Mechanisms of Brain Function in mTOR-Deficient Intellectual Disability Syndromes (mTOR-DIDS)

MID1 and alpha4 trigger the ubiquitin-dependent degradation of the microtubules-associated catalytic subunit of protein phosphatase 2A (PP2Ac) an important counteractor of many kinases including the translational master regulator mTOR. The MID1 complex is an mRNP and has been reported to move along microtubules in neurons and this movement is regulated by PP2A. The mTOR inhibitor rapamycin can disrupt the MID1 complex and lack of MID1 as observed in Opitz BBB/G syndrome (OS, midline defects like hypospadias, cleft lip/palate and mild mental disabilities) patients leads to accumulation of PP2Ac on microtubules. These increased levels of PP2Ac attenuate the formation of mTORC1 resulting in compromised mTOR signaling and hence translation. Furthermore, it was shown, that MID1 is a sequence-specific translational regulator controlling the translation of PDPK1 an important upstream regulator of mTOR (1). Besides OS, other diseases presenting with mental disabilities, like Rett syndrome (caused by Cdkl5 and MeCP2 deficiencies) also show compromized mTOR signaling, whereas in Downs and fragile X syndrome mTOR activity is enhanced (2). In neurons, mTOR is controlling cell size, axon guidance/regeneration and dendritic morphology, most probably by regulating the local translation of specific neuronal proteins. We want to study the local proteome and transcriptome in the synapses of hippocampus and cortex from mouse models for mental retardation syndromes,that have been correlated with altered mTOR signaling (available from the other teams). We want to identify specific synaptosomal proteins, the local translation of which is regulated in an mTOR dependent manner and which are involved in mental disability phenotypes. This will be achieved (i) by deep sequencing of RNAs of the synaptosome of various brain regions (e.g. cortex, hippocampus) in the mouse models versus controls, (ii) by qualitative and quantitative analysis of the synaptosomal proteome and phosphoproteome in the mouse models versus wild-type using TMT-based quantitative mass spectroscopy approach and (iii) through analysis of the role of synaptic mTOR targets in brain function (in close cooperation with the other teams). To decipher the proteome and transcriptome of the synapse, we will isolate synaptosomes from the brain regions affected in the studied syndromes, isolate synaptosomal RNA and proteins and analyze them in the omics platforms available in Berlin and Innsbruck, respectively. This should allow us to correlate the mRNA levels with the respective protein levels in a syndrome-dependent manner and enable us to identify disease relevant synaptosomal mTOR targets. These candidate targets will then be confirmed by independent methods and further studied concerning their biochemical functions and their potential roles in plasticity, learning and memory. These data will be fundamental for the differential diagnosis of ID and in the future may also open new avenues for the development of novel therapeutic strategies for patients with intellectual disability syndromes and other related disorders.

Altered activity of the mTOR kinase has been demonstrated to be an important pathobiochemical feature in several human genetic syndromes that are characterized by cognitive impairment. In this project we wanted to study the influence of mTOR-activity on the local proteome and RNome in synapses using mouse models of such syndromes to get insight into the mechanisms leading to intellectual disabilities. The joint European research consortium could provide us with brain samples of mice with Tuberous sclerosis (TSC2) and CDKL5 deficiency (CD), intellectual disability syndromes known to be associated with increased and decreased mTOR activity, respectively. After establishing a reliable method to isolate the proteome and RNome of synapses and cytosol from frozen brain samples we analysed their composition using quantitative mass spectrometry and deep-sequencing of RNA. Our protocol allowed us to identify about 1200 proteins in TSC2 synaptosomes, several of which differed significantly in their levels between wild-type and mutants. While many of these differences are in line with published data, we can also see a regulation of proteins not yet associated with TSC2 but with functions in neuronal differentiation and transmission. A pathway analysis of proteins with reduced levels in the mutants revealed their involvement in neurotransmitter metabolism. The found differences in protein levels were not high (up to 50%), but this is not unexpected because the respective mouse mutant is only heterozygous, thus one of the TSC2-genes is still intact. Specifically, we could in fact see that the levels of TSC2-protein in the synaptosomes were, probably due to a counter- regulation, identical between mutant and wild type, despite the respective RNA being only present to 50% in the mutant. However, when the mice were subjected to behavioural tests including social stress, we strikingly found the most pronounced differences between mutant and wildtype, not only in protein levels, but also in RNA levels. It turned out that the unstressed mutants show the same patterns of protein- and RNA-levels as do stressed mice. This finding suggests that the neurons in unstressed mutants are in a constant stressed state and that the stress response in the context of mTOR signaling is significantly altered in the mutants. Furthermore we found that there are gross age-dependent differences in the synaptosomal protein levels between wild-type and mutant, which is in line with the observed age-dependent aggrevation of the phenotype in the human patients suffering from Tuberous sclerosis. This age-related outcome of the project also led to a delay in the analyses because the breeding and treatment of respective mice afforded much more time than originally planned. Unfortunately, the differences in the CDKL5-deficient mice were not as pronounced and deserve further analyses to extract significant results. In summary, we were able to identify significant and informative differences in the synaptosomal proteins and RNAs in TSC2 mutants, especially in the context of stress and age. This is an important finding as it could in part explain the unusual broad phenotype variability found with this syndrome.