SHANK2 (ProSAP1) is a postsynaptic scaffolding protein of excitatory synapses in the central nervous system and implicated in the development of autism spectrum disorders (ASD). Patients with mutations in SHANK2 show autism-like behaviors, developmental delay, and intellectual disability. We generated human induced pluripotent stem cells (hiPSC) from a patient carrying a heterozygous deletion of SHANK2 and from the unaffected parents. In patient hiPSCs and derived neurons SHANK2 mRNA and protein expression was reduced. During neuronal maturation, a reduction in growth cone size and a transient increase in neuronal soma size were observed. Neuronal proliferation was increased, and apoptosis was decreased in young and mature neurons. Additionally, mature patient hiPSC-derived neurons showed dysregulated excitatory signaling and a decrease of a broad range of signaling molecules of the ERK-MAP kinase pathway. These findings could be confirmed in brain samples from Shank2(−/−) mice, which also showed decreased mGluR5 and phospho-ERK1/2 expression. Our study broadens the current knowledge of SHANK2-related ASD. We highlight the importance of excitatory-inhibitory balance and mGluR5 dysregulation with disturbed downstream ERK1/2 signaling in ASD, which provides possible future therapeutic strategies for SHANK2-related ASD.

Mutations of the postsynaptic scaffold protein Shank2 lead to autism spectrum disorders (ASD). These patients frequently suffer from higher fracture risk. Here, we investigated whether Shank2 directly regulates bone mass. We show that Shank2 is expressed in bone and that Shank2 levels are increased during osteoblastogenesis. Knockdown of Shank2 by siRNA targeting the encoding regions for PDZ and SAM domain inhibits osteoblastogenesis of primary murine calvarial osteoblasts. Shank2 knockout mice (Shank2 À/À ) have a decreased bone mass due to reduced osteoblastogenesis and bone formation, whereas bone resorption remains unaffected. Induced pluripotent stem cells (iPSCs)-derived osteoblasts from a loss-of-function Shank2 mutation in a patient showed a significantly reduced osteoblast differentiation potential. Moreover, silencing of known Shank2 interacting proteins revealed that a majority of them promote osteoblast differentiation. From this we conclude that Shank2 and interacting proteins known from the central nervous system are decisive regulators in osteoblast differentiation.

SHANK3 is a member of the SHANK family of scaffolding proteins that localize to the postsynaptic density of excitatory synapses. Mutations within the SHANK3 gene or SHANK3 haploinsufficiency is thought to be one of the major causes for Phelan-McDermid Syndrome (PMDS) that is characterized by a broad spectrum of autism-related behavioral alterations. Several approaches have already been proposed to elevate SHANK3 protein levels in PMDS patients like transcriptional activation or inhibition of SHANK3 degradation. We undertook a systematic screening approach and tested whether defined antisense oligonucleotides (ASOs) directed against the 3′ untranslated region (3′-UTR) of the human SHANK3 mRNA are suitable to elevate SHANK3 protein levels. Using human induced pluripotent stem cells (hiPSCs) and hiPSCs-derived motoneurons from controls and PMDS patients we eventually identified two 18 nucleotide ASOs (ASO 4-5.2-4 and 4-5.2-6) that were able to increase SHANK3 protein levels in vitro by about 1.3- to 1.6-fold. These findings were confirmed by co-transfection of the identified ASOs with a GFP-SHANK3-3′-UTR construct in HEK293T cells using GFP protein expression as read-out. Based on these results we propose a novel approach to elevate SHANK3 protein concentrations by 3′-UTR specific ASOs. Further research is needed to test the suitability of SHANK3 -specific ASOs as pharmacological compounds also in vivo .

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