Uncovering the Functional Link Between SHANK3 Deletions and Deficiency in Neurodevelopment Using iPSC-Derived Human Neurons

Abstract

SHANK3 mutations, including de novo deletions, have been associated with autism spectrum disorders (ASD). However, the effects of SHANK3 loss of function on neurodevelopment remain poorly understood. Here we generated human induced pluripotent stem cells (iPSC) in vitro, followed by neuro-differentiation and lentivirus-mediated shRNA expression to evaluate how SHANK3 knockdown affects the in vitro neurodevelopmental process at multiple time points (up to 4 weeks). We found that SHANK3 knockdown impaired both early stage of neuronal development and mature neuronal function, as demonstrated by a reduction in neuronal soma size, growth cone area, neurite length and branch numbers. Notably, electrophysiology analyses showed defects in excitatory and inhibitory synaptic transmission. Furthermore, transcriptome analyses revealed that multiple biological pathways related to neuron projection, motility and regulation of neurogenesis were disrupted in cells with SHANK3 knockdown. In conclusion, utilizing a human iPSC-based neural induction model, this study presented combined morphological, electrophysiological and transcription evidence that support that SHANK3 as an intrinsic, cell autonomous factor that controls cellular function development in human neurons.

Transcriptional signatures of participant-derived neural progenitor cells and neurons implicate altered Wnt signaling in Phelan McDermid syndrome and autism

“…The transcriptional signatures of PMS in iNeurons point to altered postsynaptic density, glutamatergic synaptic and GABAergic genes. Our results are in line with evidence supporting a role for SHANK3 prior to synaptogenesis and neural circuit formation, specifically in early morphogenesis and excitatory/ inhibitory balance [32, 65, 71–74]. For example, a zebrafish model of PMS that utilized morpholinos to disrupt shank3a and shank3b resulted in delayed mid- and hindbrain development, disruptions in motor behaviors, and seizure-like behaviors [73].…”

“…We also included genes that are direct targets of FMRP [64]. For the gene sets from other iPSC transcriptome studies, we curated previously described differentially expressed genes caused by: i) shRNA KD of SHANK3 in hiPSC-derived neurons [65]; ii) CRISPR/Cas9 heterozygous KO of CHD8 in hiPSC-derived NPCs and neurons [66]; iii) shRNA KD of TCF4 and EHMT1 in hiPSC-derived NPCs [67]; iv) shRNA KD of MBD5 and SATB2 in human neural stem cells [68]; v) shRNA KD of NRXN1 in human neural stem cells [69]; and vi) CRISPR/Cas9 heterozygous and homozygous KO of ten different ASD-related genes in iPSCs and iPSC-derived neurons [70]. Full gene lists are provided in Supplemental Table 4 .…”

Human in vitro models for understanding mechanisms of autism spectrum disorder

“…Two kinds of genetic modeling have been performed using cells either from individuals whose genetic contributions are unknown or undefined, so called idiopathic [59,60,[112][113][114][115][116][117][118][119][120], or from individuals harboring major effect mutations that are presumed causal or which have been engineered to carry these mutations. These mutations include ASD-associated CNVs such as 15q11q13 deletion (Angelman syndrome) [121] and duplication (Dup15q syndrome) [122], 22q11.2 deletion (DiGeorge syndrome) [123,124], 16p11.2 deletion and duplication [125], and 15q13.3 deletion [126], as well as single-gene mutations including SHANK3 [127][128][129][130], CHD8 [131,132], NRXN1 [133][134][135][136][137], NLGN4 [138], EHMT1 (Kleefstra syndrome) [139], PTCHD1-AS [140], UBE3A (Angelman's syndrome) [141], and CACNA1C (Timothy syndrome) [142] (summarized in Table 1). In this review, we will not discuss fragile X syndrome, Rett's syndrome, and tuberous sclerosis-related autism as they have all been extensively reviewed previously [148][149][150][151][152][153][154].…”

Probing disrupted neurodevelopment in autism using human stem cell‐derived neurons and organoids: An outlook into future diagnostics and drug development

“…Surprisingly, SHANK3 knockdown significantly altered neurite complexity in excitatory, but not inhibitory or dopamine neurons. 283 Intriguingly, in contrast to SHANK3, SHANK2-deficient iPSC-derived neurons obtained from individuals with ASD showed increased excitatory synaptic transmission and more elaborated dendritic arbors as compared to control neurons. 95 This suggests that downand upregulated morphogenesis and synaptogenesis could be associated with SHANK-related abnormalities and ASD in human neurons.…”

“…282 Gouder et al 263 generated iPSC-derived neurons from four patients with ASD and SHANK3 mutations, and identified abnormal dendritic spine morphology and spine density which correlated with SHANK3 mRNA levels. 263 Huang et al 283 performed shRNA-mediated SHANK3 knockdown in human iPSCs during neural differentiation, and discovered that SHANK3 knockdown led to significantly decreased dendritic lengths, neuron soma size, and excitatory and inhibitory synaptic transmission. Surprisingly, SHANK3 knockdown significantly altered neurite complexity in excitatory, but not inhibitory or dopamine neurons.…”