Intestinal dysmotility in a

zebrafish (Danio rerio)

shank3a; shank3b mutant
model of autism
DAVID M. JAMES, ROBERT A. KOZOL, YUJI KAJIWARA, ADAM L. WAHL, EMILY C.
STORRS, JOSEPH D. BUXBAUM, MASON KLEIN, BAHARAK MOSHIREE AND JULIA E.
DALLMAN

PRESENTED BY CAMRON DAVIES

Aims
INVESTIGATE THE PHYSIOLOGICAL BASIS OF GI
DISTRESS IN ASD
Background
 Autism Spectrum Disorder (ASD)
 GI distress: one of the most frequent co-morbidities experienced with ASD
 Phelan-McDermid Syndrome
 Monogenic subset of ASD
 22q13 deletion syndrome
 Global developmental delay, intellectual disability, speech abnormalities, ASD-like
behaviors, hypotonia and dysmorphic features
 GI symptoms characterized by reflux, vomiting, diarrhea and constipation
 Many genes are mutated but there is a focus on SHANK3
Background: SHANK3

 Cytoskeletal protein in the neurons, forms the

postsynaptic density (PSD)
 PSD an organized concentration of receptors in the
synaptic cleft
 Role in synapse formation and dendritic spine
maturation
 Expressed in enterocytes and nitrergic neurons
 Shown to play a role in in host/symbiont interactions
and intestinal barrier function
Background: SHANK3
Background: Zebrafish
• Genetically and physiologically
similar to humans and
mammalian models
• Similar hormonal regulation,
morphology, cell types, and
physiology, albeit simplified in
zebrafish
• Developmental stages are
transparent allowing for systems
level analysis
Validating Zebrafish & Generating
SHANK3 Knockout
 Used CRISPER/CAS9 to produce
frameshifts in shank3a and b
 Similar frameshift mutations have been
associated with ASD
 Localization to PSD and isoform
complexity of shank3 was confirmed
via western blot
 Zebrafish shank3 isoform complexity
was comparable to mouse
 Knockout displayed no visible bands
Peristalsis Frequency & Transit Time

 Shank3abΔC larvae have less frequent peristaltic contractions throughout intestine

compared to WT larvae
 Differences between heterozygous and homozygous shank3abΔC mutants were not
found to be significant
 Suggests a minimum concentration of SHANK3 needed for function
 Total DT transit time was also increased
 Indicates severely delayed but not entirely obstructed transit
 Intestinal sloshing in mutant
 Delays in two key areas: the intestinal bulb to upper-intestine transition and near the
anus, both are regions where anterograde and retrograde propulsion transition to just
retrograde propulsion
Shank3abΔC +/- Rescue

 Embryos injected with long and short isoform of human SHANK3 mRNA
 mRNA needed to be injected at the one cell stage due to its representation in diverse
tissues
 Short form did not rescue transit time
 Long form SHANK3 did partially rescue DT transit and restored anterior-posterior
movement without sloshing or pauses at transitions
 However, function was only partially restored
 Rescue likely associated with overexpression phenotypes in addition to the partial
functional rescue seen with injection
 Indicates other factors are likely involved
Cell Morphology

 WT and mutant had similar morphology at larval stage

 Similar number of goblet cells
 However, mutant one year old zebrafish had significantly increased goblet cells
 Significance of goblet cells?
 No decrease in enteric neurons compared to WT but there was a decrease in
serotonin positive enteroendocrine cells (EECs)
 EECs are mechanosensitive cells that release serotonin in response to mechanical
and/or chemical stimulation, which in turn causes the intestine to increase mucus
secretion and peristalsis, both of which facilitate transit.
Recap: Validating Zebrafish Model

 Zebrafish Shank3ab proteins show similar isoform complexity and post-synaptic

density enrichment to that seen in mammals
 The lack of Shank3ab in shank3abΔC mutants indicates mammalian antibody
specificity
 As a whole, supports functional conservation between zebrafish and mammalian
SHANK3 proteins.
 Overall zebrafish model mimics clinically relevant mutations shown previously to
cause PMS
Recap: GI Distress

 Hypomotility in mutants parallels to GI distress in PMS.

 Hypothesis: mutations in shank3ab disrupt EEC regulation of DT motility
 Mutant zebrafish show reduced peristalsis, increased transit time, and reductions in
EECs
 Increased transit and reflux are consistent with PMS, related to peristalsis
coordination
 Human SHANK3 RNA rescued transit time from the intestinal bulb (analogous to
the mammalian stomach) to the upper intestine but not overall intestinal motility
 Establishes reduced SHANK3 as causal for GI dysmotility
 Indicates other factors are involved
Recap: Tissue Morphology

 Normal tissue structure in mutants.

 ENS showed similar numbers of neurons in WT and mutants
 Mutant zebrafish exhibited an intact columnar epithelium and similar numbers of
goblet cells to their WT counterparts and to the literature
 Likely indicates there are more factors involved in GI distress
 EECs are significantly reduced in both shank3abΔC +/− and shank3abΔC −/− larvae.
EECs in the gut synthesize 95% of the body’s serotonin and, during digestion serotonin
release helps to coordinate secretion and motility as well as satiety and pain sensation
 Shank3 dependent reductions in EECs help explain dysmotility phenotypes.
 However, other cell types are also implicated in motility and need to be investigated as well,
interstitial cell of cajal for example
Strengths & Weaknesses

 Great design with a compelling case for Shank3ab as casual for GI distress
 Innovative use of new model organism
 Confounders
 A lot of emphasis on SHANK3 as the cause, however in PMS there are a lot of genes
missing
 SHANK3 is represented in many areas and interacts with many proteins, Shank3 as
proximate cause or than loss of function in other key proteins, see NOTCH
 Goblet cell increase could be due to interference from other pathways and or increased
inflammation, inflammation is a hallmark of ASD