Periodic Reporting for period 1 - MITORett (Deciphering cerebellar mitochondrial alterations in Rett Syndrome)
Okres sprawozdawczy: 2021-05-01 do 2023-04-30
Rett syndrome is a progressive neurodegenerative disorder caused by mutations in the Mecp2 gene and affecting primarily women. Although individuals with Rett syndrome initially exhibit typical development during the initial years of life, they subsequently experience a rapid regression, losing language and motor coordination skills. This regression is accompanied by the development of cognitive deficits, stereotyped hand movements, ataxia, seizures, respiratory dysrhythmias, and alterations in social behavior. Despite available treatments to alleviate symptoms, there's still no cure for Rett syndrome disease.
The MECP2 protein plays a pivotal role in regulating gene expression, and mutations in MECP2 directly impact its target genes. Animal models, particularly those focusing on specific brain cell types, have contributed valuable insights into the underlying mechanisms of the syndrome. In a study involving a mouse model of Rett Syndrome where Mecp2 was suppressed in a particular sub-type of inhibitory neurons known as Parvalbumin-neurons, motor impairments, spasticity, social and memory deficits, and premature lethality were observed in Rett Syndrome mice.
Parvalbumin-neurons, characterized by substantial energy requirements, are distributed across various brain regions, including the cerebellum, a region with significant energy demands and a substantial population of Parvalbumin-neurons. Mitochondria, essential cellular organelles, play a pivotal role in energy production. Previous studies have reported mitochondrial alterations in individuals with Rett Syndrome, despite that the specific regional and cellular identity of these alterations remain unexplored.
This project hypothesizes that cerebellar Parvalbumin-positive neurons, with their pronounced energy requirements, may be particularly vulnerable to mitochondrial alterations in Rett Syndrome. To address this hypothesis, the research proposal suggests analyzing whether the expression of mitochondrial proteins in cerebellar Parvalbumin-neurons is affected by the Mecp2 mutation. Additionally, an examination of potential modifications in mitochondrial functionality in Rett Syndrome mice is proposed. To accomplish this, histological and transcriptomic experiments have been conducted in a mouse model with Mecp2 suppression in Parvalbumin-neurons. Ongoing proteomic and functional experiments that are under compilation will complement this work.
The MECP2 protein plays a pivotal role in regulating gene expression, and mutations in MECP2 directly impact its target genes. Animal models, particularly those focusing on specific brain cell types, have contributed valuable insights into the underlying mechanisms of the syndrome. In a study involving a mouse model of Rett Syndrome where Mecp2 was suppressed in a particular sub-type of inhibitory neurons known as Parvalbumin-neurons, motor impairments, spasticity, social and memory deficits, and premature lethality were observed in Rett Syndrome mice.
Parvalbumin-neurons, characterized by substantial energy requirements, are distributed across various brain regions, including the cerebellum, a region with significant energy demands and a substantial population of Parvalbumin-neurons. Mitochondria, essential cellular organelles, play a pivotal role in energy production. Previous studies have reported mitochondrial alterations in individuals with Rett Syndrome, despite that the specific regional and cellular identity of these alterations remain unexplored.
This project hypothesizes that cerebellar Parvalbumin-positive neurons, with their pronounced energy requirements, may be particularly vulnerable to mitochondrial alterations in Rett Syndrome. To address this hypothesis, the research proposal suggests analyzing whether the expression of mitochondrial proteins in cerebellar Parvalbumin-neurons is affected by the Mecp2 mutation. Additionally, an examination of potential modifications in mitochondrial functionality in Rett Syndrome mice is proposed. To accomplish this, histological and transcriptomic experiments have been conducted in a mouse model with Mecp2 suppression in Parvalbumin-neurons. Ongoing proteomic and functional experiments that are under compilation will complement this work.
In this research proposal, the main aim was to analyze the mitochondrial alterations in the cerebellar Parvalbumin-neurons of Rett Syndrome mice. I proposed to perform proteomic and functional experiments in mitochondrial samples (mitochondrial immunoprecipitates) from cerebellar PV-positive neurons of Rett Syndrome mice.
To do so, I implemented an approach to guarantee the sufficient expression of mitochondrial-tagged organelles to successfully perform afterwards mitochondrial purification. I used the MITO-Tag mouse line, mitochondrial HA only in cells expressing Cre recombinase. During the two years of the MITORett project, I properly set up the triple mouse line named the PV-Cre;MITO-Tag;Mecp2. Currently, the cerebellar samples are already collected and prepared to perform the mitochondria immunoprecipitation and the proteomic and functional analyses.
In parallel, I have utilized the time effectively by undertaking alternative experiments to deeply analyze the cerebellar alterations in Rett Syndrome and to boost the project forward. Taking profit of the tools, techniques, animal models and methods available in Dr. Quintana Lab, I performed several experiments that perfectly complemented the initial proposed project and allowed me to gain insights into the cerebellar pathophysiology of Rett Syndorme.
Immunofluorescence and transcriptomic experiments performed in cerebellums of control and Rett Syndrome mice demonstrated that Mecp2 is preferentially expressed in Parvalbumin-positive neurons in the cerebellum. Therefore, we have performed transcriptomic analysis of the cerebellar Parvalbumin-neurons in control and Rett Syndrome symptomatic mice, which allowed us to determine that genes related with mitochondrial complex and membrane organization are changed in the cerebellar Parvalbumin-neurons of Rett Syndrome mice, suggesting alterations in the mitochondrial functionality. These results would be validated in the next months by preforming proteomic and functional studies in mitochondrial immunoprecipitates obtained from the triple mouse line PV-Cre;MITO-Tag;Mecp2. We expect to corroborate the alterations observed in the transcriptomic analysis and to find novel insights about the mitochondrial dysfunction in the cerebellum of Rett Syndrome individuals.
In addition, another group of genes found significantly modified in the cerebellum of Rett Syndrome mice, are the transcripts encoding for receptors or enzymes involved in the GABAergic signaling. These results suggest that GABAergic transmission in the cerebellar Parvalbumin-neurons is notably impaired in Rett Syndrome mice. Forthcoming experiments would be focused on targeting some of these proteins to try to ameliorate the motor and social symptoms present in Rett Syndrome.
All these results were presented in 3 international conferences, 1 workshop and 1 invited talk. Indeed, this meeting was successful since allowed me to find synergies with other cerebellar research groups and foster collaborations for this project. In addition, in May 2023, for the Brain Awareness Week, I did 2 talks in a primary school to talk about the research we were doing in the lab. Similarly, in September 2023 I did a talk as part of the event “The night for Scientific Research” for adult lay audiences. All these events, allowed me to disseminate the results of the MITORett project at scientific and non-scientific audiences, allowing me to establish important collaborations that will make this project successfully advance until completion.
To do so, I implemented an approach to guarantee the sufficient expression of mitochondrial-tagged organelles to successfully perform afterwards mitochondrial purification. I used the MITO-Tag mouse line, mitochondrial HA only in cells expressing Cre recombinase. During the two years of the MITORett project, I properly set up the triple mouse line named the PV-Cre;MITO-Tag;Mecp2. Currently, the cerebellar samples are already collected and prepared to perform the mitochondria immunoprecipitation and the proteomic and functional analyses.
In parallel, I have utilized the time effectively by undertaking alternative experiments to deeply analyze the cerebellar alterations in Rett Syndrome and to boost the project forward. Taking profit of the tools, techniques, animal models and methods available in Dr. Quintana Lab, I performed several experiments that perfectly complemented the initial proposed project and allowed me to gain insights into the cerebellar pathophysiology of Rett Syndorme.
Immunofluorescence and transcriptomic experiments performed in cerebellums of control and Rett Syndrome mice demonstrated that Mecp2 is preferentially expressed in Parvalbumin-positive neurons in the cerebellum. Therefore, we have performed transcriptomic analysis of the cerebellar Parvalbumin-neurons in control and Rett Syndrome symptomatic mice, which allowed us to determine that genes related with mitochondrial complex and membrane organization are changed in the cerebellar Parvalbumin-neurons of Rett Syndrome mice, suggesting alterations in the mitochondrial functionality. These results would be validated in the next months by preforming proteomic and functional studies in mitochondrial immunoprecipitates obtained from the triple mouse line PV-Cre;MITO-Tag;Mecp2. We expect to corroborate the alterations observed in the transcriptomic analysis and to find novel insights about the mitochondrial dysfunction in the cerebellum of Rett Syndrome individuals.
In addition, another group of genes found significantly modified in the cerebellum of Rett Syndrome mice, are the transcripts encoding for receptors or enzymes involved in the GABAergic signaling. These results suggest that GABAergic transmission in the cerebellar Parvalbumin-neurons is notably impaired in Rett Syndrome mice. Forthcoming experiments would be focused on targeting some of these proteins to try to ameliorate the motor and social symptoms present in Rett Syndrome.
All these results were presented in 3 international conferences, 1 workshop and 1 invited talk. Indeed, this meeting was successful since allowed me to find synergies with other cerebellar research groups and foster collaborations for this project. In addition, in May 2023, for the Brain Awareness Week, I did 2 talks in a primary school to talk about the research we were doing in the lab. Similarly, in September 2023 I did a talk as part of the event “The night for Scientific Research” for adult lay audiences. All these events, allowed me to disseminate the results of the MITORett project at scientific and non-scientific audiences, allowing me to establish important collaborations that will make this project successfully advance until completion.
The completion of the MITORett research proposal has an important impact at the scientific level since the results obtained pointed new mitochondrial genes and genes related with the GABAergic signaling as potential targets for the treatment of Rett Syndrome. Particularly, the fact that the methodologies used allowed us to decipher the transcriptomic and proteomic changes in a particular cell type, the Parvalbumin-neurons, is of particular interest for the forthcoming design of novel drugs, aimed at acting more precisely in the brain, with higher cell-type selectivity, greater efficacy, and reduced side effects. The next step in this project would be the use of transgenic mice in combination with the appropriate viral approaches to selectively overexpress the affected selected proteins in Rett Syndrome mice.
One of the genes found downregulated in the cerebellar parvalbumin-neurons is a GABA receptor. For others diseases with similar characteristics than Rett Syndrome (such as the Fragile X Syndrome) and with a comparable reduction in protein expression, several clinical trials to test the action of GABA receptor agonists have been successfully conducted (http://clinicaltrials.gov(odnośnik otworzy się w nowym oknie)). Thus, it is intriguing to consider the possibility of developing a similar therapy for Rett Syndrome. These preliminary results open the door to new research and potentially innovative therapeutic approaches to address the challenges associated with this condition.
One of the genes found downregulated in the cerebellar parvalbumin-neurons is a GABA receptor. For others diseases with similar characteristics than Rett Syndrome (such as the Fragile X Syndrome) and with a comparable reduction in protein expression, several clinical trials to test the action of GABA receptor agonists have been successfully conducted (http://clinicaltrials.gov(odnośnik otworzy się w nowym oknie)). Thus, it is intriguing to consider the possibility of developing a similar therapy for Rett Syndrome. These preliminary results open the door to new research and potentially innovative therapeutic approaches to address the challenges associated with this condition.