A Deficiency of Ceramide Biosynthesis Causes Cerebellar Purkinje Cell Neurodegeneration and Lipofuscin Accumulation

Sphingolipids, lipids with a common sphingoid base (also termed long chain base) backbone, play essential cellular structural and signaling functions. Alterations of sphingolipid levels have been implicated in many diseases, including neurodegenerative disorders. However, it remains largely unclear whether sphingolipid changes in these diseases are pathological events or homeostatic responses. Furthermore, how changes in sphingolipid homeostasis shape the progression of aging and neurodegeneration remains to be clarified. We identified two mouse strains, flincher (fln) and toppler (to), with spontaneous recessive mutations that cause cerebellar ataxia and Purkinje cell degeneration. Positional cloning demonstrated that these mutations reside in the Lass1 gene. Lass1 encodes (dihydro)ceramide synthase 1 (CerS1), which is highly expressed in neurons. Both fln and to mutations caused complete loss of CerS1 catalytic activity, which resulted in a reduction in sphingolipid biosynthesis in the brain and dramatic changes in steady-state levels of sphingolipids and sphingoid bases. In addition to Purkinje cell death, deficiency of CerS1 function also induced accumulation of lipofuscin with ubiquitylated proteins in many brain regions. Our results demonstrate clearly that ceramide biosynthesis deficiency can cause neurodegeneration and suggest a novel mechanism of lipofuscin formation, a common phenomenon that occurs during normal aging and in some neurodegenerative diseases

More Than 1,001 Problems with Protein Domain Databases: Transmembrane Regions, Signal Peptides and the Issue of Sequence Homology

Large-scale genome sequencing gained general importance for life science because functional annotation of otherwise experimentally uncharacterized sequences is made possible by the theory of biomolecular sequence homology. Historically, the paradigm of similarity of protein sequences implying common structure, function and ancestry was generalized based on studies of globular domains. Having the same fold imposes strict conditions over the packing in the hydrophobic core requiring similarity of hydrophobic patterns. The implications of sequence similarity among non-globular protein segments have not been studied to the same extent; nevertheless, homology considerations are silently extended for them. This appears especially detrimental in the case of transmembrane helices (TMs) and signal peptides (SPs) where sequence similarity is necessarily a consequence of physical requirements rather than common ancestry. Thus, matching of SPs/TMs creates the illusion of matching hydrophobic cores. Therefore, inclusion of SPs/TMs into domain models can give rise to wrong annotations. More than 1001 domains among the 10,340 models of Pfam release 23 and 18 domains of SMART version 6 (out of 809) contain SP/TM regions. As expected, fragment-mode HMM searches generate promiscuous hits limited to solely the SP/TM part among clearly unrelated proteins. More worryingly, we show explicit examples that the scores of clearly false-positive hits, even in global-mode searches, can be elevated into the significance range just by matching the hydrophobic runs. In the PIR iProClass database v3.74 using conservative criteria, we find that at least between 2.1% and 13.6% of its annotated Pfam hits appear unjustified for a set of validated domain models. Thus, false-positive domain hits enforced by SP/TM regions can lead to dramatic annotation errors where the hit has nothing in common with the problematic domain model except the SP/TM region itself. We suggest a workflow of flagging problematic hits arising from SP/TM-containing models for critical reconsideration by annotation users

Supplementary Material for: Induction of Regulatory T Cells as a Novel Mechanism Underlying the Therapeutic Action of Kakkonto, a Traditional Japanese Herbal Medicine, in a Murine Food Allergy Model

<b><i>Background:</i></b> The number of patients with food allergy (FA) has dramatically increased. Although satisfactory drug therapies for FA are not available, we have found that kakkonto, a traditional Japanese herbal medicine, suppressed the occurrence of allergic symptoms in an FA mouse model. Thus, we investigated whether kakkonto could regulate the activation and differentiation of T cells in the colon. <b><i>Methods:</i></b> BALB/c mice were systemically sensitized and then orally challenged with ovalbumin. FA mice were orally treated with kakkonto. Lamina propria (LP) cells from their colons were isolated and analyzed. <b><i>Results:</i></b> Kakkonto significantly reduced the proportion of CD69⁺ cells and the elevated helper T cell type 2-specific transcription factor GATA-3 mRNA expression in the LP CD4⁺ T cells, showing that kakkonto has a suppressive effect on the activation and Th2 differentiation of LP effector CD4⁺ T cells of the FA mouse colon. Furthermore, kakkonto significantly increased the proportion of Foxp3⁺CD4⁺ regulatory T cells in the LP CD4⁺ T cells of the FA mouse colon. Similarly, the number of Foxp3-positive cells was dramatically increased in the colonic mucosa of kakkonto-administered FA mice. However, the pharmacological effect and Foxp3⁺CD4⁺ regulatory T cell-inducing ability of kakkonto were not attenuated by the administration of an anti-CD25 monoclonal antibody in the FA model. <b><i>Conclusions:</i></b> The induction of Foxp3⁺CD4⁺CD25^- regulatory T cells in the colon as a novel mechanism underlying the therapeutic action of kakkonto could be utilized for the development of a novel anti-FA drug

Genetic analysis of hyl-1, the C. elegans homolog of LAG1/LASS1

Yeast LAG1 was one of the first longevity genes found. Subsequent analysis showed that it encodes a component of ceramide synthase. Homologs of LAG1 have been identified in all eukaryotes examined for their presence, and multiple homologs are the norm. In human and mouse, the LAG1 counterpart is called LASS1. The involvement of this gene in determining yeast replicative life span led us to ask whether longevity effects could be found in C. elegans. Extended longevity was seen when we used RNAi to decrease expression of the worm homolog of LAG1, termed hyl-1, for Homolog of Yeast Longevity gene. In contrast, neither deletion of the gene nor overexpression resulted in life extension. There was no evidence that hyl-1 interacts with the insulin/IGF-1 like signaling pathway to specify longevity or dauer formation, nor were effects on stress resistance detected. Gene expression of hyl-1 homologs was altered in the deletion mutant and by RNAi, showing distinct evidence for compensation at the transcript level. These regulatory changes may explain the subtle phenotypic effects found under the conditions studied here