Cellular/Molecular Differential Ubiquitination and Proteasome Regulation of Ca V 2.2 N-Type Channel Splice Isoforms

Ca V 2.2 (N-type) calcium channels control the entry of calcium into neurons to regulate essential functions but most notably presynaptic transmitter release. Ca V 2.2 channel expression levels are precisely controlled, but we know little of the cellular mechanisms involved. The ubiquitin proteasome system (UPS) is known to regulate expression of many synaptic proteins, including presynaptic elements, to optimize synaptic efficiency. However, we have limited information about ubiquitination of Ca V 2 channels. Here we show that Ca V 2.2 proteins are ubiquitinated, and that elements in the proximal C terminus of Ca V 2.2 encoded by exon 37b of the mouse Cacna1b gene predispose cloned and native channels to downregulation by the UPS. Ca V 2.2 channels containing e37b are expressed throughout the mammalian nervous system, but in some cells, notably nociceptors, sometimes e37a-not e37b-is selected during alternative splicing of Ca V 2.2 pre-mRNA. By a combination of biochemical and functional analyses we show e37b promotes a form of ubiquitination that is coupled to reduced Ca V 2.2 current density and increased sensitivity to the UPS. Cell-specific alternative splicing of e37a in nociceptors reduces Ca V 2.2 channel ubiquitination and sensitivity to the UPS, suggesting a role in pain processing

Hedgehog-driven myogenic tumors recapitulate skeletal muscle cellular heterogeneity

Hedgehog (Hh) pathway activation in R26-SmoM2;CAGGS-CreER mice, which carry a tamoxifen-inducible activated Smoothened allele (SmoM2), results in numerous microscopic tumor foci in mouse skeletal muscle. These tumors exhibit a highly differentiated myogenic phenotype and resemble human fetal rhabdomyomas. This study sought to apply previously established strategies to isolate lineally distinct populations of normal mouse myofiber-associated cells in order to examine cellular heterogeneity in SmoM2 tumors. We demonstrate that established SmoM2 tumors are composed of cells expressing myogenic, adipocytic and hematopoietic lineage markers and differentiation capacity. SmoM2 tumors thus recapitulate the phenotypic and functional hetereogeneity observed in normal mouse skeletal muscle. SmoM2 tumors also contain an expanded population of PAX7+ and MyoD+ satellite-like cells with extremely low clonogenic activity. Selective activation of Hh signaling in freshly isolated muscle satellite cells enhanced terminal myogenic differentiation without stimulating proliferation. Our findings support the conclusion that SmoM2 tumors represent an aberrant skeletal muscle state and demonstrate that, similar to normal muscle, myogenic tumors contain functionally distinct cell subsets, including cells lacking myogenic differentiation potential

Mucolipin Co-deficiency Causes Accelerated Endolysosomal Vacuolation of Enterocytes and Failure-to-Thrive from Birth to Weaning

<div><p>During the suckling period, intestinal enterocytes are richly endowed with endosomes and lysosomes, which they presumably utilize for the uptake and intracellular digestion of milk proteins. By weaning, mature intestinal enterocytes replace those rich in lysosomes. We found that mouse enterocytes before weaning express high levels of two endolysosomal cation channels, mucolipins 3 and 1 -products of <i>Trpml3</i> and <i>Trpml1</i> genes; moreover neonatal enterocytes of mice lacking both mucolipins (<i>Trpml3^−/−</i>;<i>Trpml1^−/−</i>) vacuolated pathologically within hours of birth and remained so until weaning. Ultrastructurally and chemically these fast-forming vacuoles resembled those that systemically appear in epithelial cells of mucolipidosis type IV (MLIV) patients, which bear mutations in <i>Trpml1</i>. Hence, lack of both mucolipins 1 and 3 causes an accelerated MLIV-type of vacuolation in enterocytes. The vacuoles were aberrant hybrid organelles with both endosomal and lysosomal components, and were not generated by alterations in endocytosis or exocytosis, but likely by an imbalance between fusion of lysosomes and endosomes and their subsequent scission. However, upon extensive vacuolation enterocytes displayed reduced endocytosis from the intestinal lumen, a defect expected to compromise nutrient uptake. Mice lacking both mucolipins suffered a growth delay that began after birth and continued through the suckling period but recovered after weaning, coinciding with the developmental period of enterocyte vacuolation. Our results demonstrate genetic redundancy between lysosomal mucolipins 3 and 1 in neonatal enterocytes. Furthermore, our <i>Trpml3^−/−</i>;<i>Trpml1^−/−</i> mice represent a polygenic animal model of the poorly-understood, and often intractable, neonatal failure-to-thrive with intestinal pathology. Our results implicate lysosomes in neonatal intestinal pathologies, a major cause of infant mortality worldwide, and suggest transient intestinal dysfunction might affect newborns with lysosomal storage disorders. Finally, we conclude that mucolipin-endowed lysosomes in the young play an evolutionarily-conserved role in the intracellular digestion of maternally-provided nutrients, whether milk in mammals or yolk in oviparous species.</p></div

Failure to thrive of suckling mice lacking mucolipins 3 and 1.

<p>(<b>A–D</b>) <i>Trpml3^−/−;Trpml1^−/−</i> pups, denoted by asterisks, shown next to their <i>Trpml3^−/−</i> control littermates. (<b>A</b>) A litter at P8, in which the three smallest pups have the <i>Trpml3^−/−;Trpml1^−/−</i> genotype. (<b>B</b>) Ventral view of a pair of littermates at P7, of which the one lacking mucolipins 3 and 1 is smaller and suffers diarrhea (circled). (<b>C</b>) Viscera of a pair of littermates at P7 shows that the pup lacking mucolipins 3 and 1 has the stomach filled with milk (arrows), which demonstrates its ability to suckle and ingest milk. (<b>D</b>) Extended distal intestines of a pair of littermates at P10 demonstrates diarrhea (liquid feces) of the <i>Trpml3^−/−;Trpml1^−/−</i> pup (left), compared with the pelleted feces of its control littermate (right). (<b>E</b>) Body weights of mice lacking mucolipins 1, 3 or both, normalized to the weights of wild type and heterozygote littermates, reveals that <i>Trpml3^−/−;Trpml1^−/−</i> mice become comparatively smaller after birth but partially recover after weaning (which occurs at P21, as indicated by the dashed line). Error bars denote SEM. Sample sizes (number of mice per genotype at each age) are n = 26 to 90 for wild type, n = 10 to 13 for <i>Trpml3^−/−</i>, n = 7 to 16 for <i>Trpml1^−/−</i> and n = 12 to 28 for <i>Trpml3^−/−;Trpml1^−/−</i>. Asterisks denote statistically different weights between <i>Trpml3^−/−;Trpml1^−/−</i> and <i>Trpml1^−/−</i> mice (Student's t-test, p<0.001 except at P2, with p<0.05, and P29, with p<0.01). (<b>F</b>) Daily growth rates reveal that the lower weight of <i>Trpml3^−/−;Trpml1^−/−</i> pups is not due to weight loss but to reduced growth prior to postnatal day 10. Error bars denote SEM. Sample sizes (number of pups per genotype at each age) are n = 23 to 109 for wild type, n = 20 to 78 for <i>Trpml3^−/−</i>, n = 32 to 66 for <i>Trpml1^−/−</i> and n = 8 to 28 for <i>Trpml3^−/−;Trpml1^−/−</i>. Asterisks denote statistically different rates of growth between <i>Trpml3^−/−;Trpml1^−/−</i> and all other mice (Student's t-test, p<0.01 except at day 7 to 8, with p<0.05).</p

Formation of pathological vacuolar organelles in neonatal enterocytes lacking both mucolipins 1 and 3.

<p>Ultrastructural examination of ileum from (<b>A</b>) control <i>Trpml3^−/−</i> and (<b>B–H</b>) <i>Trpml3^−/−;Trpml1^−/−</i> pups at P0. The intestinal epithelia of <i>Trpml3^−/−;Trpml1^−/−</i> mice contain normal appearing goblet cells (GC) and enterocyte nuclei (N). Their enterocytes display normally appearing nuclei (N), mitochondria and apical microvilli as well as the endocytic structures normally found at P0 (all labeled in blue and tentatively identified based on their ultrastructural features and subcellular location): invaginations presumed to be endocytic figures in the process of endocytosis (arrowheads), early endosomes (e) and late endosomes/multivesicular bodies (mv). However, <i>Trpml3^−/−;Trpml1^−/−</i> enterocytes also displayed abnormal organelles (labeled in pink), primarily a large vacuole (V) and/or multiple smaller vesicles, filled with granular material, some electron dense material and some multilamellar structures, often in the form of whorls of concentric membranes (empty arrows). There appears to be fusion (or fission) between some smaller vesicles and the large vacuole (asterisks). The dotted lines in (<b>D</b>) delineate the area magnified in (<b>H</b>). L, lumen of intestine.</p

Pathological vacuolation of neonatal, but not adult, enterocytes lacking both mucolipins 3 and 1, but not either mucolipin alone.

<p>(<b>A–D</b>) Hematoxylin and eosin (H&E) staining of paraffin-embedded sections of intestine reveals abnormal vacuolation in (<b>A</b>) <i>Trpml3^−/−;Trpml1^−/−</i> pups, but not in (<b>C</b>) wild type, (<b>B</b>) <i>Trpml3^−/−</i> or (<b>D</b>) <i>Trpml1^−/−</i> pups. (<b>E,F</b>) Periodic acid-Schiff (PAS) staining of paraffin-embedded sections of neonatal intestines reveals that (<b>E</b>) the vacuolated intestinal cells of <i>Trpml3^−/−;Trpml1^−/−</i> mice are not mucin-filled, goblet cells (labeled red), which show a distribution undistinguishable from (<b>F</b>) <i>Trpml3^−/−</i>, (<b>H</b>) <i>Trpml1^−/−</i> and (<b>G</b>) wild type controls. (<b>I,J</b>) PAS staining of adult intestines reveals (<b>I</b>) no vacuolation of enterocytes from <i>Trpml3^−/−;Trpml1^−/−</i> mice and no other signs of pathology when compared with (<b>J</b>) <i>Trpml3^−/−</i> littermate and wild type controls. All scale bars are 100 µm.</p

An enlarged, clear vacuole fills most of the intracellular space of <i>Trpml3^−/−;Trpml1^−/−</i> enterocytes by postnatal day 5.

<p>Ultrastructural examination of ileum from (<b>A–C</b>) control <i>Trpml3^−/−</i> and (<b>D–F</b>) <i>Trpml3^−/−;Trpml1^−/−</i> littermates at P5. By this stage, ileal enterocytes of control <i>Trpml3^−/−</i> pups contain their characteristic giant lysosome (GL), which is partially filled with electron dense material (presumably endocytosed milk proteins ready for intracellular degradation). By contrast, the pathological vacuoles (V, in pink) of <i>Trpml3^−/−;Trpml1^−/−</i> pups appear mostly empty, with very little electron-dense material, and are larger than at P0, occupying most of the cytoplasmic space. Smaller vesicles appear to be fusing with the larger vacuole (asterisks), which still contain some multilamellar, membranous whorls (empty arrows). Despite the aberrant deformation caused by the enlarged, pathological vacuoles, the enterocytes of <i>Trpml3^−/−;Trpml1^−/−</i> pups still display normal villi, endocytic figures in the apical membrane (arrowheads) and some early endosomes (e) (compare F with C). Additional abbreviations: L, lumen of intestine; GC, goblet cells; N, nuclei of enterocytes. Organelles present in control enterocytes are labeled in blue, whereas pathological structures are in pink.</p

Generation of mice with knockout alleles of <i>Trpml3</i>.

<p>(<b>A</b>) Schematic representation of targeting construct and Wild type, Targeted (Flox-Neo), Floxed and Knockout alleles of <i>Trpml3</i>. The Mouse Genomics Informatics (MGI) name assigned to the KO allele is <i>Mcoln3^tm1.1Jga</i>. Exons are represented by gray boxes and the area to be deleted is shaded in blue. Restriction enzymes indicated are <i>EcoR</i> I (E), <i>BamH</i> I (B), <i>Ssp I</i> (S), <i>Not</i> I (N) and <i>Hpa I</i> (H). <i>LoxP</i> sites are labeled L, <i>FRT</i> sites F, and Neomycin-expressing cassettes Neo. (<b>B</b>) Southern blots on genomic DNA from five ES cell clones demonstrating the presence of a targeted (Flox-Neo) allele of <i>Trpml3</i>, obtained by homologous recombination. Both 5′ and 3′ arms underwent homologous recombination. (<b>C</b>; left) PCR genotyping of a litter in which two mice carry the Floxed allele, created by expression of FLPe recombinase in mice bearing the Flox-Neo allele. (<b>C</b>; right) PCR genotyping of a litter in which mice carry one or both knockout alleles, created by expression of Cre recombinase in mice bearing the Flox-Neo allele.</p

The pathological vacuoles of suckling enterocytes lacking mucolipins 1 and 3 are aberrant hybrid organelles with endosomal and lysosomal components.

<p>Cryosections of intestines from 3 hour old (<b>A–C</b>) control <i>Trpml3^−/−</i> and (<b>D–F</b>) <i>Trpml3^−/−;Trpml1^−/−</i> littermates that had been fed formula with Texad Red-dextran immediately after birth. (<b>A,A′,D,D′</b>) Nomarski (<b>A,D</b>) and fluorescent (<b>A′,D′</b>) images of the same sections reveal that neonatal enterocytes endocytose the ingested Texas Red-dextran, which accumulates in the pathological vacuoles of <i>Trpml3^−/−;Trpml1^−/−</i> enterocytes (<b>D′</b>). Immunohistochemical staining with antibodies to the lysosomal marker LAMP1 (B,E) and to the early endosomal marker EEA1 (C,F) reveal that the pathological vacuoles of <i>Trpml3^−/−;Trpml1^−/−</i> enterocytes contain endosomal and lysosomal components and thus may result from the fusion of both types of organelles. (<b>G–L</b>) Confocal optical sections of immunofluorescent staining for LAMP1 (G–I) or EEA1 (J–L) on <i>Trpml3^−/−;Trpml1^−/−</i> intestines from the same P0 animal used in (D–F) reveal that endocytosed dextran accumulates in the pathological vacuoles, which contain both LAMP1 and EEA1. Due to the high level of neonatal enterocytes autofluorescence, which spans most of the detectable optical spectrum, secondary antibodies were conjugated with DyLight 405 and the 405 nm signals are pseudocolored green. Scale bars are 10 mm (A–F) or 20 µm (G–L).</p

Neonatal intestines upregulate mRNA levels of mucolipin 1 (<i>Trpml1</i>), but do not express mucolipin 2 (<i>Trpml2</i>).

<p>(<b>A–D, G</b>) In situ hybridization (ISH) on sagittal sections of postnatal day 2 (A–C) and 1 (D,G) pups reveal (<b>A–D</b>) high expression of <i>Trpml1</i> mRNA in the epithelia of the intestinal villi but (<b>G</b>) lack of detectable <i>Trpml2</i> mRNA. In situ signal for Trpml1 mRNA reveal higher levels in neonatal intestines (In) than in any other organ including kidney (Ki), adrenal gland (Ad), liver (Li), lung (Lu), heart (He), thymus (Th), inner ear (Ie), trigeminal ganglia (Tr), brain (Br) and retina (Re). (<b>E</b>) RT-qPCR reveals that the levels of <i>Trpml1</i> mRNA on small intestine are high during suckling and subside to a much lower, baseline level by weaning. (<b>F</b>) RT-qPCR on equally divided segments of small intestine also reveals that by P14 <i>Trpml1</i> mRNA is more abundant in ileum (distal) than duodenum (proximal) and jejunum (middle). Normalized <i>Trpml1</i> levels are displayed relative to the level of <i>Trpml1</i> in proximal ileum at P14. Each bar is the average of n = 3 experiments. Error bars represent the standard deviation. Scale bars are 1 mm (A–C) or 50 µm (D,G).</p