Background Cancer cachexia is a complex metabolic disease with unmet medical need. Although many rodent models are available, none are identical to the human disease. Therefore, the development of new preclinical models that simulate some of the physiological, biochemical, and clinical characteristics of the human disease is valuable. The HT-1080 human fibrosarcoma tumour cell line was reported to induce cachexia in mice. Therefore, the purpose of this work was to determine how well the HT-1080 tumour model could recapitulate human cachexia and to examine its technical performance. Furthermore, the efficacy of ghrelin receptor activation via anamorelin treatment was evaluated, because it is one of few clinically validated mechanisms. Methods Female severe combined immunodeficient mice were implanted subcutaneously or heterotopically (renal capsule) with HT-1080 tumour cells. The cachectic phenotype was evaluated during tumour development, including body weight, body composition, food intake, muscle function (force and fatigue), grip strength, and physical activity measurements. Heterotopic and subcutaneous tumour histology was also compared. Energy balance was evaluated at standard and thermoneutral housing temperatures in the subcutaneous model. The effect of anamorelin (ghrelin analogue) treatment was also examined. Results The HT-1080 tumour model had excellent technical performance and was reproducible across multiple experimental conditions. Heterotopic and subcutaneous tumour cell implantation resulted in similar cachexia phenotypes independent of housing temperature. Tumour weight and histology was comparable between both routes of administration with minimal inflammation. Subcutaneous HT-1080 tumour-bearing mice presented with weight loss (decreased fat mass and skeletal muscle mass/fibre cross-sectional area), reduced food intake, impaired muscle function (reduced force and grip strength), and decreased spontaneous activity and voluntary wheel running. Key circulating inflammatory biomarkers were produced by the tumour, including growth differentiation factor 15, Activin A, interleukin 6, and TNF alpha. Anamorelin prevented but did not reverse anorexia and weight loss in the subcutaneous model. Conclusions The subcutaneous HT-1080 tumour model displays many of the perturbations of energy balance and physical performance described in human cachexia, consistent with the production of key inflammatory factors. Anamorelin was most effective when administered early in disease progression. The HT-1080 tumour model is valuable for studying potential therapeutic targets for the treatment of cachexia.

Presynaptic CaV2.2 channels control calcium entry that triggers neurotransmitter release at both central and peripheral synapses. The Cacna1b gene encodes the α1-pore forming subunit of CaV2.2 channels. Distinct subsets of splice variants of CaV2.2 derived from cell-specific alternative splicing of the Cacna1b pre-mRNA are expressed in specific subpopulations of neurons. Four cell-specific sites of alternative splicing in Cacna1b that alter CaV2.2 channel function have been described in detail: three cassette exons (e18a, e24a, and e31a) and a pair of mutually exclusive exons (e37a/e37b). Cacna1b mRNAs containing e37a are highly enriched in a subpopulation of nociceptors where they influence nociception and morphine analgesia. E37a-Cacna1b mRNAs are also expressed in brain, but their cell-specific expression in this part of the nervous system, their functional consequences in central synapses and their role on complex behavior have not been studied. In this report, we show that e37a-Cacna1b mRNAs are expressed in excitatory projection neurons where CaV2.2 channels are known to influence transmitter release at excitatory inputs from entorhinal cortex (EC) to dentate gyrus (DG). By comparing behaviors of WT mice to those that only express e37b-CaV2.2 channels, we found evidence that e37a-CaV2.2 enhances behavioral responses to aversive stimuli. Our results suggest that alternative splicing of Cacna1b e37a influences excitatory transmitter release and couples to complex behaviors.

Presynaptic Ca V 2.2 (N‐type) channels are fundamental for transmitter release across the nervous system. The gene encoding Ca V 2.2 channels, Cacna1b , contains alternatively spliced exons that result in functionally distinct splice variants (e18a, e24a, e31a, and 37a/37b). Alternative splicing of the cassette exon 18a generates two mRNA transcripts (+e18a‐ Cacna1b and ∆e18a‐ Cacna1b ). In this study, using novel mouse genetic models and in situ hybridization (BaseScope™), we confirmed that +e18a‐ Cacna1b splice variants are expressed in monoaminergic regions of the midbrain. We expanded these studies and identified +e18a‐ Cacna1b mRNA in deep cerebellar cells and spinal cord motor neurons. Furthermore, we determined that +e18a ‐Cacna1b is enriched in cholecystokinin‐expressing interneurons. Our results provide key information to understand cell‐specific functions of Ca V 2.2 channels.

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CaV1.3 is an L-type voltage-gated calcium channel implicated in several functions including gene expression, pacemaking activity, and neurotransmitter release. The gene that encodes the CaVa1-pore forming subunit of CaV1.3 (Cacna1d) is a multi-exon gene that undergoes extensive alternative splicing, which provides functional versatility to this gene across tissues and cell-types. The function and expression of several Cacna1d splice variants within the C-terminus have been previously characterized. These splice variants differ in their voltage-dependence of activation, Ca 2+ -dependent inactivation, and their sensitivity to dihydropyridines. However, less is known about alternatively spliced exons in Cacna1d located downstream of domain I and upstream of the C-terminus (e11, e22a/e22, e31a/e31b/e32). Here, we performed a systematic study to determine the developmental and cell-specific expression of several Cacna1d splice variants. We found that the cassette e11 is upregulated during brain development, and in adult cortical tissue is more abundant in excitatory neurons relative to inhibitory interneurons. This exon is also upregulated upon nerve growth factor (NGF) induced differentiation of pheochromocytoma cells, PC12. At the functional level, the splice variants resulting from e11 alternative splicing (+e11-Cacna1d and De11-Cacna1d) form functional CaV1.3 channels with similar biophysical properties in expression mammalian systems. Of the pair of mutually exclusive exons, e22a and e22, the later dominates at all stages. However, we observed a slight upregulation of e22 from embryonic to adult human brain. A second pair of mutually exclusive exons, e31a and e31b, was also studied. We found that e31a increases during brain development. Finally, the cassette exon 32 is repressed in adult brain tissue. KEYWORDSCalcium channels, alternative splicing, splicing factors, brain development Parkinson's disease, primary aldosteronism and autism spectrum disorder (Chan et al., 2010;Baig et al., 2011;Kang et al., 2012;Reinbothe et al., 2013; Scholl et al., 2013; Pinggera et al., 2015). Interestingly, Cacna1d splice variants add functional versatility of CaV1.3 channels across tissues and cell-types.CaV1.3, like all CaVa1 subunits, is organized into four homologous domains (DI-IV). Each domain contains six transmembrane-spanning segments (S1-S6), with a reentrant loop between S5 and S6, which contains glutamates that are key for the selectivity filter. The amino and carboxyl termini, as well as linker sequences between the DI-II, DII-III, and DIII-IV are cytosolic (Fig. 1A). Sites of alternative splicing of sequences encoding for the N-terminus, DI, DI-II linker, DIII, DIV and C-terminus are present in Cacna1d pre-mRNAs. Alternative splicing of sequences encoding for the C-terminus in CaV1.3 have been well characterized, and results in splice isoforms with differences in calciumdependent inactivation, trafficking, protein-protein interactions, and sensitivity to dihydropyridines (DHPs) (Shen et al., 2006;Singh et al., 200...