Osterweil, Emily

Kind, Peter

Muscas, Melania

2020-04-01T12:19:48Z

2020-04-01T12:19:48Z

2019-12-06

Fragile X syndrome (FXS) is the most common heritable monogenic cause of intellectual disability (ID) and autism. Along with physical dysmorphic features, it is characterized by moderate to severe mental retardation, epilepsy and hyperactivity, learning disabilities, memory deficits and impairment in communication. Due to the prevalence of FXS in the general population and thanks to the availability of genetically engineered animal models, this disorder has been extensively studied for several decades and numerous treatment strategies evaluated. However, there are currently no targeted treatments and further research is essential. FXS arises from loss of the FMRP protein, a negative regulator of protein synthesis which is highly expressed in neuronal cell bodies and synapses. FMRP regulates the translation of mRNAs downstream of group I metabotropic glutamate receptors (mGluRs). With its loss, the protein-synthesis dependent long-term effects of Gp1 mGluR activity are significantly exacerbated in the hippocampus of FXS mouse and rat models (Fmr1 KO). Thus, the mGluR theory of FXS proposes the syndrome pathology arises from abnormally augmented responses to mGluR-driven protein synthesis. In support of this theory, inhibition of Gp1 mGluRs, in particular mGluR5, can rescue multiple deleterious Fmr1 KO phenotypes. One of the signalling pathways linking Gp1 mGluRs to translation is the extracellular signal-regulated kinase (ERK1/2) pathway. ERK1/2 is a major regulator of protein synthesis and its inhibition has been proven beneficial for all major phenotypes of the Fmr1 KO rodent models. This illustrates the promise of targeting over-translation for restoring function in FXS. This dissertation will describe work testing two potential treatment strategies following the idea that FXS can be ameliorated by diminishing the overtranslation of proteins resulting from the loss of FMRP, however not necessarily by targeting the ERK1/2 pathway. The two compounds are the statin simvastatin and the muscarinic receptor 4 positive allosteric modulator VU0152100 (M4 PAM). In the first part of this thesis, the investigation of statin drugs will be described. These compounds include lovastatin, which targets the mevalonate pathway upstream cholesterol synthesis, and consequently reduces the activation of the GTPase Ras that activates ERK1/2. Preliminary studies with lovastatin in rodents and humans have been encouraging, and new clinical trials with lovastatin are being considered. With respect to this, the newer statin simvastatin has been proposed as a better alternative due to its higher penetration to the brain, stronger effect on its target cholesterol, and larger availability for medical prescription worldwide. The impact of simvastatin versus lovastatin was tested using biochemical measures of protein synthesis and signalling pathway activation in acute brain slices, and behavioural assays for audiogenic seizures. Unfortunately, results from this work suggest that simvastatin is ineffective at rescuing any of the Fmr1 KO phenotypes tested. This might be due to results showing that, in contrast to lovastatin, simvastatin does not target the ERK1/2 pathway. This work was published in Muscas et al., 2019. The second part of this thesis describes behavioural experiments in Fmr1 KO mouse and rat models testing the potential of M4 PAM as a therapeutic option for FXS. Recent work shows that M4 is significantly overtranslated and overexpressed in the Fmr1 KO hippocampus, and modulating it using an M4 PAM corrects the most robust biochemical and physiological phenotypes of the Fmr1 KO mouse, including excessive protein synthesis. Thus, the experiments detailed in this project assess the effects of M4 PAM treatment on the seizure phenotype in juvenile Fmr1 KO mice and a cognitive phenotype for novelty recognition in adult Fmr1 KO rats, in both cases providing evidence that positive allosteric modulation of M4 is an effective strategy. Some of this work was published in Thomson et al., 2017. In the last part of this work, we tried to uncover the mechanisms by which this M4 PAM ameliorates FXS phenotypes. However, more research is yet to be conducted for answering to this question. Together, the results described in this dissertation provide further evidence that targeting excessive translation is a valid strategy for treating FXS.

https://hdl.handle.net/1842/36928

http://dx.doi.org/10.7488/era/229

en

The University of Edinburgh

Muscas, M., Louros, S.R. & Osterweil, E.K., 2019. Lovastatin, not simvastatin, corrects core phenotypes in the fragile X mouse model. eNeuro, 6(June), p.430348. Available at: https://www.biorxiv.org/content/early/2018/09/29/430348?rss=1

Fragile X syndrome

protein synthesis regulatory pathways

signalling cascades

FXS

elevated translation

FMRP protein

ERK1/2

VU0152100

M4 PAM

Investigating potential treatment strategies and underlying mechanisms in the Fragile X Syndrome mouse and rat models

Thesis or Dissertation

Doctoral

PhD Doctor of Philosophy