidism, concomitantly with decreased activity of hepatic DIOs. This suggested that T1AM may require the same biosynthetic factors necessary for T4 synthesis in the thyroid gland (Hackenmuller SA 2012), but the thyroid role in T1AM biosynthesis remains still open. The only tissue where T1AM synthesis from T4 metabolites was observed is the duodenal mucosa (Hoefig C 2015). Indeed, the gut sac revealed TH mucosal absorption, uptake and transport across the intestinal barrier accompanied by decarboxylation and deiodination. Moreover, experimental data showed that athyreotic patients on oral L-T4 replacement therapy exhibited higher serum T1AM concentrations as
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compared to healthy individuals (Hoefig CS 2011). Recombinantly expressed human ODC incubated with TH substrates showed to decarboxylate both T4 and 3,5-T2 to the corresponding TAM (Hoefig C 2015). Furthermore, qPCR analysis of the intestinal mucosa revealed expression of the transcript of all the three deiodinase isoenzymes and the OCD transcripts (Hoefig C 2015), thus providing a clear evidence that intestinal OCD is a good candidate to catalyze TAM formation from TH precursors.
T1AM is a promiscuous ligand for several molecular targets, indeed it binds with nanomolar affinity to the trace amine-associated receptor 1 (TAAR1), which is a G-coupled membrane receptor (Scanlan TS 2004), other TAAR subtypes, alpha2 adrenergic receptors, transient receptor potential channels, and ApoB-100, a component of VLDL and LDL lipoproteins, the latter probably accounting for high affinity protein binding in serum (Hoefig C. 2016) (Kohrle J 2019), while it does not bind TH receptors. Among the effects exerted by T1AM the best candidates appear to be regulation of metabolic homeostasis and of CNS function (Zucchi R 2014). Metabolic responses after intraperitoneal injections of T1AM in mice include a reduction in respiratory quotient, ketonuria and a significant loss of body fat (Braulke LJ 2008), together with an increase in plasma glucose and glucagon, recorded in intracerebroventricular injected mice at lower doses (Fliers E 2010), thus suggesting a neuroendocrine action on the HPA axis. Concerning effects on the CNS, T1AM has been hypothesized to be a possible modulator of monoaminergic transmission, specifically in noradrenergic, dopaminergic and histaminergic circuitries (Gordon J. T. 1995). Indeed, local applications of T1AM modify the rate of discharge in adrenergic neurons of the locus coeruleus (Gompf HS 2010), probably by inhibiting the reuptake of dopamine and norepinephrine (Snead A. N. 2007). Moreover, injections of T1AM in mice induce a significant reduction in non-REM sleep together with an increase in theta frequencies (James T. D. 2013), consistent with an increased exploratory activity observed at the behavioural
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level (Manni ME 2013). Also, experiments in which T1AM was intracerebroventricularly injected in mice evokes a pro-learning and anti-amnestic effects, as well as an improved motor performance (Manni ME 2013). T1AM showed neuroprotective actions in several models, including seizure-related excitotoxic damage, altered autophagy, amyloidosis, and ischemia-reperfusion injury (Landucci 2019) (Bellusci L. 2017) (Accorroni A 2019). In particular, it was previously shown that T1AM and other agonists of TAAR1 are able to rescue Aβ-induced synaptic dysfunctions in the entorhinal cortex and related behavioural deficits in a mouse model of AD (Accorroni A. 2020). Also, acute administration of T1AM was found to prevent ischemia-induced synaptic depression in entorhinal cortex slices exposed to oxygen-glucose deprivation (OGD). This protective effect was mediated by the trace amine-associated receptor 1 (TAAR1) (Tozzi 2018).
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1.4. Aims of my thesis work:
Clinical empirical observations and previous research in animal models indicate that L-T4 monotherapy alone is not able to fully recover the neurocognitive symptoms associated with adult-onset hypothyroidism, probably because it does not totally restore the physiological tissue levels of T3 or other metabolites, especially T1AM. Based on these premises, we hypothesised that the combined treatment of L-T4 with either L-T3 or T1AM could have beneficial effects on neurocognitive and neurobiological alterations associated with adult-onset hypothyroidism . Therefore, the aims of my thesis work were to:
Develop a pharmacological model of hypothyroidism and replacement therapy with different treatment regimens, namely: L-T4, L-T4+L-T3, L-T4+T1AM and T1AM.
Compare the different treatment regimens from a behavioural point of view assessing memory, locomotor activity, anxiety-, and depression-related behaviour.
Evaluate if the different treatment regimens have a diverse effect on neurogenesis in the SGZ of the hippocampus, assessing the expression of genes in neurogenic pathways and the neurogenetic capacity, through immunostaining for Ki67 (cellular proliferation) and doublecortin (DCX, newborn neuroblasts).23
2. Materials and Methods
2.1 In vivo experiments
2.1.1 Induction of hypothyroidism in adult mice
Six-week-old C57BL/6J male mice (n=97) were maintained in an air-conditioned animal room with a 12-h light/dark cycle. Mice were housed in plastic cages with four animals per cage and were provided with ad libitum water and basal pellet diet. All experiments were conducted in accordance with the principles of animal care and experimentation in the guidelines of the Italian Ministry of Health (Legislative Decree n. 116/92) and the European Community (European Directive 86/609/EEC). The Italian Ministry of Health approved the use of animals in this protocol (65E5B.10, n.734/2017-PR, 10/10/2017). Hypothyroidism was induced in n=80 mice by administration of Methimazole (0.20 mg/g/die, Sigma-Aldrich) and Potassium Perchlorate (0.30 mg/g/die, Sigma-Aldrich) in drinking water for 21 days, starting from postnatal day 42. Euthyroid mice (n=17) received simply tap water (Figure 4).
Body weight (BW), water intake, and food consumption were monitored once per week throughout the hypothyroidism induction period. At the end of the 21-day treatment, mice were implanted with subcutaneous ALZET® osmotic pumps, continuously delivering the different replacement compound, with reservoir volume of 100 μl and delivery rate of 0.11 μl/h. The filling solution was prepared in sterile 1% bovine serum albumin (BSA), 0.9% saline, NaOH 0.1 M. The hormonal concentrations provided were dependent on the kind of treatment: the hypothyroid and euthyroid mice received no hormones; the L-T4 treated, 0.04 μg L-T4/g BW/die; the L-T4 and L-T3 treated received 0.03 μg L-T3 and 0.007 μg L-T3/g BW/die (according to the physiological T4:T3 secretion ratio of 6:1 from the mouse thyroid) (Henning Y 2014); the L-T4 and T1AM treated, 0.04 μg L-T4 & 0.004 μg T1AM/g BW/die;
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the T1AM, 0.004 μg T1AM /g BW/die. In absence of previous data on chronic T1AM administration, the dose for the present experiment was calculated multiplying by 28 (length of replacement treatment, in days) the minimum acute dose of T1AM which was reported to improve cognition in mice, namely 4 μg/kg BW (Bellusci L 2017). For subcutaneous pump implantation, mice were anesthetised with 1.5% isoflurane in 100% oxygen in an anaesthetic chamber. The skin was disinfected over the implantation site, on the back, slightly posterior to the scapulae. Then, a 1-cm incision was made perpendicular to the spine, and a pocket was created for the insertion of the pump. The wound was closed with 2-3 sutures. After the surgery, wound healing was monitored daily for 72 h. L-T4, L-T3 and T1AM used in the replacement treatments were purchased from Sigma-Aldrich.
At the end of the treatments the experimental groups were the following:
a) Hypothyroid (n=15); b) Hypothyroid L-T4-treated (n=18); c) Hypothyroid L-T4+ L-T3-treated (n=15); d) Hypothyroid L-T4+ T1AM -treated (n=17); e) Hypothyroid T1AM -treated (n=15); f) Euthyroid (n=17).
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Figure 4. Protocol for hypothyroidism induction and substitutive therapies.
2.1.2 Hormonal determination from blood samples
Free T4 and free T3 fractions were evaluated by the automated AIA 2000LA immunoassay platform (Tosoh Bioscience, Tessenderelo, Belgium) thanks to the collaboration of Dr Prontera, in the laboratory at Gabriele Monasterio Fundation. Total T4 and total T3 quantification was performed by Dr Marco Borsò according to a method developed by Prof Alessandro Saba in the laboratory of mass spectrometry, University of Pisa (Saba A 2014).
26 2.1.3 Behavioural tests
Throughout the whole period that preceded the behavioural testing, mice were habituated to experimental handling. Mice were individually habituated for one hour to the behaviour testing room, for three consecutive days before the beginning of the experiments. Behavioural testing was performed at the end of the replacement treatment with ALZET® osmotic pumps, and included, in the following order: Elevated Plus Maze (EPM), to assess anxiety-related behaviours (Walf AA 2017); Open Field Test (OF), to assess locomotion and anxiety-related behaviours (Seibenhener ML 2015); Novel Object Recognition Test (ORT), to assess hippocampus-dependent memory (Antunes M 2012); Tail Suspension Test (TST), to assess depression-related behaviours (Can A 2012).
2.1.3.1 Elevated Plus Maze
The EPM is based on rodents’ tendency to prefer dark and circumscribed spaces as compared to enlightened and opened ones, so it is used to assess anxiety-related behaviours in rodents by measuring the conflict between the two opposite natural instincts of curiosity and avoidance. These two opposite behaviours are represented respectively by exploration of the unprotected and enclosed spaces: the higher the “anxiety” levels, the lower the proportion of explorations in the open spaces in favour of the dark spaces. The EPM apparatus was made of black Plexiglas and consisted of 4 arms (25 cm [length] x 5 cm [width]) forming a plus sign, elevated 50 cm above the floor. Two opposite facing arms have walls (16 cm [height]) and open roof (closed arms), while the other two opposite facing arms have no walls (open arms). The entire apparatus was placed in a squared arena (60 x 60 cm, normally used for the ORT) to protect the mice that fall or attempt to escape during the test (Figure 5A). Each mouse was placed in the centre facing one of the two open arms and allowed to freely explore the maze for 5 min. Mouse behaviour was monitored and recorded
27
through a camera positioned above the apparatus. The main recorded variables were: (a) time spent in open arms (expressed as percent of total time spent in both arms); (b) number of entries into open arms (expressed as percent of total number of entries into both arms); (c) number of entries into closed arms (expressed as percent of total number of entries into both arms). Entry into a given arm was considered when the mouse had all the 4 paws in that arm. As secondary measures, we scored: (d) total number of head dips; (e) number of protected/unprotected head dips (defined as head dips occurring from the closed/open arms); (f) number of end-arm explorations in open arms; (g) number of stretch-attend postures (SAP). Head dips were scored when the mouse moved the head below the level of the maze floor. End-arm explorations were defined as the mouse reaching the last portion of the open arm with all 4 paws. SAPs were scored when the mouse stretched to its full length with the forepaws toward an open arm, without moving and keeping the hind paws in the same position, and then resumed the initial position. At the end of each individual trial, the maze was cleaned thoroughly with ethanol 5% and left to dry completely before exposure to the following mouse to ensure that behaviour was not guided by odour cues.
2.1.3.2 Open field test
The OF provides information about locomotion and anxiety-related behaviours. It is based on the principle that the “anxiety” behaviour is displayed by the mice as a higher proportion of time spent close to the walls of the arena. Each mouse was placed in a 60 x 60-cm squared arena for 10 min (Figure 5 B). When mice are placed into an open field, they are more prone to explore the peripheral zone than the centre (40 x 40 cm). The scored variables were: (a) total ambulatory distance, and (b) thigmotaxis, i.e., amount of time spent in outer zones versus inner zones, presented as a function of total time in the maze.
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2.1.3.3 Novel Object Recognition Test
To investigate hippocampus-dependent memory, the ORT probably represents the best approach, since it relies on the innate curiosity of rodents which tend to explore with a higher preference what is new in the environment. By testing the rodents’ ability to discriminate a novel object in the arena, we can imply that the rodents’ memory retains a representation of the familiar object. The objects used were simple 3D objects, with size comparable to the one of the mice, and could be made of glass, plastic or metal (Figure 5 C). Testing took place in the same arena described above for the OF, taking the free exploration of the open field as the habituation phase of the ORT. After this habituation phase, the mouse was put in a cage for 1 minute before the beginning of the ORT. The first phase, called familiarization, allowed each mouse to freely explore two identical sample objects (A + A) in the open-field arena for 3 min. Then, the mouse was again put in the cage for 1 minute, while the experimenter replaced one of the two familiar objects with the novel object. During both the familiarization and the test phase, the mouse was released at the centre of the wall opposite to the objects, and the objects were located in opposite and symmetrical corners of the arena. After each session of the ORT, the arena and the objects were cleaned thoroughly with ethanol 5% and left to dry completely, to ensure that behaviour was not guided by odour cues. Mouse behaviour was monitored following the same procedure as OF. Exploration time was counted when the mice were in the object close proximity (within 2 cm) with the nose directed towards it, sniffing or touching the object with the nose. Exploration time was not counted when the nose was pointing away from the object even if the mice were beside the object, running around it, sitting or climbing on it. To determine the rates of exploration of novel relative to familiar object, observation scores were converted into discrimination indices (DI). The discrimination index represents the capability of the animal to discriminate between the novel and the familiar object taking into account the differences in the
29
exploration times. Its value can go from -1 to +1, where a positive score indicates more time spent in proximity of the novel object (i.e., better memory), and a negative one indicates more time spent close to the familiar one (i.e., worse memory). The zero score represents no preference for either novel or familiar object. The DI is calculated according to the following formula:
𝐷𝐼 =𝑡𝑖𝑚𝑒 𝑎𝑡 𝑛𝑜𝑣𝑒𝑙 − 𝑡𝑖𝑚𝑒 𝑎𝑡 𝑓𝑎𝑚𝑖𝑙𝑖𝑎𝑟 𝑡𝑖𝑚𝑒 𝑎𝑡 𝑛𝑜𝑣𝑒𝑙 + 𝑡𝑖𝑚𝑒 𝑎𝑡 𝑓𝑎𝑚𝑖𝑙𝑖𝑎𝑟
Time novel= time spent in proximity of the novel object
Time familiar= time spent in proximity of the familiar object
Time at novel + time at familiar= total time spent in proximity of both objects
2.1.3.4 Tail suspension test
The TST is used to evaluate depression-related behaviours. The TST models an unescapable stress, with the assumption, the higher is the depression level, the less will be the motility of the mouse. Mice were individually suspended to a shelf elevated 60 cm above the floor with tape, against a white background to provide optimal contrast for more reliable test scoring. In such position, with an approximate distance of 20-25 cm between the mouse’s nose and the floor, mice could neither escape nor hold to nearby surfaces. To prevent mice from climbing their tails during the test, we placed climb-stoppers around the tails, i.e. clear hollow cylinders of 2 cm length, 1.7 cm outer diameter, and 1.6 inside diameter (Figure 5 D). After each session of the TST, the suspension shelf was cleaned thoroughly with ethanol 5% and left to dry completely. Mouse behaviour was monitored and recorded through a camera. Escape-related behaviours were counted when the mouse: (a) tried to reach the walls of the apparatus and the suspension bar; (b) strongly shook the body; (c) showed movements of the limbs akin to running. Escape was not counted when the mouse
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showed small movements confined to the front legs without involvement of the hind legs, or in case of oscillations and pendulum-like swings that were due to the momentum gained during preceding mobility bouts. Lack of escape-related behaviour was considered as immobility and was taken as a measure of depression.
Figure 5. Graphical representation of the apparatus used for behavioural tests. (A) Elevated Plus Maze; (B) Open Field Test; (C) Novel Object Recognition Test; (D) Tail Suspension Test. (Copyright © 2019 Grazia Rutigliano)
31 2.1.3.5 Scoring of behavioural tests
The movies generated during the EPM, ORT, and TST were scored manually in a blind fashion. Regarding the OF, we used the open-source toolbox developed by Patel, et al. 2014 to automatically compute the total ambulatory distance as well as the amount of time spent in outer zones versus inner zones, presented as a function of total time in the maze (Patel TP 2014).
2.2 Ex vivo experiments
2.2.1 Gene expressionFollowing sacrifice, hippocampi were isolated and put in 1 ml TRIzol® Reagent for RNA isolation, followed by homogenization through the use of a rotor-stator homogenizer (TissueRuptor II, QIAGEN). The lysate was centrifuged for 5 minutes at 12,000 × g at 4°C, then the clear supernatant was transferred to a new tube. It followed 5-minute incubation to permit complete dissociation of the nucleoproteins complex. Then, 0.2 mL of chloroform was added per 1 mL of TRIzol™ Reagent and the sample was incubated for 2–3 minutes. After centrifugation for 15 minutes at 12,000 × g at 4°C, the mixture separates into a lower red phenol-chloroform, an interphase, and a colourless upper aqueous phase. The aqueous phase containing RNA was transferred to a new tube by angling the tube at 45° and pipetting the solution out. To precipitate the RNA 0.5 mL of isopropanol were added to the aqueous phase, per 1 mL of TRIzol™ Reagent used for lysis and everything was incubated for 10 minutes. After centrifugating for 10 minutes at 12,000 × g at 4°C, total RNA precipitate formed a white gel-like pellet at the bottom of the tube. The supernatant was discarded with a micropipette and the RNA was washed by resuspending the pellet in 1 mL of 75% ethanol per 1 mL of TRIzol™ Reagent used for lysis, centrifuging for 5 minutes at 7500 x g at 4°C and discarding the supernatant. Then, the RNA pellet was air dried for 10 minutes,
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resuspended in 20 μL of RNase-free water by pipetting up and down, and incubated in a heat block set at 60°C for 10 minutes. We proceeded with the determination of the RNA yield using the Qubit™ RNA BR (Broad-Range) Assay Kit with the Qubit Fluorometer (ThermoFisher Scientific). RNA samples were either stored at -80°C or used for DNase digestion.
We used the iScript Clear cDNA Synthesis kit (Bio-Rad), which includes DNase reagents for removing remaining genomic DNA (gDNA) contamination from RNA sample and reverse transcription (RT) reagents to perform cDNA synthesis. The DNase digestion was assembled on ice combining 1 µg RNA, 0.5 µl DNase, 1.5 µl DNase buffer and nuclease-free water, for a total reaction volume of 16 µl. The DNase digestion mix was incubated using a thermal cycler according to the protocol for the DNAse reaction, which consisted in DNA digestion for 5 min at 25°C, DNAse inactivation for 5 min at 75°C and storage at 4°C until the RT step.
The DNase-treated RNA template was then added with 4 µl of iScript Reverse Transcription Supermix and incubated in the thermal cycler according to the protocol in Table 3. When preparing the cDNA synthesis reaction, we added 1 µl of the PrimePCR RT control assay, as positive control for RT reaction. cDNA generated with this kit can be used directly in qPCR.
Table 1. cDNA synthesis
Step Temperature, °C Time, min
Priming 25 5
Reverse Transcription 46 20
RT inactivation 95 1
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qPCR analysis was performed with PrimePCRTM collection panel (Bio-Rad), a predesigned 96-well panel of gene targets involved in neurogenesis pathway for use with SYBR® Green (Figure 6). Apart from the genes involved in the neurogenesis pathways the plate contains housekeeping genes (12 A-C), DNA- contamination control (12 D), PrimePCR-control (12 E), two PrimePCR-quality essays (12 F-G) and a PrimePCR-reverse transcription quality essay (12 H). The SYBR Green dye is only able to emit light when it intercalates in double strand DNA, thus, in theory, the SYBR Green signal intensity doubles after every amplification cycle. qCR reaction was mixed with 10 µl of 2x Sso Advanced™ Universal Sybr® Green Supermix, 1 µl of cDNA sample, 9 µl of nuclease-free water, to reach a total volume of 20 µl. This is the volume for each sample that was distributed in each well of the 96-wells plate. Each plate was filled with only one sample aliquoted in all the respective wells. Then, the plate was put in the Bio-Rad thermal cycler and followed the cycling protocol depicted in Table 4.
Table 2. Cycling Protocol
Step Temperature Time Number of
cycles Activation 95 °C 2 min 1 Denaturation 95 °C 5 sec 40 Annealing/Extension 60 °C 30 sec 40 Melt Curve 65-95 °C (0.5 °C increments) 5 sec/step 1
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Figure 6. qPCR workflow. Hippocampus was homogenized, and total RNA was isolated by TRIzol. Genomic DNA was digested, followed by reverse transcription of mRNA to cDNA. Samples were added onto a 96-well plate containing genes involved in the neurogenesis pathway.
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After the run, gene expression analysis was performed using the CFX Maestro™ Software (Bio-Rad). The fluorescence intensity of each trace was used to determine Cq values for each well using the regression mode, that applies a multivariable, non-linear, regression model to individual well traces and then uses this model to compute an optimal Cq value. Baseline was subtracted from all fluorescence traces using the baseline subtracted curve fit option and applying the fluorescent drift correction. The relative expression of a gene is then calculated using the normalized expression ΔΔCq method according to the following formula:
𝛥𝛥𝐶𝑞 ( )
𝑅𝑄 ( )
(𝑅𝑄 ( )+ 𝑅𝑄 ( )+ 𝑅𝑄 ( ))
where, RQ is the relative quantity of the sample; and GOI is the gene of interest. The formula for relative quantity (ΔCq) for any sample (GOI) is:
𝑅𝑒𝑙𝑎𝑡𝑖𝑣𝑒 𝑞𝑢𝑎𝑛𝑡𝑖𝑡𝑦 𝑠𝑎𝑚𝑝𝑙𝑒 (𝐺𝑂𝐼) = 𝐸 ( ) ( )
where, E is the efficiency of primer and probe set that we set to 2 (100% efficiency); Cq (min) is the average Cq for the sample with the lowest average Cq for the GOI; and Cq (sample) is the Cq for the sample. Data was normalized using the expression level of three reference genes as a normalization factor. As reference genes we selected TATA-binding Protein (tbp), Gliceraldeide-3-Phosphate Dehydrogenase (gapdh) and Hypoxanthine-guanine Phosphoribosyltransferase (hprt), that are housekeeping genes assumed not to be differentially regulated in our biological system. The normalized expression for each target gene was scaled by dividing the expression level of each sample by the geometric mean level of expression of all the samples. The expression of each sample per experimental group was averaged together to determine the weighted average of each group.
36 2.2.2 Immunofluorescence
Mice were transcardially perfused with 4% paraformaldehyde (PFA). The brains were removed and fixed in 4% PFA for 24 h, then cryoprotected in 30% sucrose. Ten coronal sections of 10 µm each were cut at 182 µm intervals across the entire hippocampus, between -1.28 mm and - 2.92 mm from bregma (Figure 7 A, B). Since our interest was directed to the dorsal hippocampus, we considered the 3 sections obtained between -1.82 and -2.36 (Figure 7 A). Hippocampal slices were mounted onto polarized slides and the antigen was unmasked with 3 immersions of 1 minute each, every 3 minutes, in sodium citrate pH 6.0 at 85°C. After washing in PBS 1x and Triton 0.3% and blocking with PBS 1x, 5% BSA and 0.5% Triton, the slides were incubated overnight at 4°C with the primary antibodies in PBS 1x, 1% BSA, 0.1% Triton: Dcx 1:800 (Rabbit, Abcam Ab18723) to target new-born neuroblasts and immature neurons, and Ki67 1:500 (Rat monoclonal, Invitrogen) for cell proliferation. Afterwards, the slides were washed and incubated in the dark for 2 hours at room temperature with the secondary antibodies Donkey Anti-Rabbit, Alexa647, Abcam Ab180075 and Goat anti-rat, Alexa 488, Invitrogen, both at 1:1000. The slides were then washed and eventually dried and closed with a fluoroshield mounting medium containing DAPI for staining nuclei (Abcam Ab104139). All images were captured using a Leica confocal microscope (SP8), and analysis were performed using Fiji, a distribution of the ImageJ software (NIH). The quantification of cell numbers was performed in the dentate gyrus (Figure 7 C). The number of cells positive for Dcx and Ki67 obtained from the 3 coronal sections was summed to obtain a single estimate for each animal. All analyses were independently performed by two investigators (NG, GR), and the two estimates were averaged to obtain a single number for each animal.
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Figure 7. Immunofluorescence. A. Hippocampal coronal sections (Allen Brain Atlas). B. Sagittal view of the hippocampus showing Bregma (0 coordinate) and the regions of cut (Paxinos, George, and Keith B.J. Franklin). C. Coronal view of the dentate gyrus, bright green = dorsal part of the Dentate Gyrus (Allen Brain Atlas).
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2.3 Statistical analysis
All data were analysed to check for outliers, meaning anomalous values, performing the ROUT method with value Q=1%. The D’Agostino & Pearson normality test was used to determine if the distribution was normal. Data were reported as mean ± SEM, if normally distributed, or as median and interquartile ranges, if not normally distributed. Data deriving from the one-week monitoring of BW were analysed using two-way ANOVA for repeated measures, followed by Sidak’s post hoc tests. Differences between groups in hormonal level, behavioural performances, and gene expression were assessed with one-way ANOVAs, followed by Tukey’s post-hoc tests, in case of normal distribution of data, or with the non-parametric Kruskal-Wallis test, in case of non-normally distributed data. As regards the ORT, one-sample t-test was used to determine whether the average DI for each group was different from chance (hypothetical value = 0). Statistical significance was set at ∗p ≤ 0.05, ∗∗p ≤ 0.01, ∗∗∗p ≤ 0.001 and ∗∗∗∗p ≤ 0.0001. Statistical analyses were conducted using GraphPad Prism 6 (GraphPad Software Inc., La Jolla, CA, USA).
Table 3 depicts the number of animals for each experimental group utilized for the different purposes.
