Autism patient-derived SHANK2BY29X mutation affects the development of ALDH1A1 negative dopamine neuron.

Autism spectrum disorder (ASD) encompasses a range of neurodevelopmental conditions. Different mutations on a single ASD gene contribute to heterogeneity of disease phenotypes, possibly due to functional diversity of generated isoforms. SHANK2, a causative gene in ASD, demonstrates this phenomenon, but there is a scarcity of tools for studying endogenous SHANK2 proteins in an isoform-specific manner. Here, we report a point mutation on SHANK2, which is found in a patient with autism, located on exon of the SHANK2B transcript variant (NM_133266.5), hereby SHANK2BY29X. This mutation results in an early stop codon and an aberrant splicing event that impacts SHANK2 transcript variants distinctly. Induced pluripotent stem cells (iPSCs) carrying this mutation, from the patient or isogenic editing, fail to differentiate into functional dopamine (DA) neurons, which can be rescued by genetic correction. Available SMART-Seq single-cell data from human midbrain reveals the abundance of SHANK2B transcript in the ALDH1A1 negative DA neurons. We then show that SHANK2BY29X mutation primarily affects SHANK2B expression and ALDH1A1 negative DA neurons in vitro during early neuronal developmental stage. Mice knocked in with the identical mutation exhibit autistic-like behavior, decreased occupancy of ALDH1A1 negative DA neurons and decreased dopamine release in ventral tegmental area (VTA). Our study provides novel insights on a SHANK2 mutation derived from autism patient and highlights SHANK2B significance in ALDH1A1 negative DA neuron.

Genetic analysis of a SARS-CoV-2 Omicron variant from a Chinese traveller returning from overseas.

Since the SARS-CoV-2 Omicron variant was first reported from South Africa, it has rapidly spread in over 100 countries. Only two cases infected by the Omicron variant were recently identified in China. The one case in Guangzhou has a relatively long incubation time and mild symptoms. Analysis of the complete viral genome sequence shows three missing Omicron unique mutations and one additional mutation in the newly characterized genome. These unique mutations may be related to the clinical presentation in this case.

Insulin-like growth factor 1 partially rescues early developmental defects caused by SHANK2 knockdown in human neurons.

SHANK2 is a scaffold protein that serves as a protein anchor at the postsynaptic density in neurons. Genetic variants of SHANK2 are strongly associated with synaptic dysfunction and the pathophysiology of autism spectrum disorder. Recent studies indicate that early neuronal developmental defects play a role in the pathogenesis of autism spectrum disorder, and that insulin-like growth factor 1 has a positive effect on neurite development. To investigate the effects of SHANK2 knockdown on early neuronal development, we generated a sparse culture system using human induced pluripotent stem cells, which then differentiated into neural progenitor cells after 3-14 days in culture, and which were dissociated into single neurons. Neurons in the experimental group were infected with shSHANK2 lentivirus carrying a red fluorescent protein reporter (shSHANK2 group). Control neurons were infected with scrambled shControl lentivirus carrying a red fluorescent protein reporter (shControl group). Neuronal somata and neurites were reconstructed based on the lentiviral red fluorescent protein signal. Developmental dendritic and motility changes in VGLUT1+ glutamatergic neurons and TH+ dopaminergic neurons were then evaluated in both groups. Compared with shControl VGLUT1+ neurons, the dendritic length and arborizations of shSHANK2 VGLUT1+ neurons were shorter and fewer, while cell soma speed was higher. Furthermore, dendritic length and arborization were significantly increased after insulin-like growth factor 1 treatment of shSHANK2 neurons, while cell soma speed remained unaffected. These results suggest that insulin-like growth factor 1 can rescue morphological defects, but not the change in neuronal motility. Collectively, our findings demonstrate that SHANK2 deficiency perturbs early neuronal development, and that IGF1 can partially rescue the neuronal defects caused by SHANK2 knockdown. All experimental procedures and protocols were approved by the Laboratory Animal Ethics Committee of Jinan University, China (approval No. 20170228010) on February 28, 2017.

Inhibitor of DNA binding 2 promotes axonal growth through upregulation of Neurogenin2.

Manipulation of developmentally regulated genes presents a promising strategy to enhance the intrinsic growth capability of adult neurons. Inhibitor of DNA binding 2 (Id2), a negative regulator of bHLH transcriptional factors, promotes axonal growth after its forced expression in post-mitotic neurons. Neurogenin2 (Ngn2) is a neural specific bHLH factor which controls neuronal fate and drives neuronal differentiation during development. In this study, we investigated the mechanism of Id2 in promoting axonal growth and revealed that Ngn2 contributed to the growth-activating role of Id2 in neurons. Ngn2 expression was upregulated with increased Id2 activity by assessing RNA and protein levels. Forced expression of Id2 or Ngn2 in cortical neurons significantly promoted axonal growth with little effect on dendrites. Furthermore, knockdown of Ngn2 impaired the axonal growth promoting effect of Id2, implying that the effect of Id2 on axonal growth depends on Ngn2. These findings suggest that elevation of neuronal Ngn2 may be a new therapeutic strategy to stimulate axonal regeneration.

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

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.