The progression of Alzheimer’s disease (AD) correlates with the propagation of hyperphosphorylated tau (pTau) from the entorhinal cortex to the hippocampus and neocortex. Neutral sphingomyelinase2 (nSMase2) is critical in the biosynthesis of extracellular vesicles (EVs), which play a role in pTau propagation. We recently conjugated DPTIP, a potent nSMase2 inhibitor, to hydroxyl-PAMAM-dendrimer nanoparticles that can improve brain delivery. We showed that dendrimer-conjugated DPTIP (D–DPTIP) robustly inhibited the spread of pTau in an AAV-pTau propagation model. To further evaluate its efficacy, we tested D-DPTIP in the PS19 transgenic mouse model. Unexpectantly, D-DPTIP showed no beneficial effect. To understand this discrepancy, we assessed D-DPTIP’s brain localization. Using immunofluorescence and fluorescence-activated cell-sorting, D-DPTIP was found to be primarily internalized by microglia, where it selectively inhibited microglial nSMase2 activity with no effect on other cell types. Furthermore, D-DPTIP inhibited microglia-derived EV release into plasma without affecting other brain-derived EVs. We hypothesize that microglial targeting allowed D-DPTIP to inhibit tau propagation in the AAV-hTau model, where microglial EVs play a central role in propagation. However, in PS19 mice, where tau propagation is independent of microglial EVs, it had a limited effect. Our findings confirm microglial targeting with hydroxyl-PAMAM dendrimers and highlight the importance of understanding cell-specific mechanisms when designing targeted AD therapies.

Peripheral nerve injuries (PNIs) represent a significant clinical challenge, particularly in elderly populations where axonal remyelination and regeneration are impaired. Developing therapies to enhance these processes is crucial for improving PNI repair outcomes. Glutamate carboxypeptidase II (GCPII) is a neuropeptidase that plays a pivotal role in modulating glutamate signaling through its enzymatic cleavage of the abundant neuropeptide N-acetyl aspartyl glutamate (NAAG) to liberate glutamate. Within the PNS, GCPII is expressed in Schwann cells and activated macrophages, and its expression is amplified with aging. In this study, we explored the therapeutic potential of inhibiting GCPII activity following PNI. We report significant GCPII protein and activity upregulation following PNI, which was normalized by the potent and selective GCPII inhibitor 2-(phosphonomethyl)-pentanedioic acid (2-PMPA). In vitro, 2-PMPA robustly enhanced myelination in dorsal root ganglion (DRG) explants. In vivo, using a sciatic nerve crush injury model in aged mice, 2-PMPA accelerated remyelination, as evidenced by increased myelin sheath thickness and higher numbers of remyelinated axons. These findings suggest that GCPII inhibition may be a promising therapeutic strategy to enhance remyelination and potentially improve functional recovery after PNI, which is especially relevant in elderly PNI patients where this process is compromised.

Alzheimer’s disease(AD) progression correlates with the propagation of hyperphosphorylated Tau (pTau) from the entorhinal cortex to the hippocampus and neocortex. Neutral sphingomyelinase2(nSMase2) is critical in the biosynthesis of extracellular vesicles (EVs), which play a role in pTau propagation. We recently conjugated DPTIP, a potent nSMase2 inhibitor, to hydroxyl-PAMAM-dendrimer nanoparticles which can improve brain delivery. We showed that dendrimer-conjugated-DPTIP (D-DPTIP) robustly inhibited pTau spread in an AAV-pTau propagation model. To further evaluate its efficacy, we tested D-DPTIP in the PS19 transgenic mouse model. Unexpectantly, D-DPTIP showed no beneficial effect. To understand this discrepancy, we assessed D-DPTIP’s brain localization. Using immunofluorescence and fluorescence-activated cell-sorting, D-DPTIP was found to be primarily internalized by microglia where it selectively inhibited microglial nSMase2 activity with no effect on other cell types. Furthermore, D-DPTIP inhibited microglia-derived EV release into plasma without affecting other brain-derived EVs. We hypothesize that microglial targeting allowed D-DPTIP to inhibit tau propagation in the AAV-hTau model, where microglial EVs play a central role in propagation. However, in PS19 mice, where tau propagation is independent of microglial EVs, it had a limited effect. Our findings confirm microglial targeting with hydroxyl-PAMAM dendrimers and highlight the importance of understanding cell-specific mechanisms when designing targeted AD therapies.

Background: Cognitive decline associated with Alzheimer’s disease (AD) correlates with hyperphosphorylated tau (pTau) propagating between neurons along networks connected by synapses. It has been hypothesized this transcellular transmission occurs partially by extracellular vesicles (EVs). Both genetic and pharmacological inhibition of nSMase2 has been found to inhibit EV biogenesis and pTau propagation. However, the lack of suitable nSMase2 inhibitors for clinical development has been a challenge. In our lab, a highly selective and nM potency nSMase2 inhibitor, termed DPTIP, was identified through a high‐throughput campaign. However, it has poor oral pharmacokinetics (PK), modest brain penetration, and rapid clearance, limiting its clinical translation. To improve its PK properties, we conjugated it to a hydroxyl‐PAMAM dendrimer delivery system, creating dendrimer‐DPTIP (D‐DPTIP), which selectively targets microglia. In previous studies, using a murine AAV‐hTau brain injection propagation model, we showed that administration of D‐DPTIP significantly inhibited tau spread. In this study we extended those studies to evaluate D‐DPTIP in Tau P301S (PS19) transgenic mice.Method: PS19 mice were chronically dosed for 20 weeks. Subsequently cognitive function was evaluated using the Y‐maze and Novel Object Recognition tests, and hippocampal volume was measured through MRI. Total tau and pTau levels were quantified by immunoblotting. Immunofluorescent staining and fluorescence‐activated cell sorting (FACS) were employed to identify cell types in the brain responsible for D‐DPTIP internalization. nSMase2 target engagement assays were conducted in both CD11b+ (microglial) and CD11b‐ (non‐microglial cells). Surface plasmon resonance imaging (SPRi) was used to characterize the origin of brain extracellular vesicles (EVs) released into plasma in PS19 mice ± D‐DPTIP treatment.Result: Total tau, pTau, cognitive deficits, and hippocampal volume loss were not altered by D‐DPTIP treatment in the PS19 mice. D‐DPTIP was found to be co‐localized with microglia and selectively decreased nSMase2 activity in the CD11b+ cells. SPRi analysis showed a decrease in levels of activated microglia‐derived plasma EVs following treatment.Conclusion: D‐DPTIP selectively targets and inhibits nSMase2 activity in microglia. Consequently, in the AAV‐hTau seeded model where microglial EVs play a central role, D‐DPTIP successfully inhibits tau propagation. However, its effectiveness is limited in PS19 mice, where neuronal‐mediated tau propagation is thought to be predominant.

HIV-associated neurocognitive impairment persists despite combination antiretroviral therapy, largely driven by chronic microglial activation that sustains neuroinflammation and neuronal injury. Activated microglia contribute to HIV-associated brain pathology by releasing proinflammatory mediators that disrupt synaptic integrity and impair cognition. N-acetylaspartylglutamate (NAAG), an abundant neuropeptide that maintains glutamatergic homeostasis, is hydrolyzed by glutamate carboxypeptidase II (GCPII) to glutamate. We previously demonstrated that reduced brain and cerebrospinal fluid NAAG levels in people living with HIV correlate with cognitive impairment, and that pharmacological GCPII inhibition with 2-(phosphonomethyl)-pentanedioic acid (2-PMPA) elevates brain NAAG and improves cognition in EcoHIV-infected mice. To enhance brain delivery and preferentially target activated microglia, we conjugated 2-PMPA to a generation 4 hydroxyl poly(amidoamine) (PAMAM) dendrimer (D-2-PMPA). Our findings demonstrate that D-2-PMPA achieves preferential microglial drug delivery, resulting in a >600% increase in cerebrospinal fluid NAAG levels. At doses 8.3-fold lower than free 2-PMPA, this formulation reversed EcoHIV-induced deficits in social interaction, novel object recognition, and fear-conditioned memory without altering locomotor activity or anxiety-like behavior. D-2-PMPA also restored prefrontal cortex synaptic density and preserved dendritic architecture. Together, these findings demonstrate that microglia-targeted GCPII inhibition represents a potent nanotherapeutic strategy to restore synaptic integrity and cognitive function in HIV-associated neurocognitive impairment.