Pancortins interact with amyloid precursor protein and modulate cortical cell migration.

Neuronal precursor cell migration in the developing mammalian brain is a complex process requiring the coordinated interaction of numerous proteins. We have recently shown that amyloid precursor protein (APP) plays a role in migration into the cortical plate through its interaction with two cytosolic signaling proteins, disabled 1 (DAB1) and disrupted in schizophrenia 1 (DISC1). In order to identify extracellular factors that may signal through APP to regulate migration, we performed an unbiased mass spectrometry-based screen for factors that bind to the extracellular domain of APP in the rodent brain. Through this screen, we identified an interaction between APP and pancortins, proteins expressed throughout the developing and mature cerebral cortex. Via co-immunoprecipitation, we show that APP interacts with all four of the mammalian pancortin isoforms (AMY, AMZ, BMY, BMZ). We demonstrate that the BMZ and BMY isoforms of pancortin can specifically reduce ß-secretase- but not α-secretase-mediated cleavage of endogenous APP in cell culture, suggesting a biochemical consequence of the association between pancortins and APP. Using in utero electroporation to overexpress and knock down specific pancortin isoforms, we reveal a novel role for pancortins in migration into the cortical plate. Interestingly, we observe opposing roles for alternate pancortin isoforms, with AMY overexpression and BMZ knock down both preventing proper migration of neuronal precursor cells. Finally, we show that BMZ can partially rescue a loss of APP expression and that APP can rescue effects of AMY overexpression, suggesting that pancortins act in conjunction with APP to regulate entry into the cortical plate. Taken together, these results suggest a biochemical and functional interaction between APP and pancortins, and reveal a previously unidentified role for pancortins in mammalian cortical development.

Biochemical and functional interaction of disrupted-in-schizophrenia 1 and amyloid precursor protein regulates neuronal migration during mammalian cortical development.

Although clinically distinct, schizophrenia and Alzheimer's disease are common and devastating disorders that profoundly impair cognitive function. For Alzheimer's disease, key mechanistic insights have emerged from genetic studies that identified causative mutations in amyloid precursor protein (APP) and presenilin. Several genes have been associated with schizophrenia and other major psychoses, and understanding their normal functions will help elucidate the underlying causes of these disorders. One such gene is disrupted-in-schizophrenia 1 (DISC1). DISC1 and APP have been implicated separately in cortical development, with each having roles in both neuronal migration and neurite outgrowth. Here, we report a previously unrecognized biochemical and functional interaction between DISC1 and APP. Using in utero electroporation in the living rat brain, we show that DISC1 acts downstream of APP and Disabled-1 to regulate cortical precursor cell migration. Specifically, overexpression of DISC1 rescues the migration defect caused by a loss of APP expression. Moreover, knockdown of APP in cultured embryonic neurons results in altered subcellular localization of DISC1. Using transfected cells and normal brain tissue, we show that APP and DISC1 coimmunoprecipitate and that the intracellular domain of APP interacts with the N-terminal domain of DISC1. Based on these findings, we hypothesize that the APP cytoplasmic region transiently interacts with DISC1 to help regulate the translocation of DISC1 to the centrosome, where it plays a key role in controlling neuronal migration during cortical development.

In utero electroporation followed by primary neuronal culture for studying gene function in subset of cortical neurons.

In vitro study of primary neuronal cultures allows for quantitative analyses of neurite outgrowth. In order to study how genetic alterations affect neuronal process outgrowth, shRNA or cDNA constructs can be introduced into primary neurons via chemical transfection or viral transduction. However, with primary cortical cells, a heterogeneous pool of cell types (glutamatergic neurons from different layers, inhibitory neurons, glial cells) are transfected using these methods. The use of in utero electroporation to introduce DNA constructs in the embryonic rodent cortex allows for certain subsets of cells to be targeted: while electroporation of early embryonic cortex targets deep layers of the cortex, electroporation at late embryonic timepoints targets more superficial layers. Further, differential placement of electrodes across the heads of individual embryos results in the targeting of dorsal-medial versus ventral-lateral regions of the cortex. Following electroporation, transfected cells can be dissected out, dissociated, and plated in vitro for quantitative analysis of neurite outgrowth. Here, we provide a step-by-step method to quantitatively measure neuronal process outgrowth in subsets of cortical cells. The basic protocol for in utero electroporation has been described in detail in two other JoVE articles from the Kriegstein lab. We will provide an overview of our protocol for in utero electroporation, focusing on the most important details, followed by a description of our protocol that applies in utero electroporation to the study of gene function in neuronal process outgrowth.