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Zhang et al. Ageing Neur Dis 2023;3:24 https://dx.doi.org/10.20517/and.2023.18 Page 3 of 13

the magnitude of the deletion is positively associated with the severity of clinical manifestations [17,26-28] .
Additionally, individuals with smaller terminal deletions may exhibit more favorable developmental
trajectories compared to those with larger deletions. The size of SHANK3 deletion fragments also correlates
with disease severity.

Similarly, a cohort study conducted on a Brazilian population revealed a positive correlation between the
size of SHANK3 deletion and the severity of renal abnormalities, lymphedema, and language impairment in
PMDS patients . Another cohort study from a Chinese population reported additional significant
[29]
phenotypes, including heightened pain tolerance, impulsivity, repetitive behaviors, regression, and incessant
crying . Furthermore, imaging techniques have been extensively utilized to identify specific characteristics
[30]
[31]
and structural brain abnormalities in ASD patients . From a pathological perspective, young children
diagnosed with ASD exhibit a notable decrease in neuronal and cytoplasmic volumes in the majority of
regions examined compared to controls of the same age . Conversely, a separate study revealed a 67%
[32]
increase in the number of neurons in the entire prefrontal cortex (PFC) among male individuals with
autism . However, this datum is estimated to be the volume of reference for the regions of interest in order
[33]
to derive total cell counts. It is important to exercise caution in interpreting these findings. The pathological
and imaging reports of individuals with ASD commonly exhibit limitations such as small sample sizes and
variations in the statistical methodologies employed. Therefore, it is crucial to establish a suitable animal
model with SHANK3 mutations in order to effectively simulate ASD and investigate its pathogenesis.

SMALL ANIMAL MODELS WITH SHANK3 MUTATIONS
Various small animal models carrying shank/shank3 mutations, such as drosophila, zebrafish, mice, and
rats, have been generated to extensively investigate the molecular phenotypes related to shank3 mutations.
Due to their high efficiency in genetic manipulations and rapid generation, drosophila and zebrafish have
proven to be valuable tools for investigating the function of shank3. In one study, shank null drosophila
models were found to be fully viable and fertile, showing no apparent morphological or developmental
defects. However, these models did exhibit an abnormal structure of the central mushroom body calyx and
reduced olfactory acuity . Another shank null drosophila model demonstrated defects in synaptic bouton
[34]
number and maturation .
[35]
Similar synaptic defects related to shank3 deficiency were observed in shank3 null zebrafish models. The
adult brains of shank3b deficient zebrafish showed significantly reduced levels of both postsynaptic homer1
and presynaptic synaptophysin. These reductions were accompanied by a decrease in locomotor activity,
impaired social interaction, and significant repetitive swimming behaviors . Another shank3b mutant
[36]
zebrafish model also exhibited reductions in synaptic proteins and locomotor activity . Furthermore,
[37]
reduced sensory responsiveness in shank3ab zebrafish was associated with decreased activity in sensory
-/-
processing brain regions . In zebrafish, CRISPR-Cas9-mediated mutations in the shank3a gene led to the
[38]
downregulated expression of neuroligins, which are crucial for synaptogenesis. This downregulation could
contribute to deficits in attention .
[39]
It is important to acknowledge that zebrafish and drosophila are evolutionarily distant from humans; the
expression of shank3 in these small animals is distinct from that in mammals. For example, the zebrafish
gene corresponding to human SHANK3 exists as two duplicates. A number of mouse models carrying
shank3 mutations have been established and extensively investigated. The mouse model with exon 21
deletion displays a reduced N-methyl-D-aspartate/alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic
acid (NMDA/AMPA) ratio in the hippocampal synapses, resulting in a significant rise in mGluR5
localization to synaptosomes, motor-coordination deficits, abnormal behaviors, and social interaction .
[40]