Glucosylceramide synthase inhibition reduces ganglioside GM3 accumulation, alleviates amyloid neuropathology, and stabilizes remote contextual memory in a mouse model of Alzheimer's disease.

BACKGROUND: Gangliosides are highly enriched in the brain and are critical for its normal development and function. However, in some rare neurometabolic diseases, a deficiency in lysosomal ganglioside hydrolysis is pathogenic and leads to early-onset neurodegeneration, neuroinflammation, demyelination, and dementia. Increasing evidence also suggests that more subtle ganglioside accumulation contributes to the pathogenesis of more common neurological disorders including Alzheimer's disease (AD). Notably, ganglioside GM3 levels are elevated in the brains of AD patients and in several mouse models of AD, and plasma GM3 levels positively correlate with disease severity in AD patients. METHODS: Tg2576 AD model mice were fed chow formulated with a small molecule inhibitor of glucosylceramide synthase (GCSi) to determine whether reducing glycosphingolipid synthesis affected aberrant GM3 accumulation, amyloid burden, and disease manifestations in cognitive impairment. GM3 was measured with LC-MS, amyloid burden with ELISA and amyloid red staining, and memory was assessed using the contextual fear chamber test. RESULTS: GCSi mitigated soluble Aß42 accumulation in the brains of AD model mice when treatment was started prophylactically. Remarkably, GCSi treatment also reduced soluble Aß42 levels and amyloid plaque burden in aged (i.e., 70 weeks old) AD mice with preexisting neuropathology. Our analysis of contextual memory in Tg2576 mice showed that impairments in remote (cortical-dependent) memory consolidation preceded deficits in short-term (hippocampal-dependent) contextual memory, which was consistent with soluble Aß42 accumulation occurring more rapidly in the cortex of AD mice compared to the hippocampus. Notably, GCSi treatment significantly stabilized remote memory consolidation in AD mice-especially in mice with enhanced cognitive training. This finding was consistent with GCSi treatment lowering aberrant GM3 accumulation in the cortex of AD mice. CONCLUSIONS: Collectively, our results indicate that glycosphingolipids regulated by GCS are important modulators of Aß neuropathology and that glycosphingolipid homeostasis plays a critical role in the consolidation of remote memories.

Neutral Lipid Cacostasis Contributes to Disease Pathogenesis in Amyotrophic Lateral Sclerosis.

Amyotrophic lateral sclerosis (ALS) is a fatal neuromuscular disease characterized by motor neuron (MN) death. Lipid dysregulation manifests during disease; however, it is unclear whether lipid homeostasis is adversely affected in the in the spinal cord gray matter (GM), and if so, whether it is because of an aberrant increase in lipid synthesis. Moreover, it is unknown whether lipid dysregulation contributes to MN death. Here, we show that cholesterol ester (CE) and triacylglycerol levels are elevated several-fold in the spinal cord GM of male sporadic ALS patients. Interestingly, HMG-CoA reductase, the rate-limiting enzyme in cholesterol synthesis, was reduced in the spinal cord GM of ALS patients. Increased cytosolic phospholipase A2 activity and lyso-phosphatidylcholine (Lyso-PC) levels in ALS patients suggest that CE accumulation was driven by acyl group transfer from PC to cholesterol. Notably, Lyso-PC, a byproduct of CE synthesis, was toxic to human MNs in vitro Elevations in CE, triacylglycerol, and Lyso-PC were also found in the spinal cord of SOD1G93A mice, a model of ALS. Similar to ALS patients, a compensatory downregulation of cholesterol synthesis occurred in the spinal cord of SOD1G93A mice; levels of sterol regulatory element binding protein 2, a transcriptional regulator of cholesterol synthesis, progressively declined. Remarkably, overexpressing sterol regulatory element binding protein 2 in the spinal cord of normal mice to model CE accumulation led to ALS-like lipid pathology, MN death, astrogliosis, paralysis, and reduced survival. Thus, spinal cord lipid dysregulation in ALS likely contributes to neurodegeneration and developing therapies to restore lipid homeostasis may lead to a treatment for ALS.SIGNIFICANCE STATEMENT Neurons that control muscular function progressively degenerate in patients with amyotrophic lateral sclerosis (ALS). Lipid dysregulation is a feature of ALS; however, it is unclear whether disrupted lipid homeostasis (i.e., lipid cacostasis) occurs proximal to degenerating neurons in the spinal cord, what causes it, and whether it contributes to neurodegeneration. Here we show that lipid cacostasis occurs in the spinal cord gray matter of ALS patients. Lipid accumulation was not associated with an aberrant increase in synthesis or reduced hydrolysis, as enzymatic and transcriptional regulators of lipid synthesis were downregulated during disease. Last, we demonstrated that genetic induction of lipid cacostasis in the CNS of normal mice was associated with ALS-like lipid pathology, astrogliosis, neurodegeneration, and clinical features of ALS.

Gene delivery of a modified antibody to Aß reduces progression of murine Alzheimer's disease.

Antibody therapies for Alzheimer's Disease (AD) hold promise but have been limited by the inability of these proteins to migrate efficiently across the blood brain barrier (BBB). Central nervous system (CNS) gene transfer by vectors like adeno-associated virus (AAV) overcome this barrier by allowing the bodies' own cells to produce the therapeutic protein, but previous studies using this method to target amyloid-ß have shown success only with truncated single chain antibodies (Abs) lacking an Fc domain. The Fc region mediates effector function and enhances antigen clearance from the brain by neonatal Fc receptor (FcRn)-mediated reverse transcytosis and is therefore desirable to include for such treatments. Here, we show that single chain Abs fused to an Fc domain retaining FcRn binding, but lacking Fc gamma receptor (FcγR) binding, termed a silent scFv-IgG, can be expressed and released into the CNS following gene transfer with AAV. While expression of canonical IgG in the brain led to signs of neurotoxicity, this modified Ab was efficiently secreted from neuronal cells and retained target specificity. Steady state levels in the brain exceeded peak levels obtained by intravenous injection of IgG. AAV-mediated expression of this scFv-IgG reduced cortical and hippocampal plaque load in a transgenic mouse model of progressive ß-amyloid plaque accumulation. These findings suggest that CNS gene delivery of a silent anti-Aß scFv-IgG was well-tolerated, durably expressed and functional in a relevant disease model, demonstrating the potential of this modality for the treatment of Alzheimer's disease.

Silencing mutant huntingtin by adeno-associated virus-mediated RNA interference ameliorates disease manifestations in the YAC128 mouse model of Huntington's disease.

Huntington's disease (HD) is a fatal autosomal dominant neurodegenerative disease caused by an increase in the number of polyglutamine residues in the huntingtin (Htt) protein. With the identification of the underlying basis of HD, therapies are being developed that reduce expression of the causative mutant Htt. RNA interference (RNAi) that seeks to selectively reduce the expression of such disease-causing agents is emerging as a potential therapeutic strategy for this and similar disorders. This study examines the merits of administering a recombinant adeno-associated viral (AAV) vector designed to deliver small interfering RNA (siRNA) that targets the degradation of the Htt transcript. The aim was to lower Htt levels and to correct the behavioral, biochemical, and neuropathological deficits shown to be associated with the YAC128 mouse model of HD. Our data demonstrate that AAV-mediated RNAi is effective at transducing greater than 80% of the cells in the striatum and partially reducing the levels (~40%) of both wild-type and mutant Htt in this region. Concomitant with these reductions are significant improvements in behavioral deficits, reduction of striatal Htt aggregates, and partial correction of the aberrant striatal transcriptional profile observed in YAC128 mice. Importantly, a partial reduction of both the mutant and wild-type Htt levels is not associated with any notable overt neurotoxicity. Collectively, these results support the continued development of AAV-mediated RNAi as a therapeutic strategy for HD.

Metabolic signatures of amyotrophic lateral sclerosis reveal insights into disease pathogenesis.

Metabolic dysfunction is an important modulator of disease course in amyotrophic lateral sclerosis (ALS). We report here that a familial mouse model (transgenic mice over-expressing the G93A mutation of the Cu/Zn superoxide dismutase 1 gene) of ALS enters a progressive state of acidosis that is associated with several metabolic (hormonal) alternations that favor lipolysis. Extensive investigation of the major determinants of H(+) concentration (i.e., the strong ion difference and the strong ion gap) suggests that acidosis is also due in part to the presence of an unknown anion. Consistent with a compensatory response to avert pathological acidosis, ALS mice harbor increased accumulation of glycogen in CNS and visceral tissues. The altered glycogen is associated with fluctuations in lysosomal and neutral α-glucosidase activities. Disease-related changes in glycogen, glucose, and α-glucosidase activity are also found in spinal cord tissue samples of autopsied patients with ALS. Collectively, these data provide insights into the pathogenesis of ALS as well as potential targets for drug development.

Optimization of a histopathological biomarker for sphingomyelin accumulation in acid sphingomyelinase deficiency.

Niemann-Pick disease (types A and B), or acid sphingomyelinase deficiency, is an inherited deficiency of acid sphingomyelinase, resulting in intralysosomal accumulation of sphingomyelin in cells throughout the body, particularly within those of the reticuloendothelial system. These cellular changes result in hepatosplenomegaly and pulmonary infiltrates in humans. A knockout mouse model mimics many elements of human ASMD and is useful for studying disease histopathology. However, traditional formalin-fixation and paraffin embedding of ASMD tissues dissolves sphingomyelin, resulting in tissues with a foamy cell appearance, making quantitative analysis of the substrate difficult. To optimize substrate fixation and staining, a modified osmium tetroxide and potassium dichromate postfixation method was developed to preserve sphingomyelin in epon-araldite embedded tissue and pulmonary cytology specimens. After processing, semi-thin sections were incubated with tannic acid solution followed by staining with toluidine blue/borax. This modified method provides excellent preservation and staining contrast of sphingomyelin with other cell structures. The resulting high-resolution light microscopy sections permit digital quantification of sphingomyelin in light microscopic fields. A lysenin affinity stain for sphingomyelin was also developed for use on these semi-thin epon sections. Finally, ultrathin serial sections can be cut from these same tissue blocks and stained for ultrastructural examination by electron microscopy.

Comparative analysis of acid sphingomyelinase distribution in the CNS of rats and mice following intracerebroventricular delivery.

Niemann-Pick A (NPA) disease is a lysosomal storage disorder (LSD) caused by a deficiency in acid sphingomyelinase (ASM) activity. Previously, we reported that biochemical and functional abnormalities observed in ASM knockout (ASMKO) mice could be partially alleviated by intracerebroventricular (ICV) infusion of hASM. We now show that this route of delivery also results in widespread enzyme distribution throughout the rat brain and spinal cord. However, enzyme diffusion into CNS parenchyma did not occur in a linear dose-dependent fashion. Moreover, although the levels of hASM detected in the rat CNS were determined to be within the range shown to be therapeutic in ASMKO mice, the absolute amounts represented less than 1% of the total dose administered. Finally, our results also showed that similar levels of enzyme distribution are achieved across rodent species when the dose is normalized to CNS weight as opposed to whole body weight. Collectively, these data suggest that the efficacy observed following ICV delivery of hASM in ASMKO mice could be scaled to CNS of the rat.

Relationship between neuropathology and disease progression in the SOD1(G93A) ALS mouse.

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease characterized by the progressive loss of upper and lower motor neurons. However, recent reports suggest an active role of non-neuronal cells in the pathogenesis of the disease. Here, we examined quantitatively the temporal development of neuropathologic features in the brain and spinal cord of a mouse model of ALS (SOD1(G93A)). Four phases of the disease were studied in both male and female SOD1(G93A) mice: presymptomatic (PRE-SYM), symptomatic (SYM), endstage (ES) and moribund (MB). Compared to their control littermates, SOD1(G93A) mice showed an increase in astrogliosis in the motor cortex, spinal cord and motor trigeminal nucleus in the SYM phase that worsened progressively in ES and MB animals. Associated with this increase in astrogliosis was a concomitant increase in motor neuron cell death in the spinal cord and motor trigeminal nucleus in both ES and MB mice, as well as in the ventrolateral thalamus in MB animals. In contrast, microglial activation was significantly increased in all the same regions but only when the mice were in the MB phase. These results suggest that astrogliosis preceded or occurred concurrently with neuronal degeneration whereas prominent microgliosis was evident later (MB stage), after significant motor neuron degeneration had occurred. Hence, our findings support a role for astrocytes in modulating the progression of non-cell autonomous degeneration of motor neurons, with microglia playing a role in clearing degenerating neurons.

AAV4-mediated expression of IGF-1 and VEGF within cellular components of the ventricular system improves survival outcome in familial ALS mice.

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease characterized by motor neuron cell death in the cortex, brainstem, and spinal cord. Extensive efforts have been made to develop trophic factor-based therapies to enhance motor neuron survival; however, achievement of adequate therapeutic delivery to all regions of the corticospinal tract has remained a significant challenge. Here, we show that adeno-associated virus serotype 4 (AAV4)-mediated expression of insulin-like growth factor-1 (IGF-1) or vascular endothelial growth factor (VEGF)-165 in the cellular components of the ventricular system including the ependymal cell layer, choroid plexus [the primary cerebrospinal fluid (CSF)-producing cells of the central nervous system (CNS)] and spinal cord central canal leads to trophic factor delivery throughout the CNS, delayed motor decline and a significant extension of survival in SOD1(G93A) transgenic mice. Interestingly, when IGF-1- and VEGF-165-expressing AAV4 vectors were given in combination, no additional benefit in efficacy was observed suggesting that these trophic factors are acting on similar signaling pathways to modestly slow disease progression. Consistent with these findings, experiments conducted in a recently described in vitro cell culture model of ALS led to a similar result, with both IGF-1 and VEGF-165 providing significant motor neuron protection but in a nonadditive fashion. These findings support the continued investigation of trophic factor-based therapies that target the CNS as a potential treatment of ALS.

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.

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.

Temporal neuropathologic and behavioral phenotype of 6neo/6neo Pompe disease mice.

Pompe disease (glycogen storage disease II) is caused by mutations in the acid alpha-glucosidase gene. The most common form is rapidly progressive with glycogen storage, particularly in muscle, which leads to profound weakness, cardiac failure, and death by the age of 2 years. Although usually considered a muscle disease, glycogen storage also occurs in the CNS. We evaluated the progression of neuropathologic and behavioral abnormalities in a Pompe disease mouse model (6neo/6neo) that displays many features of the human disease. Homozygous mutant mice store excess glycogen within large neurons of hindbrain, spinal cord, and sensory ganglia by the age of 1 month; accumulations then spread progressively within many CNS cell types. "Silver degeneration" and Fluoro-Jade C stains revealed severe degeneration in axon terminals of primary sensory neurons at 3 to 9 months. These abnormalities were accompanied by progressive behavioral impairment on rotorod, wire hanging, and foot fault tests. The extensive neuropathologic alterations in this model suggest that therapy of skeletal and cardiac muscle disorders by systemic enzyme replacement therapy may not be sufficient to reverse functional deficits due to CNS glycogen storage, particularly early-onset, rapidly progressive disease. A better understanding of the basis for clinical manifestations is needed to correlate CNS pathology with Pompe disease manifestations.

Ability of adeno-associated virus serotype 8-mediated hepatic expression of acid alpha-glucosidase to correct the biochemical and motor function deficits of presymptomatic and symptomatic Pompe mice.

The availability of a murine model of Pompe disease has enabled an evaluation of the relative merits of various therapeutic paradigms, including gene therapy. We report here that administration of a recombinant adeno-associated virus serotype 8 (AAV8) vector (AAV8/DC190-GAA) encoding human acid alpha-glucosidase (GAA) into presymptomatic Pompe mice resulted in nearly complete correction of the lysosomal storage of glycogen in all the affected muscles. A relatively high dose of AAV8/DC190-GAA was necessary to attain a threshold level of GAA for inducing immunotolerance to the expressed enzyme and for correction of muscle function, coordination, and strength. Administration of AAV8/DC190-GAA into older Pompe mice with overt disease manifestations was also effective at correcting the lysosomal storage abnormality. However, these older mice exhibited only marginal improvements in motor function and no improvement in muscle strength. Examination of histologic sections showed evidence of skeletal muscle degeneration and fibrosis in aged Pompe mice whose symptoms were abated or rescued by early but not late treatment with AAV8/DC190-GAA. These results suggest that AAV8-mediated hepatic expression of GAA was effective at addressing the biochemical and functional deficits in Pompe mice. However, early therapeutic intervention is required to maintain significant muscle function and should be an important consideration in the management and treatment of Pompe disease.

Delivery of AAV-IGF-1 to the CNS extends survival in ALS mice through modification of aberrant glial cell activity.

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease of the motor system. Recent work in rodent models of ALS has shown that insulin-like growth factor-1 (IGF-1) slows disease progression when delivered at disease onset. However, IGF-1's mechanism of action along the neuromuscular axis remains unclear. In this study, symptomatic ALS mice received IGF-1 through stereotaxic injection of an IGF-1-expressing viral vector to the deep cerebellar nuclei (DCN), a region of the cerebellum with extensive brain stem and spinal cord connections. We found that delivery of IGF-1 to the central nervous system (CNS) reduced ALS neuropathology, improved muscle strength, and significantly extended life span in ALS mice. To explore the mechanism of action of IGF-1, we used a newly developed in vitro model of ALS. We demonstrate that IGF-1 is potently neuroprotective and attenuates glial cell-mediated release of tumor necrosis factor-alpha (TNF-alpha) and nitric oxide (NO). Our results show that delivering IGF-1 to the CNS is sufficient to delay disease progression in a mouse model of familial ALS and demonstrate for the first time that IGF-1 attenuates the pathological activity of non-neuronal cells that contribute to disease progression. Our findings highlight an innovative approach for delivering IGF-1 to the CNS.

Intraparenchymal injections of acid sphingomyelinase results in regional correction of lysosomal storage pathology in the Niemann-Pick A mouse.

Niemann-Pick A disease (NPD-A) is caused by a deficiency of acid sphingomyelinase (ASM) leading to the intracellular accumulation of sphingomyelin and cholesterol in lysosomes. We evaluated the effects of direct intraparenchymal brain injections of purified recombinant human ASM (hASM) at correcting the storage pathology in a mouse model of NPD-A (ASMKO). Different doses (0.1 ng to 10 mug of hASM) were injected into the right hemisphere of the hippocampus and thalamus of 12- to 14-week-old ASMKO mice. Immunohistochemical analysis after 1 week indicated that animals treated with greater than 1 mug hASM/site showed detectable levels of enzyme around the injected regions. However, localized clearance of sphingomyelin and cholesterol storage were observed in animals administered lower doses of enzyme, starting at 100 ng hASM/site. Areas of correction were also noted at distal sites such as in the contralateral hemispheres. Indications of storage re-accumulation were seen after 2 weeks post-injection. Injections of hASM did not cause any significant cell infiltration, astrogliosis, or microglial activation. These results indicate that intraparenchymal injection of hASM is associated with minimal toxicity and can lead to regional reductions in storage pathology in the ASMKO mouse.

Optimized preservation of CNS morphology for the identification of glycogen in the Pompe mouse model.

Pompe disease (glycogenosis type II) is a rare lysosomal disorder caused by a mutational deficiency of acid alpha-glucosidase (GAA). This deficiency leads to glycogen accumulation in multiple tissues: heart, skeletal muscles, and the central nervous system. A knockout mouse model mimicking the human condition has been used for histological evaluation. Currently, the best method for preserving glycogen in Pompe samples uses epon-araldite resin. Although the preservation by this method is excellent, the size of the tissue is limited to 1 mm(3). To accurately evaluate brain pathology in the Pompe mouse model, a modified glycol methacrylate (JB-4 Plus) method was developed. This approach allowed the production of larger tissue sections encompassing an entire mouse hemisphere (8 x 15 mm) while also providing a high level of morphological detail and preservation of glycogen. Application of the JB-4 Plus method is appropriate when a high level of cellular detail is desired. A modified paraffin method was also developed for use when rapid processing of multiple samples is a priority. Traditional paraffin processing results in glycogen loss. The modified paraffin method with periodic acid postfixation resulted in improved tissue morphology and glycogen preservation. Both techniques provide accurate anatomic evaluation of the glycogen distribution in Pompe mouse brain.

Combination brain and systemic injections of AAV provide maximal functional and survival benefits in the Niemann-Pick mouse.

Niemann-Pick disease (NPD) is caused by the loss of acid sphingomyelinase (ASM) activity, which results in widespread accumulation of undegraded lipids in cells of the viscera and CNS. In this study, we tested the effect of combination brain and systemic injections of recombinant adeno-associated viral vectors encoding human ASM (hASM) in a mouse model of NPD. Animals treated by combination therapy exhibited high levels of hASM in the viscera and brain, which resulted in near-complete correction of storage throughout the body. This global reversal of pathology translated to normal weight gain and superior recovery of motor and cognitive functions compared to animals treated by either brain or systemic injection alone. Furthermore, animals in the combination group did not generate antibodies to hASM, demonstrating the first application of systemic-mediated tolerization to improve the efficacy of brain injections. All of the animals treated by combination therapy survived in good health to an investigator-selected 54 weeks, whereas the median lifespans of the systemic-alone, brain-alone, or untreated ASM knockout groups were 47, 48, and 34 weeks, respectively. These data demonstrate that combination therapy is a promising therapeutic modality for treating NPD and suggest a potential strategy for treating disease indications that cause both visceral and CNS pathologies.

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.

AAV vector-mediated correction of brain pathology in a mouse model of Niemann-Pick A disease.

Niemann-Pick A disease (NPA) is a fatal lysosomal storage disorder caused by a deficiency in acid sphingomyelinase (ASM) activity. The lack of functional ASM results in cellular accumulation of sphingomyelin and cholesterol within distended lysosomes throughout the brain. In this study, we investigated the potential of AAV-mediated expression of ASM to correct the brain pathology in an ASM knockout (ASMKO) mouse model of NPA. An AAV serotype 2 vector encoding human ASM (AAV2-hASM) was injected directly into the adult ASMKO hippocampus of one hemisphere. This resulted in expression of human ASM in all major cell layers of the ipsilateral hippocampus for at least 15 weeks postinjection. Transduced cells were also present in the entorhinal cortex, medial septum, and contralateral hippocampus in a pattern consistent with retrograde axonal transport of AAV2. There was a substantial reduction of distended lysosomes and an almost complete reversal of cholesterol accumulation in all areas of the brain that were targeted by AAV2-hASM. These findings show that the ASMKO brain is responsive to ASM replacement and that retrograde transport of AAV2 functions as a platform for widespread gene delivery and reversal of pathology in affected brain.

Mouse neural progenitor cells differentiate into oligodendrocytes in the brain of a knockout mouse model of Canavan disease.

Canavan disease (CD) is an autosomal recessive disorder that leads to spongy degeneration in the white matter of the brain. Aspartoacylase (ASPA) synthesizing cells, oligodendrocytes, are lost in CD. Transplantation of neural progenitor cells (NPCs) offers an interesting therapeutic approach for treating neurodegenerative diseases by replacing the lost cells. Therefore, the NPCs transplantation to the brain of the CD mouse was studied. Injection of mouse NPCs to the striatum and cerebellum of juvenile CD mouse showed numerous BrdU positive cells at 1 month after injection. The same result was also observed in the adult CD mouse brain after 5 weeks of post-transplantation period. The implanted cells differentiated into oligodendrocytes and fibrous astrocytes, as observed using glial cell marker. This is the first report to describe the survival, distribution and differentiation of NPCs within the brain of CD mouse and a first step toward the potential clinical use of cell therapy to treat CD.