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Mucopolysaccharidosis type IIIA (MPS IIIA) is a lysosomal storage disorder caused by N-sulfoglucosamine sulfohydrolase (SGSH) deficiency. SGSH removes the sulfate from N-sulfoglucosamine residues on the nonreducing end of heparan sulfate (HS-NRE) within lysosomes. Enzyme deficiency results in accumulation of partially degraded HS within lysosomes throughout the body, leading to a progressive severe neurological disease. Enzyme replacement therapy has been proposed, but further evaluation of the treatment strategy is needed. Here, we used Chinese hamster ovary cells to produce a highly soluble and fully active recombinant human sulfamidase (rhSGSH). We discovered that rhSGSH utilizes both the CI-MPR and LRP1 receptors for uptake into patient fibroblasts. A single intracerebroventricular (ICV) injection of rhSGSH in MPS IIIA mice resulted in a tissue half-life of 9 days and widespread distribution throughout the brain. Following a single ICV dose, both total HS and the MPS IIIA disease-specific HS-NRE were dramatically reduced, reaching a nadir 2 weeks post dose. The durability of effect for reduction of both substrate and protein markers of lysosomal dysfunction and a neuroimmune response lasted through the 56 days tested. Furthermore, seven weekly 148 μg doses ICV reduced those markers to near normal and produced a 99.5% reduction in HS-NRE levels. A pilot study utilizing every other week dosing in two animals supports further evaluation of less frequent dosing. Finally, our dose-response study also suggests lower doses may be efficacious. Our findings show that rhSGSH can normalize lysosomal HS storage and markers of a neuroimmune response when delivered ICV., Competing Interests: Conflict of interest The authors were employees of BioMarin Pharmaceutical Inc at the time of the studies. The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)

Sanfilippo syndrome type B (mucopolysaccharidosis IIIB), caused by inherited deficiency of α- N -acetylglucosaminidase (NAGLU), required for lysosomal degradation of heparan sulfate (HS), is a pediatric neurodegenerative disorder with no approved treatment. Intracerebroventricular (ICV) delivery of a modified recombinant NAGLU, consisting of human NAGLU fused with insulin-like growth factor 2 (IGF2) for enhanced lysosomal targeting, was previously shown to result in marked enzyme uptake and clearance of HS storage in the  Naglu -/- mouse brain. To further evaluate regional, cell type-specific, and dose-dependent biodistribution of NAGLU-IGF2 (BMN 250) and its effects on biochemical and histological pathology, Naglu -/- mice were treated with 1-100 μg ICV doses (four times over 2 weeks). 1 day after the last dose, BMN 250 (100 μg doses) resulted in above-normal NAGLU activity levels, broad biodistribution, and uptake in all cell types, with NAGLU predominantly localized to neurons in the Naglu -/- mouse brain. This led to complete clearance of disease-specific HS and reduction of secondary lysosomal defects and neuropathology across various brain regions lasting for at least 28 days after the last dose. The substantial brain uptake of NAGLU attainable by this highest ICV dosage was required for nearly complete attenuation of disease-driven storage accumulations and neuropathology throughout the Naglu -/- mouse brain.

Alzheimer's disease (AD) is characterized by neuronal and synaptic loss. One process that could contribute to this loss is the intracellular caspase cleavage of the amyloid precursor protein (APP) resulting in release of the toxic C-terminal 31-amino acid peptide APP-C31 along with the production of APPΔC31, full-length APP minus the C-terminal 31 amino acids. We previously found that a mutation in APP that prevents this caspase cleavage ameliorated synaptic loss and cognitive impairment in a murine AD model. Thus, inhibition of this cleavage is a reasonable target for new therapeutic development. In order to identify small molecules that inhibit the generation of APP-C31, we first used an APPΔC31 cleavage site-specific antibody to develop an AlphaLISA to screen several chemical compound libraries for the level of N-terminal fragment production. This antibody was also used to develop an ELISA for validation studies. In both high throughput screening (HTS) and validation testing, the ability of compounds to inhibit simvastatin- (HTS) or cerivastatin- (validation studies) induced caspase cleavage at the APP-D720 cleavage site was determined in Chinese hamster ovary (CHO) cells stably transfected with wildtype (wt) human APP (CHO-7W). Several compounds, as well as control pan-caspase inhibitor Q-VD-OPh, inhibited APPΔC31 production (measured fragment) and rescued cell death in a dose-dependent manner. The effective compounds fell into several classes including SERCA inhibitors, inhibitors of Wnt signaling, and calcium channel antagonists. Further studies are underway to evaluate the efficacy of lead compounds - identified here using cells and tissues expressing wt human APP - in mouse models of AD expressing mutated human APP, as well as to identify additional compounds and determine the mechanisms by which they exert their effects.

Recent studies have shown that inoculation of susceptible mice with amyloid-β (Aβ) peptides accelerates Aβ deposition in the brain, supporting the idea that Aβ may be self-amplifying; however, the exact mechanism is not understood. Here we provide evidence that Aβ may self-amplify, in part, by inhibiting α-secretase ADAM10 (a disintegrin and metalloprotease) cleavage of full-length Aβ precursor protein (FL AβPP) and therefore allow greater β-secretase processing, and that Aβ itself is a substrate for ADAM10. Exposure of primary neuronal cultures from PDAβPP mice to exogenous rat Aβ1- 40 resulted in increased de novo human Aβ1-42 production and exposure of cells to Aβ decreased production of ADAM10 cleavage product soluble AβPPα (sAβPPα). In a cell-free assay, Aβ decreased ADAM10 cleavage of the chimeric substrate MBP-AβPPC125 and Aβ itself was apparently cleaved by the enzyme. The axonal guidance and trophic factor netrin-1, however, reduced the Aβ1- 40-induced Aβ1-42 increase, increased sAβPPα, and reversed the Aβ-induced sAβPPα decrease in vitro. In vivo, induction of netrin-1 expression in PDAβPPSwe/Ind transgenic mice resulted in reductions in both Aβ1-42 and Aβ1- 40, and ICV delivery of netrin-1 to PDAβPPSwe/Ind mice increased sAβPPα, decreased Aβ, and improved working memory. Finally, to support further study of netrin-1's potential as a therapeutic for Alzheimer's disease, pilot gene therapy studies were performed and a netrin mimetic peptide synthesized and tested that, like netrin, can increase sAβPPα and decrease Aβ1-42in vitro. Taken together, these data provide mechanistic insights into Aβ self-amplification and the ability of netrin-1 to disrupt it.

Tropisetron was identified in a screen for candidates that increase the ratio of the trophic, neurite-extending peptide sAPPα to the anti-trophic, neurite-retractive peptide Aβ, thus reversing this imbalance in Alzheimer's disease (AD). We describe here a hierarchical screening approach to identify such drug candidates, moving from cell lines to primary mouse hippocampal neuronal cultures to in vivo studies. By screening a clinical compound library in the primary assay using CHO-7W cells stably transfected with human APPwt, we identified tropisetron as a candidate that consistently increased sAPPα. Secondary assay testing in neuronal cultures from J20 (PDAPP, huAPP(Swe/Ind)) mice showed that tropisetron consistently increased the sAPPα/Aβ 1-42 ratio. In in vivo studies in J20 mice, tropisetron improved the sAPPα/Aβ ratio along with spatial and working memory in mice, and was effective both during the symptomatic, pre-plaque phase (5-6 months) and in the late plaque phase (14 months). This ameliorative effect occurred at a dose of 0.5mg/kg/d (mkd), translating to a human-equivalent dose of 5mg/day, the current dose for treatment of postoperative nausea and vomiting (PONV). Although tropisetron is a 5-HT3 receptor antagonist and an α7nAChR partial agonist, we found that it also binds to the ectodomain of APP. Direct comparison of tropisetron to the current AD therapeutics memantine (Namenda) and donepezil (Aricept), using similar doses for each, revealed that tropisetron induced greater improvements in memory and the sAPPα/Aβ1-42 ratio. The improvements observed with tropisetron in the J20 AD mouse model, and its known safety profile, suggest that it may be suitable for transition to human trials as a candidate therapeutic for mild cognitive impairment (MCI) and AD, and therefore it has been approved for testing in clinical trials beginning in 2014., (Copyright © 2014 Elsevier B.V. All rights reserved.)

An unbiased screen for compounds that block amyloid-β protein precursor (AβPP) caspase cleavage identified ADDN-1351, which reduced AβPP-C31 by 90%. Target identification studies showed that ADDN-1351 is a TrkA inhibitor, and, in complementary studies, TrkA overexpression increased AβPP-C31 and cell death. TrkA was shown to interact with AβPP and suppress AβPP-mediated transcriptional activation. Moreover, treatment of PDAPP transgenic mice with the known TrkA inhibitor GW441756 increased sAβPPα and the sAβPPα to Aβ ratio. These results suggest TrkA inhibition-rather than NGF activation-as a novel therapeutic approach, and raise the possibility that such an approach may counteract the hyperactive signaling resulting from the accumulation of active NGF-TrkA complexes due to reduced retrograde transport. The results also suggest that one component of an optimal therapy for Alzheimer's disease may be a TrkA inhibitor.

Studies from multiple laboratories have identified the roles of several ER stress-induced cell death modulators and effectors. Earlier, we described the role of p23 a small co-chaperone protein in preventing ER stress-induced cell death. p23 is cleaved by caspases at D142 to yield p19 (a 19 kDa product) during ER stress-induced cell death. Mutation of the caspase cleavage site not only blocks formation of the 19 kDa product but also attenuates the cell death process triggered by various ER stressors. Thus, uncleavable p23 (p23D142N) emerges as a reasonable candidate to test for potential inhibition of neurodegenerative disease phenotype that features misfolded proteins and ER stress. In the present work we report the generation of transgenic mouse lines that overexpress wild-type p23 or uncleavable p23 under the control of a ROSA promoter. These mice should prove useful for studying the role of p23 and/or uncleavable p23 in cellular stress-induced cell death., (Copyright © 2012 Elsevier B.V. All rights reserved.)

A systematic approach was used to identify AβPP-selective BACE inhibitors (ASBI) and to evaluate their in vivo ability to modulate AβPP processing selectively. We identified a bioflavonoid nutritional supplement as a molecular lead that acts as an ASBI in cell models, and show that increasing brain levels of this bioflavonoid through a pro-drug approach leads to reduction of Aβ42 in an Alzheimer's disease mouse model. ASBIs represent a novel class of candidate therapeutic agents for Alzheimer's disease.

The single methionine (Met/M) residue of amyloid-beta (Aβ) peptide, at position 35 of the 42-mer, has important relevance for Aβ-induced oxidative stress and neurotoxicity. Recent in vivo brain studies in a transgenic (Tg) Alzheimer disease (AD) mouse model with Swedish and Indiana familial AD mutations in human amyloid precursor protein (APP) (referred to as the J20 Tg mouse) demonstrated increased levels of oxidative stress. However, the substitution of the Met631 residue of APP to leucine (Leu/L) (M631L in human APP numbering, referred to as M631L Tg and corresponding to residue 35 of Aβ1-42) resulted in no significant in vivo oxidative stress levels, thereby supporting the hypothesis that Met-35 of Aβ contributes to oxidative insult in the AD brain. It is conceivable that oxidative stress mediated by Met-35 of Aβ is important in regulating numerous downstream effects, leading to differential levels of relevant biochemical pathways in AD. Therefore, in the current study using proteomics, we tested the hypothesis that several brain proteins involved in pathways such as energy and metabolism, antioxidant activity, proteasome degradation, and pH regulation are altered in J20Tg versus M631L Tg AD mice.

Background: Reduced TOR signaling has been shown to significantly increase lifespan in a variety of organisms [1], [2], [3], [4]. It was recently demonstrated that long-term treatment with rapamycin, an inhibitor of the mTOR pathway[5], or ablation of the mTOR target p70S6K[6] extends lifespan in mice, possibly by delaying aging. Whether inhibition of the mTOR pathway would delay or prevent age-associated disease such as AD remained to be determined., Methodology/principal Findings: We used rapamycin administration and behavioral tools in a mouse model of AD as well as standard biochemical and immunohistochemical measures in brain tissue to provide answers for this question. Here we show that long-term inhibition of mTOR by rapamycin prevented AD-like cognitive deficits and lowered levels of Abeta(42), a major toxic species in AD[7], in the PDAPP transgenic mouse model. These data indicate that inhibition of the mTOR pathway can reduce Abeta(42) levels in vivo and block or delay AD in mice. As expected from the inhibition of mTOR, autophagy was increased in neurons of rapamycin-treated transgenic, but not in non-transgenic, PDAPP mice, suggesting that the reduction in Abeta and the improvement in cognitive function are due in part to increased autophagy, possibly as a response to high levels of Abeta., Conclusions/significance: Our data suggest that inhibition of mTOR by rapamycin, an intervention that extends lifespan in mice, can slow or block AD progression in a transgenic mouse model of the disease. Rapamycin, already used in clinical settings, may be a potentially effective therapeutic agent for the treatment of AD.

Transplantation of neural cells is a potential approach for stroke treatment, but disruption of tissue architecture may limit transplant efficacy. One strategy for enhancing the ability of transplants to restore brain structure and function is to administer cells together with biomaterial scaffolding. We electrocoagulated the distal middle cerebral artery in adult rats and, 3 weeks later, injected one of the following into the infarct cavity: artificial cerebrospinal fluid, Matrigel scaffolding, human embryonic stem cell-derived neuronal precursor cells, scaffolding plus cells, or cells cultured in and administered together with scaffolding. Five weeks after transplantation, the latter two groups showed approximately 50% and approximately 60% reductions, respectively, in infarct cavity volume. Rats given cells cultured in and administered together with scaffolding also showed (1) survival and neuronal differentiation of transplanted cells shown by immunostaining for neuronal marker proteins and cleaved caspase-3, and by patch-clamp recording, 8 weeks after transplantation and (2) improved outcome on tests of sensorimotor and cognitive functions, 4 to 9 weeks after transplantation. These results indicate that transplantation of human neural cells together with biomaterial scaffolding has the potential to improve the outcome from stroke, even when treatment is delayed for several weeks after the ischemic event.

Numerous studies have demonstrated oxidative damage in the central nervous system in subjects with Alzheimer disease and in animal models of this dementing disorder. In this study, we show that transgenic mice modeling Alzheimer disease-PDAPP mice with Swedish and Indiana mutations in the human amyloid precursor protein (APP)-develop oxidative damage in brain, including elevated levels of protein oxidation (indexed by protein carbonyls and 3-nitrotyrosine) and lipid peroxidation (indexed by protein-bound 4-hydroxy-2-nonenal). This oxidative damage requires the presence of a single methionine residue at position 35 of the amyloid beta-peptide (Abeta), because all indices of oxidative damage in brain were completely prevented in genetically and age-matched PDAPP mice with an M631L mutation in APP. No significant differences in the levels of APP, Abeta(1-42), and Abeta(1-40) or in the ratio Abeta(1-42)/Abeta(1-40) were found, suggesting that the loss of oxidative stress in vivo in the brain of PDAPP(M631L) mice results solely from the mutation of the Met35 residue to Leu in the Abeta peptide. However, a marked reduction in Abeta-immunoreactive plaques was observed in the M631L mice, which instead displayed small punctate areas of nonplaque immunoreactivity and a microglial response. In contrast to the requirement for Met at residue 35 of the Abeta sequence (M631 of APP) for oxidative damage, indices of spatial learning and memory were not significantly improved by the M631L substitution. Furthermore, a genetically matched line with a different mutation-PDAPP(D664A)-showed the reverse: no reduction in oxidative damage but marked improvement in memory. This is the first in vivo study to demonstrate the requirement for Abeta residue Met35 for oxidative stress in the brain of a mammalian model of Alzheimer disease. However, in this specific transgenic mouse model of AD, oxidative stress is neither required nor sufficient for memory abnormalities., (Copyright 2009 Elsevier Inc. All rights reserved.)

Vascular endothelial growth factor (VEGF) is an angiogenesis factor with neurotrophic, neuroprotective and neuroproliferative effects. Depending on the dose, route and time of administration in relation to focal cerebral ischemia, VEGF can improve histological outcome and sensorimotor function in rodents. However, VEGF also increases vascular permeability, which can lead to brain edema and exacerbate ischemic brain injury. Thus, although VEGF is a candidate therapeutic for stroke and other ischemic disorders, its benefit relative to risk is uncertain. Considering that functional rather than histological measures of outcome are probably most relevant to therapeutic prospects for human stroke, we investigated the effects of VEGF after middle cerebral artery occlusion in rats using a series of behavioral tests. We report that VEGF improves functional outcome in ischemic rats, including both sensorimotor and cognitive deficiencies.