Artificial axons as a biomimetic 3D myelination platform for the discovery and validation of promyelinating compounds.

Multiple sclerosis (MS), a chronic neurodegenerative disease driven by damage to the protective myelin sheath, is currently incurable. Today, all clinically available treatments modulate the immune-mediated symptoms of the disease but they fail to stop neurodegeneration in many patients. Remyelination, the regenerative process of myelin repair by oligodendrocytes, which is considered a necessary step to protect demyelinated axons and stop neuronal death, is impaired in MS patients. One of the major obstacles to finding effective remyelinating drugs is the lack of biomimetic drug screening platforms that enable quantification of compounds' potential to stimulate 3D myelination in the physiologically relevant axon-like environment. To address this need, we built a unique myelination drug discovery platform, by expanding our previously developed technology, artificial axons (AAs), which enables 3D-printing of synthetic axon mimics with the geometry and mechanical properties closely resembling those of biological axons. This platform allows for high-throughput phenotypic myelination assay based on quantification of 3D wrapping of myelin membrane around axons in response to compounds. Here, we demonstrate quantification of 3D myelin wrapping by rat oligodendrocytes around the axon mimics in response to a small library of known pro-myelinating compounds. This assay shows pro-myelinating activity for all tested compounds consistent with the published in vitro and in vivo data, demonstrating predictive power of AA platform. We find that stimulation of myelin wrapping by these compounds is dose-dependent, providing a facile means to quantify the compounds' potency and efficacy in promoting myelin wrapping. Further, the ranking of relative efficacy among these compounds differs in this 3D axon-like environment as compared to a traditional oligodendrocyte 2D differentiation assay quantifying area of deposited myelin membrane. Together, we demonstrate that the artificial axons platform and associated phenotypic myelin wrapping assay afford direct evaluation of myelin wrapping by oligodendrocytes in response to soluble compounds in an axon-like environment, providing a predictive tool for the discovery of remyelinating therapies.

High-throughput screening for myelination promoting compounds using human stem cell-derived oligodendrocyte progenitor cells.

Promoting myelination capacity of endogenous oligodendrocyte precursor cells (OPCs) is a promising therapeutic approach for CNS demyelinating disorders such as Multiple Sclerosis (MS). To aid in the discovery of myelination-promoting compounds, we generated a genome-engineered human pluripotent stem cell (hPSC) line that consists of three reporters: identification-and-purification tag, GFP, and secreted-NanoLuc, driven by the endogenous PDGFRA, PLP1, and MBP genes, respectively. Using this cell line, we established a high-throughput drug screening platform and performed a small-molecule screen, which identified at least two myelination-promoting small-molecule (Ro1138452 and SR2211) that target prostacyclin (IP) receptor and retinoic acid receptor-related orphan receptor γ (RORγ), respectively. Single-cell-transcriptomic analysis of differentiating OPCs treated with these molecules further confirmed that they promote oligodendrocyte differentiation and revealed several pathways that are potentially modulated by them. The molecules and their target pathways provide promising targets for the possible development of remyelination-based therapy for MS and other demyelinating disorders.

Microglia ferroptosis is regulated by SEC24B and contributes to neurodegeneration.

Iron dysregulation has been implicated in multiple neurodegenerative diseases, including Parkinson's disease (PD). Iron-loaded microglia are frequently found in affected brain regions, but how iron accumulation influences microglia physiology and contributes to neurodegeneration is poorly understood. Here we show that human induced pluripotent stem cell-derived microglia grown in a tri-culture system are highly responsive to iron and susceptible to ferroptosis, an iron-dependent form of cell death. Furthermore, iron overload causes a marked shift in the microglial transcriptional state that overlaps with a transcriptomic signature found in PD postmortem brain microglia. Our data also show that this microglial response contributes to neurodegeneration, as removal of microglia from the tri-culture system substantially delayed iron-induced neurotoxicity. To elucidate the mechanisms regulating iron response in microglia, we performed a genome-wide CRISPR screen and identified novel regulators of ferroptosis, including the vesicle trafficking gene SEC24B. These data suggest a critical role for microglia iron overload and ferroptosis in neurodegeneration.

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.

Sterol auto-oxidation adversely affects human motor neuron viability and is a neuropathological feature of amyotrophic lateral sclerosis.

Aberrant cholesterol homeostasis is implicated in the pathogenesis of amyotrophic lateral sclerosis (ALS), a fatal neuromuscular disease that is due to motor neuron (MN) death. Cellular toxicity from excess cholesterol is averted when it is enzymatically oxidized to oxysterols and bile acids (BAs) to promote its removal. In contrast, the auto oxidation of excess cholesterol is often detrimental to cellular survival. Although oxidized metabolites of cholesterol are altered in the blood and CSF of ALS patients, it is unknown if increased cholesterol oxidation occurs in the SC during ALS, and if exposure to oxidized cholesterol metabolites affects human MN viability. Here, we show that in the SOD1G93A mouse model of ALS that several oxysterols, BAs and auto oxidized sterols are increased in the lumbar SC, plasma, and feces during disease. Similar changes in cholesterol oxidation were found in the cervical SC of sporadic ALS patients. Notably, auto-oxidized sterols, but not oxysterols and BAs, were toxic to iPSC derived human MNs. Thus, increased cholesterol oxidation is a manifestation of ALS and non-regulated sterol oxidation likely contributes to MN death. Developing therapeutic approaches to restore cholesterol homeostasis in the SC may lead to a treatment for ALS.

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.

Lipid Involvement in Neurodegenerative Diseases of the Motor System: Insights from Lysosomal Storage Diseases.

Lysosomal storage diseases (LSDs) are a heterogeneous group of rare inherited metabolic diseases that are frequently triggered by the accumulation of lipids inside organelles of the endosomal-autophagic-lysosomal system (EALS). There is now a growing realization that disrupted lysosomal homeostasis (i.e., lysosomal cacostasis) also contributes to more common neurodegenerative disorders such as Parkinson disease (PD). Lipid deposition within the EALS may also participate in the pathogenesis of some additional neurodegenerative diseases of the motor system. Here, I will highlight the lipid abnormalities and clinical manifestations that are common to LSDs and several diseases of the motor system, including amyotrophic lateral sclerosis (ALS), atypical forms of spinal muscular atrophy, Charcot-Marie-Tooth disease (CMT), hereditary spastic paraplegia (HSP), multiple system atrophy (MSA), PD and spinocerebellar ataxia (SCA). Elucidating the underlying basis of intracellular lipid mislocalization as well as its consequences in each of these disorders will likely provide innovative targets for therapeutic research.

Glucosylceramide synthase inhibition alleviates aberrations in synucleinopathy models.

Mutations in the glucocerebrosidase gene (GBA) confer a heightened risk of developing Parkinson's disease (PD) and other synucleinopathies, resulting in a lower age of onset and exacerbating disease progression. However, the precise mechanisms by which mutations in GBA increase PD risk and accelerate its progression remain unclear. Here, we investigated the merits of glucosylceramide synthase (GCS) inhibition as a potential treatment for synucleinopathies. Two murine models of synucleinopathy (a Gaucher-related synucleinopathy model, GbaD409V/D409V and a A53T-α-synuclein overexpressing model harboring wild-type alleles of GBA, A53T-SNCA mouse model) were exposed to a brain-penetrant GCS inhibitor, GZ667161. Treatment of GbaD409V/D409V mice with the GCS inhibitor reduced levels of glucosylceramide and glucosylsphingosine in the central nervous system (CNS), demonstrating target engagement. Remarkably, treatment with GZ667161 slowed the accumulation of hippocampal aggregates of α-synuclein, ubiquitin, and tau, and improved the associated memory deficits. Similarly, prolonged treatment of A53T-SNCA mice with GZ667161 reduced membrane-associated α-synuclein in the CNS and ameliorated cognitive deficits. The data support the contention that prolonged antagonism of GCS in the CNS can affect α-synuclein processing and improve behavioral outcomes. Hence, inhibition of GCS represents a disease-modifying therapeutic strategy for GBA-related synucleinopathies and conceivably for certain forms of sporadic disease.

Glucocerebrosidase modulates cognitive and motor activities in murine models of Parkinson's disease.

Mutations in GBA1, the gene encoding glucocerebrosidase, are associated with an enhanced risk of developing synucleinopathies such as Parkinson's disease (PD) and dementia with Lewy bodies. A higher prevalence and increased severity of motor and non-motor symptoms is observed in PD patients harboring mutant GBA1 alleles, suggesting a link between the gene or gene product and disease development. Interestingly, PD patients without mutations in GBA1 also exhibit lower levels of glucocerebrosidase activity in the central nervous system (CNS), implicating this lysosomal enzyme in disease pathogenesis. Here, we investigated whether modulation of glucocerebrosidase activity in murine models of synucleinopathy (expressing wild type Gba1) affected α-synuclein accumulation and behavioral phenotypes. Partial inhibition of glucocerebrosidase activity in PrP-A53T-SNCA mice using the covalent inhibitor conduritol-B-epoxide induced a profound increase in soluble α-synuclein in the CNS and exacerbated cognitive and motor deficits. Conversely, augmenting glucocerebrosidase activity in the Thy1-SNCA mouse model of PD delayed the progression of synucleinopathy. Adeno-associated virus-mediated expression of glucocerebrosidase in the Thy1-SNCA mouse striatum led to decrease in the levels of the proteinase K-resistant fraction of α-synuclein, amelioration of behavioral aberrations and protection from loss of striatal dopaminergic markers. These data indicate that increasing glucocerebrosidase activity can influence α-synuclein homeostasis, thereby reducing the progression of synucleinopathies. This study provides robust in vivo evidence that augmentation of CNS glucocerebrosidase activity is a potential therapeutic strategy for PD, regardless of the mutation status of GBA1.

Glycosphingolipids are modulators of disease pathogenesis in amyotrophic lateral sclerosis.

Recent genetic evidence suggests that aberrant glycosphingolipid metabolism plays an important role in several neuromuscular diseases including hereditary spastic paraplegia, hereditary sensory neuropathy type 1, and non-5q spinal muscular atrophy. Here, we investigated whether altered glycosphingolipid metabolism is a modulator of disease course in amyotrophic lateral sclerosis (ALS). Levels of ceramide, glucosylceramide, galactocerebroside, lactosylceramide, globotriaosylceramide, and the gangliosides GM3 and GM1 were significantly elevated in spinal cords of ALS patients. Moreover, enzyme activities (glucocerebrosidase-1, glucocerebrosidase-2, hexosaminidase, galactosylceramidase, α-galactosidase, and ß-galactosidase) mediating glycosphingolipid hydrolysis were also elevated up to threefold. Increased ceramide, glucosylceramide, GM3, and hexosaminidase activity were also found in SOD1(G93A) mice, a familial model of ALS. Inhibition of glucosylceramide synthesis accelerated disease course in SOD1(G93A) mice, whereas infusion of exogenous GM3 significantly slowed the onset of paralysis and increased survival. Our results suggest that glycosphingolipids are likely important participants in pathogenesis of ALS and merit further analysis as potential drug targets.

Systemic administration of a recombinant AAV1 vector encoding IGF-1 improves disease manifestations in SMA mice.

Spinal muscular atrophy is a progressive motor neuron disease caused by a deficiency of survival motor neuron. In this study, we evaluated the efficacy of intravenous administration of a recombinant adeno-associated virus (AAV1) vector encoding human insulin-like growth factor-1 (IGF-1) in a severe mouse model of spinal muscular atrophy. Measurable quantities of human IGF-1 transcripts and protein were detected in the liver (up to 3 months postinjection) and in the serum indicating that IGF-1 was secreted from the liver into systemic circulation. Spinal muscular atrophy mice administered AAV1-IGF-1 on postnatal day 1 exhibited a lower extent of motor neuron degeneration, cardiac and muscle atrophy as well as a greater extent of innervation at the neuromuscular junctions compared to untreated controls at day 8 posttreatment. Importantly, treatment with AAV1-IGF-1 prolonged the animals' lifespan, increased their body weights and improved their motor coordination. Quantitative polymerase chain reaction and western blot analyses showed that AAV1-mediated expression of IGF-1 led to an increase in survival motor neuron transcript and protein levels in the spinal cord, brain, muscles, and heart. These data indicate that systemically delivered AAV1-IGF-1 can correct several of the biochemical and behavioral deficits in spinal muscular atrophy mice through increasing tissue levels of survival motor neuron.

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.

Augmenting CNS glucocerebrosidase activity as a therapeutic strategy for parkinsonism and other Gaucher-related synucleinopathies.

Mutations of GBA1, the gene encoding glucocerebrosidase, represent a common genetic risk factor for developing the synucleinopathies Parkinson disease (PD) and dementia with Lewy bodies. PD patients with or without GBA1 mutations also exhibit lower enzymatic levels of glucocerebrosidase in the central nervous system (CNS), suggesting a possible link between the enzyme and the development of the disease. Previously, we have shown that early treatment with glucocerebrosidase can modulate α-synuclein aggregation in a presymptomatic mouse model of Gaucher-related synucleinopathy (Gba1(D409V/D409V)) and ameliorate the associated cognitive deficit. To probe this link further, we have now evaluated the efficacy of augmenting glucocerebrosidase activity in the CNS of symptomatic Gba1(D409V/D409V) mice and in a transgenic mouse model overexpressing A53T α-synuclein. Adeno-associated virus-mediated expression of glucocerebrosidase in the CNS of symptomatic Gba1(D409V/D409V) mice completely corrected the aberrant accumulation of the toxic lipid glucosylsphingosine and reduced the levels of ubiquitin, tau, and proteinase K-resistant α-synuclein aggregates. Importantly, hippocampal expression of glucocerebrosidase in Gba1(D409V/D409V) mice (starting at 4 or 12 mo of age) also reversed their cognitive impairment when examined using a novel object recognition test. Correspondingly, overexpression of glucocerebrosidase in the CNS of A53T α-synuclein mice reduced the levels of soluble α-synuclein, suggesting that increasing the glycosidase activity can modulate α-synuclein processing and may modulate the progression of α-synucleinopathies. Hence, increasing glucocerebrosidase activity in the CNS represents a potential therapeutic strategy for GBA1-related and non-GBA1-associated synucleinopathies, including PD.

Merits of combination cortical, subcortical, and cerebellar injections for the treatment of Niemann-Pick disease type A.

Niemann-Pick disease Type A (NPA) is a neuronopathic lysosomal storage disease (LSD) caused by the loss of acid sphingomyelinase (ASM). The goals of the current study are to ascertain the levels of human ASM that are efficacious in ASM knockout (ASMKO) mice, and determine whether these levels can be attained in non-human primates (NHPs) using a multiple parenchymal injection strategy. Intracranial injections of different doses of AAV1-hASM in ASMKO mice demonstrated that only a small amount of enzyme (<0.5 mg hASM/g tissue) was sufficient to increase survival, and that increasing the amount of hASM did not enhance this survival benefit until a new threshold level of >10 mg hASM/g tissue was reached. In monkeys, injection of 12 tracts of AAV1-hASM resulted in efficacious levels of enzyme in broad regions of the brain that was aided, in part, by axonal transport of adeno-associated virus (AAV) and movement through the perivascular space. This study demonstrates that a combination cortical, subcortical, and cerebellar injection protocol could provide therapeutic levels of hASM to regions of the NHP brain that are highly affected in NPA patients. The information from this study might help design new AAV-mediated enzyme replacement protocols for NPA and other neuronopathic LSDs in future clinical trials.

Gene transfer to the CNS is efficacious in immune-primed mice harboring physiologically relevant titers of anti-AAV antibodies.

Central nervous system (CNS)-directed gene therapy with recombinant adeno-associated virus (AAV) vectors has been used effectively to slow disease course in mouse models of several neurodegenerative diseases. However, these vectors were typically tested in mice without prior exposure to the virus, an immunological scenario unlikely to be duplicated in human patients. Here, we examined the impact of pre-existing immunity on AAV-mediated gene delivery to the CNS of normal and diseased mice. Antibody levels in brain tissue were determined to be 0.6% of the levels found in systemic circulation. As expected, transgene expression in brains of mice with relatively high serum antibody titers was reduced by 59-95%. However, transduction activity was unaffected in mice that harbored more clinically relevant antibody levels. Moreover, we also showed that markers of neuroinflammation (GFAP, Iba1, and CD3) and histopathology (hematoxylin and eosin (H&E)) were not enhanced in immune-primed mice (regardless of pre-existing antibody levels). Importantly, we also demonstrated in a mouse model of Niemann Pick Type A (NPA) disease that pre-existing immunity did not preclude either gene transfer to the CNS or alleviation of disease-associated neuropathology. These findings support the continued development of AAV-based therapies for the treatment of neurological disorders.

IGF-1 delivery to CNS attenuates motor neuron cell death but does not improve motor function in type III SMA mice.

The efficacy of administering a recombinant adeno-associated virus (AAV) vector encoding human IGF-1 (AAV2/1-hIGF-1) into the deep cerebellar nucleus (DCN) of a type III SMA mouse model was evaluated. High levels of IGF-1 transcripts and protein were detected in the spinal cord at 2 months post-injection demonstrating that axonal connections between the cerebellum and spinal cord were able to act as conduits for the viral vector and protein to the spinal cord. Mice treated with AAV2/1-hIGF-1 and analyzed 8 months later showed changes in endogenous Bax and Bcl-xl levels in spinal cord motor neurons that were consistent with IGF-1-mediated anti-apoptotic effects on motor neurons. However, although AAV2/1-hIGF-1 treatment reduced the extent of motor neuron cell death, the majority of rescued motor neurons were non-functional, as they lacked axons that innervated the muscles. Furthermore, treated SMA mice exhibited abnormal muscle fibers, aberrant neuromuscular junction structure, and impaired performance on motor function tests. These data indicate that although CNS-directed expression of IGF-1 could reduce motor neuron cell death, this did not translate to improvements in motor function in an adult mouse model of type III SMA.

Disease progression in a mouse model of amyotrophic lateral sclerosis: the influence of chronic stress and corticosterone.

Amyotrophic lateral sclerosis (ALS) is a progressive neurodegenerative disease characterized by motor neuron cell loss, muscular atrophy, and a shortened life span. Survival is highly variable, as some patients die within months, while others live for many years. Exposure to stress or the development of a nonoptimal stress response to disease might account for some of this variability. We show in the SOD1(G93A) mouse model of ALS that recurrent exposure to restraint stress led to an earlier onset of astrogliosis and microglial activation within the spinal cord, accelerated muscular weakness, and a significant decrease in median survival (105 vs. 122 d) when compared to nonstressed animals. Moreover, during normal disease course, ALS mice display a cacostatic stress response by developing an aberrant serum corticosterone circadian rhythm. Interestingly, we also found that higher corticosterone levels were significantly correlated with both an earlier onset of paralysis (males: r(2)=0.746; females: r(2)=0.707) and shorter survival times (males: r(2)=0.680; females: r(2)=0.552) in ALS mice. These results suggest that stress is capable of accelerating disease progression and that strategies that modulate glucocorticoid metabolism might be a viable treatment approach for ALS.

Distribution of acid sphingomyelinase in rodent and non-human primate brain after intracerebroventricular infusion.

One treatment approach for lysosomal storage diseases (LSDs) is the systemic infusion of recombinant enzyme. Although this enzyme replacement is therapeutic for the viscera, many LSDs have central nervous system (CNS) components that are not adequately treated by systemic enzyme infusion. Direct intracerebroventricular (ICV) infusion of a high concentration of recombinant human acid sphingomyelinase (rhASM) into the CNS over a prolonged time frame (hours) has shown therapeutic efficacy in a mouse model of Niemann-Pick A (NP/A) disease. To evaluate whether such an approach would translate to a larger brain, rhASM was infused into the lateral ventricles of both rats and Rhesus macaques, and the resulting distribution of enzyme characterized qualitatively and quantitatively. In both species, ICV infusion of rhASM resulted in parenchymal distribution of enzyme at levels that were therapeutic in the NP/A mouse model. Enzyme distribution was global in nature and exhibited a relatively steep gradient from the cerebrospinal fluid compartment to the inner parenchyma. Additional optimization of an ICV delivery approach may provide a therapeutic option for LSDs with neurologic involvement.

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.