Acid Sphingomyelinase Knockout Mice Research Articles

Intracerebroventricular infusion of acid sphingomyelinase corrects CNS manifestations in a mouse model of Niemann–Pick A disease

Niemann–Pick A (NPA) disease is a lysosomal storage disorder (LSD) caused by a deficiency in acid sphingomyelinase (ASM) activity. Previously, we showed that the storage pathology in the ASM knockout (ASMKO) mouse brain could be corrected by intracerebral injections of cell, gene and protein based therapies. However, except for instances where distal areas were targeted with viral vectors, correction of lysosomal storage pathology was typically limited to a region within a few millimeters from the injection site. As NPA is a global neurometabolic disease, the development of delivery strategies that maximize the distribution of the enzyme throughout the CNS is likely necessary to arrest or delay progression of the disease. To address this challenge, we evaluated the effectiveness of intracerebroventricular (ICV) delivery of recombinant human ASM into ASMKO mice. Our findings showed that ICV delivery of the enzyme led to widespread distribution of the hydrolase throughout the CNS. Moreover, a significant reduction in lysosomal accumulation of sphingomyelin was observed throughout the brain and also within the spinal cord and viscera. Importantly, we demonstrated that repeated ICV infusions of ASM were effective at improving the disease phenotype in the ASMKO mouse as indicated by a partial alleviation of the motor abnormalities. These findings support the continued exploration of ICV delivery of recombinant lysosomal enzymes as a therapeutic modality for LSDs such as NPA that manifests substrate accumulation within the CNS.

Lipid content of brain, brain membrane lipid domains, and neurons from acid sphingomyelinase deficient mice

The cholesterol, sphingolipid, and glycerophospholipid content of total brain, of detergent-resistant membranes prepared from the total brain, and of cerebellar granule cells differentiated in culture from wild type (WT) and acid sphingomyelinase knockout (ASMKO) were studied. Brains derived from 7-month-old ASMKO animals showed a fivefold higher level of sphingomyelin and a significant increase in ganglioside content, mainly because of monosialogangliosides GM3 and GM2 accumulation, while the cholesterol and glycerophospholipid content was unchanged with respect to WT animals. An increase in sphingomyelin, but not in gangliosides, was also detected in cultured cerebellar granule neurons from ASMKO mice, indicating that ganglioside accumulation is not a direct consequence of the enzyme defect. When a detergent-resistant membrane fraction was prepared from ASMKO brains, we observed that a higher detergent-to-protein ratio was needed than in WT animals. This likely reflects a reduced fluidity in restricted membrane areas because of a higher enrichment in sphingolipids in the case of ASMKO brain.

Acid Sphingomyelinase Overexpression Enhances the Antineoplastic Effects of Irradiation In Vitro and In Vivo

Exposure of cells or animals to stress frequently induces acid sphingomyelinase (ASM)-mediated ceramide production that leads to cell death. Consistent with this, overexpression of ASM in subcutaneous B16-F10 mouse melanomas, in combination with irradiation, resulted in tumors that were up to 12-fold smaller than irradiated control melanomas. Similarly, when irradiated melanomas were pretreated with a single, peritumoral injection of recombinant ASM (rhASM), the tumors were up to threefold smaller. The in vivo effect of ASM was likely due to enhanced cell death of the tumor cells themselves, as well as the surrounding microvascular endothelial cells. In vitro, rhASM had little or no effect on the growth of tumor cells, even in combination with irradiation. However, when the culture media was acidified to mimic the acidic microenvironment of solid tumors, rhASM-mediated cell death was markedly enhanced when combined with irradiation. Microscopic analysis suggested that this was associated with an increase in autophagy. rhASM has been produced for the treatment of the lysosomal storage disorder, type B Niemann-Pick disease, and is currently being evaluated in a phase-1 clinical trial. Based on the data presented in this article, we propose that further investigation of this protein and gene as antineoplastic agents also is warranted.

Neuropathology of the acid sphingomyelinase knockout mouse model of Niemann-Pick A disease including structure–function studies associated with cerebellar Purkinje cell degeneration

Niemann-Pick A (NP-A) is an inherited metabolic (lysosomal storage) disease characterized by neurovisceral accumulation of sphingomyelin due to deficiency of acid sphingomyelinase (ASM). An ASM knockout (ASMKO) mouse model of NP-A is available through targeted disruption of the parent gene. This study presents the pattern and time course of lysosomal pathology and neurodegeneration in the ASMKO mouse nervous system. Cells throughout the nervous system developed the classic foamy appearance associated with lysosomal storage disorders. Despite this, neurons were capable of retrogradely transporting dyes within established brain pathways comparable to control animals. A silver degeneration staining method demonstrated widespread damage in the form of ‘classic’ impregnation of cells, fibers and synaptic terminals. Of particular interest was the degeneration of Purkinje cells (PC) within the cerebellum, beginning by 7 weeks of age in parasagittal bands and culminating with near complete degeneration of this cell type by 20 weeks. In parallel, ASMKO mice had progressively deteriorating motor performance on two versions of the rotating rod test (accelerating and rocking). ASMKO mice at 5–7 weeks of age performed similarly to controls on both rotating rod tests, but performance sharply deteriorated between 7 and 20 weeks of age. This study further characterized the neuropathology associated with ASM deficiency, and identifies quantitative histological and behavioral endpoints for evaluation of therapeutic intervention in this authentic NP-A mouse model.

Delivery of Acid Sphingomyelinase in Normal and Niemann-Pick Disease Mice Using Intercellular Adhesion Molecule-1-Targeted Polymer Nanocarriers

Type B Niemann-Pick disease (NPD) is a multiorgan system disorder caused by a genetic deficiency of acid sphingomyelinase (ASM), for which lung is an important and challenging therapeutic target. In this study, we designed and evaluated new delivery vehicles for enzyme replacement therapy of type B NPD, consisting of polystyrene and poly(lactic-coglycolic) acid polymer nanocarriers targeted to intercellular adhesion molecule (ICAM)-1, an endothelial surface protein up-regulated in many pathologies, including type B NPD. Real-time vascular imaging using intravital microscopy and postmortem imaging of mouse organs showed rapid, uniform, and efficient binding of fluorescently labeled ICAM-1-targeted ASM nanocarriers (anti-ICAM/ASM nanocarriers) to endothelium after i.v. injection in mice. Fluorescence microscopy of lung alveoli actin, tissue histology, and 125I-albumin blood-to-lung transport showed that anti-ICAM nanocarriers cause neither detectable lung injury, nor abnormal vascular permeability in animals. Radioisotope tracing showed rapid disappearance from the circulation and enhanced accumulation of anti-ICAM/125I-ASM nanocarriers over the nontargeted naked enzyme in kidney, heart, liver, spleen, and primarily lung, both in wild-type and ASM knockout mice. These data demonstrate that ICAM-1-targeted nanocarriers may enhance enzyme replacement therapy for type B NPD and perhaps other lysosomal storage disorders.

Influence of Amitriptyline on Eryptosis, Parasitemia and Survival of Plasmodium Berghei-Infected Mice

Plasmodia express a sphingomyelinase, which is apparently required for their development. On the other hand, the sphingomyelinase product ceramide has previously been shown to delay parasite development. Moreover, ceramide triggers suicidal erythrocyte death or eryptosis, characterized by exposure of phosphatidylserine at the erythrocyte surface and cell shrinkage. Accelerated eryptosis of infected erythrocytes is considered to clear infected erythrocytes from circulating blood and, thus, to favourably influence the clinical course of malaria. The present experiments explored whether the sphingomyelinase inhibitor amitriptyline or genetic knockout of host acid sphingomyelinase influence in vitro parasite growth, eryptosis of Plasmodium falciparum-infected human erythrocytes, in vivo parasitemia and survival of P. berghei-infected mice. Phosphatidylserine exposure was determined by annexin V-binding and cell volume by forward scatter in FACS analysis. In vitro infection of human erythrocytes increased annexin- binding, an effect blunted in the presence of amitriptyline (≥50 μM). Amitriptyline did not significantly alter intraerythrocytic parasite development but significantly (≥ 1 μM) delayed the increase in parasitemia in vitro. Most importantly, amitriptyline treatment (1 mM in drinking water) resulted in a significant delay of parasitemia and death of infected mice. However, upon infection, ceramide formation was stimulated in both, acid sphingomyelinase knockout mice (Smpd1^-/-) and their wild type littermates (Smpd1^+/+). Parasitemia following P. berghei infection was significantly lower in Smpd1^-/- than in Smpd1^+/+ mice but did not significantly extend the life span of infected animals. In conclusion, mammalian and parasite sphingomyelinase contribute to ceramide formation during malaria, whereby the parasite sphingomyelinase ultimately determines the course of the infection. Amitriptyline presumably blocks both sphingomyelinases and, thus, its use might be a novel strategy to treat malaria.

Sperm Abnormalities in Heterozygous Acid Sphingomyelinase Knockout Mice Reveal a Novel Approach for the Prevention of Genetic Diseases

Acid sphingomyelinase knockout mice are a model of the inherited human disorder types A and B Niemann-Pick disease. Herein, we show that heterozygous (ASMKO(+/-)) mice have two distinct sperm populations resembling those found in normal and mutant animals, respectively, and that these two populations could be distinguished by their morphology, ability to undergo capacitation or the acrosome reaction, and/or mitochondrial membrane potential (MMP). The abnormal morphology of the mutant sperm could be normalized by demembranation with detergents or by the addition of recombinant acid sphingomyelinase to the culture media, and the corrected sperm also had an enhanced fertilization capacity. Methods were then explored to enrich for normal sperm from the mixed ASMKO(+/-) population, and flow cytometric sorting based on MMP provided the best results. In vitro fertilization was performed using ASMKO(+/-) oocytes and sperm before and after MMP sorting, and it was found that the sorted sperm produced significantly more wild-type pups than nonsorted sperm. Sperm sorting is much less invasive and more cost-effective than egg isolation, and offers several advantages over the existing assisted reproduction options for Niemann-Pick disease carrier couples. It therefore could have a major impact on the prevention of this and perhaps other genetic diseases.

Identification of Novel Biomarkers for Niemann–Pick Disease Using Gene Expression Analysis of Acid Sphingomyelinase Knockout Mice

Although several therapies are available or being developed for lysosomal storage disorders (LSDs), assessment of therapeutic efficacy is challenged by the lack of markers to assess disease progression and severity. This is particularly true for rare diseases such as LSDs, since natural history data from human populations are often lacking. Herein we describe the use of gene expression analysis in the acid sphingomyelinase-deficient mouse model (ASMKO) of Types A and B Niemann–Pick disease (NPD) to identify novel serum biomarkers. We used microarray and real-time PCR analyses to compare mRNA expression in ASMKO and normal mice in two important sites of pathology, lung and brain, and from these data identified and validated several potential biomarkers. The cytokine MIP-1α was markedly elevated in ASMKO mouse serum, and following enzyme replacement therapy (ERT) it was reduced to normal levels. Total iron levels were similarly elevated in ASMKO mice, reflective of the elevated ferritin light chain transcript, and decreased to normal after ERT. Serum growth hormone levels were also elevated in ASMKO mice and were reduced to normal after brain-directed gene therapy, but not ERT. These studies illustrate the value of gene expression analysis for the identification of biomarkers, and provide new insight into the pathobiology of NPD.

417. Combination Brain and Systemic Injections of AAV Results in Whole Body Therapy and Extension of Lifespan in the Niemann-Pick Mouse

The majority of lysosomal storage diseases contain both CNS and visceral pathology. Many experimental designs use either intracranial injection of viral vectors to treat the neurodegenerative phenotype of the disease, or systemic delivery to treat the viscera. In this study, we tested whether a combination of brain and systemic injections of adeno-associated virus (AAV) vectors could increase lifespan, provide whole body reversal of pathology, and improve motor and cognitive function in the acid sphingomyelinase knock out (ASMKO) mouse model of Niemann-Pick disease. ASMKO mice at 4 weeks of age were injected systemically with AAV8- hASM, and subsequently, the same animals were injected into the brain with AAV2-hASM. All the mice treated by combination injections survived to 54 weeks of age. This was a significant improvement over untreated ASMKO mice, animals that had been treated by systemic injection only or by brain injection only, which had median life spans of 34 weeks, 45 weeks, and 43 weeks, respectively. Thus the brain alone injections did not protect animals from dying. Animals treated by the combination therapy also displayed normal weight gain, and significant functional recovery on the rotarod (motor task) and the Barnes maze (cognitive task) throughout the time course of the study. These data support the contention that combination therapy can improve the quality of life before and within the time frame of extended survival.

Gene transfer of human acid sphingomyelinase corrects neuropathology and motor deficits in a mouse model of Niemann-Pick type A disease

Niemann-Pick type A disease is a lysosomal storage disorder caused by a deficiency in acid sphingomyelinase (ASM) activity. Previously we showed that storage pathology in the ASM knockout (ASMKO) mouse brain can be corrected by adeno-associated virus serotype 2 (AAV2)-mediated gene transfer. The present experiment compared the relative therapeutic efficacy of different recombinant AAV serotype vectors (1, 2, 5, 7, and 8) using histological, biochemical, and behavioral endpoints. In addition, we evaluated the use of the deep cerebellar nuclei (DCN) as a site for injection to facilitate global distribution of the viral vector and enzyme. Seven-week-old ASM knockout mice were injected within the DCN with different AAV serotype vectors encoding human ASM (hASM) and then killed at either 14 or 20 weeks of age. Results showed that AAV1 was superior to serotypes 2, 5, 7, and 8 in its relative ability to express hASM, alleviate storage accumulation, and correct behavioral deficits. Expression of hASM was found not only within the DCN, but also throughout the cerebellum, brainstem, midbrain, and spinal cord. This finding demonstrates that targeting the DCN is an effective approach for achieving widespread enzyme distribution throughout the CNS. Our results support the continued development of AAV based vectors for gene therapy of the CNS manifestations in Niemann-Pick type A disease.

AAV8-Mediated Hepatic Expression of Acid Sphingomyelinase Corrects the Metabolic Defect in the Visceral Organs of a Mouse Model of Niemann–Pick Disease

Acid sphingomyelinase deficiency is a lysosomal storage disorder in which the defective lysosomal hydrolase fails to degrade sphingomyelin. The resulting accumulation of substrate in the lysosomes of histiocytic cells leads to hepatosplenomegaly and severe pulmonary inflammation. Administration of a recombinant AAV1 vector encoding human acid sphingomyelinase to acid sphingomyelinase knockout (ASMKO) mice effectively reduced the accumulated substrate in all of the affected visceral organs. However, more complete and rapid clearance of sphingomyelin was observed when an AAV8-based serotype vector was used in lieu of AAV1. Importantly, AAV8-mediated hepatic expression of higher and sustained levels of the enzyme also corrected the abnormal cellularity, cell differentials, and levels of the chemokine MIP-1alpha in the bronchoalveolar lavage fluids of the ASMKO mice. Treatment also reversed the morphological aberrations associated with the alveolar macrophages of ASMKO mice and restored their phagocytic activity. No antibodies to the expressed enzyme were detected when the viral vectors were used in conjunction with a transcription cassette harboring a liver-restricted enhancer/promoter. Together, these data support the continued development of AAV8-mediated hepatic gene transfer as an approach to treat the visceral manifestations observed in individuals with acid sphingomyelinase deficiency.

110. Performance of Different AAV Serotype Vectors Following Injection into the Deep Cerebellar Nuclei of ASMKO Mouse Brain

Niemann-Pick A disease (NPA) is a lysosomal storage disorder caused by a deficiency in acid sphingomyelinase (ASM) activity. Consequent accumulation of sphingomyelin and other lipids in the CNS results in the development of a rapidly progressive neurodegenerative disease with death occurring by 2 to 3 years of age. Previously we have shown that the storage pathology in the ASM knockout (ASMKO) mouse brain is amenable to AAV2/2-mediated gene therapy. Interestingly, correction of storage pathology occurred not only at the injection site, but also in regions that send and/or receive input from the injection site|[ndash]|suggesting that AAV vector and/or expressed ASM protein underwent transport. The present experiment evaluated the relative ability of recombinant AAV2/1, AAV2/2, AAV2/5, AAV2/7 and AAV2/8 serotype vectors encoding human ASM to facilitate gene transduction, express ASM protein, correct cholesterol storage pathology, undergo transport, rescue Purkinje cells, and initiate functional recovery in the ASMKO mouse. Male ASMKO mice (|[sim]|7 weeks old) were unilaterally injected with the different AAV serotype vectors within the deep cerebellar nuclei of the cerebellum (DCN). The DCN was targeted because it is highly connected with the CNS; and therefore, may provide a means to achieve widespread ASM expression throughout the brain. Mice were sacrificed at 14 weeks of age after undergoing rotarod testing. Mice injected with AAV2/1 and AAV2/8 demonstrated significant functional improvement on the rotarod, whereas mice injected with AAV2/2, AAV2/5 and AAV2/7 did not. Consistent with the behavioral results, cerebellar ASM protein levels (as detected by ELISA) were significantly higher in mice injected with AAV2/1 and AAV2/8, than mice injected with AAV2/2, AAV2/5, AAV2/7 and control mice. Preservation of Purkinje cells based on calbindin immunostaining was greatest in mice injected with AAV2/1 and AAV2/8. In all AAV-ASM treated mice, expression of ASM led to widespread clearance of filipin/cholesterol staining in the cerebellum, brainstem, and midbrain|[ndash]|indicating that AAV vector and/or expressed ASM underwent transport from the DCN. Overall, a positive relationship between ASM protein levels, filipin clearance, Purkinje cell survival, and rotarod performance was observed. These results support the further evaluation of AAV2/1 and AAV2/8-based vectors for gene therapy of the CNS manifestations in Niemann-Pick A disease.

249. Minimal Dose of AAV8/DC190-ASM Necessary for Therapeutic Efficacy in a Mouse Model of Niemann-Pick Disease

Niemann-Pick disease type B (NPD) is a congenital defect caused by a deficiency of the lysosomal hydrolase, acid sphingomyelinase (ASM). This panethnic disorder results in the pathological accumulation of sphingomyelin (SPM) in the macrophages of lung, spleen, liver and bone marrow. While a broad continuum of clinical manifestations occurs in NPD patients, significant morbidity results as a consequence of progressively worsening pulmonary involvement. Previously, we reported that systemic administration of 3x1011 drp of a recombinant adeno-associated virus vector (AAV8) encoding human ASM under the transcriptional control of a liver-restricted promoter (DC190) into acid sphingomyelinase knockout mice (ASMKO) resulted in expression and secretion of therapeutic levels of the enzyme, as measured by SPM clearance in visceral tissues. Furthermore, substrate depletion in lung was accompanied by significant reduction in the levels of proinflammatory cytokines and complete reversal of lung pathology. The goal of the present study was to define the minimal dose of AAV8/DC190-ASM necessary for therapeutic efficacy in the ASMKO mice. As expected, administration of lower doses (3X1010 to 1X1011 ) of AAV8/DC190-ASM resulted in expression and secretion of correspondingly lower levels of ASM. Encouragingly, while uptake of ASM was found to be dose-dependent, all doses were capable of substantially reducing SPM levels in the affected tissues, including the lung, by day 60 post-treatment. To assess the kinetics of clearance, ASMKO were administered various doses of AAV8/DC190-ASM and groups were sacrificed at day 14 or day 28 post-administration. Maximal tissue levels of ASM were achieved as early as 14 days post-injection. At the highest dose (3X1011 drp), SPM levels in the liver, spleen, lung and bone marrow were largely normalized by day 14. Importantly, as an extension of our previous observations, we found that decreased SPM levels in the lung coincided with a reduction in the levels of pro-inflammatory mediators (e.g. MIP-1|[alpha]|) in the bronchoalveolar lavages of ASMKO mice. It was also noted that liver-restricted expression of human ASM promoted immune tolerance to the expressed enzyme in the ASMKO mice at all the doses tested. Hence, AAV8-mediated liver-restricted expression of ASM was capable of correcting the visceral storage disease and abrogating the chronic pulmonary inflammatory manifestations shown associated with NPD. Therapeutic efficacy could be attained with a 10-fold lower dose of vector (3X1010 drp/mouse) than previously studied and near-complete correction was observed within two weeks post-administration. Based on these findings, it is anticipated that yet lower doses of AAV8/DC190-ASM may be therapeutically efficacious in the ASMKO mice.

Intracerebral Transplantation of Adult Mouse Neural Progenitor Cells into the Niemann-Pick-A Mouse Leads to a Marked Decrease in Lysosomal Storage Pathology

Niemann-Pick disease is caused by a genetic deficiency in acid sphingomyelinase (ASM) leading to the intracellular accumulation of sphingomyelin and cholesterol in lysosomes. In the present study, we evaluated the effects of direct intracerebral transplantation of neural progenitor cells (NPCs) on the brain storage pathology in the ASM knock-out (ASMKO) mouse model of Type A Niemann-Pick disease. NPCs derived from adult mouse brain were genetically modified to express human ASM (hASM) and were transplanted into multiple regions of the ASMKO mouse brain. Transplanted NPCs survived, migrated, and showed region-specific differentiation in the host brain up to 10 weeks after transplantation (the longest time point examined). In vitro, gene-modified NPCs expressed up to 10 times more and released five times more ASM activity into the culture media compared with nontransduced NPCs. In vivo, transplanted cells expressed hASM at levels that were barely detectable by immunostaining but were sufficient for uptake and cross-correction of host cells, leading to reversal of distended lysosomal pathology and regional clearance of sphingomyelin and cholesterol storage. Within the host brain, the area of correction closely overlapped with the distribution of the hASM-modified NPCs. No correction of pathology occurred in brain regions that received transplants of nontransduced NPCs. These results indicate that the presence of transduced NPCs releasing low levels of hASM within the ASMKO mouse brain is necessary and sufficient to reverse lysosomal storage pathology. Potentially, NPCs may serve as a useful gene transfer vehicle for the treatment of CNS pathology in other lysosomal storage diseases and neurodegenerative disorders.

Effects of acid sphingomyelinase deficiency on male germ cell development and programmed cell death.

Deficiency of acid sphingomyelinase (ASM), an enzyme responsible for producing a pro-apoptotic second messenger ceramide, has previously been shown to promote the survival of fetal mouse oocytes in vivo and to protect oocytes from chemotherapy-induced apoptosis in vitro. Here we investigated the effects of ASM deficiency on testicular germ cell development and on the ability of germ cells to undergo apoptosis. At the age of 20 weeks, ASM knock-out (ASMKO) sperm concentrations were comparable with wild-type (WT) sperm concentrations, whereas sperm motility was seriously affected. ASMKO testes contained significantly elevated levels of sphingomyelin at the age of 8 weeks as detected by high-performance, thin-layer chromatography. Electron microscopy revealed that the testes started to accumulate pathological vesicles in Sertoli cells and in the interstitium at the age of 21 days. Irradiation of WT and ASMKO mice did not elevate intratesticular ceramide levels at 16 h after irradiation. In situ end labeling of apoptotic cells also showed a similar degree of cell death in both groups. After a 21-day recovery period, the numbers of primary spermatocytes and spermatogonia at G2 as well as spermatids were essentially the same in the WT and ASMKO testes, as detected by flow cytometry. In serum-free cultures both ASMKO and WT germ cells showed a significant increase in the level of ceramide, as well as massive apoptosis. In conclusion, ASM is required for maintenance of normal sphingomyelin levels in the testis and for normal sperm motility, but not for testicular ceramide production or for the ability of the germ cells to undergo apoptosis.

210. AAV Vector-Mediated Enzyme Replacement Therapy Is Efficacious in Reversing Storage Pathology and Preventing Purkinje Cell Death in a Mouse Model of Niemann-Pick A Disease

Top of pageAbstract Type A Niemann-Pick disease (NPA) is a fatal neurometabolic childhood disorder caused by a genetic deficiency of acid sphingomyelinase (ASM). The lack of functional ASM results in sphingomyelin and cholesterol accumulation within the lysosomal compartment of cells throughout the brain, leading to neurodegeneration. In this study, we investigated the efficacy of using AAV gene therapy as a form of ASM enzyme replacement to correct lysosomal pathology and to prevent Purkinje cell death in a mouse ASM knockout (ASMKO) model of NPA. An AAV serotype-2 vector encoding human ASM under the control of CMV promoter (AAV2-CMV-ASM) was injected into the adult hippocampus of one hemisphere. This resulted in human ASM mRNA and protein expression in all major cell layers of the ipsilateral hippocampus for up to 15 weeks post-injection. Transduced cells were also present in the entorhinal cortex, medial septum, supramammillary nucleus, and contralateral hippocampus in a pattern consistent with retrograde transport of AAV2. There was a substantial reduction of distended lysosomal pathology and an almost complete reversal of cholesterol accumulation in all brain regions expressing human ASM. Following injection of AAV2-CMV-ASM into other brain regions (e.g. striatal and cerebellar circuits), we observed a similar pattern of local and distant (retrograde) transduction and reversal of pathology. Within the cerebellum of ASMKO mice, there is an almost complete loss of Purkinje cells between 7 and 20 weeks of age. AAV2-CMV-ASM was injected into the ASMKO cerebellum between 5 and 7 weeks, and treated mice were sacrificed 15 weeks later (20–22 weeks old). These injections resulted in neuroprotection as evidenced by the preservation of large numbers of human ASM mRNA- and protein-positive Purkinje cells within treated lobules. We also found that intraventricular injection of juvenile (3 week old) ASMKO mice resulted in Purkinje cell transduction, human ASM expression and survival for up to 18 weeks post-injection. These results demonstrate that viral vector delivery via the cerebrospinal fluid is an effective way to target Purkinje cells for genetic manipulation. We are currently investigating different promoters and AAV serotypes for vector optimization. To this end, we have generated an AAV serotype-1 vector encoding human ASM under the control of chicken beta-actin promoter (AAV1-CBA-ASM). Administration into the cerebrum resulted in long-term human ASM expression and widespread clearance of cholesterol deposits for up to 26 weeks. Preliminary results have also shown that injection of AAV1-CBA-ASM into the cerebellum of young ASMKO mice preserved rotorod performance, a test sensitive to proprioceptive deficits caused, in part, by Purkinje cell degeneration. In conclusion, our data show that AAV-mediated human ASM enzyme replacement therapy leads to long-lasting, widespread reversal of lysosomal storage pathology, neuroprotection and functional sparing in the ASMKO mouse model of NPA.

853. Efficacy of AAV-Mediated Expression of Acid Sphingomyelinase at Correcting the Visceral and Pulmonary Manifestations of Niemann-Pick B Disease

Niemann-Pick B disease (NPD) is an inheritable lysosomal storage disease in which the deficient activity of the lysosomal hydrolase, acid sphingomyelinase (ASM), results in the intracellular accumulation of sphingomyelin. Accumulation is most strikingly evidenced by the presence of enlarged, often multinucleated myeloid-lineage cells in bone marrow, lung, liver, and spleen. In particular, the presence of lung infiltrates of engorged alveolar macrophages is frequently referenced in human lung biopsies and at autopsy. In NPD patients, significant morbidity is attributed to protracted pulmonary decline and recurrent respiratory infections, underscoring the importance of achieving efficacy in this organ. Systemic administration of either an AAV2/1 or 2/8 pseudotyped vector encoding human ASM under the transcriptional control of a liver-restricted promoter into acid sphingomyelinase knockout mice (ASMKO) resulted in expression of therapeutic levels of the enzyme. Transduction and subsequent expression, which was primarily hepatic-restricted, was approximately 50–100-fold higher with the AAV2/8 than with the AAV2/1 vector. Furthermore, the high levels attained using the AAV2/8 vector were readily detected in the circulation by day 4 and were sustained at the end of the 120-day study. Subsequent extrahepatic uptake of the secreted enzyme, presumably via the mannose and mannose 6-phosphate receptors, was associated with a reduction in sphingomyelin levels in all the affected tissues, including the lung. As may be expected, since the levels of expression attained with the AAV2/8 vector were significantly higher, the kinetics of sphingomyelin clearance was more rapid than with the AAV2/1 vector. Biochemical and histological analysis of ASMKO mice at days 60 and 120 post-administration demonstrated a profound reversal of lung pathology concomitant with substrate depletion to basal levels. Correction was also associated with reduced levels of the proinflammatory chemokine, MIP-1, and normalization of the cellularity and cell differentials in the bronchoalveolar lavage fluids (BALF) of treated animals. In contrast to the severe storage inclusions and enzyme-uptake defects shown associated with alveolar macrophages from ASMKO BALF, those isolated from AAV2/8-treated mice demonstrated improved phagocytic activity and displayed morphologies that were more akin to normal macrophages. Hepatic-restricted expression of the human ASM also promoted immune tolerance to the expressed enzyme in the ASMKO mice. No antibodies to the human enzyme were detected in the animals throughout the 120-day study. Hence, AAV2/8-mediated, hepatic-restricted expression of ASM was capable of correcting the visceral storage disease and abrogating the chronic pulmonary inflammatory manifestations shown associated with NPD. This data supports the continued evaluation of the gene delivery system for the treatment of NPD.

Expression of the immunoglobulin superfamily neuroplastin adhesion molecules in adult and developing mouse cerebellum and their localisation to parasagittal stripes.

Neuroplastin (np) 55 and 65 are immunoglobulin superfamily members that arise by alternative splicing of the same gene and have been implicated in long-term activity-dependent synaptic plasticity. Both biochemical and immunocytochemical data suggest that np55 is the predominant isoform (>95% of total neuroplastin) in cerebellum. Neuroplastin immunoreactivity is concentrated in the molecular layer and synaptic glomeruli in the granule cell layer. Expression in the molecular layer appears to be postsynaptic. First, neuroplastin is associated with Purkinje cell dendrites in two mouse granuloprival cerebellar mutants, disabled and cerebellar deficient folia. Second, in an acid sphingomyelinase knockout mouse with widespread protein trafficking defects, neuroplastin accumulates in the Purkinje cell somata. Finally, primary cerebellar cultures show neuroplastin expression in Purkinje cell dendrites and somata lacking normal histotypic organization and synaptic connections, and high-magnification views indicate a preferential association with dendritic spines. In the molecular layer, differences in neuroplastin expression levels present as a parasagittal array of stripes that alternates with that revealed by the expression of another compartmentation antigen, zebrin II/aldolase c. Neuroplastin immunoreactivity is first detected weakly at postnatal day 3 (P3) in the anterior lobe vermis. By P5, parasagittal stripes are already apparent in the immature molecular layer. At this stage, punctate deposits are also localised at the perimeter of the Purkinje cell perikarya; these are no longer detected by P15. The data suggest a role for neuroplastins in the development and maintenance of normal synaptic connections in the cerebellum.

Alveolar lipoproteinosis in an acid sphingomyelinase-deficient mouse model of Niemann-Pick disease.

Types A and B Niemann-Pick disease (NPD) are lipid storage disorders caused by the deficient activity of acid sphingomyelinase (ASM). In humans, NPD is associated with the dysfunction of numerous organs including the lung. Gene targeting of the ASM gene in transgenic mice produced an animal model with features typical of NPD, including pulmonary inflammation. To assess mechanisms by which ASM perturbed lung function, we studied lung morphology, surfactant content, and metabolism in ASM-deficient mice in vivo. Pulmonary inflammation, with increased cellular infiltrates and the accumulation of alveolar material, was associated with alterations in surfactant content. Saturated phosphatidylcholine (SatPC) content was increased twofold, and sphingomyelin content was increased 5.5-fold in lungs of the ASM knockout (ASMKO) mice. Additional sphingomyelin enhanced the sensitivity of surfactant inhibition by plasma proteins. Clearance of SatPC from the lungs of ASMKO mice was decreased. Catabolism of SatPC by alveolar macrophages from the ASMKO mouse was significantly decreased, likely accounting for decreased pulmonary SatPC in vivo. In summary, ASM is required for normal surfactant catabolism by alveolar macrophages in vivo. Alterations in surfactant composition, including increased sphingomyelin content, contributed to the abnormal surfactant function observed in the ASM-deficient mouse.

Acid Sphingomyelinase-deficient Macrophages Have Defective Cholesterol Trafficking and Efflux

Cholesterol efflux from macrophage foam cells, a key step in reverse cholesterol transport, requires trafficking of cholesterol from intracellular sites to the plasma membrane. Sphingomyelin is a cholesterol-binding molecule that transiently exists with cholesterol in endosomes and lysosomes but is rapidly hydrolyzed by lysosomal sphingomyelinase (L-SMase), a product of the acid sphingomyelinase (ASM) gene. We therefore hypothesized that sphingomyelin hydrolysis by L-SMase enables cholesterol efflux by preventing cholesterol sequestration by sphingomyelin. Macrophages from wild-type and ASM knockout mice were incubated with [(3)H]cholesteryl ester-labeled acetyl-LDL and then exposed to apolipoprotein A-I or high density lipoprotein. In both cases, [(3)H]cholesterol efflux was decreased substantially in the ASM knockout macrophages. Similar results were shown for ASM knockout macrophages labeled long-term with [(3)H]cholesterol added directly to medium, but not for those labeled for a short period, suggesting defective efflux from intracellular stores but not from the plasma membrane. Cholesterol trafficking to acyl-coenzyme A:cholesterol acyltransferase (ACAT) was also defective in ASM knockout macrophages. Using filipin to probe cholesterol in macrophages incubated with acetyl-LDL, we found there was modest staining in the plasma membrane of wild-type macrophages but bright, perinuclear fluorescence in ASM knockout macrophages. Last, when wild-type macrophages were incubated with excess sphingomyelin to "saturate" L-SMase, [(3)H]cholesterol efflux was decreased. Thus, sphingomyelin accumulation due to L-SMase deficiency leads to defective cholesterol trafficking and efflux, which we propose is due to sequestration of cholesterol by sphingomyelin and possibly other mechanisms. This model may explain the low plasma high density lipoprotein found in ASM-deficient humans and may implicate L-SMase deficiency and/or sphingomyelin enrichment of lipoproteins as novel atherosclerosis risk factors.