Andrés D. Klein

RIPK3 as a potential therapeutic target for Gaucher's disease

Gauchers disease (GD), an inherited metabolic disorder caused by mutations in the glucocerebrosidase gene (GBA), is the most common lysosomal storage disease. Heterozygous mutations in GBA are a major risk factor for Parkinsons disease. GD is divided into three clinical subtypes based on the absence (type 1) or presence (types 2 and 3) of neurological signs. Type 1 GD was the first lysosomal storage disease (LSD) for which enzyme therapy became available, and although infusions of recombinant glucocerebrosidase (GCase) ameliorate the systemic effects of GD, the lack of efficacy for the neurological manifestations, along with the considerable expense and inconvenience of enzyme therapy for patients, renders the search for alternative or complementary therapies paramount. Glucosylceramide and glucosylsphingosine accumulation in the brain leads to massive neuronal loss in patients with neuronopathic GD (nGD) and in nGD mouse models. However, the mode of neuronal death is not known. Here, we show that modulating the receptor-interacting protein kinase-3 (Ripk3) pathway markedly improves neurological and systemic disease in a mouse model of GD. Notably, Ripk3 deficiency substantially improved the clinical course of GD mice, with increased survival and motor coordination and salutary effects on cerebral as well as hepatic injury.

Imatinib therapy blocks cerebellar apoptosis and improves neurological symptoms in a mouse model of Niemann-Pick type C disease

Niemann‐Pick type C (NPC) disease is a fatal autosomal recessive disorder characterized by the accumulation of free cholesterol and glycosphingolipids in the endosomal‐lysosomal system. Patients with NPC disease have markedly progressive neuronal loss, mainly of cerebellar Purkinje neurons. There is strong evidence indicating that cholesterol accumulation and trafficking defects activate apoptosis in NPC brains. The purpose of this study was to analyze the relevance of apoptosis and particularly the proapoptotic c‐Abl/ p73 system in cerebellar neuron degeneration in NPC disease. We used the NPC1 mouse model to evaluate c‐Abl/p73 expression and activation in the cerebellum and the effect of therapy with the c‐Abl‐specific inhibitor imatinib. The proapoptotic c‐Abl/p73 system and the p73 target genes are expressed in the cerebellums of NPC mice. Furthermore, inhibition of c‐Abl with imatinib preserved Purkinje neurons and reduced general cell apoptosis in the cerebellum, improved neurological symptoms, and increased the survival of NPC mice. Moreover, this prosurvival effect correlated with reduced mRNA levels of p73 proapoptotic target genes. Our results suggest that the c‐Abl/p73 pathway is involved in NPC neurodegeneration and show that treatment with c‐Abl inhibitors is useful in delaying progressive neurodegeneration, supporting the use of imatinib for clinical treatment of patients with NPC disease.—Alvarez, A. R., Klein, A., Castro, J., Cancino, G. I., Amigo, J., Mosqueira, M., Vargas, L. M., Yévenes, L. F., Bronfman, F. C., Zanlungo, S. Imatinib therapy blocks cerebellar apoptosis and improves neurological symptoms in a mouse model of Niemann Pick type C disease. FASEB J. 22, 3617–3627 (2008)

NPC2 is expressed in human and murine liver and secreted into bile: Potential implications for body cholesterol homeostasis†

The liver plays a critical role in the metabolism of lipoprotein cholesterol and in controlling its elimination through the bile. Niemann‐Pick type C 2 (NPC2), a cholesterol‐binding protein, is key for normal intracellular trafficking of lipoprotein cholesterol, allowing its exit from the endolysosomal pathway into the metabolically active pool of the cell. In addition, NPC2 is a secretory protein from astrocytes and epididymal cells. Although NPC2 mRNA is detected in the liver, plasma and biliary NPC2 protein levels and function have not been reported. This study demonstrates that NPC2 is present in murine and human plasma and bile. In addition, hepatic NPC2 protein expression was dramatically increased in NPC1‐deficient mice but not regulated by cholesterol feeding or pharmacological modulation of various nuclear receptors involved in cholesterol and bile acid metabolism. Interestingly, biliary NPC2 levels were 3‐fold increased in gallstone‐susceptible C57BL6/J versus gallstone‐resistant BALB/c mice. Furthermore, NPC2 was exclusively found in the cholesterol pro‐nucleating ConA‐binding fraction of human bile. In conclusion, NPC2 is secreted from the liver into bile and plasma, where it may have a functional role in cholesterol transport in normal and disease conditions. (HEPATOLOGY 2006;43:126–133.)

Reduced ceramide synthase 2 activity causes progressive myoclonic epilepsy.

Ceramides are precursors of complex sphingolipids (SLs), which are important for normal functioning of both the developing and mature brain. Altered SL levels have been associated with many neurodegenerative disorders, including epilepsy, although few direct links have been identified between genes involved in SL metabolism and epilepsy.

Npc1 deficiency in the C57BL/6J genetic background enhances Niemann-Pick disease type C spleen pathology.

Niemann-Pick type C (NPC) disease is an autosomal recessive neurovisceral lipid storage disorder. The affected genes are NPC1 and NPC2. Mutations in either gene lead to intracellular cholesterol accumulation. There are three forms of the disease, which are categorized based on the onset and severity of the disease: the infantile form, in which the liver and spleen are severely affected, the juvenile form, in which the liver and brain are affected, and the adult form, which affects the brain. In mice, a spontaneous mutation in the Npc1 gene originated in the BALB/c inbred strain mimics the juvenile form of the disease. To study the influence of genetic background on the expression of NPC disease in mice, we transferred the Npc1 mutation from the BALB/c to C57BL/6J inbred background. We found that C57BL/6J-Npc1(-/-) mice present with a much more aggressive form of the disease, including a shorter lifespan than BALB/c-Npc1(-/-) mice. Surprisingly, there was no difference in the amount of cholesterol in the brains of Npc1(-/-) mice of either mouse strain. However, Npc1(-/-) mice with the C57BL/6J genetic background showed striking spleen damage with a marked buildup of cholesterol and phospholipids at an early age, which correlated with large foamy cell clusters. In addition, C57BL/6J Npc1(-/-) mice presented red cell abnormalities and abundant ghost erythrocytes that correlated with a lower hemoglobin concentration. We also found abnormalities in white cells, such as cytoplasmic granulation and neutrophil hypersegmentation that included lymphopenia and atypias. In conclusion, Npc1 deficiency in the C57BL6/J background is associated with spleen, erythrocyte, and immune system abnormalities that lead to a reduced lifespan.

Delineating pathological pathways in a chemically induced mouse model of Gaucher disease

Great interest has been shown in understanding the pathology of Gaucher disease (GD) due to the recently discovered genetic relationship with Parkinsons disease. For such studies, suitable animal models of GD are required. Chemical induction of GD by inhibition of acid β‐glucosidase (GCase) using the irreversible inhibitor conduritol B‐epoxide (CBE) is particularly attractive, although few systematic studies examining the effect of CBE on the development of symptoms associated with neurological forms of GD have been performed. We now demonstrate a correlation between the amount of CBE injected into mice and levels of accumulation of the GD substrates, glucosylceramide and glucosylsphingosine, and show that disease pathology, indicated by altered levels of pathological markers, depends on both the levels of accumulated lipids and the time at which their accumulation begins. Gene array analysis shows a remarkable similarity in the gene expression profiles of CBE‐treated mice and a genetic GD mouse model, the Gbaflox/flox;nestin‐Cre mouse, with 120 of the 144 genes up‐regulated in CBE‐treated mice also up‐regulated in Gbaflox/flox;nestin‐Cre mice. We also demonstrate that various aspects of neuropathology and some behavioural abnormalities can be arrested upon cessation of CBE treatment during a specific time window. Together, our data demonstrate that injection of mice with CBE provides a rapid and relatively easy way to induce symptoms typical of neuronal forms of GD. This is particularly useful when examining the role of specific biochemical pathways in GD pathology, since CBE can be injected into mice defective in components of putative pathological pathways, alleviating the need for time‐consuming crossing of mice. Copyright

Identification of Modifier Genes in a Mouse Model of Gaucher Disease

Diseases caused by single-gene mutations can display substantial phenotypic variability, which may be due to genetic, environmental, or epigenetic modifiers. Here, we induce Gaucher disease (GD), a rare inherited metabolic disorder, by injecting 15 inbred mouse strains with a low dose of a chemical inhibitor of acid β-glucosidase, the enzyme defective in GD. Different mouse strains exhibit widely different lifespans, which is unrelated to levels of acid β-glucosidases substrate accumulation. Genome-wide association reveals a number of candidate risk loci, including a marker within Grin2b, which in combination with another marker allows us to predict the lifespan of additional mouse strains. An antagonist of the NMDA receptor (encoded by Grin2b) significantly increases the lifespan of GD mice that would otherwise have lived for a short time. Our data identify putative modifier genes that may be involved in determining GD severity, which might help elucidate phenotypic variability between patients with similar GD mutations.

Lack of Activation of the Unfolded Protein Response in Mouse and Cellular Models of Niemann-Pick Type C Disease

Background: Niemann-Pick type C (NPC) disease is a fatal lysosomal storage disease related to progressive neurodegeneration secondary to abnormal intracellular accumulation of cholesterol. Signs of endoplasmic reticulum (ER) stress have been reported in other lipidoses. Adaptation to ER stress is mediated by the unfolded protein response (UPR), an integrated signal transduction pathway that attenuates stress or triggers apoptosis of irreversibly damaged cells. Objective: To investigate the possible engagement of ER stress responses in NPC models. Methods: We used NPC1 deficient mice and an NPC cell-based model by knocking down the expression of NPC1 to measure several UPR markers through different approaches. Results: Despite expectations that the UPR will be activated in NPC, our results indicate a lack of ER stress reactions in the cerebellum of symptomatic mice. Similarly, knocking down NPC1 in Neuro2a cells leads to clear cholesterol accumulation without evidence of UPR activation. Conclusion: Our results suggest that cholesterol overload and neuronal dysfunction in NPC is not associated with ER stress, which contrasts with recent reports suggesting the activation of the UPR in other lysosomal storage diseases.

Modeling diseases in multiple mouse strains for precision medicine studies

The genetic basis of the phenotypic variability observed in patients can be studied in mice by generating disease models through genetic or chemical interventions in many genetic backgrounds where the clinical phenotypes can be assessed and used for genome-wide association studies (GWAS). This is particularly relevant for rare disorders, where patients sharing identical mutations can present with a wide variety of symptoms, but there are not enough number of patients to ensure statistical power of GWAS. Inbred strains are homozygous for each loci, and their single nucleotide polymorphisms catalogs are known and freely available, facilitating the bioinformatics and reducing the costs of the study, since it is not required to genotype every mouse. This kind of approach can be applied to pharmacogenomics studies as well.

Controversies on the potential therapeutic use of rapamycin for treating a lysosomal cholesterol storage disease

Precision medicine aims at developing tailored interventions for each patient based on their biology. This is particularly relevant for lysosomal storage disorders (LSDs), such as Niemann-Pick type C (NPC), where disease symptoms are highly heterogeneous among patients and paradoxically one-size-fits-all therapeutic approaches are usually proposed. Under this logic, the use of rapamycin has been explored in cell-based models of NPC disease showing contradictory results. On one hand, induction of autophagy with rapamycin increased the levels of cholesterol buildup in human skin NPC cells and its inhibition decreased cholesterol accumulation, indicating that autophagy is an important source of stored cholesterol in NPC lysosomes [1]. In addition, NPC-induced mitochondrial fragmentation can be rescued by the autophagy inhibitor 3-methyladenine in human NPC neurons [2], suggesting that inhibition of autophagy could be a therapeutic option for NPC. On the other hand, stimulating autophagy with rapamycin restored the autophagic flux in human NPC iPSC-derived hepatic and neuronal cells, leading to increased cellular viability [3], suggesting that promotion of autophagy may be a strategy for treating NPC disease. Can both lines of evidence be correct? To test the therapeutic outcomes of rapamycin for NPC disease progression, male Npc1 mice from C57BL6/J (C57) and FVB/NJ (FVB) genetic backgrounds, which have been previously generated [4,5] were treated starting from postnatal day 20. Average lifespan of PBS-treated FVB-NPC mice is ∼75-days, while C57-NPC survive ∼30 days. Rapamycin, which was prepared as previously described [6], led to an amazing 100% increase in lifespan in the C57 background (p= 0.00592); however it was toxic for the FVB-NPC mice, reducing their lifespans (p=0.00265) (Fig. 1).