Alteraciones en la homeostasis de calcio y estrés oxidativo en neuronas deficientes en la esfingomielinasa ácida. Implicaciones en la enfermedad de Niemann Pick de tipo A

Tesis doctoral inédita leída en la Universidad Autónoma de Madrid, Facultad de Ciencias, Departamento de Biología Molecular. Fecha de lectura: 15-06-2015La esfigomielina (SM) es el esfingolópido más abundante que está especialmente enriquecido en las membranas de las neuronas. Su importancia en la fisiología neuronal se pone de manifiesto en las graves alteraciones neurológicas que caracterizan enfermedades donde los niveles de este lípido están alterados. Este es el caso de la enfermedad de Niemann Pick tipo A (NPA), provocada por mutaciones en el gen que codifica para la esfingomielinasa ácida (ASM) conduciendo a la acumulación de SM en los lisosomas y en la membrana plasmática. La NPA pertenece al grupo de enfermedades de depósito lisosomal (LSDs). A pesar de la naturaleza diferente de los sustratos que se acumulan en las LSDs, éstas comparten características patológicas y clínicas. En este trabajo de tesis hemos caracterizado dos de las vías patológicas comunes a varias LSDs que no habían sido exploradas en la NPA: la homeostasis de calcio y el estrés oxidativo. Nuestros resultados demuestran que el estrés oxidativo es una característica patógica en cerebros de pacientes de NPA y de ratones carentes de ASM (ASM-/-) que son modelo para la enfermedad. El análisis de las neuronas de estos últimos nos ha permitido determinar que el incremento de los niveles de calcio intracelular es el responsable de la aparición del estrés oxidativo. A su vez, el aumento de calcio es consecuencia de la acumulación de SM en la membrana plasmática que reduce la actividad de la ATPasa de calcio de membrana, PMCA. Esta información nos llevó a ensayar in vitro e in vivo una estrategia farmacológica para evitar estas anomalías basada en el uso de un inhibidor de deacetilasas de histonas, SAHA, con demostrada capacidad para aumentar los niveles de PMCA y activar la extrusión de calcio en un modelo celular de cáncer de mama. El tratamiento oral con SAHA en ratones ASM-/- consiguió aumentar los niveles de proteína PMCA en cerebro, prevenir el estrés oxidativo y la neurodegeneración y mejorar su memoria y su coordinación motora. Consideramos que estos resultados revelan un nuevo papel de la SM en la regulación de la homeostasis del calcio neuronal y el estrés oxidativo. Además, identifican un nuevo mecanismo patológico en NPA abriendo perspectivas terapéuticas para esta enfermedad hoy en día incurable. .Sphingomyelin (SM) is the most abundant sphingolipid, which is specially enriched in neuronal membranes. The severe neurological alterations that su er patients of di erent diseases in which the levels of SM are altered reinforce the relevant role of this lipid in neuronal physiology. One of this diseases is Niemann Pick disease type A (NPA), which is caused by mutations in the gene that encodes for acid sphingomyelinase (ASM), promoting SM accumulation in the lysosomes and in the plasma membrane. NPA is classi ed as a lysosomal storage disorder (LSD). Despite the distinctive types of substrates that accumulate in LSDs they share common pathological and clinical features. In this PhD work we have characterized two pathological pathways that are common to many LSDs but have not been explored in NPA: alterations in calcium homeostasis and oxidative stress. The results obtained demonstrate that oxidative stress is a hallmark in the brain of NPA patients and of the mouse model of the disease, the ASM knockout mice (ASM-/-). Analyzing ASM-/- hippocampal neurons we have determined that the increase in basal calcium levels causes the appearance of oxidative stress. In turn, calcium increase is a consequence of SM accumulation in the plasma membrane that reduces the activity of the plasma membrane calcium-ATPase (PMCA). Using a pharmacological strategy in vitro and in vivo we have been able to prevent these anomalies. This strategy is based in the use of SAHA, a histone deacetylase inhibitor that was shown to increase PMCA levels and to enhance calcium clearance in a breast cancer cell model. Oral treatment of ASM-/- mice with SAHA increased PMCA levels in the brain, prevented oxidative stress and neurodegeneration, and improved memory and motor coordination. Taken together, these results unveil the role of SM in the regulation of neuronal calcium homeostasis and oxidative stress. Moreover, they identify a novel pathological mechanism in NPA, opening therapeutical perspectives for this currently untreatable disease

Adeno-associated viral vector serotype 9-based gene therapy for Niemann-Pick disease type A

Niemann-Pick disease type A (NPD-A) is a lysosomal storage disorder characterized by neurodegeneration and early death. It is caused by loss-of-function mutations in the gene encoding for acid sphingomyelinase (ASM), which hydrolyzes sphingomyelin into ceramide. Here, we evaluated the safety of cerebellomedullary (CM) cistern injection of adeno-associated viral vector serotype 9 encoding human ASM (AAV9-hASM) in nonhuman primates (NHP). We also evaluated its therapeutic benefit in a mouse model of the disease (ASM-KO mice). We found that CM injection in NHP resulted in widespread transgene expression within brain and spinal cord cells without signs of toxicity. CM injection in the ASM-KO mouse model resulted in hASM expression in cerebrospinal fluid and in different brain areas without triggering an inflammatory response. In contrast, direct cerebellar injection of AAV9-hASM triggered immune response. We also identified a minimally effective therapeutic dose for CM injection of AAV9-hASM in mice. Two months after administration, the treatment prevented motor and memory impairment, sphingomyelin (SM) accumulation, lysosomal enlargement, and neuronal death in ASM-KO mice. ASM activity was also detected in plasma from AAV9-hASM CM-injected ASM-KO mice, along with reduced SM amount and decreased inflammation in the liver. Our results support CM injection for future AAV9-based clinical trials in NPD-A as well as other lysosomal storage brain disorders.Nation Foundation and by grants from the Spanish Ministry of Economy and Competitivity (SAF-2014-57539-R and SAF2017-87698-R) to M.D.L. and from NIH-NINDS (R01NS073940) to K.S.B. A.P.-C. was a recipient of the FPU predoctoral fellowship from the Spanish Ministry of Economy and Competitivity and Fundación Ramón Areces to the Centro Biología Molecular Severo Ochoa

Adeno-associated viral vector serotype 9–based gene therapy for Niemann-Pick disease type A

Niemann-Pick disease type A (NPD-A) is a lysosomal storage disorder characterized by neurodegeneration and early death. It is caused by loss-of-function mutations in the gene encoding for acid sphingomyelinase (ASM), which hydrolyzes sphingomyelin into ceramide. Here, we evaluated the safety of cerebellomedullary (CM) cistern injection of adeno-associated viral vector serotype 9 encoding human ASM (AAV9-hASM) in nonhuman primates (NHP). We also evaluated its therapeutic benefit in a mouse model of the disease (ASM-KO mice). We found that CM injection in NHP resulted in widespread transgene expression within brain and spinal cord cells without signs of toxicity. CM injection in the ASM-KO mouse model resulted in hASM expression in cerebrospinal fluid and in different brain areas without triggering an inflammatory response. In contrast, direct cerebellar injection of AAV9-hASM triggered immune response. We also identified a minimally effective therapeutic dose for CM injection of AAV9-hASM in mice. Two months after administration, the treatment prevented motor and memory impairment, sphingomyelin (SM) accumulation, lysosomal enlargement, and neuronal death in ASM-KO mice. ASM activity was also detected in plasma from AAV9-hASM CM-injected ASM-KO mice, along with reduced SM amount and decreased inflammation in the liver. Our results support CM injection for future AAV9-based clinical trials in NPD-A as well as other lysosomal storage brain disorders

Prion Protein Accumulation In Lipid Rafts of Mouse Aging Brain

The cellular form of the prion protein (PrP(C)) is a normal constituent of neuronal cell membranes. The protein misfolding causes rare neurodegenerative disorders known as transmissible spongiform encephalopathies or prion diseases. These maladies can be sporadic, genetic or infectious. Sporadic prion diseases are the most common form mainly affecting aging people. In this work, we investigate the biochemical environment in which sporadic prion diseases may develop, focusing our attention on the cell membrane of neurons in the aging brain. It is well established that with aging the ratio between the most abundant lipid components of rafts undergoes a major change: while cholesterol decreases, sphingomyelin content rises. Our results indicate that the aging process modifies the compartmentalization of PrP(C). In old mice, this change favors PrP(C) accumulation in detergent-resistant membranes, particularly in hippocampi. To confirm the relationship between lipid content changes and PrP(C) translocation into detergent-resistant membranes (DRMs), we looked at PrP(C) compartmentalization in hippocampi from acid sphingomyelinase (ASM) knockout (KO) mice and synaptosomes enriched in sphingomyelin. In the presence of high sphingomyelin content, we observed a significant increase of PrP(C) in DRMS. This process is not due to higher levels of total protein and it could, in turn, favor the onset of sporadic prion diseases during aging as it increases the PrP intermolecular contacts into lipid rafts. We observed that lowering sphingomyelin in scrapie-infected cells by using fumonisin B1 led to a 50% decrease in protease-resistant PrP formation. This may suggest an involvement of PrP lipid environment in prion formation and consequently it may play a role in the onset or development of sporadic forms of prion diseases

Dopaminergic neuron loss in mice due to increased levels of wild-type human α-Synuclein only takes place under conditions of accelerated aging

Abstract Understanding the intricate pathogenic mechanisms behind Parkinson's disease (PD) and its multifactorial nature presents a significant challenge in disease modeling. To address this, we explore genetic models that better capture the disease's complexity. Given that aging is the primary risk factor for PD, this study investigates the impact of aging in conjunction with overexpression of wild-type human α-synuclein (α-Syn) in the dopaminergic system. This is achieved by introducing a novel transgenic mouse strain overexpressing α-Syn under the TH-promoter within the senescence-accelerated SAMP8 (P8) genetic background. Behavioral assessments, conducted at both 10 and 16 months of age, unveil motor impairments exclusive to P8 α-SynTg mice, a phenomenon conspicuously absent in α-SynTg mice. These findings suggest a synergistic interplay between heightened α-Syn levels and the aging process, resulting in motor deficits. These motor disturbances correlate with reduced dopamine (DA) levels, increased DA turnover, synaptic terminal loss, and notably, the depletion of dopaminergic neurons in the substantia nigra and noradrenergic neurons in the locus coeruleus. Furthermore, P8 α-SynTg mice exhibit alterations in gut transit time, mirroring early PD symptoms. In summary, P8 α-SynTg mice effectively replicate parkinsonian phenotypes by combining α-Syn transgene expression with accelerated aging. This model offers valuable insights into the understanding of PD and serves as a valuable platform for further research

PrP^C in DRMs from hippocampal membrane of young wild-type mice compared with age-matched ASMKO mice.

<div><p>Western blot analysis of DRMs prepared from equal amounts of hippocampal extracts (100 µg of protein) from young (4-5 months old) wild-type and ASMKO mice. Each lane corresponds to a single animal. Antibodies used: D18 (1:1,000; InPro Biotechnology, Inc, South San Francisco), mouse monoclonal anti flotillin1 (1:1,000; BD Biosciences). Quantification of relative PrP^C amounts from 3 control mice and 6 ASMKO mice. Each data point represents the relative PrP level normalized over flotillin1 ± SD. PrP^C levels are 20% higher in ASMKO mice compared to age-matched wild-type mice.</p> <p>**: <i>p</i><0.01.</p></div

A diet enriched with plant sterols prevents the memory impairment induced by cholesterol loss in senescence-accelerated mice

Cholesterol reduction at the neuronal plasma membrane has been related to age-dependent cognitive decline. We have used senescent-accelerated mice strain 8 (SAMP8), an animal model for aging, to examine the association between cholesterol loss and cognitive impairment and to test strategies to revert this process. We show that the hippocampus of SAMP8 mice presents reduced cholesterol levels and enhanced amount of its degrading enzyme Cyp46A1 (Cyp46) already at 6 months of age. Cholesterol loss accounts for the impaired long-term potentiation in these mice. Plant sterol (PSE)–enriched diet prevents long-term potentiation impairment and cognitive deficits in SAMP8 mice without altering cholesterol levels. PSE diet also reduces the abnormally high amyloid peptide levels in SAMP8 mice brains and restores membrane compartmentalization of presenilin1, the catalytic component of the amyloidogenic γ-secretase. These results highlight the influence of cholesterol loss in age-related cognitive decline and provide with a noninvasive strategy to counteract it. Our results suggest that PSE overtake cholesterol functions in the brain contributing to reduce deleterious consequences of cholesterol loss during aging.This work was financed by grants from Ministerio Español de Economía y CompetitividadSAF2014-57539-R to Maria Dolores Ledesma, SAF2012-39852-C02-02 to Coral Sanfeliu, CSD2010-00045 to Maria Dolores Ledesma, Coral Sanfeliu, and Jose A. Esteban, and the European Regional Development Fund. Azucena Pérez-Cañamás and Sara Sarroca hold a predoctoral fellowship (FPU) from Ministerio Español de Ciencia e Innovación.Peer Reviewe

PrP^C co-immunolabeling with Tau and MAP2.

<div><p>Confocal images of 21 DIV hippocampal primary neurons. Surface immunolabeling of PrP^C (green) coupled with Tau staining (red) (left) or MAP2 staining (red) (right). Antibodies used: see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0074244#pone-0074244-g006" target="_blank">figures 6</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0074244#pone-0074244-g007" target="_blank">7</a>.</p> <p>Scale bar = 10 µm.</p></div

PrP^C co-immunolabeling with Tau.

<div><p>Confocal images of hippocampal primary neurons at different developmental stages. Normal (left) and surface (right) immunolabeling of PrP^C (green) coupled with Tau staining (red). Antibodies used: D18 (10 µg/mL and 20 µg/mL in surface immunolabeling; InPro Biotechnology, Inc, South San Francisco), MN7.51, mouse monoclonal anti Tau (1:10; previously described in Novak et al., 1991).</p> <p>Scale bar = 10 µm.</p></div

PrP^C in DRM preparation of young (3-4 months) vs. old (20-21 months) mice.

<p>Western blot analysis of DRMs prepared from equal amounts of total hippocampal protein extracts (100 µg of total protein) at the indicated ages. Young = 3-4 months old; old = 21-22 months old. Each lane corresponds to a single animal. Antibodies used: D18 (1:1,000; InPro Biotechnology, Inc, South San Francisco), mouse monoclonal anti flotillin1 (1:1,000; BD Biosciences). Relative PrP^C amounts from 3 mice per time point were analyzed. Each data point represents the relative protein level normalized over flotillin1 ± SD.</p