Carboxypeptidase Effects on Early Brain Development

Role of Carboxypeptidase E in Regulation of Cortical Neuron Migration and Dendrite Morphology

Short Title: CPE regulates neurodevelopment

Abstract

Higher brain function relies on proper development of the cerebral cortex, including correct positioning of neurons and dendrite morphology. Disruptions in these processes may result in various neurocognitive disorders. Mutations in the CPE gene, which encodes carboxypeptidase E, have been linked to depression and intellectual disability. However, it remains unclear whether CPE is involved in early brain development, and in turn, contributes to the pathophysiology of neurocognitive disorders. Here, we investigate the effects of CPE knockdown on early brain development and explore the functional significance of the interaction between CPE and its binding partner p150^Glued. We demonstrate that CPE is required for cortical neuron migration and dendrite arborization. Furthermore, we show that expression of CPE-C10 redistributes p150^Glued from the centrosome and that disruption of its interaction with p150^Glued leads to abnormal neuronal migration and dendrite morphology, suggesting that a complex between CPE and p150^Glued is necessary for proper neurodevelopment.

Introduction

Functional connectivity between neurons in the brain depends on where the neurons are positioned and how their dendrites are shaped during development. Abnormal neuronal migration and dendrite patterning may result in various neurocognitive disorders due to developmental defects. Often, aberrant brain structure results from improper function or expression of a complex of proteins rather than of a single protein. Thus, studying the interaction between multiple proteins that regulate neuronal development will aid in our understanding of processes that result in abnormal brain function.

Carboxypeptidase E (CPE) was identified as a prohormone-processing enzyme (Fricker & Snyder, 1982; Hook, Eiden, & Brownstein, 1982). Recent studies have reported non-enzymatic functions of CPE protein, including prohormone sorting and vesicle transport, in the endocrine and nervous systems (reviewed in (Cawley et al., 2012)). One neuropeptide sorted and transported by CPE is brain-derived neurotrophic factor (BDNF). Disruption of CPE binding to BDNF-containing vesicles results in reduced BDNF localization to neurites of hippocampal neurons (Lou et al., 2005; Park, Cawley, & Loh, 2008a). The importance of CPE function in the brain has been further supported by studies in CPE^-/- mice and humans. Knockout mice exhibit aberrant dendritic architecture and spine morphology, deficits in learning and memory, and neurodegeneration under stress (Woronowicz et al., 2010; Woronowicz et al., 2008). Studies on a spontaneous mutant mouse model, Cpe^fat/fat, which lacks CPE enzymatic activity due to a point mutation in the Cpe gene (Coleman & Eicher, 1990; Fricker, Berman, Leiter, & Devi, 1996), revealed anxiety and depressive-like behaviors in these mice (Rodriguiz et al., 2013). Similarly, a truncation mutation of carboxypeptidase E (CPE) was identified in a morbidly obese human female with intellectual disability (Alsters et al., 2015), suggesting that this protein plays a role in brain function in both species.

The mechanism by which CPE regulates neural development is still poorly understood, but it is known that one of its binding partners, p150^Glued, may play an important role in CPE function. The cytoplasmic tail of CPE interacts with p150^Glued (Park, Cawley, & Loh, 2008b), the major subunit of the dynactin complex, which binds directly to microtubules and motor proteins (reviewed in (Schroer, 2004)). Dynactin is involved in coordination of bi-directional cargo transport by regulating both retrograde transport mediated by dynein and anterograde transport mediated by kinesin-2 (Berezuk & Schroer, 2007; King & Schroer, 2000). In addition to regulation of motor activities, dynactin functions at the centrosome to anchor microtubules and recruit cell cycle regulators (Quintyne et al., 1999; Quintyne & Schroer, 2002). Mutations in the dynactin gene (DCTN1), encoding p150^Glued, are associated with Perry syndrome, characterized by early-onset Parkinsonism, depression, weight loss, and hypoventilation (Farrer et al., 2009; Wider et al., 2010). In addition, it was recently reported that full-length p150^Glued is a neuron-specific anti-catastrophe factor of microtubules, and one of the causal mutations in p150^Glued found in patients with Perry syndrome abolishes this anti-catastrophe activity (Lazarus, Moughamian, Tokito, & Holzbaur, 2013). Thus, by regulating microtubule dynamics and molecular motors, p150^Glued may play a role in shaping brain structure and function.

As such, understanding the roles of CPE and p150^Glued and their interaction in regulating brain development can provide insight into mechanisms underlying neurocognitive disorders, such as major depressive disorder and Perry syndrome, and elucidate potential therapeutic targets for these disorders. We previously reported the involvement of CPE in mediating Nitric Oxide Synthase 1 Adaptor Protein (NOS1AP)-mediated decreases in dendritic arborization (Carrel et al., 2009), but an understanding of the mechanism by which CPE itself regulates dendrite branching has not been fully elucidated. In addition, since CPE interacts with p150^Glued, and members of the dynactin complex regulate cortical development (Dujardin et al., 2003; Smith et al., 2000; Tai, Dujardin, Faulkner, & Vallee, 2002), we asked whether CPE plays a role in cortical neuron migration. We also investigated the effects of CPE knockdown on dendrite branching and explored the functional significance of its carboxyl terminal interaction with p150^Glued. We found that CPE is required for proper cortical neuron migration and dendrite morphology. Furthermore, we identify the carboxyl terminus of CPE as an important domain in mediating these CPE functions via its interaction with p150^Glued. These results underscore the importance of a role for CPE during brain development and suggest that disruption of the interaction between CPE and p150^Glued may result in impaired brain function, such as that observed in patients with major depressive disorder and Perry syndrome.

Results

 

CPE is expressed in neurons of the developing rodent cortex

To address the role of CPE in brain development, we examined the expression profile of CPE protein at different developmental stages. We studied CPE expression in mouse brain from embryonic day (E)12 to postnatal day (P)10, which is a crucial time window for positioning of neurons in different layers of the neocortex and in single neuron development (Jiang & Nardelli, 2016). Cortices from mice at E12, E14, E16, P0, P10, and P60-90 (adult) were isolated and examined by Western Blot analysis. We detected the expression of both a membrane-associated form (molecular mass, ~53 KDa) (Hook, 1985) and a soluble form (molecular mass, ~50 KDa) (Fricker & Devi, 1993) of CPE as early as E12, and the expression level remains stable from E12 to E16. CPE expression significantly increases from E16 to P0 and from P0 to P10. During adulthood, CPE protein levels drop and remain at a similar level as seen in P0 (Figure 1A, B). These results suggest that CPE may play an important role during embryonic and postnatal brain development.

To further investigate CPE expression in specific cell types, we dissected brains from rats at E18 and immunostained for CPE and neuron-specific class III β-tubulin (Tuj1) in the lateral neocortex. We observed that cells expressing high levels of CPE protein are, for the most part, positioned in the cortical plate (CP), while cells in the intermediate zone (IZ) and ventricular zone (VZ) show much lower CPE expression levels (Figure 1C). Moreover, cells that express CPE are Tuj1-positive (Figure 1C), indicating that CPE is expressed specifically in neurons of the neocortex.

Finally, we examined the subcellular distribution of CPE in cultured primary hippocampal neurons. CPE protein is evenly distributed throughout the dendrites and co-localizes with β-III tubulin (Figure 1D), suggesting a function for CPE other than its previously reported role in vesicle transport (Park et al., 2008a).

CPE is required for proper cortical neuron migration

It has been reported that dynactin and associated proteins regulate cortical cell radial migration (Tsai & Gleeson, 2005). Since CPE associates with p150^Glued, we investigated whether CPE deficiency alters neuronal migration. Thus, we designed a short-hairpin (sh)RNA against CPE to examine the effect of CPE knockdown in vivo. To test the specificity of the shRNA, we employed both Western blot analysis and immunohistochemistry. Rat cortical neurons expressing CPE shRNA demonstrate a 50% decrease in CPE levels compared to neurons expressing control shRNA (Figure 2A,B). Immunostaining for CPE in coronal brain sections after in utero electroporation (IUE) further support knockdown of CPE in single transfected neurons (Figure 2C).

To examine cortical neuron migration, we performed IUE in mice at E14.5, introducing either CPE shRNA or scramble control shRNA together with cDNA encoding red fluorescent protein (RFP) and analyzed the distribution of RFP-positive cells in each cortical area at E17.5. In brain sections from mice expressing control shRNA, ~45% of transfected cells migrate to the cortical plate (CP) and ~40% of transfected cells are located in the intermediate zone (IZ). In contrast, only ~25% of transfected cells reach the CP when CPE is knocked down and a significant number of transfected cells stall in the IZ (Figure 2C, D).

 

Multipolar neurons transition to a bipolar morphology when localized to the IZ so that they can continue locomotion to the CP (Nishimura et al., 2010; Ohtaka-Maruyama & Okado, 2015; Rakic, 2006). Thus, we asked whether CPE regulates this transition, which may explain the observed defects in radial migration when CPE is knocked down. We analyzed the morphology of transfected cells in the IZ of coronal brain sections after IUE and found that knockdown of CPE significantly increases the percentage of multipolar cells in the IZ (Figure 2E, F). Thus, the migration defects observed may be explained by the failure to transition from multipolar to bipolar morphology.

 

CPE regulates dendrite morphology in vivo and in cultured hippocampal neurons

 

CPE knockout mice show aberrant dendritic architecture in cortical and hippocampal neurons at 14-weeks of age (Woronowicz et al., 2010). We find that that expression of CPE increases significantly from E16 to P10 in mice and that CPE is expressed in differentiating neurons in the CP, suggesting a role of CPE in post-migratory neuronal development. Thus, we examined the potential involvement of CPE in regulating dendrite morphology in vivo from E14.5 to P7. We observed that in cortical layer II/III, neurons electroporated with CPE shRNA at E14.5 exhibit less number of dendrites and shorter total dendrite length than neurons in control condition at P7 (Figure 3A-C). To demonstrate the specificity of CPE shRNA, we generated an shRNA-resistant CPE construct and co-electroporated this construct with CPE shRNA. As expected, the shRNA-resistant CPE construct rescues the decreases in the number and total length of dendrites caused by CPE knockdown (Figure 3A-C), supporting specificity of the shRNA for CPE knockdown.

To support the idea that CPE knockdown disrupts dendritogenesis, we performed similar experiments in hippocampal neuronal cultures between day in vitro (DIV) 7 and DIV 10, as both primary dendrite extension and higher order dendrite formation actively take place during this time window (Banker & Goslin, 1988; Dotti, Sullivan, & Banker, 1988). To knockdown CPE in culture, we used a commercially purchased siRNA against rat CPE, and its specificity was demonstrated by immunocytochemistry (Figure 3G). Transfected neurons show ~50% reduction in CPE protein levels (Figure 3H). Results of Sholl analysis show that knockdown of CPE results in decreased dendrite branching at a distance of 0-42μm from the soma (Figure 3D-F). These results are in agreement with our in vivo data, indicating that CPE is required for dendritic arborization.

 

CPE interacts with p150^Glued and overexpression of the carboxyl terminal 10 amino acids of CPE (CPE-10) redistributes p150^Glued from the centrosome

 

It has been reported that in pituitary corticotrope tumor (AtT20) cells (Park et al., 2008b), the carboxyl terminal 10 amino acids of CPE interact with p150^Glued, the largest subunit in the Dynactin protein complex. To confirm whether this interaction between CPE and p150^Glued exists in brain, we performed a co-immunoprecipitation assay using rat brain lysates. As expected, p150^Glued co-immunoprecipitates with CPE (Figure 4A, B).

p150^Glued predominantly localizes to and anchors microtubules to the centrosome and recruits cell cycle regulators (Quintyne et al., 1999; Quintyne & Schroer, 2002). Given that CPE interacts with p150^Glued (Park et al., 2008b), it is possible that CPE affects the subcellular localization of p150^Glued, which in turn, alters the cytoskeleton and other downstream processes. Excess CPE-C10, a peptide containing the carboxyl terminal 10 amino acids of CPE, competes endogenous CPE binding to p150^Glued (Park et al., 2008a, 2008b). Therefore, we overexpressed CPE-C10 in COS-7 cells to disrupt the normal interaction between CPE and p150^Glued and analyzed the subcellular distribution of p150^Glued. Centrosomes were labeled by immunolabeling with an antibody to pericentrin, an integral component of the pericentriolar material (Doxsey, Stein, Evans, Calarco, & Kirschner, 1994). In cells expressing GFP, there was abundant co-localization of p150^Glued with pericentrin at the centrosome, while overexpression of CPE-C10 resulted in a significant decrease in the percentage of p150^Glued localized at the centrosome (Figure 4C, D), suggesting a dominant-negative effect of CPE-C10 on p150^Glued localization. Taken together, these results support our hypothesis that the interaction between CPE and p150^Glued is important for proper localization of p150^Glued, which may in turn influence p150^Glued function in regulating the microtubule cytoskeleton.

Overexpression of CPE-C10 disrupts neuronal migration and dendritic arborization

 

We next tested the role of the interaction between the carboxyl terminus of CPE and p150^Glued in regulating neuronal migration and dendritic arborization. GFP-tagged CPE-C10 or GFP (control) was expressed in neural progenitor cells using IUE at E14.5, and neuronal migration was examined at E17.5. We observe that expression of CPE-C10 results in a significant decrease in the percentage of cells reaching the CP (Figure 5A, B), suggesting that binding of a CPE interacting protein, such as p150^Glued, may be necessary for proper cortical migration and that disruption of this interaction attenuates the function of CPE in neurodevelopment. Together, our data suggest that CPE is required for proper cortical neuron migration and that its carboxyl terminus, and hence interaction with p150^Glued, may be involved in this process.

We also analyzed the morphology of transfected neurons in the IZ to determine the mechanism underlying the failure of neurons to reach the CP. We observed that, similar to CPE knock down, expression of CPE-C10 disrupts neuronal multipolar to bipolar transition (Figure 5C, D), which may explain the migration defects mediated by CPE-C10 expression.

To further support the idea that the interaction between CPE and p150^Glued regulates dendritic arborization, cultured hippocampal neurons were co-transfected with cDNA encoding mOrange and GFP or GFP-CPE-C10 at DIV 7. Neurons positive for both mOrange and GFP were analyzed for dendrite branching at DIV 10. Overexpression of CPE-C10 results in significantly decreased dendrite branching close to soma (0-18μm; Figure 5E-G), suggesting that the effects of CPE on dendrite morphology are dependent on its carboxyl terminal interaction with p150^Glued.

Discussion

We have identified a role for CPE and its interaction with p150^Glued in cortical neuron migration and dendritogenesis. Knock down of CPE or disruption of the CPE-p150^Glued interaction results in decreased migration of neurons to the CP. We show that this is due to disrupted neuronal multipolar to bipolar transition. Furthermore, we show that both in vivo and in vitro, expression of CPE and its interaction with p150^Glued are necessary for proper development and branching of proximal dendrites in cortical and hippocampal neurons. We also found that overexpression of the carboxyl terminal 10 amino acids of CPE decreases localization of p150^Glued at the centrosome, providing a potential mechanism for the role of the CPE-p150^Glued interaction in proper brain development. Our results suggest for the first time that in addition to vesicle sorting and transport, CPE acts via p150^Glued, and hence microtubule organization, to regulate neuronal placement and shape.

Expression of CPE during embryonic brain development

 

CPE protein is detected in all major areas of the adult rat brain (Birch, Rodriguez, Dixon, & Mezey, 1990), and its roles in prohormone processing and sorting and vesicle transport have been well-studied (reviewed in (Cawley et al., 2012)). In addition to its expression in the adult nervous system, CPE has been reported to be present in mammalian embryos (Selvaraj et al., 2017; Zheng, Streck, Scott, Seidah, & Pintar, 1994). Studies of CPE expression during mouse brain development show that CPE is expressed in neural tissues earlier than other prohormone convertases, such as PC1 and PC2, suggesting a distinct role for CPE during embryonic neurodevelopment. In this study, we elucidate the expression profile of CPE, including the membrane-associated and soluble forms, in the mouse brain at different developmental stages. In mouse cortices, levels of both forms of CPE protein increase during embryonic and early postnatal development with a peak of expression at P10, and higher CPE expression is detected in neurons in the CP than in the IZ or VZ. These observations suggest that in addition to its roles in neuronal migration and dendrite arborization during developmental stages examined in this study, CPE may also regulate the development of later-born neurons or regulate later processes in neuronal development, such as spine formation and synaptogenesis.

Using Western blot analysis, we did not detect expression of CPEΔN (molecular mass, ~40 KDa), a CPE isoform lacking the signaling peptide within the amino terminus transiently expressed during mouse embryonic development (Lee et al., 2011; Qin, Cheng, Murthy, Selvaraj, & Loh, 2014). At the cellular level, endogenous CPE is evenly distributed throughout the soma and neurites, and it co-localizes with β-III tubulin throughout the neurites. It was previously reported that overexpressed CPE in hippocampal neurons localizes in punctate vesicles and mediates vesicle transport (Park et al., 2008a). Our findings suggest CPE may have other functions besides sorting and transport of vesicles in neurons. Taken together, we propose that CPE contributes to early brain development, and impairments in neuronal positioning and morphology resulting from CPE gene mutations and decreased CPE protein levels may underlie neurocognitive deficits exhibited by CPE^-/- and Cpe^fat/fat mice models (Rodriguiz et al., 2013; Woronowicz et al., 2008). Furthermore, in human subjects, CPE gene mutations found in patients with dementia, CPE-QQ mutation in an Alzheimer’s patient with depression (Cheng et al., 2016), and truncation mutation of CPE in a morbidly obese female with intellectual disability (Alsters et al., 2015), may disrupt CPE-mediated neurodevelopment.

Roles of CPE in cortical development and dendritic arborization

 

It has been well-established that abnormalities in cortical neuron migration and dendrite morphology higher brain functions, such as learning, memory, emotion, and cognition, and may cause neurocognitive disorders, including depression, bipolar disorders, and schizophrenia (Barkovich, Guerrini, Kuzniecky, Jackson, & Dobyns, 2012; Brennand, Simone, Tran, & Gage, 2012; Kulkarni & Firestein, 2012; Valiente & Marin, 2010). It is of importance to understand how our data fit into the current understanding of neurodevelopmental disorders. During development of the cerebral cortex, pyramidal neurons are generated by neural progenitor cells in the VZ or subventricular zone (SVZ) and migrate towards the pial surface. Here, we show that neurons electroporated with a CPE shRNA construct fail to migrate to the CP, suggesting that CPE is required for proper cortical neuron migration. Normally in the IZ, newly differentiated neurons possess multipolar morphology and undergo transition to bipolar morphology, which is critical for subsequent glial-guided radial migration, and this transition relies heavily on microtubule dynamics (Ohtaka-Maruyama & Okado, 2015). Knockdown of CPE protein increases the percentage of multipolar cells in the IZ, indicating the involvement of CPE in mediating neuronal multipolar to bipolar transition.

As neurons reach their final destination, they differentiate into a multipolar morphology with defined axons and dendritic arbors (Takano, Xu, Funahashi, Namba, & Kaibuchi, 2015). Proper dendrite morphology, including shape, size, and patterning of the dendritic arbor, is critical for many aspects of neuronal function, such as the number and type of synapses that will be formed along the dendrites, electrical properties of a neuron, and the positioning of particular signaling molecules (London & Hausser, 2005; Spruston, 2008). Here, we report that CPE knockdown results in decreased number and total length of dendrites of pyramidal neurons in P7 mouse cortex and decreased proximal dendrite branching in developing hippocampal neuronal cultures, suggesting the necessity of CPE protein for proper dendritogenesis. Similarly, 14-week-old CPE^-/- mice exhibit increased dendritic branching proximal to the soma and decreased branching in distal dendritic arbor (Woronowicz et al., 2010). Taken together with this report, our findings further demonstrate the importance of CPE in dendritogenesis and indicate that loss of CPE at different developmental stages may have distinct impacts on dendrite morphology.

Mechanism by which CPE regulates cortical development and dendrite morphology

 

Neuronal migration and dendrite morphogenesis are dependent on continuous morphological changes mediated by the cytoskeleton, and in specific, the centrosome and microtubule network (reviewed in (Dent, 2017; Fukuda & Yanagi, 2017)). A number of risk genes have been identified for psychiatric disorders, and many are involved in cytoskeletal regulation (Copf, 2016; Fukuda & Yanagi, 2017). p150^Glued plays an important role in regulating the microtubule network (Askham, Vaughan, Goodson, & Morrison, 2002; Berezuk & Schroer, 2007; Deacon et al., 2003; King & Schroer, 2000; Lazarus et al., 2013; Ligon, Shelly, Tokito, & Holzbaur, 2006; Lloyd et al., 2012; Schroer, 2004). In the present study, we confirm an interaction between CPE and p150^Glued in rat brain lysate. We identify, for the first time, a function for the CPE carboxyl terminus in regulating the localization of p150^Glued at the centrosome. The observation that CPE-C10, which blocks the normal interaction between CPE and p150^Glued in a dominant negative manner, alters p150^Glued localization, neuronal migration, and dendrite branching suggests that the carboxyl terminus is the major domain responsible for CPE-mediated effects in neurodevelopment. Multiple lines of evidence from previous studies support the idea that p150^Glued regulates cortical development and dendrite branching, giving further evidence that CPE may act in a p150^Glued-dependent manner. First, accumulation of p150^Glued at the centrosome and microtubule plus-end is critical for maintaining microtubule stability and dynamics (Lazarus et al., 2013; Quintyne et al., 1999). Therefore, disrupted p150^Glued localization may affect microtubule organization, and in turn, alter neuronal migration and dendrite branching. The fact that mutations of the DCTN1 gene, encoding p150^Glued, within the glycine-rich (CAP-Gly) domain affect the affinity of dynactin for microtubules and cause Perry syndrome further supports a role for the CPE-p150^Glued interaction in neurodevelopmental processes (Farrer et al., 2009). Second, several cytoskeletal regulators, such as Disrupted in Schizophrenia – 1 (DISC1), regulate cortical migration and are associated with psychiatric disorders (Fukuda & Yanagi, 2017). In fact, DISC1 is a risk gene for various neurodevelopmental disorders, regulates the localization of dynein-dynactin complex at the centrosome, and hence, contributes to cortical development (Kamiya et al., 2005). Lastly, CPE interacts with p150^Glued to recruit motor proteins and transport BDNF-containing vesicles, which increases the number of proximal dendrites in pyramidal neurons (Horch, Kruttgen, Portbury, & Katz, 1999; Kwinter, Lo, Mafi, & Silverman, 2009; McAllister, Lo, & Katz, 1995). The involvement of BDNF and its receptor TrkB in neuronal migration has also been reported in different brain regions (Borghesani et al., 2002; Chiaramello et al., 2007; Medina et al., 2004; Ringstedt et al., 1998). BDNF promotes migration of cerebellar granule cells and neural precursors of the olfactory bulb. In the cerebral cortex, overexpression of BNDF causes aberrant cortical lamination.

In addition, the fact that excess CPE-C10 competes endogenous CPE but not motor proteins that bind to p150^Glued (Park et al., 2008a) strongly suggests that CPE directly interacts with p150^Glued via its carboxyl terminus. However, current evidence cannot rule out the possibility of an indirect interaction with other proteins that exist in a complex with p150^Glued. Thus, CPE may regulate the localization of p150^Glued indirectly by influencing other components of the dynactin and motor protein complex.

Model of the role for CPE and p150^Glued interaction in neuronal development

Taken together, we propose a model (Figure 6) for understanding the mechanism underlying CPE-mediated effects on neuronal migration and dendrite branching. A balance of CPE-p150^Glued protein complex and free CPE protein is required to maintain microtubule stability and dynamics, and in turn, to promote proper neuronal migration and dendrite branching. Decreased CPE protein or expression of CPE-C10 disrupts the normal CPE-p150^Glued interaction and alters proper subcellular distribution of p150^Glued to the centrosome or microtubules. This results in abnormal cytoskeletal function and disrupted neurodevelopment. In summary, our findings provide new insight for the functions of CPE during early brain development and potential involvement in neurocognitive disorders and demonstrate the importance of the interaction between the carboxyl terminus of CPE and p150^Glued in mediating proper cortical neuron migration and dendrite morphology.

Materials and Methods

DNA constructs and RNA interference

cDNA encoding the carboxyl terminal 10 amino acids of the rat full-length CPE protein was subcloned into pEGFP-C1 and pCAG-GFP vectors. siRNA against CPE and negative control siRNA (CPE siRNA, Mouse S64324; CPE siRNA Rat S220210; negative control #1, cat# 4390743) were purchased from Life Technologies (Carlsbad, CA). shRNAs targeting the CPE transcript (5’-GGTGGAATGCAAGACTTCA-3’) and an unrelated sequence (5’-GAGCATTTGTATGAGCGCG-3’) were designed and ligated into the pGE2hrGFPII vector (Agilent Technologies, Santa Clara, CA). The CPE shRNA target sequence we designed and the purchased siRNA targets both the membrane-associated and soluble forms of CPE. pCAG-IRES-EGFP plasmid (pCIG) was a gift from Dr. Gabriella D’Arcangelo (Rutgers University, United States), and pCAG-IRES-TagRFP plasmid (pCIR) was a gift from Dr. Marie-Catherine Tiveron (Institut de Biologie du Développement de Marseille, France).

Antibodies and reagents

Mouse CPE (BD 610758) and mouse MAP2 (BD 556320) primary antibodies were purchased from BD Biosciences (San Jose, CA). Mouse Dynactin p150^Glued antibody (SC-135890) was from Santa Cruz (Dalla, TX). Rabbit anti-pericentrin (ab4448) was from Abcam (Cambridge, UK). Chicken anti-GFP (PA-9533) was from Thermo Fisher (Waltham, MA). Mouse GAPDH (MAB374) and chicken β-III tubulin (AB9354) primary antibodies were from EMD Millipore (Billerica, MA). Rabbit anti-Tuj1 (MRB-435P) was from Covance Antibody Products (Dedham, MA).

COS-7 cell transfection and immunocytochemistry

COS-7 cells were plated at 15,800 cells/cm² on coverslips 0.1mg/ml poly-D-lysine and transfected 24hrs after plating with pEGFP-C1 or pEGFP-C1-CPE-C10 using Lipofectmine 2000 (Thermo Fisher, Waltham, MA) following the manufacturer’s protocol. Cells were fixed 2 days after transfection with incubation in 4% paraformaldehyde (PFA) in phosphate-buffered saline (PBS) for 15min and immunostained for GFP, pericentrin, and Dynactin p150^Glued, followed by nuclear staining with Hoechst dye. Coverslips were mounted onto glass slides with Fluoromount G (Southern Biotechnology; Birmingham, AL).

Immunofluorescent microscopy and p150^Glued localization analysis

Slides of COS-7 cells were prepared as described above. Images were taken at 600X using an Olympus Optical (Tokyo, Japan) IX50 microscope and fluorescent imaging system. For assessment of p150^Glued localization, a straight line was drawn from one side of the cell through the centrosome (positive for pericentrin immunostaining) and the nucleus (labeled by Hoechst dye) to the other end of the cell. To quantify the percentage of p150^Glued concentrated on the centrosome, the intensities of p150^Glued and pericentrin were plotted along the line. p150^Glued intensity within the peak of pericentrin was measured (value A), and its intensity within 1 micron away on each side was measured (value B). The ratio of A to B was calculated to get the percentage of p150^Glued localized to the centrosome.

Primary neuronal culture and dendrite branching analysis

Neuronal cultures were prepared from hippocampi of rat embryos at 18 days of gestation as we described previously (Firestein et al., 1999). Cells dissociated from the hippocampi were plated at 10,500 cells/cm² onto coverslips previously coated with 0.1mg/ml poly-D-lysine-coated and maintained in Neurobasal medium (Thermo Fisher, Waltham, MA) supplemented with B27 (Thermo Fisher, Waltham, MA), GlutaMAX (Thermo Fisher, Waltham, MA), penicillin, and streptomycin. Cultures were co-transfected with pCAG-mOrange and indicated CPE constructs or siRNA at day in vitro (DIV) 7 using Lipofectamine LTX with Plus (Thermo Fisher, Waltham, MA), fixed at DIV 10 with 4% PFA in PBS, and immunostained for GFP and MAP2. Images of neurons were taken using Olympus Optical (Tokyo, Japan) IX50 microscope and fluorescent imaging system, and dendrite morphology was assessed as previously described using our Bonfire program (Kutzing, Langhammer, Luo, Lakdawala, & Firestein, 2010; Langhammer et al., 2010).

Co-immunoprecipitation and Western blot analysis

Adult rat cortices were dissected and homogenized in hydroxyethyl piperazineethanesulfonic acid (HEPES)/Sucrose buffer (20 mM HEPES (pH 7.5), 320 mM sucrose, 1 mM EDTA, 5 mM DTT, 1 mM PMSF). Rat cortical brain extracts were centrifuged at 350 x g to pellet unbroken cells or tissue debris. The supernatant was collected and centrifuged at 100,000 x g at 4C for 1hr to fractionate cytosolic proteins. Equal amounts of cytosolic proteins were pre-cleared with protein G agarose (50% slurry (GE Healthcare, Piscataway, NJ)) for 1 hour and then subjected to immunoprecipitation with monoclonal Dynactin p150^Glued antibody at 4C overnight after addition of 0.05% bovine serum albumin (BSA) to the protein extract. Samples were then incubated with protein G agarose for 1 hour, and immunoprecipitates were washed with buffer (0.1% Triton X-100, 50mM Tris, PH 7.4, 300mM Nacl, 5mM EDTA, 0.02% NaN₃). Proteins were eluted with protein loading buffer (50mM Tris-HCl (pH 6.8), 2% sodium dodecyl sulfate, 5% glycerol, 0.01% bromophenol blue, 5% β-Mercaptoethanol), resolved by SDS-PAGE, and transferred to PVDF membranes. Membranes were probed for CPE and p150^Glued. Immunoreactive bands were visualized with HyGlo quick spray (Denville Scientific; South Plainfield, NJ), and resulting films were quantified with ImageJ software (http://rsb.info.nih.gov/ij/; National Institutes of Health, Bethesda, Maryland).

In utero electroporation, immunohistochemistry, and microscopy

In utero electroporation was performed as we previously described (Carrel et al., 2015). Pregnant Swiss mice at gestation day 14.5 (E14.5) were anesthetized with isoflurane. The abdominal cavity was opened to expose the uterine horns. 1-3 μl of plasmids (2-2.5 µg/µl) with 1 mg/mL Fast Green (Sigma) were microinjected through the uterus into the lateral ventricles of embryos by pulled glass capillaries (Drummond Scientific, Broomall, PA). Electroporation was performed by placing the heads of the embryos between tweezer-type electrodes. Square electric pulses (35 V, 50 ms) were passed five times at 1-s intervals using a NEPA21 electroporator (SONIDEL Limited, Dublin, Ireland). Embryos were allowed to develop in utero for 3 days after electroporation (until E17.5) or were kept until 7 days after birth (postnatal day (P) 7). Embryonic mouse brains were dissected and fixed for 48 h in 4% PFA in PBS at 4°C. P7 mouse brains were fixed by transcardial perfusion of 4% PFA in PBS and postfixed for 24 hours in 4% PFA in PBS. Brains were cryoprotected in 30% sucrose in PBS, frozen in PolyFreeze (Sigma), and sectioned coronally at 16 µm (E17.5 brains) and 30 µm and 80 µm (P7 brains) using a cryostat.

For analysis of migration at E17.5 and P7 and cell morphology at E17.5, images of fluorescent mouse brain sections were taken on a Zeiss Axio Observer.Z1 microscope using a 20× numerical aperture (NA) 0.8 objective, with a Clara E CCD Camera (Andor Technology Limited, Belfast, UK).

For analysis of neuronal morphology at P7, 30- to 50-mm-thick z-stacks were acquired on a Zeiss LSM 510 confocal laser-scanning microscope (Zeiss) using a 20× NA .5 objective, and z-series were projected to two-dimensional representations.

Analysis of neuronal migration and dendritic branching in vivo

TagRFP-positive cells were counted using the image analysis software ImageJ (http://rsb.info.nih.gov/ij/; National Institutes of Health, Bethesda, Maryland). For each section analyzed, cortical regions of interest containing positive cells were manually selected using Hoechst staining of the nuclei. Then, for each region, we used a combination of ImageJ built-in minimum and unsharp mask filters to enhance the signal due to fluorescent cell bodies while lowering the signal due to fluorescent processes. Cells were automatically counted as local maxima, while keeping the same level of noise tolerance for a given set of experiments (and after validation of this level by manual counting of three to four sections). To normalize the analysis, we used median sections in a series of sections containing transfected cells (two sections per brain at E20, separated by at least 48 mm). Therefore, brains that did not meet these two criteria were discarded.

For analysis of neuronal morphology at P7, neurites were traced and quantified with NeuronJ software (http://rsb.info.nih.gov/ij/) (Meijering et al., 2004). Total neurite length and number of branches for each individual neuron were measured.

Figure 1. CPE is expressed in neurons in the developing rodent brain. (A) CPE protein expression increases developmentally in mouse cortex. Proteins were extracted from cortices of mice at the indicated embryonic (E), postnatal (P) or adult ages and were resolved by SDS-PAGE. Proteins were transferred to membranes and immunoblotted for CPE and GAPDH. Representative blots are shown. (B) Quantitation of CPE expression levels. Intensities of CPE bands were quantitated and normalized to GAPDH intensities. Ratio of CPE levels at indicated developmental ages to adult levels is shown. Error bars indicate ± S.E.M. n=3 for all conditions. ^†p < 0.0001 (E12, E14 and E16 vs. P0 or P10) as determined by one-way ANOVA followed by Tukey’s multiple comparisons test. ^*p< 0.05 as determined by one-way ANOVA followed by Tukey’s multiple comparisons test. (C) CPE is expressed predominantly in neurons in the cortical plate (CP). Coronal sections of E18 rat lateral neocortex were immunostained with anti-CPE (red) and anti-Tuj 1 (neuron-specific Class III β-tubulin; green). Images were taken using confocal microscopy. Scale bar = 50μm. VZ, ventricular zone; IZ, intermediate zone; CP, cortical plate. (D) Representative images showing endogenous CPE protein distribution in rat hippocampal neurons at DIV 10. Neurons were immunostained for CPE (green) and β-III tubulin (red). Scale bar = 50μm.

Figure 2. CPE is required for proper cortical neuron migration. (A) CPE shRNA knockdown efficiency in cortical neurons. E18 cortical neurons were electroporated with pGE-control shRNA (CTL shRNA) or pGE-CPE shRNA (CPE shRNA) before plating using Amaxa Rat Neuron transfection kit. After 96 h, cells were lysed, and extracted proteins were resolved using SDS-PAGE. Proteins were transferred to membrane and probed with antibodies to CPE and GAPDH. Representative blots are shown. (B) Quantitation of CPE protein levels relative to control (normalized to GAPDH). Error bars indicate ± S.E.M. n=3. ^*p < 0.05 by Student’s t-test. (C) CPE shRNA knockdown specificity in embryonic neocortex. CPE shRNA construct including GFP marker was electroporated into the lateral ventricle of E14.5 mice and analyzed 3 days later. Transfected cells were GFP-positive (green), CPE protein (red) was detected by immunostaining. Representative images of coronal sections of the lateral neocortex are shown. GFP-positive cells (marked by the dotted circles) in the left image show low levels of CPE protein as demonstrated on the right. Scale bar = 25μm. (D) Mice at E14.5 were electroporated in utero with constructs as indicated, and brains were analyzed at E17.5. Representative images of coronal brain sections are shown for each condition. Red, transfected cells; Blue, Hoechst dye. VZ, ventricular zone; IZ, intermediate zone; CP, cortical plate. Scale bar = 50μm. (E) Quantitation of the percentage of transfected cells in each cortical area. Error bars indicate ± S.E.M. n=7, CTL shRNA; n=7, CPE shRNA. ^****p < 0.0001 as determined by two-way ANOVA followed by Sidak’s multiple comparisons test. (F) Representative images of transfected cells in the IZ are shown for each condition. Arrows point to unipolar or bipolar cells; Arrowheads point to multipolar cells. Scale bar = 20μm. (G) Quantitation of percentage of multipolar cells in each condition. Error bars indicate ± S.E.M. n=7, CTL shRNA; n=7, CPE shRNA. ^**p < 0.01 as determined by unpaired Student’s t-test.

Figure 3. Knockdown of CPE decreases dendritic branching in cortical neurons in vivo and in cultured hippocampal neurons in vitro. (A) Mice at E14.5 were electroporated in utero with constructs as indicated, and neurons within cortical layer II/III were analyzed at P7. Representative tracings of dendrites are shown for each condition. Scale bar = 50μm. (B, C) Quantitation of the number of dendrites (B) and total dendrite length (C) of neurons in each condition. Error bars indicate ± S.E.M. n (neurons) = 26, CTL shRNA; n=29, CPE shRNA; n=50, CPE shRNA+Rescue. ^*p < 0.05, ^**p < 0.01 as determined by one-way ANOVA followed by Dunnett’s multiple comparisons test. (D) Hippocampal neurons were co-transfected with pCAG-mOrange and negative control siRNA (CTL siRNA) or CPE siRNA as indicated at DIV 7. Neurons were fixed and immunostained for GFP and MAP2 at DIV 10. Neurons positive for both GFP and mOrange were assessed for Sholl analysis. Representative mOrange fluorescent images of neurons are shown as inverted black images. Scale bar = 50μm. (E) Sholl analysis of neurons transfected with the indicated siRNA. Purple line indicates p value is at least less than 0.001 as determined by two-way ANOVA followed by Sidak’s multiple comparisons test. (F) Sholl analysis within 60μm from soma. n (neurons)=84, CTL siRNA; n=95, CPE siRNA. ^*p < 0.05, ^**p< 0.01 as determined by two-way ANOVA followed by Sidak’s multiple comparisons test. (G) Representative images showing CPE siRNA knockdown efficiency in transfected neurons. Hippocampal neurons were co-transfected with pCAG-mOrange (red) and either CTL siRNA or CPE siRNA at DIV 7, fixed at DIV 10, and immunostained for CPE (green). Scale bar = 50μm. (H) Quantitation of CPE fluorescence intensity in the cell body of transfected neurons. Error bars indicate ± S.E.M. n (neurons)=12, CTL siRNA; n=19, CPE siRNA. ^*p < 0.05 as determined by Student’s t-test.
 

Figure 4. CPE interacts with p150^Glued and overexpression of the carboxyl terminus of CPE redistributes p150^Glued from the centrosome. (A) CPE and p150^Glued co-immunoprecipitate from rat cortical lysate. Adult rat cortices were homogenized. Proteins were extracted and subjected to immunoprecipitation with anti-p150^Glued or rabbit-IgG. Cortical protein extract (Load), p150^Glued immunoprecipitates (IP; p150^Glued) and rabbit-IgG immunoprecipitates (IP; IgG) were resolved by SDS-PAGE. Proteins were transferred to membrane and immunoblotted for CPE and p150^Glued. Load represents 5% of extracted proteins. Representative blots are shown. (B) Quantitation of CPE band intensities, represented in panel A. Error bars indicate ± S.E.M. n=3. ^*p< 0.05 as determined by paired Student’s t-test. (C) Representative images showing p150^Glued localization in COS-7 cells. COS-7 cells were transfected with pEGFP (GFP) or a construct encoding the carboxyl terminal 10 amino acids of CPE (pEGFP-CPE-C10 referred to as CPE-C10) as indicated. Cells were fixed after 48 h and immunostained with anti-GFP (green), anti-p150^Glued (red), anti-pericentrin (white), and nuclei were labeled with Hoechst dye (blue). Scale bar = 10μm. (D) Quantitation of the percentage of p150^Glued protein (pixels) localized at the centrosome. For each independent experiment, 15-25 individual transfected cells of each condition were analyzed as described in Methods and Materials. Error bars indicate ± S.E.M. n=4 for both conditions. ^*p< 0.05 as determined by unpaired Student’s t-test.

 

Figure 5. Overexpression of the carboxyl terminus of CPE disrupts neuronal migration and dendritic arborization. (A) Mice at E14.5 were electroporated in utero with constructs as indicated, and brains were analyzed at E17.5. Representative images of coronal brain sections are shown for each condition. Red, transfected cells; Blue, Hoechst dye. VZ, ventricular zone; IZ, intermediate zone; CP, cortical plate. Scale bar = 50μm. (B) Quantitation of the percentage of transfected cells in each cortical area. Error bars indicate ± S.E.M. n=8, GFP; n=6, CPE-C10 (carboxyl terminal 10 amino acids of CPE).^**p < 0.01 as determined by two-way ANOVA followed by Sidak’s multiple comparisons test. (C) Representative images of transfected cells in the IZ are shown for each condition. Arrows point to unipolar or bipolar cells; Arrowheads point to multipolar cells. Scale bar =20μm. (D) Quantitation of percentage of multipolar cells in each condition. Error bars indicate ± S.E.M. n=10, GFP; n=6, CPE-C10. ^**p < 0.01 as determined by Student’s t-test. (E) Hippocampal neurons were co-transfected with pCAG-mOrange and pEGFP (GFP) or pEGFP-CPE-C10 (CPE-C10) as indicated at DIV 7. Neurons were fixed and immunostained for GFP and MAP2 at DIV 10. Neurons positive for both GFP and mOrange were assessed for Sholl analysis. Representative mOrange fluorescent images of neurons are shown as inverted black images. Scale bar = 50μm. (F) Sholl analysis of neurons expressing GFP or CPE-C10 on dendrite branching. Red line indicates p value is at least less than 0.01 as determined by two-way ANOVA followed by Sidak’s multiple comparisons test. (G) Sholl analysis within 60μm from soma. Error bars indicate ± S.E.M. n (neurons)=95, GFP; n=90, CPE-C10. ^*p < 0.05, ^**p < 0.01, ^***p< 0.001 as determined by two-way ANOVA followed by Sidak’s multiple comparisons test.

Figure 6. Model of CPE and p150^Glued interaction in regulating cortical neuron migration and dendrite morphology. (A) Under normal conditions, CPE regulates p150^Glued localization via its carboxyl terminus to maintain necessary stability and dynamics of microtubules (MTs), thus ensuring proper cortical migration and dendrite morphology. (B) When CPE protein is knocked down or when CPE-C10 is overexpressed and competes endogenous CPE for binding to p150^Glued, the normal interaction between CPE and p150^Glued is disrupted, resulting in abnormal neuronal migration and decreased dendrite branching.