Identification of novel apolipoprotein E receptor 2 splice variants and their role in synaptic transmission

Apolipoprotein E (APOE) is one of the most important genetic risk factors for late-onset sporadic Alzheimer’s disease (LOAD). APOE is a 35 kDa glycoprotein and ligand known to bind to members of the low-density lipoprotein receptor (LDLR) family, including APOE receptor 2 (apoER2; official gene name LRP8). ApoER2 is a type I transmembrane protein with a large extracellular domain (ECD) and a short cytoplasmic tail that can be proteolytic cleaved. In addition, apoER2 is enriched in the brain and plays an important role in synaptic function and plasticity. Interestingly, the ECD of apoER2 contains several ligand binding repeats that are organized into exons with aligning phase junctions, which allows exon skipping during alternative splicing to retain protein fidelity. The amount of alternative spliced isoforms distinguishes apoER2 from the rest of the LDLR family members. In fact, mouse Apoer2 has been identified as one of the top ten neuronal genes related to cell-type exon skipping events. Regarding human APOER2, we have identified over 40 different APOER2 isoforms from human brain using gene-specific primers and amplifying the N- and C-terminal open reading frame of APOER2. The majority of APOER2 variants consist of a diverse array of exon skipping events within the ligand binding domain (LBD). We therefore, hypothesized that human APOER2 splice variants act as functionally divergent isoforms that can influence ligand binding properties, receptor proteolysis and changes to synaptic function. ApoER2 undergoes sequential proteolytic cleavage in response to ligand binding, resulting in the release of C-terminal fragments (CTFs) and transcriptionally active intracellular domain (ICD). We therefore, systematically tested whether the diversity of human N-terminal APOER2 splice variants lacking various LBDs affects APOER2 cleavage and signaling events. We found that alternative splicing of certain APOER2 exons generated different amounts of CTFs compared to full-length APOER2 (APOER2-FL). The pattern was not simply based on the number of ligand binding domains suggesting that excision of certain exons may alter the tertiary structure of the receptor sufficiently to make the receptor more or less accessible to cleavage and generation of CTFs. To further characterize APOER2 splice variants, we specifically examined APOER2 splice variants that generated the highest and lowest amounts of CTF generation compared to APOER2-FL and focused on APOER2 splice variant lacking exons 5-8 (Δ5-8) and lacking exons 4-6 (Δ4-6), respectively. The differential CTF generation of APOER2 Δ5-8 and Δ4-6 reflects the proteolytic release of the APOER2-ICD. This APOER2-ICD mediates transcriptional activation, facilitated by the Mint1 adaptor protein. To investigate whether human N-terminal APOER2 splice variants influence APOE binding and receptor cleavage properties, we used microscale thermophoresis and tested the well-validated human APOE mimetic peptide. We found that specific exons or ligand-binding cassettes differentially affect APOE peptide binding to APOER2 splice variants. In addition, APOE peptide induces generation of APOER2-CTF acutely within one hour. Functionally, we demonstrated that APOER2 is required for spontaneous neurotransmitter release in mature neurons. Loss of mouse Apoer2 robustly decreased miniature event frequency in excitatory synapses compared to heterozygous Apoer2 neurons. We found APOER2-FL fully restored the miniature event frequency in excitatory synapses but not APOER2 Δ5-8. APOER2 Δ4-6 restored the miniature event frequency similar to heterozygous Apoer2 neurons. These results suggest that different human N-terminal APOER2 splice variants have distinct and differential synaptic properties signifying a role of APOER2 splice variants as regulators of synaptic function

CLASP2 links Reelin to the cytoskeleton during neocortical development

Published in final edited form as: Neuron. 2017 March 22; 93(6): 1344–1358.e5. doi:10.1016/j.neuron.2017.02.039.INTRODUCTION The complex architecture of the brain requires precise control over the timing of neurogenesis, neuron migration, and differentiation. These three developmental processes are exquisitely controlled during the expansion of the mammalian neocortex. The six morphologically distinct layers of the neocortex form in an “inside-out” pattern with early-born neurons forming deeper layers and later-born neurons migrating past them to form superficial layers of the cortical plate (Rakic, 1974). The Reelin signaling pathway plays a crucial role in cortical lamination. Reelin is a secreted glycoprotein that exerts its function by binding to the lipoprotein receptors ApoER2 and VLDLR and inducing tyrosine phosphorylation of the intracellular adaptor protein Disabled (Dab1) (Howell et al., 2000, Bock and Herz, 2003). Phosphorylated Dab1 then recruits downstream signaling molecules to promote cytoskeletal changes necessary for neuronal migration, final positioning, and morphology (D’Arcangelo, 2005). Mutations of Reelin, the dual ApoER2/VLDLR receptor, or Dab1 lead to an inversion of the normal inside-out pattern of cortex development (D’Arcangelo et al., 1995, Howell et al., 1997, Trommsdorff et al., 1999). In addition, a number of mutations in cytoskeletal-encoded genes produce deficits in neuron migration and cortical lamination phenotypically similar to Reelin mutants, firmly establishing a mechanistic and developmentally critical connection between Reelin and the cytoskeleton. For example, human mutations in lissencephaly-1, doublecortin, and tubulin, integral components of the microtubule cytoskeleton, cause severe cortical lamination defects with later-born neurons failing to migrate past previously born neurons (Reiner et al., 1993, Gleeson et al., 1998, Romaniello et al., 2015). The culmination of these genetic studies indicates that several signaling pathways, including the Reelin pathway, converge on downstream cytoskeletal proteins to affect proper neuronal migration and brain development. However, the molecular effectors of these pathways have not been fully characterized. CLASPs (cytoplasmic linker associated proteins) belong to a heterogeneous family of plus-end tracking proteins (+TIPs) that specifically accumulate at the growth cone. This localization strategically places them in a position to control neurite growth, directionality, and the crosstalk between microtubules and the actin cytoskeleton (Akhmanova and Hoogenraad, 2005, Basu and Chang, 2007, Akhmanova and Steinmetz, 2008). Previous evidence showed that CLASPs accumulate asymmetrically toward the leading edge of migrating fibroblasts, indicating a role for CLASPs in cell polarity and movement (Akhmanova et al., 2001, Wittmann and Waterman-Storer, 2005). We found that CLASP2 protein levels steadily increase throughout neuronal development and are specifically enriched at the growth cones of extending neurites. In particular, short-hairpin RNA (shRNA)-mediated knockdown of CLASP2 in primary mouse neurons decreases neurite length, whereas overexpression of human CLASP2 causes the formation of multiple axons, enhanced dendritic branching, and Golgi condensation (Beffert et al., 2012). These results implicate a role for CLASP2 in neuronal morphogenesis and polarization; however, the function of CLASP2 during brain development is unknown. Here we demonstrate that CLASP2 is a modifier of the Reelin signaling pathway during cortical development. In vivo knockdown experiments demonstrate that CLASP2 plays significant roles in neural precursor proliferation, neuronal migration, and morphogenesis. In addition, we show that GSK3β-mediated phosphorylation of CLASP2 controls its binding to the Reelin adaptor protein Dab1, a required molecular step governing CLASP2’s regulatory effects on neuron morphology and movement. RESULTS CLASP2 Expression Is Functionally Associated with the Reelin Signaling Pathway To identify novel genes downstream of Reelin signaling, we examined the expression of mRNA transcripts by microarray between adult brain cortices from mice deficient in either Reelin, the double ApoER2/VLDLR receptor mutant, or Dab1 and compared Affymetrix gene expression profiles against age-matched, wild-type mice. Importantly, each of these mutant mouse models present a similar phenotype that includes severe neuronal migration defects (D’Arcangelo et al., 1995, Howell et al., 1997, Trommsdorff et al., 1999). We defined a large network of genes perturbed above a threshold of 1.5-fold in response to deficient Reelin signaling, identifying 832 upregulated and 628 downregulated genes that were common to all three mouse models (Figure 1A). Ingenuity Pathway Analysis revealed a network of genes that is functionally related to cytoskeleton organization, microtubule dynamics, neurogenesis, and migration of cells (Figure 1B). Of the few cytoskeletal candidate genes identified, CLASP2 was the only microtubule +TIP. Specifically, CLASP2 mRNA expression was increased in all three Reelin mutant phenotypes, while CLASP1 mRNA expression remained unchanged (Figure 1B). Consistent with the microarray data, CLASP2 protein levels were ∼2.8-fold higher in Dab1 knockout mice (Figure 1C). These findings suggest that Reelin signaling controls CLASP2 expression and establishes the first molecular link between a plus-end, microtubule binding protein downstream of extracellular Reelin signaling.We thank Drs. Thomas C. Sudhof, Joachim Herz, Santos Franco, and Torsten Wittmann for plasmids and antibodies. We thank Alicia Dupre, Elias Fong, and Christine Learned for technical support. This work was supported by grants from the National Institutes of Health (R21 MH100581 to T.F.H., U.B., and A.H.). (R21 MH100581 - National Institutes of Health)Accepted manuscrip

COE Loss-of-Function Analysis Reveals a Genetic Program Underlying Maintenance and Regeneration of the Nervous System in Planarians

<div><p>Members of the COE family of transcription factors are required for central nervous system (CNS) development. However, the function of COE in the post-embryonic CNS remains largely unknown. An excellent model for investigating gene function in the adult CNS is the freshwater planarian. This animal is capable of regenerating neurons from an adult pluripotent stem cell population and regaining normal function. We previously showed that planarian <i>coe</i> is expressed in differentiating and mature neurons and that its function is required for proper CNS regeneration. Here, we show that <i>coe</i> is essential to maintain nervous system architecture and patterning in intact (uninjured) planarians. We took advantage of the robust phenotype in intact animals to investigate the genetic programs <i>coe</i> regulates in the CNS. We compared the transcriptional profiles of control and <i>coe</i> RNAi planarians using RNA sequencing and identified approximately 900 differentially expressed genes in <i>coe</i> knockdown animals, including 397 downregulated genes that were enriched for nervous system functional annotations. Next, we validated a subset of the downregulated transcripts by analyzing their expression in <i>coe</i>-deficient planarians and testing if the mRNAs could be detected in <i>coe⁺</i> cells. These experiments revealed novel candidate targets of <i>coe</i> in the CNS such as ion channel, neuropeptide, and neurotransmitter genes. Finally, to determine if loss of any of the validated transcripts underscores the <i>coe</i> knockdown phenotype, we knocked down their expression by RNAi and uncovered a set of <i>coe-</i>regulated genes implicated in CNS regeneration and patterning, including orthologs of <i>sodium channel alpha-subunit</i> and <i>pou4</i>. Our study broadens the knowledge of gene expression programs regulated by COE that are required for maintenance of neural subtypes and nervous system architecture in adult animals.</p></div

CNS regeneration defects following knockdown of COE-regulated genes.

<p>(<b>A–D</b>) Animals were fed control, <i>scna-2</i>, <i>nkx2l</i> and <i>pou4l-1</i> bacterially-expressed dsRNA (indicated to the left of each panel), amputated pre-pharyngeally and allowed to regenerate. Ten-day regenerates were imaged live (A–D), killed and immunostained with anti-SYNAPSIN or processed for fluorescent <i>in situ</i> hybridization to <i>ChAT</i> or <i>npl</i> (N≥4). (<b>E–F</b>) Brain size estimated by measuring head area stained by anti-SYNAPSIN or <i>in situ</i> hybridization to <i>ChAT</i> and normalized by the length of animal for <i>control</i>, <i>scna-2</i>, <i>nkx2l</i>, <i>and pou4l-1</i> RNAi planarians. (<b>G</b>) Quantification of <i>npl⁺</i> cells normalized by brain size measured from <i>ChAT</i> stain in F (N≥4 animals in each group); the total number of <i>npl⁺</i> cells counted is indicated within each bar. Error bars in all graphs are s.d. from the mean; *P<0.05, Student's t-test. Anterior is up in A–D. Scale bars = 100 µm.</p

COE function is required for differentiation and maintenance of diverse neuron types.

<p>(<b>A</b>) <i>coe</i> is expressed in lineage-committed neoblasts (<i>smedwi⁺</i>) and early progeny <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004746#pgen.1004746-Cowles1" target="_blank">[24]</a>, and diverse neuron types, including cholinergic (<i>ChAT</i>), GABAergic (<i>gad</i>), octopaminergic (<i>tbh</i>), dopaminergic (<i>th</i>), serotonergic (<i>tph</i>), and neuropeptidergic (<i>cpp-1</i>, <i>npl</i>, <i>spp-18</i>, <i>spp-19</i>, <i>spp-2</i>) neurons. Genes in green were identified in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004746#pgen.1004746-Cowles1" target="_blank">[24]</a>. (<b>B</b>) To gain insights into how loss of COE function contributes to defects in nervous system differentiation, we analyzed the function of genes that were downregulated in <i>coe(RNAi)</i> animals. These analyses identified additional genes required for CNS regeneration (<i>gbrb1</i>, <i>npl</i>, <i>scna-2</i>, <i>scna-3</i>, <i>pou4l-1</i>) and patterning (<i>nkx2l</i>). In <i>coe(RNAi)</i> animals, we also detected upregulated genes enriched for GO terms associated with muscle development (<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004746#pgen-1004746-t001" target="_blank">Table 1</a>), suggesting that COE may also function to repress the expression of mesoderm-specific genes.</p

Annotation of genes differentially expressed in <i>coe(RNAi)</i> animals using DAVID software.

<p>Annotation of genes differentially expressed in <i>coe(RNAi)</i> animals using DAVID software.</p

Functional analysis of genes downregulated following <i>coe</i> RNAi.

<p>The number of animals showing the phenotype(s) among the total number examined from at least two independent experiments is indicated in parentheses.</p><p>Functional analysis of genes downregulated following <i>coe</i> RNAi.</p

<i>coe</i> RNAi strongly inhibits the expression of <i>ChAT</i> in intact planarians.

<p>(<b>A–C</b>) <i>coe</i> RNAi-treated animals were processed for fluorescent <i>in situ</i> hybridization (FISH) to <i>ChAT</i> (N = 10 for each treatment), <i>mat</i> (N = 3 control and 4 RNAi planarians), or <i>collagen</i> (N = 7 control and 5 RNAi). White dashed boxes in A denote regions imaged at higher magnification shown in the panels to the right. Black dashed boxes in C denote regions imaged at higher magnification shown in top right insets. (<b>D</b>) RT-qPCR experiments measuring the relative expression of <i>coe</i>, <i>ChAT</i>, <i>mat</i>, or <i>collagen</i> in <i>control(RNAi)</i> or <i>coe(RNAi)</i> planarians following the 6^th RNAi treatment. Graph shows the mean ± s.d. expression levels relative to the controls. *P<0.05, Student's t-test.</p

<i>coe</i> is expressed in the nervous system and a subset of cycling stem cells.

<p>(<b>A</b>) <i>In situ</i> hybridization to <i>coe</i> in <i>S. mediterranea</i> (vn, ventral nerve cords; p, pharynx). Dashed boxes show regions imaged in B–C (N≥10). (<b>B–C</b>) Double-fluorescent <i>in situ</i> hybridization to <i>coe</i> and <i>h2b</i>. Arrowheads mark examples of double-labeled cells (N = 14). Anterior is up in all panels. Scale bars, A = 200 µm, B = 100 µm.</p

Identification of genes expressed <i>coe⁺</i> neurons.

<p>Fluorescent <i>in situ</i> hybridization to <i>coe</i> and either <i>spp-19</i>, <i>spp18</i>, <i>npl</i>, <i>spp-2</i>, <i>ncam-2</i>, or <i>netrin-1</i>. Percentages indicate the proportion ± s.d. of cells that were also <i>coe⁺</i> (N = 110 <i>spp-19⁺</i>, 319 <i>spp-18⁺</i>, 173 <i>npl⁺</i>, 202 <i>spp-2</i>, 236 <i>ncam-2</i>, and 141 <i>netrin-1</i> cells counted from 2–3 animals per group). Arrowheads mark double-labeled cells. Anterior is up in all panels. Scale bars = 100 µm.</p