Periodic Reporting for period 1 - EDiPSPrevent (Using a novel animal model of the dopaminergic dysfunction of schizophrenia to trial an innovative treatment approach)
Période du rapport: 2022-01-31 au 2024-01-30
Schizophrenia affects around 1% of the population and leads to dramatically reduced quality of life, as well as premature mortality in many instances. The current treatments for schizophrenia – antipsychotic drugs – show some efficacy at treating the hallucinations and delusions of this disorder. However, these drugs are far from ideal, as they result in side-effects which discourage adherence, and are ineffective in around 30% of patients. Improving treatment options for schizophrenia is important for patients, but also for their carers, on whom the burden of financial and emotional support falls heavily. This disorder also results in additional economic cost via the public health system.
Therefore, this fellowship had two primary objectives:
- To improve our understanding of two critical models of schizophrenia-relevant neurobiology: the ketamine model, and the Enhanced Dopamine in Prodromal Schizophrenia Model
- To use these models and other neurobiological techniques to understand more about a certain class of novel treatment options – trace amine-associated receptor 1 (TAAR1) agonists.
As a result of this fellowship, I developed a novel method to analyse preclinical neuroimaging data, to look at the midbrain – a region which is highly implicated in schizophrenia. I then applied this method to the ketamine model of schizophrenia. I also trialled the Enhanced Dopamine in Prodromal Schizophrenia Model in mice (whereas it has been developed in rats). While these results were not conclusive, they suggest that this model can be established in mice, and this work will form the pilot data for future funding applications. Finally, I used mouse brain slices to look at the effects of a TAAR1 agonist – ulotaront on dopamine release in the striatum. Patients with schizophrenia show elevated dopamine release in this region, and therefore an effective treatment could target this dysfunction. I found that ulotaront did indeed decrease dopamine release in this region, supporting its use a novel treatment option for schizophrenia.
Therefore, this fellowship had two primary objectives:
- To improve our understanding of two critical models of schizophrenia-relevant neurobiology: the ketamine model, and the Enhanced Dopamine in Prodromal Schizophrenia Model
- To use these models and other neurobiological techniques to understand more about a certain class of novel treatment options – trace amine-associated receptor 1 (TAAR1) agonists.
As a result of this fellowship, I developed a novel method to analyse preclinical neuroimaging data, to look at the midbrain – a region which is highly implicated in schizophrenia. I then applied this method to the ketamine model of schizophrenia. I also trialled the Enhanced Dopamine in Prodromal Schizophrenia Model in mice (whereas it has been developed in rats). While these results were not conclusive, they suggest that this model can be established in mice, and this work will form the pilot data for future funding applications. Finally, I used mouse brain slices to look at the effects of a TAAR1 agonist – ulotaront on dopamine release in the striatum. Patients with schizophrenia show elevated dopamine release in this region, and therefore an effective treatment could target this dysfunction. I found that ulotaront did indeed decrease dopamine release in this region, supporting its use a novel treatment option for schizophrenia.
The work undertaken in this project falls under 5 sub-projects:
1. Midbrain method: I developed a method for analysing the midbrain region from preclinical PET scans. I re-analysed a control cohort of animals using a template to place a midbrain ROI, based on the mouse brain atlas. I then applied this method to the ketamine model, and showed that the ketamine model animals show elevated pre-synaptic dopamine function in both the striatum (previously demonstrated) but also the midbrain. This work was presented at the international conferences of Schizophrenia International Research Society (SIRS) and the European College of Neuropsychopharmacology (ECNP), and these findings were published in the Journal of Molecular Imaging and Biology in 2023.
2. EDiPS in mice: I conducted two pilot experiments to assess the feasibility of using the EDiPS construct (originally used in rats) in mice. I delivered the AAV-packaged construct (or a control construct) to mice. I assessed the locomotor response to amphetamine – a psychomimetic, known to release dopamine from the synapse. These results were not conclusive. However, immunohistochemistry of brain slices from these animals showed that the construct-derived proteins were indeed present, indicating the feasibility of this project. This data will be used as pilot data for future funding applications to continue this project.
3. Long-term ketamine: As part of the ketamine model, ketamine (or saline) is delivered over 5 days, and then behavioural testing or PET scanning is conducted on day 7. However, it is unclear how long this effect of ketamine treatment is maintained. I conducted the final PET scans to complete this project. Although the ketamine treatment appeared to result in increased dopamine synthesis capacity beyond the 7-day timepoint, this was not statistically increased relative to the saline-treated animals. Turther work may be conducted to clarify whether the effect in saline-treated control animals is a true effect, or an artefact of a technical issue.
4. Ketamine immunohistochemistry: I generated animals for the ketamine model (treated for 5 days with either ketamine or saline), and collected brain tissue from these animals for immunohistochemistry. I then undertook staining to examine the expression levels of two excitatatory-related proteins – VGLUT1 and VGLUT2 – in the midbrain. I found no significant differences in the expression of VGLUT1 or VGLUT2 proteins in the midbrain. Although this does not preclude changes in functional activity of excitatory transmission to this region, it suggests that glutamatergic projections from the cortex are not a key factor in this model. Future work is planned to identify potential changes in inhibitory function in this region.
5. Voltammetry: During my secondment period (3 months), I undertook ex vivo voltammetry to unpick the actions of the TAAR1 agonist ulotaront on dopamine function in the striatum. In this technique, I was able to look at dopamine release from wild-type (untreated) mouse brain slices. I firstly applied ulotaront alone, and found that it decreased normal levels of dopamine release in a dose-dependent manner. This effect was still present even when acetylcholine signalling - which modulates dopamine release – was blocked. I also used a TAAR1 antagonist to confirm that the effect of ulotaront were indeed mediated by the TAAR1, rather than via its actions at, for instance, a serotonergic receptor. This data is being included in a manuscript under preparation.
1. Midbrain method: I developed a method for analysing the midbrain region from preclinical PET scans. I re-analysed a control cohort of animals using a template to place a midbrain ROI, based on the mouse brain atlas. I then applied this method to the ketamine model, and showed that the ketamine model animals show elevated pre-synaptic dopamine function in both the striatum (previously demonstrated) but also the midbrain. This work was presented at the international conferences of Schizophrenia International Research Society (SIRS) and the European College of Neuropsychopharmacology (ECNP), and these findings were published in the Journal of Molecular Imaging and Biology in 2023.
2. EDiPS in mice: I conducted two pilot experiments to assess the feasibility of using the EDiPS construct (originally used in rats) in mice. I delivered the AAV-packaged construct (or a control construct) to mice. I assessed the locomotor response to amphetamine – a psychomimetic, known to release dopamine from the synapse. These results were not conclusive. However, immunohistochemistry of brain slices from these animals showed that the construct-derived proteins were indeed present, indicating the feasibility of this project. This data will be used as pilot data for future funding applications to continue this project.
3. Long-term ketamine: As part of the ketamine model, ketamine (or saline) is delivered over 5 days, and then behavioural testing or PET scanning is conducted on day 7. However, it is unclear how long this effect of ketamine treatment is maintained. I conducted the final PET scans to complete this project. Although the ketamine treatment appeared to result in increased dopamine synthesis capacity beyond the 7-day timepoint, this was not statistically increased relative to the saline-treated animals. Turther work may be conducted to clarify whether the effect in saline-treated control animals is a true effect, or an artefact of a technical issue.
4. Ketamine immunohistochemistry: I generated animals for the ketamine model (treated for 5 days with either ketamine or saline), and collected brain tissue from these animals for immunohistochemistry. I then undertook staining to examine the expression levels of two excitatatory-related proteins – VGLUT1 and VGLUT2 – in the midbrain. I found no significant differences in the expression of VGLUT1 or VGLUT2 proteins in the midbrain. Although this does not preclude changes in functional activity of excitatory transmission to this region, it suggests that glutamatergic projections from the cortex are not a key factor in this model. Future work is planned to identify potential changes in inhibitory function in this region.
5. Voltammetry: During my secondment period (3 months), I undertook ex vivo voltammetry to unpick the actions of the TAAR1 agonist ulotaront on dopamine function in the striatum. In this technique, I was able to look at dopamine release from wild-type (untreated) mouse brain slices. I firstly applied ulotaront alone, and found that it decreased normal levels of dopamine release in a dose-dependent manner. This effect was still present even when acetylcholine signalling - which modulates dopamine release – was blocked. I also used a TAAR1 antagonist to confirm that the effect of ulotaront were indeed mediated by the TAAR1, rather than via its actions at, for instance, a serotonergic receptor. This data is being included in a manuscript under preparation.
This project progressed the field of preclinical neuroimaging analysis by the development of a novel method for PET scan analysis. This method could be applied widely to other models of dopaminergic disorders. It may prove especially useful in models of Parkinson’s disorder, for which degeneration of dopamine in the midbrain is a critical factor. This aspect of the project has resulted in a publication which can be utilized by groups internationally who are undertaking preclinical PET.
Through the neuroimaging experiments as well as the immunohistochemistry performed, this project advanced the understanding of the ketamine model of schizophrenia. This work revealed that dopamine dysfunction which has already been shown in the striatum in this model is also present in the midbrain. However, I also demonstrated that the excitatory stimulation to the midbrain is unlikely to be altered by treatment with ketamine. This suggests that future work understanding the impact of ketamine (either to induce a model, or as a potential treatment) could explore changes in inhibitory factors in this region. As the ketamine model is widely used, this has implications beyond this project, and beyond this research group.
The voltammetry data acquired during the secondment period has perhaps the greatest real-world potential impact. This work demonstrated that a TAAR1 agonist ulotaront, which has already shown some efficacy in small-scale clinical trials, may indeed function through decreasing dopamine release in the striatum. This provides a mechanistic explanation for its positive effects in these trials. This further lays a foundation for future drug development to explore other compounds which may modulate the function of TAAR1. It also supports the use of ulotaront in a clinical setting for the treatment of schizophrenia.
Through the neuroimaging experiments as well as the immunohistochemistry performed, this project advanced the understanding of the ketamine model of schizophrenia. This work revealed that dopamine dysfunction which has already been shown in the striatum in this model is also present in the midbrain. However, I also demonstrated that the excitatory stimulation to the midbrain is unlikely to be altered by treatment with ketamine. This suggests that future work understanding the impact of ketamine (either to induce a model, or as a potential treatment) could explore changes in inhibitory factors in this region. As the ketamine model is widely used, this has implications beyond this project, and beyond this research group.
The voltammetry data acquired during the secondment period has perhaps the greatest real-world potential impact. This work demonstrated that a TAAR1 agonist ulotaront, which has already shown some efficacy in small-scale clinical trials, may indeed function through decreasing dopamine release in the striatum. This provides a mechanistic explanation for its positive effects in these trials. This further lays a foundation for future drug development to explore other compounds which may modulate the function of TAAR1. It also supports the use of ulotaront in a clinical setting for the treatment of schizophrenia.