Single molecule, long-read Apoer2 sequencing identifies conserved and species-specific splicing patterns

Apolipoprotein E receptor 2 (Apoer2) is a synaptic receptor in the brain that binds disease-relevant ligand Apolipoprotein E (Apoe) and is highly alternatively spliced. We examined alternative splicing (AS) of conserved Apoer2 exons across vertebrate species and identified gain of exons in mammals encoding functional domains such as the cytoplasmic and furin inserts, and loss of an exon in primates encoding the eighth LDLa repeat, likely altering receptor surface levels and ligand-binding specificity. We utilized single molecule, long-read RNA sequencing to profile full-length Apoer2 isoforms and identified 68 and 48 unique full-length Apoer2 transcripts in the mouse and human cerebral cortex, respectively. Furthermore, we identified two exons encoding protein functional domains, the third EGF-precursor like repeat and glycosylation domain, that are tandemly skipped specifically in mouse. Our study provides new insight into Apoer2 isoform complexity in the vertebrate brain and highlights species-specific differences in splicing decisions that support functional diversity.Published versio

ApoER2: functional tuning through splicing

Alternative splicing occurs in over 95% of protein-coding genes and contributes to the diversity of the human proteome. Apolipoprotein E receptor 2 (apoER2) is a critical modulator of neuronal development and synaptic plasticity in the brain and is enriched in cassette exon splicing events, in which functional exons are excluded from the final transcript. These alternative splicing events affect apoER2 function, as individual apoER2 exons tend to encode distinct protein functional domains. Although several apoER2 splice variants have been characterized, much work remains to understand how apoER2 splicing events modulate distinct apoER2 activities, including ligand binding specificity, synapse formation and plasticity. Additionally, little is known about how apoER2 splicing events are regulated. Often, alternative splicing events are regulated through the combinatorial action of RNA-binding proteins and other epigenetic mechanisms, however, the regulatory pathways corresponding to each specific exon are unknown in most cases. In this mini-review, we describe the structure of apoER2, highlight the unique functions of known isoforms, discuss what is currently known about the regulation of apoER2 splicing by RNA-binding proteins and pose new questions that will further our understanding of apoER2 splicing complexity.R01 AG059762 - NIA NIH HHSPublished versio

Human APOER2 isoforms have differential cleavage events and synaptic properties

Human APOER2 is a type I transmembrane protein with a large extracellular domain (ECD) and a short cytoplasmic tail. APOER2-ECD contains several ligand binding domains (LBD) that are organized into exons with aligning phase junctions, which allows for in-frame exon cassette splicing events. We have identified 25 human APOER2 isoforms from cerebral cortex using gene-specific APOER2 primers, where the majority are exon-skipping events within the N-terminal LBD regions in comparison to 6 identified in the heart. APOER2 undergoes proteolytic cleavage in response to ligand binding that releases a C-terminal fragment (CTF) and transcriptionally active intracellular domain (ICD). We therefore tested whether the diversity of human brain-specific APOER2 variants affects APOER2 cleavage. We found exclusion of different ligand binding repeats from splicing generated different amounts of CTFs compared to full-length APOER2 (APOER2-FL). Specifically, APOER2 isoforms lacking exons 5-8 (Δex5-8) and lacking exons 4-6 (Δex4-6) generated the highest and lowest amounts of CTF generation respectively in response to APOE peptide compared to APOER2-FL. The differential CTF generation of APOER2 Δex5-8 and Δex4-6 coincides with the proteolytic release of the ICD which mediates transcriptional activation facilitated by the Mint1 adaptor protein. Functionally, we demonstrated loss of mouse Apoer2 decreased miniature event frequency in excitatory synapses suggesting that Apoer2 is required for spontaneous neurotransmitter release in mature neurons. Lentiviral rescue with human APOER2-FL or Δex4-6 isoform in Apoer2 knockout neurons fully restored the miniature event frequency but not Δex5-8 isoform. These results suggest that human APOER2 isoforms have differential cleavage events and synaptic properties.R01 AG059762 - NIA NIH HHSFirst author draf

The Pafah1b Complex Interacts with the Reelin Receptor VLDLR

Reelin is an extracellular protein that directs the organization of cortical structures of the brain through the activation of two receptors, the very low-density lipoprotein receptor (VLDLR) and the apolipoprotein E receptor 2 (ApoER2), and the phosphorylation of Disabled-1 (Dab1). Lis1, the product of the Pafah1b1 gene, is a component of the brain platelet-activating factor acetylhydrolase 1b (Pafah1b) complex, and binds to phosphorylated Dab1 in response to Reelin. Here we investigated the involvement of the whole Pafah1b complex in Reelin signaling and cortical layer formation and found that catalytic subunits of the Pafah1b complex, Pafah1b2 and Pafah1b3, specifically bind to the NPxYL sequence of VLDLR, but not to ApoER2. Compound Pafah1b1(+/−);Apoer2(−/−) mutant mice exhibit a reeler-like phenotype in the forebrain consisting of the inversion of cortical layers and hippocampal disorganization, whereas double Pafah1b1(+/−);Vldlr(−/−) mutants do not. These results suggest that a cross-talk between the Pafah1b complex and Reelin occurs downstream of the VLDLR receptor

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

Neuronal LRP1 functionally associates with postsynaptic proteins and is required for normal motor function in mice

The LDL receptor-related protein 1 (LRP1) is a multifunctional cell surface receptor that is highly expressed on neurons. Neuronal LRP1 in vitro can mediate ligand endocytosis, as well as modulate signal transduction processes. However, little is known about its role in the intact nervous system. Here, we report that mice that lack LRP1 selectively in differentiated neurons develop severe behavioral and motor abnormalities, including hyperactivity, tremor, and dystonia. Since their central nervous systems appear histoanatomically normal, we suggest that this phenotype is likely attributable to abnormal neurotransmission. This conclusion is supported by studies of primary cultured neurons that show that LRP1 is present in close proximity to the N-methyl-Daspartate (NMDA) receptor in dendritic synapses and can be coprecipitated with NMDA receptor subunits and the postsynaptic density protein PSD-95 from neuronal cell lysates. Moreover, treatment with NMDA, but not dopamine, reduces the interaction of LRP1 with PSD-95, indicating that LRP1 participates in transmitterdependent postsynaptic responses. Together, these findings suggest that LRP1, like other ApoE receptors, can modulate synaptic transmission in the brain.Published versio

The Reelin Receptors Apoer2 and Vldlr Coordinate the Patterning of Purkinje Cell Topography in the Developing Mouse Cerebellum

The adult cerebellar cortex is comprised of reproducible arrays of transverse zones and parasagittal stripes of Purkinje cells. Adult stripes are created through the perinatal rostrocaudal dispersion of embryonic Purkinje cell clusters, triggered by signaling through the Reelin pathway. Reelin is secreted by neurons in the external granular layer and deep cerebellar nuclei and binds to two high affinity extracellular receptors on Purkinje cells-the Very low density lipoprotein receptor (Vldlr) and apolipoprotein E receptor 2 (Apoer2). In mice null for either Reelin or double null for Vldlr and Apoer2, Purkinje cell clusters fail to disperse. Here we report that animals null for either Vldlr or Apoer2 individually, exhibit specific and parasagittally-restricted Purkinje cell ectopias. For example, in mice lacking Apoer2 function immunostaining reveals ectopic Purkinje cells that are largely restricted to the zebrin II-immunonegative population of the anterior vermis. In contrast, mice null for Vldlr have a much larger population of ectopic Purkinje cells that includes members from both the zebrin II-immunonegative and -immunopositive phenotypes. HSP25 immunoreactivity reveals that in Vldlr null animals a large portion of zebrin II-immunopositive ectopic cells are probably destined to become stripes in the central zone (lobules VI–VII). A small population of ectopic zebrin II-immunonegative Purkinje cells is also observed in animals heterozygous for both receptors (Apoer2+/−: Vldlr+/−), but no ectopia is present in mice heterozygous for either receptor alone. These results indicate that Apoer2 and Vldlr coordinate the dispersal of distinct, but overlapping subsets of Purkinje cells in the developing cerebellum

FOXR1 regulates stress response pathways and is necessary for proper brain development

The forkhead box (Fox) family of transcription factors are highly conserved and play essential roles in a wide range of cellular and developmental processes. We report an individual with severe neurological symptoms including postnatal microcephaly, progressive brain atrophy and global developmental delay associated with a de novo missense variant (M280L) in the FOXR1 gene. At the protein level, M280L impaired FOXR1 expression and induced a nuclear aggregate phenotype due to protein misfolding and proteolysis. RNAseq and pathway analysis showed that FOXR1 acts as a transcriptional activator and repressor with central roles in heat shock response, chaperone cofactor-dependent protein refolding and cellular response to stress pathways. Indeed, FOXR1 expression is increased in response to cellular stress, a process in which it directly controls HSPA6, HSPA1A and DHRS2 transcripts. The M280L mutant compromises FOXR1's ability to respond to stress, in part due to impaired regulation of downstream target genes that are involved in the stress response pathway. Quantitative PCR of mouse embryo tissues show Foxr1 expression in the embryonic brain. Using CRISPR/Cas9 gene editing, we found that deletion of mouse Foxr1 leads to a severe survival deficit while surviving newborn Foxr1 knockout mice have reduced body weight. Further examination of newborn Foxr1 knockout brains revealed a decrease in cortical thickness and enlarged ventricles compared to littermate wild-type mice, suggesting that loss of Foxr1 leads to atypical brain development. Combined, these results suggest FOXR1 plays a role in cellular stress response pathways and is necessary for normal brain development.R21 GM114629 - NIGMS NIH HHSPublished versio

Apolipoprotein E in Alzheimer's disease

Alzheimer's disease (AD) is the most important cause of dementia, but the mechanism of pathogenesis remains unknown. The epsilon4 allele of apolipoprotein E (apoE) has been confirmed as the most important genetic risk factor for most cases of AD and is associated with Abeta-containing senile plaques, one of the neuropathological hallmarks of AD. We determined the neuronal binding, internalization and degradation of human apoE isoforms apoE3 and apoE4 in the presence and absence of Abeta peptides using primary hippocampal neurons from rat. The findings demonstrate apoE binding and internalization in neurons without endogenous synthesis. Increased apoE3 degradation was observed relative to the apoE4 isoform. Lipophilic Abeta peptides increased apoE binding to hippocampal neurons; the increase was greater for the apoE4 isoform than for the apoE3 isoform. Abeta uptake by hippocampal neurons was increased in the presence of apoE, more so in the presence of the apoE4 than apoE3 isoform. Further, apoE was shown to reduce the extracellular level of Abeta in hippocampal cultures. We also find that all three major apoE isoforms were equally potent inhibitors of Abeta fibril formation, which is thought to be necessary for in vitro neurotoxicity and possibly the neuronal degeneration observed in AD. Further, when apoE was associated with cholesterol-containing liposomes or very low density lipoproteins, its ability to prevent Abeta fibril formation was diminished. In two brain regions affected by AD neuropathology, the hippocampus and frontal cortex, apoE levels were found to be decreased while Abeta levels were increased. Levels of apoE and Abeta1--40 were found to be apoE genotype dependent, with lowest levels of apoE and highest levels of Abeta occurring in epsilon4 allele carriers, suggesting that a reduction in apoE levels may give rise to increased deposition of amyloid peptides in AD brain. The low density lipoprotein receptor-related protein, an important neuronal a