Tissue Proteome of 2-Hydroxyacyl-CoA Lyase Deficient Mice Reveals Peroxisome Proliferation and Activation of ω-Oxidation.

Peroxisomal fatty acid α-oxidation is an essential pathway for the degradation of ß-carbon methylated fatty acids such as phytanic acid. One enzyme in this pathway is 2-hydroxyacyl CoA lyase (HACL1), which is responsible for the cleavage of 2-hydroxyphytanoyl-CoA into pristanal and formyl-CoA. Hacl1 deficient mice do not present with a severe phenotype, unlike mice deficient in other α-oxidation enzymes such as phytanoyl-CoA hydroxylase deficiency (Refsum disease) in which neuropathy and ataxia are present. Tissues from wild-type and Hacl1-/- mice fed a high phytol diet were obtained for proteomic and lipidomic analysis. There was no phenotype observed in these mice. Liver, brain, and kidney tissues underwent trypsin digestion for untargeted proteomic liquid chromatography-mass spectrometry analysis, while liver tissues also underwent fatty acid hydrolysis, extraction, and derivatisation for fatty acid gas chromatography-mass spectrometry analysis. The liver fatty acid profile demonstrated an accumulation of phytanic and 2-hydroxyphytanic acid in the Hacl1-/- liver and significant decrease in heptadecanoic acid. The liver proteome showed a significant decrease in the abundance of Hacl1 and a significant increase in the abundance of proteins involved in PPAR signalling, peroxisome proliferation, and omega oxidation, particularly Cyp4a10 and Cyp4a14. In addition, the pathway associated with arachidonic acid metabolism was affected; Cyp2c55 was upregulated and Cyp4f14 and Cyp2b9 were downregulated. The kidney proteome revealed fewer significantly upregulated peroxisomal proteins and the brain proteome was not significantly different in Hacl1-/- mice. This study demonstrates the powerful insight brought by proteomic and metabolomic profiling of Hacl1-/- mice in better understanding disease mechanism in fatty acid α-oxidation disorders.

T regulatory cells control susceptibility to invasive pneumococcal pneumonia in mice.

Streptococcus pneumoniae is an important human pathogen responsible for a spectrum of diseases including pneumonia. Immunological and pro-inflammatory processes induced in the lung during pneumococcal infection are well documented, but little is known about the role played by immunoregulatory cells and cytokines in the control of such responses. We demonstrate considerable differences in the immunomodulatory cytokine transforming growth factor (TGF)-ß between the pneumococcal pneumonia resistant BALB/c and susceptible CBA/Ca mouse strains. Immunohistochemistry and flow cytometry reveal higher levels of TGF-ß protein in BALB/c lungs during pneumococcal pneumonia that correlates with a rapid rise in lung Foxp3(+)Helios(+) T regulatory cells. These cells have protective functions during pneumococcal pneumonia, because blocking their induction with an inhibitor of TGF-ß impairs BALB/c resistance to infection and aids bacterial dissemination from lungs. Conversely, adoptive transfer of T regulatory cells to CBA/Ca mice, prior to infection, prolongs survival and decreases bacterial dissemination from lungs to blood. Importantly, strong T regulatory cell responses also correlate with disease-resistance in outbred MF1 mice, confirming the importance of immunoregulatory cells in controlling protective responses to the pneumococcus. This study provides exciting new evidence for the importance of immunomodulation during pulmonary pneumococcal infection and suggests that TGF-ß signalling is a potential target for immunotherapy or drug design.

Pleiotropic brain function of whirlin identified by a novel mutation.

Despite some evidence indicating diverse roles of whirlin in neurons, the functional corollary of whirlin gene function and behavior has not been investigated or broadly characterized. A single nucleotide variant was identified from our recessive ENU-mutagenesis screen at a donor-splice site in whirlin, a protein critical for proper sensorineural hearing function. The mutation (head-bob, hb) led to partial intron-retention causing a frameshift and introducing a premature termination codon. Mutant mice had a head-bobbing phenotype and significant hyperactivity across several phenotyping tests. Lack of complementation of head-bob with whirler mutant mice confirmed the head-bob mutation as functionally distinct with compound mutants having a mild-moderate hearing defect. Utilizing transgenics, we demonstrate rescue of the hyperactive phenotype and combined with the expression profiling data conclude whirlin plays an essential role in activity-related behaviors. These results highlight a pleiotropic role of whirlin within the brain and implicate alternative, central mediated pathways in its function.

Identification of a Z-band associated protein complex involving KY, FLNC and IGFN1.

The KY protein underlies a form of muscular dystrophy in the mouse but its role in muscle remains elusive. Immunodetection of endogenous KY protein in C2C12-derived myotubes and expression of a recombinant form in neonatal cardiomyocytes indicated that KY is a Z-band associated protein. Moreover, characterization of a KY interacting protein fragment led to the identification of Igfn1 (Immunoglobulin-like and fibronectin type 3 domain containing 1). Igfn1 is a transcriptionally complex locus encoding many protein variants. A yeast two-hybrid screen identified the Z-band protein filamin C (FLNC) as an interacting partner. Consistent with this, expression of an IGFN1 recombinant fragment showed that the three N-terminal globular domains, common to at least five IGFN1 variants, are sufficient to provide Z-band targeting. Taken together, the yeast two-hybrid, biochemical and immunofluorescence data support the notion that KY, IGFN1 and FLNC are part of a Z-band associated protein complex likely to provide structural support to the skeletal muscle sarcomere.

IGFN1_v1 is required for myoblast fusion and differentiation.

Igfn1 is a complex locus that codes for multiple splicing variants of Immunoglobulin- and Fibronectin-like domain containing proteins predominantly expressed in skeletal muscle. To reveal possible roles for Igfn1, we applied non-selective knock-down by shRNAs as well as specific targeting of Igfn1 exon 13 by CRISPR/Cas9 mutagenesis in C2C12 cells. Decreased expression of Igfn1 variants via shRNAs against the common 3'-UTR region caused a total blunting of myoblast fusion, but did not prevent expression of differentiation markers. Targeting of N-terminal domains by elimination of exon 13 via CRISPR/Cas9 mediated homologous recombination, also resulted in fusion defects as well as large multinucleated cells. Expression of IGFN1_v1 partially rescued fusion and myotube morphology in the Igfn1 exon 13 knock-out cell line, indicating a role for this variant in myoblast fusion and differentiation. However, in vivo overexpression of IGFN1_v1 or the Igfn1 Exon 13 CRISPR/Cas9 targeting vector did not result in significant size changes in transfected fibres.