Protoporphyrin IX accumulation disrupts mitochondrial dynamics and function in ABCG2-deficient hepatocytes

Targeted inhibition of multidrug ABCG2 transporter is believed to improve cancer therapeutics. However, the consequences of ABCG2 inhibition have not been systematically evaluated since ABCG2 is expressed in several organs including the liver. Here, we demonstrate that ABCG2-deficient hepatocytes have increased amounts of fragmental mitochondria accompanied by disruption of mitochondrial dynamics and functions. This disruption was due to ABCG2 knockout elevating intracellular protoporphyrin IX, which led to upregulation of DRP-1-mediated mitochondrial fission. The finding that ABCG2 deficiency can generate dysfunctional mitochondria in hepatocytes raises concerns regarding the systematic use of ABCG2 inhibitor in cancer patients.     

Modest actomyosin energy conservation increases myocardial postischemic function

We have proposed that pharmacological preconditioning, leading to PKC-epsilon activation, in hearts improves postischemic functional recovery through a decrease in actomyosin ATPase activity and subsequent ATP conservation. The purpose of the present study was to determine whether moderate PKC-independent decreases in actomyosin ATPase are sufficient to improve myocardial postischemic function. Rats were given propylthiouracil (PTU) for 8 days to induce a 25% increase in beta-myosin heavy chain with a 28% reduction in actomyosin ATPase activity. Recovery of postischemic left ventricular developed pressure (LVDP) was significantly higher in PTU-treated rat hearts subjected to 30 min of global ischemia than in control hearts: 57.9 +/- 6.2 vs. 32.6 +/- 5.1% of preischemic values. In addition, PTU-treated hearts exhibited a delayed onset of rigor contracture during ischemia and a higher global ATP content after ischemia. In the second part of our study, we demonstrated a lower maximal actomyosin ATPase and a higher global ATP content after ischemia in human troponin T (TnT) transgenic mouse hearts. In mouse hearts with and without a point mutation at F110I of human TnT, recovery of postischemic LVDP was 55.4 +/- 5.5 and 62.5 +/- 14.5% compared with 20.0 +/- 2.9% in nontransgenic mouse hearts after 35 min of global ischemia. These results are consistent with the hypothesis that moderate decreases in actomyosin ATPase activity result in net ATP conservation that is sufficient to improve postischemic contractile function.     

Cholesterol accumulation sequesters Rab9 and disrupts late endosome function in NPC1-deficient cells

Niemann-Pick type C disease is an autosomal recessive disorder that leads to massive accumulation of cholesterol and glycosphingolipids in late endosomes and lysosomes. To understand how cholesterol accumulation influences late endosome function, we investigated the effect of elevated cholesterol on Rab9-dependent export of mannose 6-phosphate receptors from this compartment. Endogenous Rab9 levels were elevated 1.8-fold in Niemann-Pick type C cells relative to wild type cells, and its half-life increased 1.6-fold, suggesting that Rab9 accumulation is caused by impaired protein turnover. Reduced Rab9 degradation was accompanied by stabilization on endosome membranes, as shown by a reduction in the capacity of Rab9 for guanine nucleotide dissociation inhibitor-mediated extraction from Niemann-Pick type C membranes. Cholesterol appeared to stabilize Rab9 directly, as liposomes loaded with prenylated Rab9 showed decreased extractability with increasing cholesterol content. Rab9 is likely sequestered in an inactive form on Niemann-Pick type C membranes, as cation-dependent mannose 6-phosphate receptors were missorted to the lysosome for degradation, a process that was reversed by overexpression of GFP-tagged Rab9. In addition to using primary fibroblasts isolated from Niemann-Pick type C patients, RNA interference was utilized to recapitulate the disease phenotype in cultured cells, greatly facilitating the analysis of cholesterol accumulation and late endosome function. We conclude that cholesterol contributes directly to the sequestration of Rab9 on Niemann-Pick type C cell membranes, which in turn, disrupts mannose 6-phosphate receptor trafficking.     

Impaired striatal Akt signaling disrupts dopamine homeostasis and increases feeding

Background

The prevalence of obesity has increased dramatically worldwide. The obesity epidemic begs for novel concepts and therapeutic targets that cohesively address “food-abuse” disorders. We demonstrate a molecular link between impairment of a central kinase (Akt) involved in insulin signaling induced by exposure to a high-fat (HF) diet and dysregulation of higher order circuitry involved in feeding. Dopamine (DA) rich brain structures, such as striatum, provide motivation stimuli for feeding. In these central circuitries, DA dysfunction is posited to contribute to obesity pathogenesis. We identified a mechanistic link between metabolic dysregulation and the maladaptive behaviors that potentiate weight gain. Insulin, a hormone in the periphery, also acts centrally to regulate both homeostatic and reward-based HF feeding. It regulates DA homeostasis, in part, by controlling a key element in DA clearance, the DA transporter (DAT). Upon HF feeding, nigro-striatal neurons rapidly develop insulin signaling deficiencies, causing increased HF calorie intake.

Methodology/Principal Findings

We show that consumption of fat-rich food impairs striatal activation of the insulin-activated signaling kinase, Akt. HF-induced Akt impairment, in turn, reduces DAT cell surface expression and function, thereby decreasing DA homeostasis and amphetamine (AMPH)-induced DA efflux. In addition, HF-mediated dysregulation of Akt signaling impairs DA-related behaviors such as (AMPH)-induced locomotion and increased caloric intake. We restored nigro-striatal Akt phosphorylation using recombinant viral vector expression technology. We observed a rescue of DAT expression in HF fed rats, which was associated with a return of locomotor responses to AMPH and normalization of HF diet-induced hyperphagia.

Conclusions/Significance

Acquired disruption of brain insulin action may confer risk for and/or underlie “food-abuse” disorders and the recalcitrance of obesity. This molecular model, thus, explains how even short-term exposure to “the fast food lifestyle” creates a cycle of disordered eating that cements pathological changes in DA signaling leading to weight gain and obesity.     

Multivitamin restriction increases adiposity and disrupts glucose homeostasis in mice

A strong association between obesity and low plasma concentrations of vitamins has been widely reported; however, the causality of this relationship is still not established. Our goal was to evaluate the impact of a multivitamin restriction diet (MRD) on body weight, adiposity and glucose homeostasis in mice. The mice were given a standard diet or a diet containing 50 % of the recommended vitamin intake (MRD) for 12 weeks. At the end of the experiment, total body weight was 6 % higher in MRD animals than in the control group, and the adiposity of the MRD animals more than doubled. The HOMA-IR index of the MRD animals was significantly increased. The adipose tissue of MRD animals had lower expression of mRNA encoding adiponectin and Pnpla2 (47 and 32 %, respectively) and 43 % higher leptin mRNA levels. In the liver, the mRNA levels of Pparα and Pgc1α were reduced (29 and 69 %, respectively) in MRD mice. Finally, the level of β-hydroxybutyrate, a ketonic body reflecting fatty acid oxidation, was decreased by 45 % in MRD mice. Our results suggest that MRD promotes adiposity, possibly by decreasing adipose tissue lipolysis and hepatic β-oxidation. These results could highlight a possible role of vitamin deficiency in the etiology of obesity and associated disorders.

Electronic supplementary material

The online version of this article (doi:10.1007/s12263-014-0410-x) contains supplementary material, which is available to authorized users.     

Insulin structure and function

Throughout much of the last century insulin served a central role in the advancement of peptide chemistry, pharmacology, cell signaling and structural biology. These discoveries have provided a steadily improved quantity and quality of life for those afflicted with diabetes. The collective work serves as a foundation for the development of insulin analogs and mimetics capable of providing more tailored therapy. Advancements in patient care have been paced by breakthroughs in core technologies, such as semisynthesis, high performance chromatography, rDNA-biosynthesis and formulation sciences. How the structural and conformational dynamics of this endocrine hormone elicit its biological response remains a vigorous area of study. Numerous insulin analogs have served to coordinate structural biology and biochemical signaling to provide a first level understanding of insulin action. The introduction of broad chemical diversity to the study of insulin has been limited by the inefficiency in total chemical synthesis, and the inherent limitations in rDNA-biosynthesis and semisynthetic approaches. The goals of continued investigation remain the delivery of insulin therapy where glycemic control is more precise and hypoglycemic liability is minimized. Additional objectives for medicinal chemists are the identification of superagonists and insulins more suitable for non-injectable delivery. The historical advancements in the synthesis of insulin analogs by multiple methods is reviewed with the specific structural elements of critical importance being highlighted. The functional refinement of this hormone as directed to improved patient care with insulin analogs of more precise pharmacology is reported.     

Tebufenozide disrupts ovarian development and function in silkmoths

Adult development and production of up to 400 eggs within the pupal case of female silkmoths are both dependent on 20-hydroxyecdysone (20E), the steroid hormone of insects. When adult development was initiated with tebufenozide, the non-steroidal ecdysteroid agonist, instead of 20E, full development of all epidermal tissues like the wing was witnessed, but ovarian growth and egg formation was minimal. Administration of tebufenozide to female pharate adults caused disruption of the follicular epithelium, produced nurse cell damage, and inhibited oogenesis. Reduced ability to synthesize RNA and protein accompanied these tebufenozide induced morphological disturbances of the follicles. In vivo accumulation of vitellogenin (Vg) from the hemolymph was reduced in tebufenozide treated female ovaries as well as their ability to accumulate Vg in vitro. Determination of protein staining intensity and antibody reactivity of Vg pointed out that hemolymph Vg level remained fairly constant all through adult development whether induced by 20E or tebufenozide. Measurement of hemolymph volumes and hemolymph Vg levels of control and experimental animals allowed us to conclude that egg development involves the uptake of all the hemolymph proteins and not Vg alone. The loss of hemolymph that accompanies egg maturation was considerably reduced in tebufenozide initiated female pharate adults. 20E could not overcome ovarian growth inhibitory effects of tebufenozide. Dual mechanisms, one involving ecdysteroid antagonist action at the beginning of development, and the other unrelated to that function during heightened egg formation, are needed explain the biphasic inhibitory actions of tebufenozide on silkmoth ovaries.     

Testosterone treatment increases thromboxane function in rat cerebral arteries

We previously showed that testosterone, administered in vivo, increases the tone of cerebral arteries. A possible underlying mechanism is increased vasoconstriction through the thromboxane A2 (TxA2) pathway. Therefore, we investigated the effect of chronic testosterone treatment (4 wk) on TxA2 synthase levels and the contribution of TxA2 to vascular tone in rat middle cerebral arteries (MCAs). Using immunofluorescence and confocal microscopy, we demonstrated that TxA2 synthase is present in MCA segments in both smooth muscle and endothelial layers. Using Western blot analysis, we found that TxA2 synthase protein levels are higher in cerebral vessel homogenates from testosterone-treated orchiectomized (ORX + T) rats compared with orchiectomized (ORX) control animals. Functional consequences of changes in cerebrovascular TxA2 synthase were determined using cannulated, pressurized MCA segments in vitro. Constrictor responses to the TxA2 mimetic U-46619 were not different between the ORX + T and ORX groups. However, dilator responses to either the selective TxA2 synthase inhibitor furegrelate or the TxA2-endoperoxide receptor (TP) antagonist SQ-29548 were greater in the ORX + T compared with ORX group. In endothelium-denuded arteries, the dilation to furegrelate was attenuated in both the ORX and ORX + T groups, and the difference between the groups was abolished. These data suggest that chronic testosterone treatment enhances TxA2-mediated tone in rat cerebral arteries by increasing endothelial TxA2 synthesis without altering the TP receptors mediating constriction. The effect of in vivo testosterone on cerebrovascular TxA2 synthase, observed here after chronic hormone administration, may contribute to the risk of vasospasm and thrombosis related to cerebrovascular disease.     

Manganese disrupts astrocyte glutamine transporter expression and function

Glutamine (Gln) plays an important role in brain energy metabolism and as a precursor for the synthesis of neurotransmitter glutamate and GABA. Previous studies have shown that astrocytic Gln transport is impaired following manganese (Mn) exposure. The present studies were performed to identify the transport routes and the respective Gln transporters contributing to the impairment. Rat neonatal cortical primary astrocytes treated with Mn displayed a significant decrease in Gln uptake mediated by the principle Gln transporting systems, N and ASC. Moreover, systems N, ASC and L were less efficient in Gln export after Mn treatment. Mn treatment caused a significant reduction of both in mRNA expression and protein levels of SNAT3 (system N), SNAT2 (system A) and LAT2 (system L), and lowered the protein but not mRNA expression of ASCT2 (system ASC). Mn exposure did not affect the expression of the less abundant systems N transporter SNAT5 and the system L transporter LAT1, at either the mRNA or protein level. Hence, Mn-induced decrease of inward and outward Gln transport can be largely ascribed to the loss of the specific Gln transporters. Consequently, deregulation of glutamate homeostasis and its diminished availability to neurons may lead to impairment in glutamatergic neurotransmission, a phenomenon characteristic of Mn-induced neurotoxicity.     

Insulin increases macrophage triglyceride accumulation under diabetic conditions through the down regulation of hormone sensitive lipase and adipose triglyceride lipase

Diabetes mellitus (DM) is a major risk factor for the development of atherosclerosis, and high-serum levels of insulin are strongly associated with type 2 DM. Atherosclerosis is characterized by lipid-laden macrophage foam cell formations, which contain substantial amount of cholesterol and triglycerides (TG). This study analyzed for the first time, the effects of insulin on TG metabolism in macrophages under normal and diabetic conditions. Mouse peritoneal macrophages from C57BL6 mice were cultured under normal (5 mM) or high (diabetic condition, 25 mM) glucose concentration, with or without insulin, followed by the assessment of TGs metabolism in these cells. Under diabetic condition, insulin increased TG accumulation in macrophages by 100%, decreased cellular TG degradation by 21%, and increased C-reactive protein levels in macrophages by 83%. Insulin decreased hormone-sensitive lipase mRNA and protein expression by 28 and 60%, respectively, and adipose TG lipase (ATGL) protein expression by 36%, with no significant reduction in ATGL mRNA levels. The inhibition of insulin-mediated phosphorylation, and the addition of cyclic adenosine 3'5'-monoposphate, abolished the insulin-mediated inhibition of TGs degradation in cells. Insulin increases macrophage TGs accumulation only under diabetic conditions, suggesting that impaired glycemic control in diabetic patients treated with insulin may contribute to foam cell formations and enhanced inflammation in macrophages.     

Insulin inhibits myocardial ischemia-induced apoptosis and alleviates chronic adverse changes in post-ischemic cardiac structure and function

Insulin has been shown to possess significant anti-apoptotic effect in myocardial ischemia/reperfusion (MI/R). However, the contribution by this protection of insulin to the prolonged cardiac function in rats subjected to ischemia remains unclear. The present study attempted to test whether early insulin treatment influences adverse prolonged post-ischemic cardiac structural and functional changes. Adult male rats were subjected to left anterior descending coronary artery occlusion and were randomized to receive one of the following treatments: saline (4 ml/kg/h i.v. injection beginning 10 min before the ischemia and continuing for 2 h), insulin (60 U/l, i.v. injection following the same routine, and hypodermic injection of insulin (0.5 U/ml, 1 ml/kg/d) for 3 days after the ischemia surgery) or insulin plus wortmannin (15 μg/kg i.v. injection 15 min before each insulin administration). Treatment with insulin significantly reduced infarct size, decreased plasma creatine kinase and lactate dehydrogenase activities, decreased apoptosis index and caspase-3 activity (all P < 0.01 vs. saline), and improved cardiac function 24 h after ischemia. Importantly, at the end of 4 weeks after the ischemia surgery, MI rats receiving insulin treatment showed smaller left ventricle (LV) cavity and thicker systolic interventricular septum, and increased cardiac ejection fraction and LV fractional shortening (all P < 0.05 vs. saline). Inhibition of insulin signaling with wortmannin not only blocked insulin’s anti-apoptotic effect, but also almost completely abolished effects of insulin on cardiac structure and function. These data indicate that inhibition of apoptosis by early insulin treatment alleviates chronic adverse changes in post-ischemic cardiac structure and function. Wenjuan Xing and Wenjun Yan contributed equally to this study.     

Hindlimb unloading increases oxidative stress and disrupts antioxidant capacity in skeletal muscle

Skeletal muscle disuse with space-flight and ground-based models (e.g., hindlimb unloading) results in dramatic skeletal muscle atrophy and weakness. Pathological conditions that cause muscle wasting (i.e., heart failure, muscular dystrophy, sepsis, COPD, cancer) are characterized by elevated "oxidative stress," where antioxidant defenses are overwhelmed by oxidant production. However, the existence, cellular mechanisms, and ramifications of oxidative stress in skeletal muscle subjected to hindlimb unloading are poorly understood. Thus we examined the effects of hindlimb unloading on hindlimb muscle antioxidant enzymes (e.g., superoxide dismutase, catalase, glutathione peroxidase), nonenzymatic antioxidant scavenging capacity (ASC), total hydroperoxides, and dichlorohydrofluorescein diacetate (DCFH-DA) oxidation, a direct indicator of oxidative stress. Twelve 6 month old Sprague Dawley rats were divided into two groups: 28 d of hindlimb unloading (n = 6) and controls (n = 6). Hindlimb unloading resulted in a small decrease in Mn-superoxide dismutase activity (10.1%) in the soleus muscle, while Cu,Zn-superoxide dismutase increased 71.2%. In contrast, catalase and glutathione peroxidase, antioxidant enzymes that remove hydroperoxides, were significantly reduced in the soleus with hindlimb unloading by 54.5 and 16.1%, respectively. Hindlimb unloading also significantly reduced ASC. Hindlimb unloading increased soleus lipid hydroperoxide levels by 21.6% and hindlimb muscle DCFH-DA oxidation by 162.1%. These results indicate that hindlimb unloading results in a disruption of antioxidant status, elevation of hydroperoxides, and an increase in oxidative stress.     

DNA damage induces down-regulation of UDP-glucose ceramide glucosyltransferase, increases ceramide levels and triggers apoptosis in p53-deficient cancer cells

DNA damaging agents typically induce an apoptotic cascade in which p53 plays a central role. However, absence of a p53-mediated response does not necessarily abrogate programmed cell death, due to the existence of p53-independent apoptotic pathways, such as those mediated by the pro-apoptotic molecule ceramide. We compared ceramide levels before and after DNA damage in human osteosarcoma (U2OS) and colon cancer (HCT116) cells that were either expressing or deficient in p53. When treated with mitomycin C, p53-deficient cells, but not p53-expressing cells, showed a marked increase in ceramide levels. Microarray analysis of genes involved in ceramide metabolism identified acid ceramidase (ASAH1, up-regulated), ceramide glucosyltransferase (UGCG, down-regulated), and galactosylceramidase (GALC, up-regulated) as the three genes most affected. Experiments employing pharmacological and siRNA agents revealed that inhibition of UGCG is sufficient to increase ceramide levels and induce cell death. When inhibition of UGCG and treatment with mitomycin C were combined, p53-deficient, but not p53-expressing cells, showed a significant increase in cell death, suggesting that the regulation of sphingolipid metabolism could be used to sensitize cells to chemotherapeutic drugs.     

Aberrant intracellular cholesterol transport disrupts pituitary and ovarian function

Cholesterol is imported and processed to provide substrate for ovarian steroidogenesis. The Niemann Pick type C-1 gene codes for a glycoprotein that processes low-density lipoproteinimported cholesterol. Mutation of this gene causes marked impairment of export of low-density lipoprotein-derived cholesterol from endosomes, and consequent lysosomal accumulation of the sterol. The BALB/c npc(nih-/-) mouse line, bearing spontaneous mutation of the NPC-1 gene, provides a model for investigation of aberrant endosomal cholesterol transfer in the ovary. Female homozygote mutant mice are infertile, with underdeveloped ovarian follicles, reduced steroidogenesis, no ovulation, and no corpora lutea. Mutant ovaries transplanted under wild-type kidney capsules display both ovulation and formation of corpora lutea. Gonadotropin treatment induces ovulation and restores expression of steroidogenic proteins. Pituitary glands of mutants are hypoplastic, and prolactin expression is dramatically reduced compared with wild-type mice. Both long and short splice variants of the dopamine-D2 receptors are overexpressed in the pituitary of BALB/c npc(nih-/-) mice. Chronic treatment of mutant mice with 17beta-estradiol restores pituitary volume, prolactin expression, and folliculogenetic capability. We conclude that inactivating mutation of Niemann Pick C-1 perturbs the hypothalamic-pituitary-ovarian feedback loop. Reduced estrogens attenuate prolactin expression and alter gonadotropin secretion patterns and interfere with normal ovarian follicular development and ovulation.     

Vaccinia virus infection disrupts microtubule organization and centrosome function

We examined the role of the microtubule cytoskeleton during vaccinia virus infection. We found that newly assembled virus particles accumulate in the vicinity of the microtubule-organizing centre in a microtubule- and dynein-dynactin complex-dependent fashion. Microtubules are required for efficient intracellular mature virus (IMV) formation and are essential for intracellular enveloped virus (IEV) assembly. As infection proceeds, the microtubule cytoskeleton becomes dramatically reorganized in a fashion reminiscent of overexpression of microtubule-associated proteins (MAPs). Consistent with this, we report that the vaccinia proteins A10L and L4R have MAP-like properties and mediate direct binding of viral cores to microtubules in vitro. In addition, vaccinia infection also results in severe reduction of proteins at the centrosome and loss of centrosomal microtubule nucleation efficiency. This represents the first example of viral-induced disruption of centrosome function. Further studies with vaccinia will provide insights into the role of microtubules during viral pathogenesis and regulation of centrosome function.     

D,L-Threo-1-phenyl-2-decanoylamino-3-morpholino-1-propanol (DL-PDMP) increases endoplasmic reticulum stress, autophagy and apoptosis accompanying ceramide accumulation via ceramide synthase 5 protein expression in A549 cells

In A549 cells, the addition of D,L-threo-1-phenyl-2-decanoylamino-3-morpholino-1-propanol (DL-PDMP) led to marked autophagy with massive microtubule-associated protein 1 light chain 3B (LC3B)-II protein expression as an indication of autophagy and a steep decrease of p62 protein as a co-indication of autophagy. The addition of DL-PDMP caused massive autophagy with an increase of CAAT/enhancer binding protein homologous protein (CHOP) expression as the marker of endoplasmic reticulum (ER) stress, lactate dehydrogenase (LDH) release without caspase 3 activation and many autophagic vacuoles/devoid of a cell membrane on morphology. On the other hand, the addition of DL-PDMP caused an increase in cellular or subcellular ceramides (Cers), especially palmitoyl-Cer, based on de novo synthesis of Cer, and led to caspase-independent apoptosis. Marked increases of Cer levels in the nuclear envelope were observed 17 h after the addition. The elevations of Cer synthase activity and longevity-assurance homologue (LASS)5 protein expression were observed in subcellular fractions from 30 min until 2 h after the addition. However, the elevations of Cer synthase activity were independent of reactive oxygen species generation or cytochrome P450 4F2 activity. Since an increase in LASS5 protein expression in subcellular fraction occur in preference to the variation of LC3B-II protein expression via CHOP expression after the addition and Cer accumulation induced by the addition contributes to ER stress, it is thought that an elevation of Cer synthase activity via LASS5 protein expression associate to autophagy via CHOP expression (ER stress) with the addition.     

Delta 9-tetrahydrocannabinol disrupts mitochondrial function and cell energetics

We have observed rapid and extensive depletion of cellular energy stores by Delta(9)-tetrahydrocannabinol (THC) in the pulmonary transformed cell line A549. ATP levels declined dose dependently with an IC(50) of 7.5 microg/ml of THC after 24-h exposure. Cell death was observed only at concentrations >10 microg/ml. Studies using JC-1, a fluorescent probe for mitochondrial membrane potential, revealed diminished mitochondrial function at THC concentrations as low as 0.5 microg/ml. At concentrations of 2.5 or 10 microg/ml of THC, a decrease in mitochondrial membrane potential was observed as early as 1 h after THC exposure. Mitochondrial function remained diminished for at least 30 h after THC exposure. Flow cytometry studies on cells exposed to particulate smoke extracts indicate that JC-1 red fluorescence was fivefold lower in cells exposed to marijuana smoke extract relative to cells exposed to tobacco smoke extract. Comparison with a variety of mitochondrial inhibitors demonstrates that THC produced effects similar to that of carbonyl cyanide p-trifluoromethoxyphenylhydrazone, suggesting uncoupling of electron transport. Loss of red JC-1 fluorescence by THC was suppressed by cyclosporin A, suggesting mediation by the mitochondrial permeability transition pore. This disruption of mitochondrial function was sustained for at least 24 h after removal of THC by extensive washing. These results suggest that exposure of the bronchopulmonary epithelium to THC may have important health and physiological consequences.     

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.