Cognitive decline in Alzheimer's disease (AD) is associated with hyperphosphorylated tau (pTau) propagation between neurons along synaptically connected networks, in part via extracellular vesicles ...(EVs). EV biogenesis is triggered by ceramide enrichment at the plasma membrane from neutral sphingomyelinase2 (nSMase2)-mediated cleavage of sphingomyelin. We report, for the first time, that human tau expression elevates brain ceramides and nSMase2 activity.
To determine the therapeutic benefit of inhibiting this elevation, we evaluated PDDC, the first potent, selective, orally bioavailable, and brain-penetrable nSMase2 inhibitor in the transgenic PS19 AD mouse model. Additionally, we directly evaluated the effect of PDDC on tau propagation in a mouse model where an adeno-associated virus (AAV) encoding P301L/S320F double mutant human tau was stereotaxically-injected unilaterally into the hippocampus. The contralateral transfer of the double mutant human tau to the dentate gyrus was monitored. We examined ceramide levels, histopathological changes, and pTau content within EVs isolated from the mouse plasma.
Similar to human AD, the PS19 mice exhibited increased brain ceramide levels and nSMase2 activity; both were completely normalized by PDDC treatment. The PS19 mice also exhibited elevated tau immunostaining, thinning of hippocampal neuronal cell layers, increased mossy fiber synaptophysin immunostaining, and glial activation, all of which were pathologic features of human AD. PDDC treatment reduced these changes. The plasma of PDDC-treated PS19 mice had reduced levels of neuronal- and microglial-derived EVs, the former carrying lower pTau levels, compared to untreated mice. In the tau propagation model, PDDC normalized the tau-induced increase in brain ceramides and significantly reduced the amount of tau propagation to the contralateral side.
PDDC is a first-in-class therapeutic candidate that normalizes elevated brain ceramides and nSMase2 activity, leading to the slowing of tau spread in AD mice.
Alzheimer's disease (AD) is characterized by the progressive accumulation of amyloid-β and hyperphosphorylated tau (pTau), which can spread throughout the brain via extracellular vesicles (EVs). ...Membrane ceramide enrichment regulated by the enzyme neutral sphingomyelinase 2 (nSMase2) is a critical component of at least one EV biogenesis pathway. Our group recently identified 2,6-Dimethoxy-4-(5-Phenyl-4-Thiophen-2-yl-1H-Imidazol-2-yl)-Phenol (DPTIP), the most potent (30 nM) and selective inhibitor of nSMase2 reported to date. However, DPTIP exhibits poor oral pharmacokinetics (PK), modest brain penetration, and rapid clearance, limiting its clinical translation. To enhance its PK properties, we conjugated DPTIP to a hydroxyl-PAMAM dendrimer delivery system, creating dendrimer-DPTIP (D-DPTIP). In an acute brain injury model, orally administered D-DPTIP significantly reduced the intra-striatal IL-1β-induced increase in plasma EVs up to 72 h post-dose, while oral DPTIP had a limited effect. In a mouse tau propagation model, where a mutant hTau (P301L/S320F) containing adeno-associated virus was unilaterally seeded into the hippocampus, oral D-DPTIP (dosed 3× weekly) significantly inhibited brain nSMase2 activity and blocked the spread of pTau to the contralateral hippocampus. These data demonstrate that dendrimer conjugation of DPTIP improves its PK properties, resulting in significant inhibition of EV propagation of pTau in mice. Dendrimer-based delivery of DPTIP has the potential to be an exciting new therapeutic for AD.
Background
Alzheimer’s Disease (AD) is the most common form of dementia worldwide and is characterized by progressive neurodegeneration and cognitive decline, putatively driven by the accumulation ...Amyloid‐b and hyperphosphorylated Tau. Clinical trial successes focusing on reducing Amyloid‐b plaque levels in patient brains have been modest, spurring a renewed focus on tau. Tau spreads throughout the brain along anatomical pathways, correlating strongly with the disease progression severity. Recent evidence has highlighted extracellular vesicles (EVs) in enabling transcellular transmission of pathological tau in the brain in a ‘prion‐like’ manner and identified the inhibition of EV biogenesis via small‐molecule inhibitors of nSMase2 as a potential therapeutic avenue. However, presently available tool compounds are unsuitable for clinical development.
Method
To improve upon current inhibitors, we performed high‐throughput screening followed by extensive chemistry and identified two classes of compounds. The first, PDDC, has excellent potency, oral bioavailability, and brain penetration, as well as in vitro and in vivo EV inhibition. The second, DPTIP, has higher potency, but poor oral pharmacokinetics and modest brain penetration. To circumvent these limitations, we conjugated DPTIP to a brain‐targeting hydroxyl‐dendrimer delivery system (D‐DPTIP) and demonstrated CNS target engagement following oral dosing. We then chronically administered PDDC and D‐DPTIP to PS19 transgenic mice and WT mice seeded with an AAV‐hTau vector unilaterally stereotaxically injected into the CA1 hippocampal region. After chronic dosing, we quantified the levels of phosphorylated tau in the hippocampus in the PS19s and the contralateral dentate gyrus in the AAV‐hTau seeded model. We also characterized isolated neuronally‐derived plasma EVs from these mice.
Result
PS19 mice treated with PDDC had significantly reduced hippocampal tau and increased neuronal counts compared with vehicle. Additionally, PDDC treatment reduced the release of neuronally‐derived EVs into circulation with lower tau level trends. Similarly, seeded mice treated with either PDDC or D‐DPTIP had reduced tau staining intensity in the contralateral dentate gyrus. Importantly, neither compound induced significant toxicity in either model.
Conclusion
Observing significant reductions in tau burden in two separate AD models provides proof of concept for nSMase2 inhibition in slowing tau propagation, highlighting the promise of tested compounds as potential AD therapeutics.
