SHANK protein biology, mutant mice and autism spectrum disorders (ASDs)

As much attention as autism spectrum disorders (ASDs) have received in recent years, their underlying genetic, pathophysiological and neurological bases are still largely unknown. Mouse models could be very important in solving the very complex nature of ASDs and in developing effective ASD therapies. Models with Shank gene mutations may be particularly helpful, especially in light of the evidence that SHANK mutations are associated with human ASDs. In a recent review, Drs. Jiang Yong-hui from the Departments of Pediatrics and Neurobiology, Duke University School of Medicine, Durham, NC, and Michael D. Ehlers, from Pfizer Worldwide Research and Development, Neuroscience Research Unit, Cambridge, MA, discuss the molecular genetics of SHANK mutations in human ASDs and orthologous mutations in ASD mouse models (Jiang and Ehlers 2013). This article gives but a glimpse of the intricate SHANK gene biology reviewed by Yong-hui and Ehelers and summarizes the key features of the Shank mouse models distributed by The Jackson Laboratory.

The complexity of SHANK gene biology

There are three human SHANK genes: SHANK1, SHANK2, and SHANK3. They encode synaptic scaffolding proteins whose major function is to organize extensive protein complexes at the postsynaptic densities (PSDs) of excitatory glutamatergic synapses. The web of biochemical pathways involved in SHANK protein biology almost defies description. All three SHANK proteins occur in many isoforms and participate in a dizzying array of synaptic functions. They have five major domains that interact with more than 30 other synaptic proteins, including other scaffolding proteins, receptors (among them being all the major glutamate receptors), ion channels, cytoskeletal proteins, enzymes and signaling molecules. The large complexes organized by SHANK protein interactions with these proteins play key roles in cytoskeletal remodeling, synapse, endocytosis and regulation of synaptic transmission and plasticity. The expression of different SHANK protein isoforms is cell-, tissue-, organ- and developmental stage-specific. Epigenetic factors like methylation also regulate their expression.

The SHANK3 gene is the most characterized and typifies the complexity of SHANK gene biology. Its five intragenic promoters account for its ability to undergo extensive alternative splicing, producing many isoforms. A variety of SHANK3 mutations – including large and small deletions, point mutations, translocations, duplications and copy number variations – have been associated with human ASDs. Many of the deletions are associated with the very rare – only about 600 cases worldwide – chromosome 22q13.3 deletion syndrome, also known as the Phelan-McDermid syndrome. Some of the mutations have been associated with schizophrenia. The autism-like phenotypes resulting from SHANK3 mutations range from mild to severe.

Mouse models with Shank3 mutations

The Jackson Laboratory distributes mutant mice for the five Shank3 mutations produced so far. The key features of these mutants are listed below:

B6(Cg)-Shank3^tm1.2Bux/J (017890)

• Knockout - exons 4-9 of the Shank3 gene are deleted, preventing full-length Shank3 expression.
• Heterozygotes are viable and fertile and have no observable gross structural brain defects or seizures; homozygotes are viable with subtle motor abnormalities.
• Shank3 mRNA/protein is absent in homozygous PSD fractions and reduced by 50% in heterozygous PSD fractions.
• Heterozygotes have low basal neurotransmission.
• Heterozygous males display less social sniffing and emit fewer ultrasonic vocalizations during interactions with estrus female mice.
• Heterozygotes have impaired long-term potentiation and altered spine remodeling.

B6(Cg)-Shank3^tm1.1Bux/J (017889)

• Floxed strain - loxP sites flank exons 4-9 of the Shank3 gene.
• When bred to a strain that expresses Cre recombinase, produces offspring in which the sequences encoding the ankyrin repeat domains are deleted in the Cre-expressing tissues.
• May be used to generate tissue-specific Shank3 deletions for studying synaptic glutamate receptor development/function, transmission, and plasticity, Shank3 haploinsufficiency, Philan-McDermid syndrome and other ASDs.

B6.129-Shank3^tm2Gfng/J (017688)

• A neo cassette replaces exons 13-16 of the Shank3 gene, disrupting the PDZ domain, resulting in a deficiency of Shank3a and Shank3b and reducing expression of the Shank3g isoform.
• Social behaviors are abnormal, grooming excessive and repetitive behaviors self-injurious.
• Striatal synapses, cortical-striatal circuits and medium spiny neuron morphology and density are defective or altered.

B6.129S7-Shank3^tm1Yhj/J (017442)

• A neo cassette replaces exons 4-9 of the Shank3 gene: the neo cassette is inserted downstream of promoter 2, making this mouse deficient in SHANK3a-b (transcripts from promoters 1-2).
• Produces SHANK3c-e transcripts from downstream promoters 3-5.
• Social behaviors, communication, learning and memory are abnormal; exhibits repetitive behaviors; motor coordination is severely impaired in males.
• Dendritic spines are morphologically altered and long-term potentiation is defective.
• Homozygotes are viable and fertile.

STOCK Shank3^tm1.1Pfw/J (018398)

• Lacks exon 21 of the Shank3 gene, causing a frame shift that results in a SHANK3 protein missing the entire C-terminal region – including the Homer-binding site in the sterile alpha motif (SAM) domain.
• The truncated protein associates with wild-type protein, reducing wild-type SHANK3 by 75% in cortical cultures and 90% in synaptic cultures.
• Exhibits autism-like behaviors, including aberrant social behavior and aggression.
• NMDA receptor function in hippocampal neurons is low, reducing long term potential and depression.
• Learning, memory, PSD proteins, spine morphology and synapse numbers are normal.
• Heterozygotes are viable and fertile; homozygotes may be sub-fertile.

Mouse models with Shank1 mutations

The Jackson Laboratory distributes two mouse models, each on a different background harboring the only Shank1 mutation thus far produced. The key features of that mutation are listed below:

B6.129S4-Shank1^tm1Shng/J (008108)

129S4/SvJae-Shank1^tm1Shng/J (008109)

• Shank1 exons 14-15 are deleted (may not be a complete Shank1 knockout).
• Expression of Shank-associated proteins guanylate kinase-associated protein (GKAP) and homer homolog 1 (HOMER) is reduced.
• Dendritic spines are small, the largest PSD's are lost and basal synaptic transmission in the brain is weak.
• Synaptic plasticity – including long-term potentiation long-term depression, and late-phase long term potentiation – is normal.
• Anxiety-related behavior is high, contextual fear memory is impaired.
• Homozygotes perform above normal in spatial learning task but below normal in long-term memory retention.

The diversity of SHANK proteins, their interactions, their expression patterns and the variety of ways they can be mutated result in a wide range of ASD phenotypes. Modeling these phenotypes in mice is difficult enough; extrapolating mouse Shank biology to humans is even more difficult. Nevertheless, Shank mouse mutants will likely continue to help scientists dissect the highly involved biology of, and develop more effective treatments for, human ASDs.

Recent research using JAX^® Mice models of autism

• Chronic Cell Danger Response Signaling Tied to Autism in Mice: A research team led by Dr. Robert Naviaux, from the San Diego School of Medicine, San Diego, Calif., found that a purinergic signaling inhibitor corrects all the ASD-associated abnormalities in an ASD mouse model.
• Autism-Like Behavior Corrected in Mice: A research team comprised of scientists from the National Institute of Mental Health (NIMH), Bethesda, Md., and Pfizer Worldwide Research and Development, Groton, Conn., used a mGluR5 antagonist to mitigate autism-like behaviors in a well-known model of autism, BTBR T⁺ Itpr3^tf/J (002282), and C58/J (000669).