Alternative splicing of APOER2 across the evolution of the vertebrate brain and its relevance to Alzheimer's disease

Alternative splicing is a key mechanism by which eukaryotes generate phenotypic complexity without increasing genomic load. In vertebrate evolution, cassette exon alternative splicing is prominent with increasing phenotypic complexity and is specifically enriched in the brain. Apolipoprotein receptor 2 (Apoer2) is a neuronal alternatively spliced transmembrane receptor that binds critical extracellular ligands such as neuroprotective Reelin and Alzheimer’s disease (AD) related risk factor APOE4. Inclusion and exclusion of single exons in Apoer2 regulates isoform specific roles in neuronal processes, such as long-term potentiation (LTP) and neuronal survival. Alternative splicing of APOER2 exon 18, which encodes a functional domain critical for LTP, has been reported as dysregulated in AD. However, the full repertoire and function of APOER2 isoforms in physiological and AD conditions is not well understood. We hypothesize that combinatorial APOER2 alternative splicing events generate a diverse pool of isoforms in the human brain that can become dysregulated in AD and alter receptor function in neurons. Our overall goal is to define the APOER2 transcript pool and understand whether isoform proportions and functions are altered in AD, potentially contributing to synaptic dysfunction. In this work, we observed that Apoer2 has evolved over the course of vertebrate evolution, gaining new exons that alter function at the protein level and increasing the complexity of its alternative splicing events from zebrafish to humans. We generated the first APOER2 specific long-read RNA sequencing dataset in the human cerebral cortex, which identified 48 full-length APOER2 isoforms, some of which are unique compared to full-length murine Apoer2 isoforms and indicate that Apoer2 is spliced in a species specific manner. To determine whether splicing of APOER2 is dysregulated in AD, we generated full-length APOER2 isoform maps in Control and AD parietal cortex and hippocampus. We identified over 200 unique APOER2 isoforms in each brain region with 151 isoforms common between the two brain regions. We also identified region and disease specific APOER2 isoforms suggesting APOER2 splicing is spatially regulated and altered in AD. We found AD and Control-specific APOER2 isoforms exhibited alterations in receptor processing and cleavage patterns, indicating combinatorial splicing across APOER2 dictates protein function and is changed in AD. Sequential cleavage of Apoer2 in response to Reelin generates an intracellular domain (ICD) that translocates to the nucleus and affects transcription; however, whether APOE influences Apoer2 cleavage is unclear. We found Apoer2-ICD is generated in an APOE isoform specific manner and is generated regardless of exon 19 inclusion, which encodes part of the ICD. We generated four novel mouse lines to examine the effects of Apoer2 exon 19 inclusion and APOE isoforms (APOE3 and APOE4) on hippocampal gene expression. We found Apoer2 exon 19 inclusion modulates upregulation of genes such as Serpina3n known to be induced by APOE4 expression, which has strong implications for understanding molecular mechanisms underlying APOE4 as a risk factor in AD. Lastly, since Apoer2 exon 19 confers critical functions at the protein level, including adaptor protein binding and association with the NMDA receptor, as well as potentially modulating APOE4’s transcriptional effects, we were interested in how an RNA binding protein, Srsf1, may influence Apoer2 exon 19 splicing. We and others have found SRSF1 partially represses exon 19 inclusion in primary murine neurons. Because splicing is often modulated by neuronal activity, we examined whether Apoer2 exon 19 and Srsf1 are altered in response to activity stimulation. We found upregulation of exon 19 exclusion and no strong changes in SRSF1 expression or phosphorylation, suggesting modulation of SRSF1 is not a potent regulatory mechanism of activity induced changes in Apoer2 exon 19 splicing. Overall, we have examined the Apoer2 splicing landscape in the brain across multiple vertebrate species. We identified a rich diversity of alternatively spliced APOER2 isoforms in Control and AD brains providing novel APOER2 variants that are significantly changed in AD. These AD related APOER2 isoforms have differential functional impacts on APOER2 biology that may contribute to AD pathogenesis

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

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

Application of error-prone PCR to functionally probe the morbillivirus Haemagglutinin protein.

The enveloped morbilliviruses utilise conserved proteinaceous receptors to enter host cells: SLAMF1 or Nectin-4. Receptor binding is initiated by the viral attachment protein Haemagglutinin (H), with the viral Fusion protein (F) driving membrane fusion. Crystal structures of the prototypic morbillivirus measles virus H with either SLAMF1 or Nectin-4 are available and have served as the basis for improved understanding of this interaction. However, whether these interactions remain conserved throughout the morbillivirus genus requires further characterisation. Using a random mutagenesis approach, based on error-prone PCR, we targeted the putative receptor binding site for SLAMF1 interaction on peste des petits ruminants virus (PPRV) H, identifying mutations that inhibited virus-induced cell-cell fusion. These data, combined with structural modelling of the PPRV H and ovine SLAMF1 interaction, indicate this region is functionally conserved across all morbilliviruses. Error-prone PCR provides a powerful tool for functionally characterising functional domains within viral proteins

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

Characterization of 23 polymorphic SSR markers in Salix humboldtiana (Salicaceae) using next‐generation sequencing and cross‐amplification from related species

Premise of the study: We present a set of 23 polymorphic nuclear microsatellite loci, 18 of which are identified for the first time within the riparian species Salix humboldtiana (Salicaceae) using next‐generation sequencing. Methods and Results: To characterize the 23 loci, up to 60 individuals were sampled and genotyped at each locus. The number of alleles ranged from two to eight, with an average of 4.43 alleles per locus. The effective number of alleles ranged from 1.15 to 3.09 per locus, and allelic richness ranged from 2.00 to 7.73 alleles per locus. Conclusions: The new marker set will be used for future studies of genetic diversity and differentiation as well as for unraveling spatial genetic structures in S. humboldtiana populations in northern Patagonia, Argentina.EEA BarilocheFil: Bozzi, Jorge Alfredo. Instituto Nacional de Tecnología Agropecuaria (INTA). Estación Experimental Agropecuaria Bariloche; ArgentinaFil: Liepelt, Sascha. University of Marburg. Faculty of Biology. Conservation Biology Group; AlemaniaFil: Ohneiser, Sebastian. University of Marburg. Faculty of Biology. Conservation Biology Group; AlemaniaFil: Gallo, Leonardo Ariel. Instituto Nacional de Tecnología Agropecuaria (INTA). Estación Experimental Agropecuaria Bariloche; ArgentinaFil: Marchelli, Paula. Instituto Nacional de Tecnología Agropecuaria (INTA). Estación Experimental Agropecuaria Bariloche; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Leyer, Ilona. University of Marburg. Faculty of Biology. Conservation Biology Group; Alemania. University of Geisenheim. Institute of Botany, Plant Ecology and Nature Conservation; AlemaniaFil: Ziegenhagen, Birgit. University of Marburg. Faculty of Biology. Conservation Biology Group; AlemaniaFil: Mengel, Christina. University of Marburg. Faculty of Biology. Conservation Biology Group; Alemani

Concurrent Measurement of O2 Production and Isoprene Emission During Photosynthesis: Pros, Cons and Metabolic Implications of Responses to Light, CO2 and Temperature

Traditional leaf gas exchange experiments have focused on net CO2 exchange (Anet). Here, using California poplar (Populus trichocarpa), we coupled measurements of net oxygen production (NOP), isoprene emissions and δ18O in O2 to traditional CO2/H2O gas exchange with chlorophyll fluorescence, and measured light, CO2 and temperature response curves. This allowed us to obtain a comprehensive picture of the photosynthetic redox budget including electron transport rate (ETR) and estimates of the mean assimilatory quotient (AQ = Anet/NOP). We found that Anet and NOP were linearly correlated across environmental gradients with similar observed AQ values during light (1.25 ± 0.05) and CO2 responses (1.23 ± 0.07). In contrast, AQ was suppressed during leaf temperature responses in the light (0.87 ± 0.28), potentially due to the acceleration of alternative ETR sinks like lipid synthesis. Anet and NOP had an optimum temperature (Topt) of 31°C, while ETR and δ18O in O2 (35°C) and isoprene emissions (39°C) had distinctly higher Topt. The results confirm a tight connection between water oxidation and ETR and support a view of light-dependent lipid synthesis primarily driven by photosynthetic ATP/NADPH not consumed by the Calvin-Benson cycle, as an important thermotolerance mechanism linked with high rates of (photo)respiration and CO2/O2 recycling

Proper Sterol Distribution Is Required for Candida albicans Hyphal Formation and Virulence

Candida albicans is an opportunistic fungus responsible for the majority of systemic fungal infections. Multiple factors contribute to C. albicans pathogenicity. C. albicans strains lacking CaArv1 are avirulent. Arv1 has a conserved Arv1 homology domain (AHD) that has a zinc-binding domain containing two cysteine clusters. Here, we explored the role of the CaAHD and zinc-binding motif in CaArv1-dependent virulence. Overall, we found that the CaAHD was necessary but not sufficient for cells to be virulent, whereas the zinc-binding domain was essential, as Caarv1/Caarv1 cells expressing the full-length zinc-binding domain mutants, Caarv1C3S and Caarv1C28S, were avirulent. Phenotypically, we found a direct correlation between the avirulence of Caarv1/Caarv1, Caarrv1AHD, Caarv1C3S, and Caarv1C28S cells and defects in bud site selection, septa formation and localization, and hyphal formation and elongation. Importantly, all avirulent mutant strains lacked the ability to maintain proper sterol distribution. Overall, our results have established the importance of the AHD and zinc-binding domain in fungal invasion, and have correlated an avirulent phenotype with the inability to maintain proper sterol distribution

Characterizing Sterol Defect Suppressors Uncovers a Novel Transcriptional Signaling Pathway Regulating Zymosterol Biosynthesis

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