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Zhang et al. Ageing Neur Dis 2023;3:24 https://dx.doi.org/10.20517/and.2023.18 Page 9 of 13

[52]
heterozygous SHANK3-mutant monkeys to ensure germline transmission of the SHANK3 mutations .
The behavioral assessment scores showed that overall sleep efficiency and muscle strength were reduced in
SHANK3-mutant monkeys. However, the stereotyped or repetitive behaviors (such as licking fingers and
cage bars) revealed a substantial increase compared to controls. Meanwhile, SHANK3-mutant monkeys
displayed reduced levels of exploration, social interaction, and vocalization, which parallels some of the
phenotypes found in children with ASD or PMDS [61,62] . Contrary to the previous SHANK3-targeted monkey
model , structural MRI analysis of the new SHANK3-mutant monkeys revealed that the volume of gray
[57]
matter had decreased. In addition, reduced global connectivity and greater local connectivity were also
observed in the new SHANK3 mutant monkeys.

Although there are small differences in behavioral phenotypes between the two SHANK3-targeted monkey
models, most of the behavioral alterations in SHANK3-targeted monkeys were similar to the clinical
[51]
characteristics of autism patients. However, unlike monkeys with a large SHANK3 gene deletion , the
[52]
monkeys with exon 21 targeting were not reported to have neuronal loss . Thus, it is likely that complete
loss of SHANK3 can more severely affect neuronal survival and development, whereas partial loss of
SHANK3 or depletion of some isoforms of SHANK3 may lead to haploinsufficiency as seen in heterozygous
SHANK3 mutations in humans.

Behavioral phenotypes of SHANK3 mutant monkey models highlight the complex regulation of SHANK3
expression and its multifaceted functions in large animals. Thus, large animals with SHANK3 mutations
may provide unique pathogenesis insights that have not been identified in small animals. Indeed, dog
models with SHANK3 mutations were also created and provided some interesting findings. The
electrocorticograms (ECoG) revealed that SHANK3 mutant dogs responded to sound stimulation more
intensely and were more sensitive to frequencies (1-4 kHz) that are sensitive to the human auditory
system . Stem cells from human exfoliated deciduous teeth transplantation improved social novelty
[63]
preference, reduced social stress, and decreased serum IF-γ levels in heterozygous SHANK3 mutant beagle
dogs . Considering that non-human primates are closer to humans than other animals, SHANK3-mutant
[64]
monkeys would be a more faithful animal model for investigating the pathogenesis of human ASD.

SPECIES-SPECIFIC NEURODEGENERATION
In heterozygous shank3 knockout mice, there are no or only very mild phenotypes, which is in contrast to
+/-
the more severe clinical symptoms observed in patients with SHANK3 heterozygous mutations. Abnormal
behavioral phenotypes in mice are almost exclusively found in homozygous mutants [44,47,48] . However,
homozygous SHANK3 mutations have not been reported in humans, possibly because such mutations are
embryonic lethal or can severely affect early brain development. In support of this idea, a large deletion of
[51]
the monkey SHANK3 gene can cause remarkable neuronal loss . The absence of such neuronal loss in
complete shank3 knockout mice clearly indicates that species-dependent differences are important for the
development of neurodegeneration seen in humans.

Analogous to other neurodegenerative diseases such as Alzheimer’s, Parkinson’s, and Huntington’s diseases,
genetically modified mouse models fail to recapitulate the overt and selective neurodegeneration observed
[65]
in patient brains . For example, homozygous mutations in PINK1, a kinase that is thought to be involved
in mitophagy, can cause neurodegeneration in Parkinson’s disease via the loss of function mechanism.
However, the complete loss of Pink1 in knockout mice does not lead to neurodegeneration or obvious
phenotypes [66-68] . In contrast, CRISPR/Cas9-mediated deletion of the PINK1 gene in monkeys results in
severe neurodegeneration . Furthermore, this primate-specific neurodegeneration caused by PINK1
[69]
deficiency is due to reduced protein phosphorylation rather than mitochondrial homeostasis defects .
[65]