By Geraldine Bliss,   Director, Phelan-McDermid Syndrome Foundation 

July 28, 2017

Over the last several years there have been many important discoveries about PMS resulting from mice, rats, zebrafish, and fruit flies.  Those animal models will continue to have great importance as the scientific community increases efforts towards drug development, however, evolutionary differences from humans mean there are some limitations in how they can be used to understand human disease.  This week the first scientific paper reporting a SHANK3-deficient non-human primate was published.  Below is a lay summary of the paper.  Dr. Yong-hui Jiang, one of the lead authors, reviewed the summary and kindly answered a few questions for PMSF.

Who did the work?

Several research teams from China, Duke, and Emory worked together.  Dr. Yong-hui Jiang, who has long been involved with PMSF, has participated in PMSF meetings, and developed the complete SHANK3 mouse model that PMSF funded, is one of the lead authors.

Why are non-human primates needed? 

There have been 14 independent lines of Shank3 mutant mice, which have been helpful for understanding the underlying molecular and cellular mechanisms of ASD.  However, there are evolutionary differences in brain structures and behaviors between mice and humans, which limit the uses of mouse models for understanding human behavior.  For that reason, there has been great interest in developing non-human primate models of neurodevelopmental disorders (NDD).  So far, the only other ASD/NDD-associated primate models that have been developed have been for a gene called MECP2, associated with Rett Syndrome.  This paper reports the first SHANK3-deficient primate model.

How did they make SHANK3-deficient monkeys?

The team used CRISPR/Cas9 to disrupt the SHANK3 gene in cynomolgus monkeys (a type of macaque that has sophisticated social and adaptive behaviors).  Because SHANK3 is a large gene with multiple intragenic promoters, the team targeted two sites in SHANK3, exons 6 and 12, to induce a large deletion that would disrupt all SHANK3 isoforms.

Pregnancies

Out of 116 embryos that were transferred to 37 surrogate mothers, only three survived.  The pregnancy rate of 8.1% was lower than was expected, and the authors speculate that SHANK3 expression might be important for early (prenatal) development in primates.

Offspring

One of the offspring, a female, (M1) was lost as a result of a late miscarriage.  One of the offspring, a male, (M2) was lost during a difficult labor.  The third offspring, a male, (M3) is still alive.  There were no apparent problems of fetal development in M1 or M2 that explained their deaths.

The offspring had different types of mutations in the region between exons 6 and 12.  The team confirmed the mutations through sequencing and semi-quantitative PCR.  No off-target mutations were revealed.

Expression of SHANK3

To determine if the offspring did, indeed, have a deficiency of SHANK3, the team examined somatic (non-brain) tissue from all three babies and brain tissue from M1 and M2.  SHANK3 is expressed in different parts of the brain at different times of development.  In mice its peak expression occurs in the striatum at day 14, and in the monkeys, the peak expression occurs in the prefrontal cortex at birth, a developmental time point that corresponds to day 14 in mice.

When the team looked at the expression of SHANK3 isoforms (different “flavors” of SHANK3 that are normally expressed), none were detected in the cortex of M1, which indicated a complete loss of SHANK3 there, but when they looked at the striatum, there was a short isoform still present.

In M2, the full length SHANK3 isoform was decreased in the striatum, but in the cortex, the majority of SHANK3 isoforms were mildly increased.  The authors postulate the increase in some isoforms in the cortex was through some compensatory mechanism.

The authors note that the variability of SHANK3 expression in the brain could be the result of isoform-specific expression of SHANK3 in different brain regions or because of the mosaic nature of CRISPR/Cas9 gene editing.

Analysis of other postsynaptic proteins

Studies of other animal models have shown that when SHANK3 is disrupted, the proteins that interact with SHANK3 also become disrupted.  In the monkeys, there were decreases in mGLuR5, GluN2B and PSD95 in the pre-frontal cortex (this is important, because it can suggest potential new drug targets).  The researchers did not see changes in the levels of GluN1, GluA2, Homer1b/c, or pan-Homer in the pre-frontal cortex but observed similar accumulation of Homer1b/c as shown in Shank3 mutant mice, which is important because it indicates that the molecular mechanism may be conserved between rodent and non-human primates.

Neuronal markers

The researchers looked more closely at the pre-frontal cortex of M1.  They found that the cell bodies were smaller and there were fewer NeuN+ neurons.  There was also an increase in GFAP+ astrocytes.  These findings have not been reported in mice, which suggests a unique role for SHANK3 in the early development of non-human primates. (In M2, where there was no change in SHANK3 expression in the prefrontal cortex, there were also no histological changes.)

Dendritic spine changes

M1, but not M2, had reduced dendritic spine density, a finding that has been reported in mice.  The dendritic spine length was normal.

Questions & Answers with Dr. Jiang

Q:  Dr. Jiang, what happens next?

A:  We will continue monitoring the M3 animal at the behavioral level

Q:  Will you grow a colony of SHANK3-deficient monkeys?  How long will it take?

A:  We are planning to grow the colony of M3 and produce the next generation which may take 3-5 years or produce more new mutant animals by performing additional injections. These will depend on the resources.

Q:  Will you start behavioral tests of M3 soon?

A:  We are closely monitoring the behavior of the M3 animal

Q:  What is the potential of using primate models for drug screening?  What is the approximate timeline for that?

A:  The differences in the expression of Shank3 in mice and monkey support the value of non-human primate model.   The Shank3 monkey model will be valuable for the future drug screening and trials. It may take a longer time to develop a paradigm.

Link to the full publication:
Zhao, H. et al. Altered neurogenesis and disrupted expression of synaptic proteins in prefrontal cortex of SHANK3-deficient non-human primate. Cell Res (2017).  PubMed