Neurodevelopmental disorders (NDD) encompass a group of conditions that typically manifest during early stages of development. They are characterized by impairments in personal, social, academic, and occupational functioning. One recurring genetic variation known as 16p11.2, which involves a copy number variant (CNV) spanning 600kb, is one of the most common causes of NDD. This variation can result in both deletions and duplications and is associated with symptoms found in autism spectrum disorder (ASD), intellectual disability (ID), attention deficit hyperactivity disorder (ADHD), schizophrenia (SZ), and epilepsy. Within the affected region of chromosome 16p11.2 lies a gene called MAPK3, which codes for ERK1, a protein kinase involved in modulating the intracellular signalling pathway known as mitogen-activated protein kinase/extracellular signal-regulated kinase (MAPK/ERK). Previous research from our laboratory has indicated that the ERK signalling pathway is disrupted in a mouse model with a 16p11.2 deletion (16p11.2+/-). Additionally, we have found that pharmacological inhibition of this intracellular pathway can alleviate some of the associated brain and behavioural symptoms. In order to gain insight into the potential role of ERK signalling in the pathophysiology of NDD, I conducted immunostaining experiments using samples from two different mouse strains: one with heterozygous ERK1 (ERK1+/-) and another modelling the 16p11.2+/- deletion. These experiments were performed at two different developmental stages: postnatal day 60 (P60) and embryonic day 14.5 (E14.5). To assess ERK signalling activity in the brain, I utilized specific antibodies that recognize the phosphorylated forms of both ERK1 and ERK2 (pERK) in immunohistochemistry. This analysis was conducted in two key brain areas implicated in NDD etiology: the prefrontal cortex (PFC) and the striatum. Additionally, I employed an antibody against c-Fos solely in PFC sections to measure neuronal activity. Furthermore, I incorporated markers such as Cyclin D1 (CD1), which are indicative of cell cycle progression, as well as the intermediate progenitor and differentiation marker Tbr2 in the PFC sections. These developmental markers were chosen due to previous reports suggesting that ERK activation promotes G1 cell cycle progression and the accumulation of CD1. Moreover, aberrant neural progenitor proliferation associated with ERK activity often leads to dysregulation in the generation of various neuronal cell types within the cortex. Collectively, the gathered data indicate that having a single copy of ERK1, as observed in both ERK1+/- and 16p11.2+/- mice, is sufficient to maintain normal basal ERK activity in the examined brain regions. However, when subjected to a pharmacological challenge using a selective dopamine D1 receptor agonist, SKF38393, both the prefrontal cortex (PFC) and striatum exhibited a significant increase in downstream signalling, indicating hyperactivation. Furthermore, during the developmental stages, abnormalities were observed in the staining patterns of CD1 and Tbr2 in both ERK1+/- and 16p11.2+/- mice, suggesting a similar regulation of ERK signalling during embryonic development. Overall, this analysis provides preliminary evidence of cellular and signalling deficits present in mouse models with the 16p11.2 CNV. These findings may have broader implications for future studies involving behavioural rescue experiments and the development of potential therapeutics in preclinical settings.
I disturbi del neurosviluppo sono un gruppo di condizioni che si verificano tipicamente nelle prime fasi dello sviluppo e sono caratterizzati da difficoltà nella sfera personale, sociale, accademica e occupazionale. 16p11.2 è una alterazione cromosomica di 600 kb (CNV) ed è una delle cause più comuni nei disordini del neurosviluppo. Si può trovare sotto forma di delezione o duplicazione, ed entrambe presentano sintomi associati allo spettro autistico (ASD), alla disabilità intellettiva (ID), al disturbo da deficit di attenzione e iperattività (ADHD), alla schizofrenia (SZ) e all'epilessia. Il gene MAPK3, che codifica per ERK1, si trova in questa regione cromosomica 16p11.2. ERK1 è una proteina chinasi capace di regolare la cascata di segnalazione intracellulare mitogen-activated protein kinase/extracellular signal-regulated kinase (MAPK/ERK). Precedenti studi del nostro laboratorio indicano che l’attività di ERK è deregolata in un modello murino di delezione 16p11.2 (16p11.2+/-), e che l'inibizione farmacologica di questa via intracellulare ripristina alcuni fenotipi cerebrali e comportamentali associati. Per comprendere il potenziale ruolo della segnalazione di ERK nella fisiopatologia dei disturbi del neurosviluppo, abbiamo utilizzato tecniche di immunoistochimica su due ceppi di topi, eterozigoti per ERK1 (ERK1+/-) e 16p11.2+/-, in due diversi stadi dello sviluppo, giorno postnatale (P) 60 e giorno embrionale (E) 14.5. Per comprendere l'attività di ERK nel cervello, sono stati utilizzati anticorpi che riconoscono specificamente la versione fosforilata di ERK1 e ERK2 (pERK) sia nella corteccia prefrontale (PFC) che nello striato, due aree chiave implicate nell'eziologia dei disordini del neurosviluppo. Inoltre, nella PFC sono stati utilizzati, un anticorpo contro c-Fos per misurare l'attività neuronale, un marcatore del ciclo cellulare (CiclineD1, CD1), e Tbr2, un marcatore di differenziamento e dei progenitori intermedi. Abbiamo utilizzato questi marcatori poiché in letteratura è riportato che l'attivazione di ERK promuove la progressione del ciclo cellulare G1 e l'accumulo di CD1. Inoltre, l'attività di ERK è spesso associata a una proliferazione aberrante dei progenitori neurali, che porta a una perturbazione nel numero di tipi di cellule neuronali generate nella corteccia. Complessivamente, i dati raccolti indicano che una singola copia di ERK1 presente nei topi ERK1+/- e nei topi 16p11.2+/- è sufficiente a mantenere una normale attività basale di ERK in entrambe le regioni cerebrali. Tuttavia, una stimolazione farmacologica sia nella PFC che nello striato, con un agonista selettivo del recettore della dopamina D1, SKF38393, è sufficiente per evidenziare una significativa iperattivazione della segnalazione a valle. Inoltre, durante lo sviluppo, CD1 e Tbr2 sono risultati alterati sia nei topi ERK1+/- che nei topi 16p11.2+/-, suggerendo una simile regolazione della segnalazione ERK durante lo sviluppo embrionale. Complessivamente, questa analisi fornisce evidenze preliminari di deficit cellulari e di segnalazione riscontrati nei modelli murini CNV di 16p11.2, con potenziali implicazioni più ampie per successivi esperimenti di recupero comportamentale e per lo sviluppo preclinico di terapie future.
Attivazione cerebrale della cascata di segnalazione ERK in un modello murino della delezione 16p11.2
ZUGLIAN, CECILIA
2021/2022
Abstract
Neurodevelopmental disorders (NDD) encompass a group of conditions that typically manifest during early stages of development. They are characterized by impairments in personal, social, academic, and occupational functioning. One recurring genetic variation known as 16p11.2, which involves a copy number variant (CNV) spanning 600kb, is one of the most common causes of NDD. This variation can result in both deletions and duplications and is associated with symptoms found in autism spectrum disorder (ASD), intellectual disability (ID), attention deficit hyperactivity disorder (ADHD), schizophrenia (SZ), and epilepsy. Within the affected region of chromosome 16p11.2 lies a gene called MAPK3, which codes for ERK1, a protein kinase involved in modulating the intracellular signalling pathway known as mitogen-activated protein kinase/extracellular signal-regulated kinase (MAPK/ERK). Previous research from our laboratory has indicated that the ERK signalling pathway is disrupted in a mouse model with a 16p11.2 deletion (16p11.2+/-). Additionally, we have found that pharmacological inhibition of this intracellular pathway can alleviate some of the associated brain and behavioural symptoms. In order to gain insight into the potential role of ERK signalling in the pathophysiology of NDD, I conducted immunostaining experiments using samples from two different mouse strains: one with heterozygous ERK1 (ERK1+/-) and another modelling the 16p11.2+/- deletion. These experiments were performed at two different developmental stages: postnatal day 60 (P60) and embryonic day 14.5 (E14.5). To assess ERK signalling activity in the brain, I utilized specific antibodies that recognize the phosphorylated forms of both ERK1 and ERK2 (pERK) in immunohistochemistry. This analysis was conducted in two key brain areas implicated in NDD etiology: the prefrontal cortex (PFC) and the striatum. Additionally, I employed an antibody against c-Fos solely in PFC sections to measure neuronal activity. Furthermore, I incorporated markers such as Cyclin D1 (CD1), which are indicative of cell cycle progression, as well as the intermediate progenitor and differentiation marker Tbr2 in the PFC sections. These developmental markers were chosen due to previous reports suggesting that ERK activation promotes G1 cell cycle progression and the accumulation of CD1. Moreover, aberrant neural progenitor proliferation associated with ERK activity often leads to dysregulation in the generation of various neuronal cell types within the cortex. Collectively, the gathered data indicate that having a single copy of ERK1, as observed in both ERK1+/- and 16p11.2+/- mice, is sufficient to maintain normal basal ERK activity in the examined brain regions. However, when subjected to a pharmacological challenge using a selective dopamine D1 receptor agonist, SKF38393, both the prefrontal cortex (PFC) and striatum exhibited a significant increase in downstream signalling, indicating hyperactivation. Furthermore, during the developmental stages, abnormalities were observed in the staining patterns of CD1 and Tbr2 in both ERK1+/- and 16p11.2+/- mice, suggesting a similar regulation of ERK signalling during embryonic development. Overall, this analysis provides preliminary evidence of cellular and signalling deficits present in mouse models with the 16p11.2 CNV. These findings may have broader implications for future studies involving behavioural rescue experiments and the development of potential therapeutics in preclinical settings.È consentito all'utente scaricare e condividere i documenti disponibili a testo pieno in UNITESI UNIPV nel rispetto della licenza Creative Commons del tipo CC BY NC ND.
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https://hdl.handle.net/20.500.14239/16065