Date of Degree

10-2014

Document Type

Dissertation

Degree Name

Ph.D.

Program

Biology

Advisor(s)

Probal Banerjee

Subject Categories

Neuroscience and Neurobiology

Abstract

The function of the phospholipid flippase Atp8a1 in neurotransmission, brain development, and autistic behavior

By

Daniel J. Kerr

The plasma membrane consists of lipids and proteins. Among the integral membrane proteins are P-type ATPases, widely expressed in both prokaryotes and eukaryotes, which use ATP to translocate ions across a plasma membrane. Type IV enzymes are putative aminophospholipid translocases (APLTs) and catalyze phosphatidylserine (PS) transfer into the cytosolic leaflet of a lipid bilayer. Previously, our group showed pronounced PS externalization in Atp8a1 (-/-) mice but not wild type (WT). Subsequent behavioral testing demonstrated that these Atp8a1 (-/-) mice display significant deficiency in spatial learning (p = 0.0001), increased hyperactivity, and decreased anxiety, all indicating aberrant hippocampus-dependent behavior. The putative flippase ATP10C gene, located within chromosome 15q11-q13 has been identified as an autism susceptibility locus and that the Atp8a1 gene is located in the middle of an autism-associated 4p12-15.3 inversion domain also containing a chromosome 4p GABAA receptor gene cluster. Based on such information, I asked whether there is a link between Atp8a1 and autism. Using Western blotting analysis of human brain homogenates (tissue specimens from brain bank), I demonstrated a pronounced induction of Atp8a1 in the hippocampus of juvenile autistic subjects compared to control. The difference was more evident when only the juvenile males were examined. This increase in the putative flippase was also observed in the temporal lobe of autistic children. I saw no significant differences in the level of post-synaptic density 95 (PSD95), a well-known marker for synaptic connectivity, in neither the hippocampus nor temporal lobe of autistic juvenile individuals. The role of Atp8a1 was further investigated by using mouse models. Using electron microscopy (EM), Atp8a1 (-/-) mice were shown to have fewer and weaker glutamatergic excitatory synapses in the CA1 hippocampus region compared to wild type controls. Paired-pulse analysis of the Schaffer collateral pathway demonstrates inhibition at 20-ms inter-stimulus interval of the Atp8a1 (-/-) mice but not controls. No differences in social interaction were observed between the two groups. In separate experiments the level of Atp8a1 was boosted by injecting a lenti virus-Atp8a1 construct into the hippocampi of C57/BL6 mouse pups at the early developmental stage of postnatal day 6 (P6). EM analysis revealed that the mice with elevated Atp8a1 had fewer and weaker glutamatergic excitatory synapses in the hippocampal CA1 region compared to mice injected with the empty construct. Although not significant, a trend toward inhibition of the Schaffer collateral pathway at 20-ms inter-stimulus was observed in the mice receiving the Atp8a1 construct. Social interaction tests indicated possible autistic-like behavior in the mice with increased Atp8a1 in the hippocampus. These findings suggest that either enhanced or diminished levels of the flippase Atp8a1 may be detrimental to brain connectivity. Furthermore, increased levels of Atp8a1 in the mouse hippocampus may be associated with deficits in social behavior. It is therefore possible that mice with enhanced hippocampus Atp8a1 may serve as a future model in autism research.

 
 

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