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Aim We have previously reported that cambinol (DDL-112), a known inhibitor of neutral sphingomyelinase-2 (nSMase2), suppressed extracellular vesicle (EV)/exosome production in vitro in a cell model and reduced tau seed propagation. The enzyme nSMase2 is involved in the production of exosomes carrying proteopathic seeds and could contribute to cell-to-cell transmission of pathological protein aggregates implicated in neurodegenerative diseases such as Parkinson’s disease (PD). Here, we performed in vivo studies to determine if DDL-112 can reduce brain EV/exosome production and proteopathic alpha synuclein (αSyn) spread in a PD mouse model. Methods The acute effects of single-dose treatment with DDL-112 on interleukin-1β-induced extracellular vesicle (EV) release in brain tissue of Thy1-αSyn PD model mice and chronic effects of 5 week DDL-112 treatment on behavioral/motor function and proteinase K-resistant αSyn aggregates in the PD model were determined. Results/discussion In the acute study, pre-treatment with DDL-112 reduced EV/exosome biogenesis and in the chronic study, treatment with DDL-112 was associated with a reduction in αSyn aggregates in the substantia nigra and improvement in motor function. Inhibition of nSMase2 thus offers a new approach to therapeutic development for neurodegenerative diseases with the potential to reduce the spread of disease-specific proteopathic proteins.

It is shown that the formation of amyloid-β oligomers, one of the histopathological signatures of Alzheimer’s disease, can be triggered by small quantities of a specifically truncated and post-translationally modified version of amyloid-β. Hypertoxic amyloid variants Here it is demonstrated that the formation of hypertoxic amyloid-β (Aβ) oligomers can be triggered by small quantities of a specifically truncated and post-translationally modified (pyroglutamylated) version of Aβ, called pEAβ. Previous studies have shown that pE modification of Aβ enhances its aggregation kinetics, toxicity and resistance to degradation, but a mechanistic explanation for these observations was lacking. This study shows that pEAβ causes template-induced misfolding of Aβ 1–42 into small hypertoxic structurally distinct oligomers that propagate through a prion-like mechanism. Tau expression is required for the cytotoxicity of these oligomers, and similar molecules can be isolated from the brains of people with Alzheimer's disease. Extracellular plaques of amyloid-β and intraneuronal neurofibrillary tangles made from tau are the histopathological signatures of Alzheimer’s disease. Plaques comprise amyloid-β fibrils that assemble from monomeric and oligomeric intermediates, and are prognostic indicators of Alzheimer’s disease. Despite the importance of plaques to Alzheimer’s disease, oligomers are considered to be the principal toxic forms of amyloid-β 1 , 2 . Interestingly, many adverse responses to amyloid-β, such as cytotoxicity 3 , microtubule loss 4 , impaired memory and learning 5 , and neuritic degeneration 6 , are greatly amplified by tau expression. Amino-terminally truncated, pyroglutamylated (pE) forms of amyloid-β 7 , 8 are strongly associated with Alzheimer’s disease, are more toxic than amyloid-β, residues 1–42 (Aβ 1–42 ) and Aβ 1–40 , and have been proposed as initiators of Alzheimer’s disease pathogenesis 9 , 10 . Here we report a mechanism by which pE-Aβ may trigger Alzheimer’s disease. Aβ 3(pE)–42 co-oligomerizes with excess Aβ 1–42 to form metastable low- n oligomers (LNOs) that are structurally distinct and far more cytotoxic to cultured neurons than comparable LNOs made from Aβ 1–42 alone. Tau is required for cytotoxicity, and LNOs comprising 5% Aβ 3(pE)–42 plus 95% Aβ 1–42 (5% pE-Aβ) seed new cytotoxic LNOs through multiple serial dilutions into Aβ 1–42 monomers in the absence of additional Aβ 3(pE)–42 . LNOs isolated from human Alzheimer’s disease brain contained Aβ 3(pE)–42 , and enhanced Aβ 3(pE)–42 formation in mice triggered neuron loss and gliosis at 3 months, but not in a tau-null background. We conclude that Aβ 3(pE)–42 confers tau-dependent neuronal death and causes template-induced misfolding of Aβ 1–42 into structurally distinct LNOs that propagate by a prion-like mechanism. Our results raise the possibility that Aβ 3(pE)–42 acts similarly at a primary step in Alzheimer’s disease pathogenesis.

We describe here the results from the testing of a small molecule first-in-class apolipoprotein E4 (ApoE4)-targeted sirtuin1 (SirT1) enhancer, A03, that increases the levels of the neuroprotective enzyme SirT1 while not affecting levels of neurotoxic sirtuin 2 (SirT2) in vitro in ApoE4-transfected cells. A03 was identified by high-throughput screening (HTS) and found to be orally bioavailable and brain penetrant. In vivo , A03 treatment increased SirT1 levels in the hippocampus of 5XFAD-ApoE4 (E4FAD) Alzheimer’s disease (AD) model mice and elicited cognitive improvement while inducing no observed toxicity. We were able to resolve the enantiomers of A03 and show using in vitro models that the L-enantiomer was more potent than the corresponding D-enantiomer in increasing SirT1 levels. ApoE4 expression has been shown to decrease the level of the NAD-dependent deacetylase and major longevity determinant SirT1 in brain tissue and serum of AD patients as compared to normal controls. A deficiency in SirT1 level has been recently implicated in increased tau acetylation, a dominant post-translational modification and key pathological event in AD and tauopathies. Therefore, as a novel approach to therapeutic development for AD, we targeted identification of compounds that enhance and normalize brain SirT1 levels.

We have previously reported that cambinol (DDL-112), a known inhibitor of neutral sphingomyelinase-2 (nSMase2), suppressed extracellular vesicle (EV)/exosome production in vitro in a cell model and reduced tau seed propagation. The enzyme nSMase2 is involved in the production of exosomes carrying proteopathic seeds and could contribute to cell-to-cell transmission of pathological protein aggregates implicated in neurodegenerative diseases such as Parkinson's disease (PD). Here, we performed in vivo studies to determine if DDL-112 can reduce brain EV/exosome production and proteopathic alpha synuclein ([alpha]Syn) spread in a PD mouse model. The acute effects of single-dose treatment with DDL-112 on interleukin-1[beta]-induced extracellular vesicle (EV) release in brain tissue of Thy1-[alpha]Syn PD model mice and chronic effects of 5 week DDL-112 treatment on behavioral/motor function and proteinase K-resistant [alpha]Syn aggregates in the PD model were determined. In the acute study, pre-treatment with DDL-112 reduced EV/exosome biogenesis and in the chronic study, treatment with DDL-112 was associated with a reduction in [alpha]Syn aggregates in the substantia nigra and improvement in motor function. Inhibition of nSMase2 thus offers a new approach to therapeutic development for neurodegenerative diseases with the potential to reduce the spread of disease-specific proteopathic proteins.

The aggregation of misfolded amyloid proteins has been linked to the pathogenesis of a large number of degenerative diseases, including but not limited to Alzheimer's disease, Parkinson's disease, Huntington's disease, amyotrophic lateral sclerosis, prion diseases, and diabetes. While the misfolded proteins implicated in these diseases are unique in terms of their primary sequence, they all tend to aggregate into amyloid structures, which are in turn hypothesized to mediate the toxicity and pathological effects of these proteins. Amyloid structures may be divided into prefibrillar oligomeric species containing anti-parallel β-sheets and fibrillar species characterized by parallel in-register cross-β sheets. The OC and A11 polyclonal antisera are able to immunologically distinguish the two broad groups of amyloids. Here, we introduce a series of 23 unique OC-type monoclonal antibodies developed in our lab, which display conformation-specific reactivity patterns against distinct fibrillar amyloid aggregates made from Aβ, along with aggregates formed by other amyloidogenic proteins. The wide range of reactivity profiles of these monoclonal antibodies highlights the remarkably diverse nature of the immune response against fibrillar amyloids, which in turn is a reflection of the heterogeneity of fibrillar structures formed by amyloid proteins. Interestingly, many of these monoclonal antibodies react with linear segments of the Aβ peptide while reacting in a conformational manner with amyloid aggregates of proteins with no sequence homology with Aβ. This observation indicates that antibody reactivity with linear segments of an amyloid peptide is not a reliable predictor of an antibody's specificity toward the protein in question. In fact, here we report conformational reactivity patterns observed with 2 commercially available monoclonal antibodies proposed to be sequence-specific to Aβ and -synuclein. Since the mOC antibodies are able to discern a wide range of unique conformations within fibrillar aggregates of WT Aβ, we decided to use these antibodies to study potential differences in the immunological reactivity patterns and aggregation kinetics of Aβ peptides containing familial Alzheimer's disease (FAD) mutations. Here we present data demonstrating clear differences in the amyloid conformations adopted by 11 Aβ isotypes containing FAD mutations. Furthermore, we report significant differences in the aggregation kinetics and β-sheet stacking conformation of these peptides. These data suggest that the heterogeneity of FAD cases due to mutations localized to the Aβ sequence may be due to differences in the amyloid conformations adopted by these peptides. Taken together, our data demonstrate that the immune response to fibrillar amyloid structures is diverse and leads to the production of monoclonal antibodies with a wide range of specificities. This observation in turn highlights the importance of the selection of the appropriate antibody in passive immunization therapeutic strategies, as well as raising the possibility of the use of these antibodies in imaging studies and as important in vitro tools in distinguishing different strains of fibrillar amyloid.

Extracellular plaques of amyloid-b and intraneuronal neurofibrillary tangles made from tau are the histopathological signatures of Alzheimer's disease. Plaques comprise amyloid-β fibrils that assemble from monomeric and oligomeric intermediates, and are prognostic indicators of Alzheimer's disease. Despite the importance of plaques to Alzheimer's disease, oligomers are considered to be the principal toxic forms of amyloid-β1,2. Interestingly, many adverse responses to amyloid-β, such as cytotoxicity3,microtubule loss4, impairedmemory and learning5, and neuritic degeneration6, are greatly amplified by tau expression. Amino-terminally truncated, pyroglutamylated (pE) forms of amyloid-β7,8 are strongly associated with Alzheimer's disease, are more toxic than amyloid-β, residues 1-42 (Aβ^sub 1-42^) and Aβ^sub 1-40^, and have been proposed as initiators of Alzheimer's disease pathogenesis9,10. Here we report a mechanism by which pE-Aβ may trigger Alzheimer's disease. Aβ^sub 3(pE)-42^ co-oligomerizes with excess Aβ^sup 1-42^ to form metastable low-n oligomers (LNOs) that are structurally distinct and far more cytotoxic to cultured neurons than comparable LNOs made from Aβ^sub 1-42^ alone. Tau is required for cytotoxicity, and LNOs comprising 5% Aβ^sub 3(pE)-42^ plus 95% Aβ^sub 1-42^ (5% pE-Aβ) seed new cytotoxic LNOs through multiple serial dilutions into Aβ^sub 1-42^ monomers in the absence of additional Aβ^sub 3(pE)-42^. LNOs isolated from human Alzheimer's disease brain contained Aβ^sub 3(pE)-42^, and enhanced Aβ^sub 3(pE)-42^ formation in mice triggered neuron loss and gliosis at 3months,but not in a tau-null background. We conclude that Aβ^sub 3(pE)-42^ confers tau-dependent neuronal death and causes template-induced misfolding of Aβ^sub 1-42^ into structurally distinctLNOs that propagate by a prion-likemechanism.Our results raise the possibility that Aβ^sub 3(pE)-42^ acts similarly at a primary step in Alzheimer's disease pathogenesis. [PUBLICATION ABSTRACT]