Alternative splicing of Lymnaea Cav3 and NALCN ion channel genes serves to alter biophysical properties, membrane expression, and ion selectivity

Evidence is presented that Lymnaea contains homologues for mammalian Cav3 and NALCN 4-domain ion channels, which retain key amino acid sequence motifs that differentiate these channels from other 4-domain types. Molecular cloning and heterologous expression of the first invertebrate Cav3 channel cDNA from Lymnaea confirms that it indeed is a true homologue to mammalian Cav3 channels, retaining some hallmark biophysical and pharmacological features1. Interestingly, the Lymnaea Cav3 channel gene also exhibits alternative splicing that is conserved with mammalian Cav3.1 and Cav3.2 channels, with homologous exons 8b in the I-II linker (Cav3.1) and 25c in the III-IV linker (Cav3.1 and Cav3.2), that can selectively be included or omitted from the full length channel. We show that the developmental and spatial expression patterns of these splice variants are remarkably conserved, and that these splice variants produce analogous changes in membrane localization and biophysical properties when channels are expressed in HEK-293T cells. The Lymnaea Cav3 channel gene also undergoes alternative splicing in the domain II P-loop, with mutually exclusive exons 12A and 12B that code for a large portion of the P-loop just upstream of the selectivity filter. Such splicing is a novel discovery that is not conserved with vertebrates or any other deuterostome animal, all of which only contain 12A homologues of exon 12. However, protostome animals including Lymnaea stagnalis, Drosophila melanogaster, and C. elegans all have mutually exclusive 12A and 12B exons in their Cav3 channel genes. Evidence is presented that exon 12A is likely the ancestral exon for the domain II P-loop, and that alternate exon 12B evolved later. Furthermore, although the two Lymnaea variants possess the same selectivity filter motifs characteristic for Cav3 channels (i.e. EEDD), they exhibit dramatic differences in calcium vs. sodium selectivity, without significant differences in biophysical properties. This is the first account of alternative splicing used to modulate ion selectivity in a Cav3 channel homologue, and given that calcium is such an important electrogenic signaling molecule, these alterations are expected to have profound physiological implications. Amazingly, Lymnaea NALCN was also found to undergo alternative splicing in the domain II P-loop, but in this case, the entire P-loop is replaced by mutually exclusive exons 15a and 15b such that the selectivity filter is converted from the proposed non-selective sodium-permeable configuration (15b/EKEE; EEKE in mammals, nematodes and insects), to a calcium channel-like pore (15a/EEEE). Thorough phylogenetic analysis reveals that NALCN is extremely unconventional, in that alternative splicing has frequently and independently evolved to alter the selectivity filter in domains II or III, in multiple animal clades. Furthermore, the ancestral NALCN channel most likely contained an EEEE pore. This work brings into question NALCN’s proposed role as a major leak sodium conductance that depolarizes neurons to help set the resting membrane potential, since some species possess only an EEEE variant, and based on homology to other 4-domain ion channels, this should render the channel calcium-selective. Unfortunately, heterologous expression and electrophysiological characterization of the two Lymnaea NALCN isoforms was unsuccessful, corroborating with others the inability to record NALCN ionic currents in heterologous systems.1 yea

Gene Splicing of an Invertebrate Beta Subunit (LCav?) in the N-Terminal and HOOK Domains and Its Regulation of LCav1 and LCav2 Calcium Channels

The accessory beta subunit (Cavβ) of calcium channels first appear in the same genome as Cav1 L-type calcium channels in single-celled coanoflagellates. The complexity of this relationship expanded in vertebrates to include four different possible Cavβ subunits (β1, β2, β3, β4) which associate with four Cav1 channel isoforms (Cav1.1 to Cav1.4) and three Cav2 channel isoforms (Cav2.1 to Cav2.3). Here we assess the fundamentally-shared features of the Cavβ subunit in an invertebrate model (pond snail Lymnaea stagnalis) that bears only three homologous genes: (LCav1, LCav2, and LCavβ). Invertebrate Cavβ subunits (in flatworms, snails, squid and honeybees) slow the inactivation kinetics of Cav2 channels, and they do so with variable N-termini and lacking the canonical palmitoylation residues of the vertebrate β2a subunit. Alternative splicing of exon 7 of the HOOK domain is a primary determinant of a slow inactivation kinetics imparted by the invertebrate LCavβ subunit. LCavβ will also slow the inactivation kinetics of LCav3 T-type channels, but this is likely not physiologically relevant in vivo. Variable N-termini have little influence on the voltage-dependent inactivation kinetics of differing invertebrate Cavβ subunits, but the expression pattern of N-terminal splice isoforms appears to be highly tissue specific. Molluscan LCavβ subunits have an N-terminal “A” isoform (coded by exons: 1a and 1b) that structurally resembles the muscle specific variant of vertebrate β1a subunit, and has a broad mRNA expression profile in brain, heart, muscle and glands. A more variable “B” N-terminus (exon 2) in the exon position of mammalian β3 and has a more brain-centric mRNA expression pattern. Lastly, we suggest that the facilitation of closed-state inactivation (e.g. observed in Cav2.2 and Cavβ3 subunit combinations) is a specialization in vertebrates, because neither snail subunit (LCav2 nor LCavβ) appears to be compatible with this observed property

Glial activation in prion diseases is selectively triggered by neuronal PrPSc

Although prion infections cause cognitive impairment and neuronal death, transcriptional and translational profiling shows progressive derangement within glia but surprisingly little changes within neurons. Here we expressed PrP(C) selectively in neurons and astrocytes of mice. After prion infection, both astrocyte and neuron-restricted PrP(C) expression led to copious brain accumulation of PrPSc. As expected, neuron-restricted expression was associated with typical prion disease. However, mice with astrocyte-restricted PrP(C) expression experienced a normal life span, did not develop clinical disease, and did not show astro- or microgliosis. Besides confirming that PrPSc is innocuous to PrP(C)-deficient neurons, these results show that astrocyte-born PrPSc does not activate the extreme neuroinflammation that accompanies the onset of prion disease and precedes any molecular changes of neurons. This points to a nonautonomous mechanism by which prion-infected neurons instruct astrocytes and microglia to acquire a specific cellular state that, in turn, drives neural dysfunction

Gene Transcription and Splicing of T-Type Channels Are Evolutionarily-Conserved Strategies for Regulating Channel Expression and Gating

T-type calcium channels operate within tightly regulated biophysical constraints for supporting rhythmic firing in the brain, heart and secretory organs of invertebrates and vertebrates. The snail T-type gene, LCav3 from Lymnaea stagnalis, possesses alternative, tandem donor splice sites enabling a choice of a large exon 8b (201 aa) or a short exon 25c (9 aa) in cytoplasmic linkers, similar to mammalian homologs. Inclusion of optional 25c exons in the III–IV linker of T-type channels speeds up kinetics and causes hyperpolarizing shifts in both activation and steady-state inactivation of macroscopic currents. The abundant variant lacking exon 25c is the workhorse of embryonic Cav3 channels, whose high density and right-shifted activation and availability curves are expected to increase pace-making and allow the channels to contribute more significantly to cellular excitation in prenatal tissue. Presence of brain-enriched, optional exon 8b conserved with mammalian Cav3.1 and encompassing the proximal half of the I–II linker, imparts a ∼50% reduction in total and surface-expressed LCav3 channel protein, which accounts for reduced whole-cell calcium currents of +8b variants in HEK cells. Evolutionarily conserved optional exons in cytoplasmic linkers of Cav3 channels regulate expression (exon 8b) and a battery of biophysical properties (exon 25c) for tuning specialized firing patterns in different tissues and throughout development

A conformational switch controlling the toxicity of the prion protein

Prion infections cause conformational changes of the cellular prion protein (PrPC) and lead to progressive neurological impairment. Here we show that toxic, prion-mimetic ligands induce an intramolecular R208-H140 hydrogen bond (‘H-latch’), altering the flexibility of the α2–α3 and β2–α2 loops of PrPC. Expression of a PrP2Cys mutant mimicking the H-latch was constitutively toxic, whereas a PrPR207A mutant unable to form the H-latch conferred resistance to prion infection. High-affinity ligands that prevented H-latch induction repressed prion-related neurodegeneration in organotypic cerebellar cultures. We then selected phage-displayed ligands binding wild-type PrPC, but not PrP2Cys. These binders depopulated H-latched conformers and conferred protection against prion toxicity. Finally, brain-specific expression of an antibody rationally designed to prevent H-latch formation prolonged the life of prion-infected mice despite unhampered prion propagation, confirming that the H-latch is an important reporter of prion neurotoxicity

ITALIAN CANCER FIGURES - REPORT 2015: The burden of rare cancers in Italy = I TUMORI IN ITALIA - RAPPORTO 2015: I tumori rari in Italia

OBJECTIVES: This collaborative study, based on data collected by the network of Italian Cancer Registries (AIRTUM), describes the burden of rare cancers in Italy. Estimated number of new rare cancer cases yearly diagnosed (incidence), proportion of patients alive after diagnosis (survival), and estimated number of people still alive after a new cancer diagnosis (prevalence) are provided for about 200 different cancer entities. MATERIALS AND METHODS: Data herein presented were provided by AIRTUM population- based cancer registries (CRs), covering nowadays 52% of the Italian population. This monograph uses the AIRTUM database (January 2015), which includes all malignant cancer cases diagnosed between 1976 and 2010. All cases are coded according to the International Classification of Diseases for Oncology (ICD-O-3). Data underwent standard quality checks (described in the AIRTUM data management protocol) and were checked against rare-cancer specific quality indicators proposed and published by RARECARE and HAEMACARE (www.rarecarenet.eu; www.haemacare.eu). The definition and list of rare cancers proposed by the RARECAREnet "Information Network on Rare Cancers" project were adopted: rare cancers are entities (defined as a combination of topographical and morphological codes of the ICD-O-3) having an incidence rate of less than 6 per 100,000 per year in the European population. This monograph presents 198 rare cancers grouped in 14 major groups. Crude incidence rates were estimated as the number of all new cancers occurring in 2000-2010 divided by the overall population at risk, for males and females (also for gender-specific tumours).The proportion of rare cancers out of the total cancers (rare and common) by site was also calculated. Incidence rates by sex and age are reported. The expected number of new cases in 2015 in Italy was estimated assuming the incidence in Italy to be the same as in the AIRTUM area. One- and 5-year relative survival estimates of cases aged 0-99 years diagnosed between 2000 and 2008 in the AIRTUM database, and followed up to 31 December 2009, were calculated using complete cohort survival analysis. To estimate the observed prevalence in Italy, incidence and follow-up data from 11 CRs for the period 1992-2006 were used, with a prevalence index date of 1 January 2007. Observed prevalence in the general population was disentangled by time prior to the reference date (≤2 years, 2-5 years, ≤15 years). To calculate the complete prevalence proportion at 1 January 2007 in Italy, the 15-year observed prevalence was corrected by the completeness index, in order to account for those cancer survivors diagnosed before the cancer registry activity started. The completeness index by cancer and age was obtained by means of statistical regression models, using incidence and survival data available in the European RARECAREnet data. RESULTS: In total, 339,403 tumours were included in the incidence analysis. The annual incidence rate (IR) of all 198 rare cancers in the period 2000-2010 was 147 per 100,000 per year, corresponding to about 89,000 new diagnoses in Italy each year, accounting for 25% of all cancer. Five cancers, rare at European level, were not rare in Italy because their IR was higher than 6 per 100,000; these tumours were: diffuse large B-cell lymphoma and squamous cell carcinoma of larynx (whose IRs in Italy were 7 per 100,000), multiple myeloma (IR: 8 per 100,000), hepatocellular carcinoma (IR: 9 per 100,000) and carcinoma of thyroid gland (IR: 14 per 100,000). Among the remaining 193 rare cancers, more than two thirds (No. 139) had an annual IR <0.5 per 100,000, accounting for about 7,100 new cancers cases; for 25 cancer types, the IR ranged between 0.5 and 1 per 100,000, accounting for about 10,000 new diagnoses; while for 29 cancer types the IR was between 1 and 6 per 100,000, accounting for about 41,000 new cancer cases. Among all rare cancers diagnosed in Italy, 7% were rare haematological diseases (IR: 41 per 100,000), 18% were solid rare cancers. Among the latter, the rare epithelial tumours of the digestive system were the most common (23%, IR: 26 per 100,000), followed by epithelial tumours of head and neck (17%, IR: 19) and rare cancers of the female genital system (17%, IR: 17), endocrine tumours (13% including thyroid carcinomas and less than 1% with an IR of 0.4 excluding thyroid carcinomas), sarcomas (8%, IR: 9 per 100,000), central nervous system tumours and rare epithelial tumours of the thoracic cavity (5%with an IR equal to 6 and 5 per 100,000, respectively). The remaining (rare male genital tumours, IR: 4 per 100,000; tumours of eye, IR: 0.7 per 100,000; neuroendocrine tumours, IR: 4 per 100,000; embryonal tumours, IR: 0.4 per 100,000; rare skin tumours and malignant melanoma of mucosae, IR: 0.8 per 100,000) each constituted <4% of all solid rare cancers. Patients with rare cancers were on average younger than those with common cancers. Essentially, all childhood cancers were rare, while after age 40 years, the common cancers (breast, prostate, colon, rectum, and lung) became increasingly more frequent. For 254,821 rare cancers diagnosed in 2000-2008, 5-year RS was on average 55%, lower than the corresponding figures for patients with common cancers (68%). RS was lower for rare cancers than for common cancers at 1 year and continued to diverge up to 3 years, while the gap remained constant from 3 to 5 years after diagnosis. For rare and common cancers, survival decreased with increasing age. Five-year RS was similar and high for both rare and common cancers up to 54 years; it decreased with age, especially after 54 years, with the elderly (75+ years) having a 37% and 20% lower survival than those aged 55-64 years for rare and common cancers, respectively. We estimated that about 900,000 people were alive in Italy with a previous diagnosis of a rare cancer in 2010 (prevalence). The highest prevalence was observed for rare haematological diseases (278 per 100,000) and rare tumours of the female genital system (265 per 100,000). Very low prevalence (<10 prt 100,000) was observed for rare epithelial skin cancers, for rare epithelial tumours of the digestive system and rare epithelial tumours of the thoracic cavity. COMMENTS: One in four cancers cases diagnosed in Italy is a rare cancer, in agreement with estimates of 24% calculated in Europe overall. In Italy, the group of all rare cancers combined, include 5 cancer types with an IR>6 per 100,000 in Italy, in particular thyroid cancer (IR: 14 per 100,000).The exclusion of thyroid carcinoma from rare cancers reduces the proportion of them in Italy in 2010 to 22%. Differences in incidence across population can be due to the different distribution of risk factors (whether environmental, lifestyle, occupational, or genetic), heterogeneous diagnostic intensity activity, as well as different diagnostic capacity; moreover heterogeneity in accuracy of registration may determine some minor differences in the account of rare cancers. Rare cancers had worse prognosis than common cancers at 1, 3, and 5 years from diagnosis. Differences between rare and common cancers were small 1 year after diagnosis, but survival for rare cancers declined more markedly thereafter, consistent with the idea that treatments for rare cancers are less effective than those for common cancers. However, differences in stage at diagnosis could not be excluded, as 1- and 3-year RS for rare cancers was lower than the corresponding figures for common cancers. Moreover, rare cancers include many cancer entities with a bad prognosis (5-year RS <50%): cancer of head and neck, oesophagus, small intestine, ovary, brain, biliary tract, liver, pleura, multiple myeloma, acute myeloid and lymphatic leukaemia; in contrast, most common cancer cases are breast, prostate, and colorectal cancers, which have a good prognosis. The high prevalence observed for rare haematological diseases and rare tumours of the female genital system is due to their high incidence (the majority of haematological diseases are rare and gynaecological cancers added up to fairly high incidence rates) and relatively good prognosis. The low prevalence of rare epithelial tumours of the digestive system was due to the low survival rates of the majority of tumours included in this group (oesophagus, stomach, small intestine, pancreas, and liver), regardless of the high incidence rate of rare epithelial cancers of these sites. This AIRTUM study confirms that rare cancers are a major public health problem in Italy and provides quantitative estimations, for the first time in Italy, to a problem long known to exist. This monograph provides detailed epidemiologic indicators for almost 200 rare cancers, the majority of which (72%) are very rare (IR<0.5 per 100,000). These data are of major interest for different stakeholders. Health care planners can find useful information herein to properly plan and think of how to reorganise health care services. Researchers now have numbers to design clinical trials considering alternative study designs and statistical approaches. Population-based cancer registries with good quality data are the best source of information to describe the rare cancer burden in a population

The biological function of the cellular prion protein: an update

The misfolding of the cellular prion protein (PrPC) causes fatal neurodegenerative diseases. Yet PrPC is highly conserved in mammals, suggesting that it exerts beneficial functions preventing its evolutionary elimination. Ablation of PrPC in mice results in well-defined structural and functional alterations in the peripheral nervous system. Many additional phenotypes were ascribed to the lack of PrPC, but some of these were found to arise from genetic artifacts of the underlying mouse models. Here, we revisit the proposed physiological roles of PrPC in the central and peripheral nervous systems and highlight the need for their critical reassessment using new, rigorously controlled animal models. The cellular prion protein (PrPC) is a cell surface protein expressed in a variety of different organs and tissues with high expression levels in the central and peripheral nervous systems [1]. It is mainly known for its infamous role in prion diseases, where its misfolding and aggregation cause inevitably fatal neurodegenerative conditions [2]. Prion diseases are transmissible and misfolded prion protein (PrPSc) is—according to the “protein-only hypothesis’”—the only disease-causing agent [3]. Under this view, it is puzzling that a protein underlying such severe diseases is highly conserved throughout mammals [4]. This suggests the existence of distinct benefits and, potentially, important physiological functions. A definitive, fully satisfactory understanding of the physiological function of PrPC has been lacking for a long time. Very recently, we identified a native function of PrPC in the peripheral nervous system and the underlying mechanism of that function [5]. However, PrPC is also highly expressed in the central nervous system (CNS) and its biological activity there is still far from being clear. This review will focus on the proposed roles of cellular prion protein in the central and peripheral nervous systems

<Résumés of Lectures at the Annual Meeting>

<div><p>Background</p><p>The sea slug <i>Tritonia diomedea</i> (Mollusca, Gastropoda, Nudibranchia), has a simple and highly accessible nervous system, making it useful for studying neuronal and synaptic mechanisms underlying behavior. Although many important contributions have been made using <i>Tritonia</i>, until now, a lack of genetic information has impeded exploration at the molecular level.</p><p>Results</p><p>We performed Illumina sequencing of central nervous system mRNAs from <i>Tritonia</i>, generating 133.1 million 100 base pair, paired-end reads. <i>De novo</i> reconstruction of the RNA-Seq data yielded a total of 185,546 contigs, which partitioned into 123,154 non-redundant gene clusters (unigenes). BLAST comparison with RefSeq and Swiss-Prot protein databases, as well as mRNA data from other invertebrates (gastropod molluscs: <i>Aplysia californica</i>, <i>Lymnaea stagnalis</i> and <i>Biomphalaria glabrata</i>; cnidarian: <i>Nematostella vectensis</i>) revealed that up to 76,292 unigenes in the <i>Tritonia</i> transcriptome have putative homologues in other databases, 18,246 of which are below a more stringent E-value cut-off of 1x10-6. <i>In silico</i> prediction of secreted proteins from the <i>Tritonia</i> transcriptome shotgun assembly (TSA) produced a database of 579 unique sequences of secreted proteins, which also exhibited markedly higher expression levels compared to other genes in the TSA.</p><p>Conclusions</p><p>Our efforts greatly expand the availability of gene sequences available for <i>Tritonia diomedea</i>. We were able to extract full length protein sequences for most queried genes, including those involved in electrical excitability, synaptic vesicle release and neurotransmission, thus confirming that the transcriptome will serve as a useful tool for probing the molecular correlates of behavior in this species. We also generated a neurosecretome database that will serve as a useful tool for probing peptidergic signalling systems in the <i>Tritonia</i> brain.</p></div

Physiology and evolution of voltage-gated calcium channels in early diverging animal phyla: Cnidaria, Placozoa, Porifera and Ctenophora

Voltage-gated calcium (Cav) channels serve dual roles in the cell, where they can both depolarize the membrane potential for electrical excitability, and activate transient cytoplasmic Ca2+ signals. In animals, Cav channels play crucial roles including driving muscle contraction (excitation-contraction coupling), gene expression (excitation-transcription coupling), pre-synaptic and neuroendocrine exocytosis (excitation-secretion coupling), regulation of flagellar/ciliary beating, and regulation of cellular excitability, either directly or through modulation of other Ca2+-sensitive ion channels. In recent years, genome sequencing has provided significant insights into the molecular evolution of Cav channels. Furthermore, expanded gene datasets have permitted improved inference of the species phylogeny at the base of Metazoa, providing clearer insights into the evolution of complex animal traits which involve Cav channels, including the nervous system. For the various types of metazoan Cav channels, key properties that determine their cellular contribution include: ion selectivity, pore gating, and, importantly, cytoplasmic protein-protein interactions that direct sub-cellular localization and functional complexing. It is unclear when many of these defining features, many of which are essential for nervous system function, evolved. In this review, we highlight some experimental observations that implicate Cav channels in the physiology and behavior of the most early-diverging animals from the phyla Cnidaria, Placozoa, Porifera and Ctenophora. Given our limited understanding of the molecular biology of Cav channels in these basal animal lineages, we infer insights from better-studied vertebrate and invertebrate animals. We also highlight some apparently conserved cellular functions of Cav channels, which might have emerged very early on during metazoan evolution, or perhaps predated it

Summary of biophysical parameters of LCav3 channel variants containing exons 8b and 25c expressed in HEK-293T cells, with one-way analysis of variance to assess statistical significance.

<p>n.s. not significant;</p>*<p>p<0.05;</p>**<p>p<0.005;</p>***<p>p<0.001.</p