Proteomic Analysis of the Autism Candidate Gene, Jakmip1, Suggests Its Role In Brain Translational Regulation

Background : Janus kinase and microtubule-interacting protein 1 (JAKMIP1) is dysregulated in the lymphoblastoid cells of autistic subjects with Fragile X, maternally inherited 15q duplication, and idiopathic autism spectrum disorder.  Studies suggest JAKMIP1 is downstream of both fragile X mental retardation 1 (FMR1) and cytoplasmic FMR1 interacting protein 1 (CYFIP1), a gene within the 15q duplication region, in mouse and human.  Jakmip1 is predominantly expressed in neural tissues and is enriched in neurons.

Objectives : We determined JAKMIP1’s expression during development and adulthood in the neocortex, a brain region associated with autism, and characterized JAKMIP1’s neuronal sub-type and cortical layer expression pattern.  We subsequently performed Multidimensional Protein Identification Technology (MudPIT) to annotate JAKMIP1's in vivo proteomic interactome so as to better understand JAKMIP1 function.

Methods : Developmental expression profiling was determined using Western Blotting.  The neuron-specific and cortical layer expression patterning was ascertained using double-label immunohistochemistry.  MudPIT was used to discover JAKMIP1’s in vivo proteomic interactome in mouse cerebral neocortex. MudPIT read-out values were compared between JAKMIP1 and rabbit preimmune serum immunoprecipitated neocortices. Ingenuity Pathway Analysis was used to determine the gene ontology categories of JAKMIP1’s interactome.  We implemented permutation analysis to test the statistical significance of protein-protein interactions in the top gene ontology network.  PatternLab ACFold analysis was conducted to determine statistically significant protein interactors.  We used bidirectional co-immunoprecipitation to validate top protein interactions.  

Results : We found that JAKMIP1 is expressed predominantly in glutamatergic projection neurons of mouse neocortex and broadly throughout neocortical layers.  Its peak expression occurs from p8 to p12 in mouse cerebral cortex, concomitant with neuronal maturation and synaptogenesis.  We identified a group of 36 JAKMIP1 protein interactors by MudPIT. Additional analysis using PatternLab ACFold generated a conservative core of 11 proteins within this list of 36.  The most predominant gene ontology theme of JAKMIP1’s protein binding partners is protein synthesis, being a common denominator between networks and the most significant molecular and cellular function.  Permutation analysis indicates a statistically significant enrichment of “protein-protein interactions” in the top gene ontology network of JAKMIP1 binders, suggesting JAKMIP1’s membership in a protein complex.  We were able to experimentally validate 4/6 MudPIT-identified interactors, some of which are known to regulate protein expression.  JAKMIP1 and validated protein binding partners fractionate with polyribosomes in independent mouse postnatal neocortices.  Some of JAKMIP1’s protein associations in mouse neocortices depend on single stranded RNA, suggesting select associations may exist in the polyribosome. 

Conclusions : Using a combined approach, multiple lines of evidence implicate a role for JAKMIP1 in protein translation during cortical development.  Understanding JAKMIP1's dynamic relationship with key players of this essential cellular process will advance our knowledge of this autism candidate gene's role in neural development and further suggests the importance of translational control in the pathophysiology of autism spectrum disorders.