Final Report Summary - MECPST-IPD (Identification of inflammation pathways involved in the predisposition to decreased neurogenesis and depression)
As outlined in the recent Nature special issue, The Great Depression, depression causes greater disability than any other condition. The special issue also underlines how research seems to be chronically running aground as little has been achieved in terms of new treatments exemplified by the fact that no new drugs employing novel mechanisms have been released despite these massive efforts. The pharmaceutical industry has for example for decades focused resources on targeting the monoamine system in the treatment of depression. These neuropsychopharmacological studies have resulted in the development of commercially and clinically successful drugs including serotonin specific reuptake inhibitors and serotonin noradrenaline reuptake inhibitors. However, these drugs have several notable disadvantages, including a delayed on-set of action, detrimental side effects, and are furthermore are not efficacious in a large portion of those suffering from depression. In this project I aimed to focus on depression, not as a homogeneous disorder, but as a complex multi-factorial disorder. To this end I combined cutting-edge ideas hypotheses regarding the pathophysiology of depression from the disciplines of neuroscience, neuropsychopharmacology, and psychoneuroimmunology in order to shed new light on the understanding of depression as a whole and indicate a new treatment strategy for depression.
To realize these aims I developed several objectives to model pathophysiological elements of depression. In our first objective I modelled inflammation- a process which has recently been proposed to be important pathophysiological element in depression. In particular, it has been hypothesized that childhood trauma can cause increases in inflammation that may increase the susceptibility of individuals to develop depression in later life (Danese et al. 2008). Mice were therefore injected with the inflammation inducing substance lipopolysaccharide after several postnatal days to mimic early life trauma leading to increases in inflammation. Another important pathophysiological element of depression is stress which often precipitates depressive episodes in humans. To model stress I subjected mice to unpredictable chronic mild stress (UCMS) to mimic a later life stress. As a part of the first objective I exposed these inflammatory predisposed mice to UCMS in later life to see if the predisposition affected their response to stress. In a second objective I introduced a third pathophysiological element which is decreases in adult neurogenesis. Adult neurogenesis, the production of new neurons in the adult hippocampus has been suggested to be important for the development (decreases) and recovery (restoration) from depression. I therefore used the anti-mitotic drug temozolomide to decrease levels of neurogenesis to mimic events which decrease adult neurogenesis. As a part of this objective I waited until the inflammatory predisposed mice reached adulthood and then treated them with temozolomide after which I finally exposed the mice to UCMS. In a third objective I looked at the mechanisms which may be underlying the pathophysiology by examining the inflammatory components of blood in mice at different time points after exposure to the inflammatory insult.
At the beginning of this project in 2013 I performed pilot experiments with doses of LPS at the same time as I set up the UCMS protocol in the lab. Once a working protocol for both of these was established I proceeded with experiments examining the role of the pathophysiological element on affective behaviour. Results from this were analysed with several sub-optimal caveats found. I then proceeded to correct these in a subsequent protocol of UCMS which produced satisfactory results. In addition to this I concomitantly began looking at the dynamics of the inflammatory system as potential underlying mechanisms responsible for behavioural effects. From initial experiments it was clear that of the various pathophysiological elements, decreases in adult neurogenesis had substantial effects. I therefore investigated this individually under optimized conditions.
Results from these studies found several notable changes. In particular mice with all 3 pathophysiological elements combined (the “triple hit” mouse group) displayed the least amount of weight gain which was statistically significant when compared to control (p<0.05). This triple hit group also displayed a significantly higher coat score, a measure of stress susceptibility, after 3 weeks of stress in comparison with other stress treated groups (p<0.05). When evaluating mice in the anxiety testing open field test after 6 weeks of UCMS, both the double and triple hit mice display a significant increase in locomotion when in the groups exposed to UCMS (p < 0.05 n=10). This hyper-locomotion is indicative of an increases in dopaminergic tone as a result of the early life insult. In my initial UCMS triple hit study, 6 weeks of UCMS induced resilience in the mice rather than a depressive phenotype. Resilience in mice has recently indicated to result from enhancement of depression mechanisms, suggesting that mice in this initial study were stressed beyond what was necessary to induce depression (Friedman et al. 2014). In the mentioned study, depression susceptible mice had decreases in mid-brain dopamine activity in comparison to controls but surprisingly the resilient mice, rather than having normal levels of dopamine activity, had even more pronounced decreases. This correlates well with our finding as our most hyperactive and hence hyper-dopaminergic mice, the triple hit group, had the lowest levels of anxiety in the open field with an 80% increase in centre time in comparison to controls (p < 0.05 n=10). This was similarly reflected in the anhedonia measuring, sucrose preference test, another behaviour closely associated with the dopaminergic system. In this test UCMS control mice had a decrease in sucrose preference but the triple hit resilient mice had an slightly increased (insignificant) sucrose preference. Thus in summary my results show that the triple hit of pathophysiological elements causes behavioural results reflective of an increased activation of the dopaminergic system.
In order to examine the potential mechanism we used devised an study to look at inflammatory cytokine dynamics in early life. Results from these experiments demonstrated that an early life inflammatory insult alters the dynamics throughout the life of the mouse. In particular, we find post-natal day 6 mice have hypo-responsive immune system. In addition, early life inflammatory insult causes P14 mice to have an prolonged IL-6 response while at P46 they have an attenuated response .
During the initial experiment it was discovered that a decrease in neurogenesis alone, using temozolomide, induced a depressive phenotype in several tests. An experiment was thus designed to investigate this as a model of depression. Results from this experiment showed that decreased neurogenesis induced increases in depressive-associated behaviour in the novelty supressed feeding test and transient increases anhedonia. However, neurogenesis depletion did not affect anxiety-like behaviour. In contrast the stress response, measured using the stress hormone glucocorticoid, was increased in the neurogenesis deficient mice after an acute stressor. The results of the NSF indicate that adult neurogenesis is important in the processing of novel situations. The increased stress response further indicates that neurogenesis deficient mice have an increased activation of the limbic system, suggesting a deficiency in emotional processing. Therefore, the deficiencies seen in the NSF may also be a result of deficits in emotional processing of a novel context.
As described above there is currently a crisis with regards to the treatment of depression. The expected final result of this project is that it will provide insight into the pathophysiology of depression and allow future treatments to be developed based on these findings. My results described have shown that early life inflammatory insults may induce changes which alter an individuals reaction to future stress. In addition results from the Temozolomide study indicate that adult neurogenesis may be important during the emotional processing of novel situations. These studies indicate thus may be important clues for developing future treatments.
To realize these aims I developed several objectives to model pathophysiological elements of depression. In our first objective I modelled inflammation- a process which has recently been proposed to be important pathophysiological element in depression. In particular, it has been hypothesized that childhood trauma can cause increases in inflammation that may increase the susceptibility of individuals to develop depression in later life (Danese et al. 2008). Mice were therefore injected with the inflammation inducing substance lipopolysaccharide after several postnatal days to mimic early life trauma leading to increases in inflammation. Another important pathophysiological element of depression is stress which often precipitates depressive episodes in humans. To model stress I subjected mice to unpredictable chronic mild stress (UCMS) to mimic a later life stress. As a part of the first objective I exposed these inflammatory predisposed mice to UCMS in later life to see if the predisposition affected their response to stress. In a second objective I introduced a third pathophysiological element which is decreases in adult neurogenesis. Adult neurogenesis, the production of new neurons in the adult hippocampus has been suggested to be important for the development (decreases) and recovery (restoration) from depression. I therefore used the anti-mitotic drug temozolomide to decrease levels of neurogenesis to mimic events which decrease adult neurogenesis. As a part of this objective I waited until the inflammatory predisposed mice reached adulthood and then treated them with temozolomide after which I finally exposed the mice to UCMS. In a third objective I looked at the mechanisms which may be underlying the pathophysiology by examining the inflammatory components of blood in mice at different time points after exposure to the inflammatory insult.
At the beginning of this project in 2013 I performed pilot experiments with doses of LPS at the same time as I set up the UCMS protocol in the lab. Once a working protocol for both of these was established I proceeded with experiments examining the role of the pathophysiological element on affective behaviour. Results from this were analysed with several sub-optimal caveats found. I then proceeded to correct these in a subsequent protocol of UCMS which produced satisfactory results. In addition to this I concomitantly began looking at the dynamics of the inflammatory system as potential underlying mechanisms responsible for behavioural effects. From initial experiments it was clear that of the various pathophysiological elements, decreases in adult neurogenesis had substantial effects. I therefore investigated this individually under optimized conditions.
Results from these studies found several notable changes. In particular mice with all 3 pathophysiological elements combined (the “triple hit” mouse group) displayed the least amount of weight gain which was statistically significant when compared to control (p<0.05). This triple hit group also displayed a significantly higher coat score, a measure of stress susceptibility, after 3 weeks of stress in comparison with other stress treated groups (p<0.05). When evaluating mice in the anxiety testing open field test after 6 weeks of UCMS, both the double and triple hit mice display a significant increase in locomotion when in the groups exposed to UCMS (p < 0.05 n=10). This hyper-locomotion is indicative of an increases in dopaminergic tone as a result of the early life insult. In my initial UCMS triple hit study, 6 weeks of UCMS induced resilience in the mice rather than a depressive phenotype. Resilience in mice has recently indicated to result from enhancement of depression mechanisms, suggesting that mice in this initial study were stressed beyond what was necessary to induce depression (Friedman et al. 2014). In the mentioned study, depression susceptible mice had decreases in mid-brain dopamine activity in comparison to controls but surprisingly the resilient mice, rather than having normal levels of dopamine activity, had even more pronounced decreases. This correlates well with our finding as our most hyperactive and hence hyper-dopaminergic mice, the triple hit group, had the lowest levels of anxiety in the open field with an 80% increase in centre time in comparison to controls (p < 0.05 n=10). This was similarly reflected in the anhedonia measuring, sucrose preference test, another behaviour closely associated with the dopaminergic system. In this test UCMS control mice had a decrease in sucrose preference but the triple hit resilient mice had an slightly increased (insignificant) sucrose preference. Thus in summary my results show that the triple hit of pathophysiological elements causes behavioural results reflective of an increased activation of the dopaminergic system.
In order to examine the potential mechanism we used devised an study to look at inflammatory cytokine dynamics in early life. Results from these experiments demonstrated that an early life inflammatory insult alters the dynamics throughout the life of the mouse. In particular, we find post-natal day 6 mice have hypo-responsive immune system. In addition, early life inflammatory insult causes P14 mice to have an prolonged IL-6 response while at P46 they have an attenuated response .
During the initial experiment it was discovered that a decrease in neurogenesis alone, using temozolomide, induced a depressive phenotype in several tests. An experiment was thus designed to investigate this as a model of depression. Results from this experiment showed that decreased neurogenesis induced increases in depressive-associated behaviour in the novelty supressed feeding test and transient increases anhedonia. However, neurogenesis depletion did not affect anxiety-like behaviour. In contrast the stress response, measured using the stress hormone glucocorticoid, was increased in the neurogenesis deficient mice after an acute stressor. The results of the NSF indicate that adult neurogenesis is important in the processing of novel situations. The increased stress response further indicates that neurogenesis deficient mice have an increased activation of the limbic system, suggesting a deficiency in emotional processing. Therefore, the deficiencies seen in the NSF may also be a result of deficits in emotional processing of a novel context.
As described above there is currently a crisis with regards to the treatment of depression. The expected final result of this project is that it will provide insight into the pathophysiology of depression and allow future treatments to be developed based on these findings. My results described have shown that early life inflammatory insults may induce changes which alter an individuals reaction to future stress. In addition results from the Temozolomide study indicate that adult neurogenesis may be important during the emotional processing of novel situations. These studies indicate thus may be important clues for developing future treatments.