Dendrimer-2PMPA selectively blocks upregulated microglial GCPII activity and improves cognition in a mouse model of multiple sclerosis

Abstract

Cognitive impairment is a common aspect of multiple sclerosis (MS) for which there are no treatments. Reduced brain N-acetylaspartylglutamate (NAAG) levels are linked to impaired cognition in various neurological diseases, including MS. NAAG levels are regulated by glutamate carboxypeptidase II (GCPII), which hydrolyzes the neuropeptide to N-acetyl-aspartate and glutamate. GCPII activity is upregulated multifold in microglia following neuroinflammation. Although several GCPII inhibitors, such as 2-PMPA, elevate brain NAAG levels and restore cognitive function in preclinical studies when given at high systemic doses or via direct brain injection, none are clinically available due to poor bioavailability and limited brain penetration. Hydroxyl-dendrimers have been successfully used to selectively deliver drugs to activated glia. Methods: We attached 2-PMPA to hydroxyl polyamidoamine (PAMAM) dendrimers (D-2PMPA) using a click chemistry approach. Cy5-labelled-D-2PMPA was used to visualize selective glial uptake in vitro and in vivo. D-2PMPA was evaluated for anti-inflammatory effects in LPS-treated glial cultures. In experimental autoimmune encephalomyelitis (EAE)-immunized mice, D-2PMPA was dosed biweekly starting at disease onset and cognition was assessed using the Barnes maze, and GCPII activity was measured in CD11b+ hippocampal cells. Results: D-2PMPA showed preferential uptake into microglia and robust anti-inflammatory activity, including elevations in NAAG, TGFβ, and mGluR3 in glial cultures. D-2PMPA significantly improved cognition in EAE mice, even though physical severity was unaffected. GCPII activity increased >20-fold in CD11b+ cells from EAE mice, which was significantly mitigated by D-2PMPA treatment. Conclusions: Hydroxyl dendrimers facilitate targeted drug delivery to activated microglia. These data support further development of D-2PMPA to attenuate elevated microglial GCPII activity and treat cognitive impairment in MS.

Keywords: GCPII; NAAG; cognitive impairment; dendrimer; multiple sclerosis.

Figure 1

Synthesis and characterization of D-2PMPA. A. Synthetic protocol for the attachment of cleavable linker to 2-PMPA; B. The synthetic scheme for the conjugation of 2-PMPA on the surface of hydroxyl PAMAM dendrimer; C. Comparison of ¹H NMR spectra for the intermediates and the final D-2PMPA conjugate; D. HPLC chromatogram showing distinct shifts at each reaction step; E. Size distribution by number of D-2PMPA conjugate, and F. Zeta potential distribution of D-2PMPA as analyzed by the dynamic light scattering.

Figure 2

Synthesis and characterization of Cy5-D-2PMPA. A. Synthetic protocol for the synthesis of fluorescently-labeled dendrimer-2-PMPA conjugate (Cy5-D-2PMPA); B. HPLC chromatogram of Cy5-D-2PMPA at dendrimer absorption wavelength (210nm) and Cy5 absorption wavelength (650nm); and C. UV/Vis profile of Cy5-D-2PMPA showing absorption at both dendrimer and Cy5 wavelengths.

Figure 3

The comparative IC₅₀s of the free drug, drug linker and dendrimers determined using human recombinant GCPII. A. 2-PMPA; B. 2-PMPA-PEG-azide; C. D-2PMPA; and D. D-OH.

Figure 4

Confocal images of glial cultures demonstrating Cy5-D-2PMPA uptake. Microglia were stained using Iba-1 (green), astrocytes were stained using GFAP (orange), nuclei were stained using DAPI (blue), and the D-2PMPA conjugates were labelled with Cy5 (red). Cells were treated with 50 µg/mL of Cy5-D-2PMPA to evaluate cellular uptake. At 3 and 6 hours post treatment, Cy5-D-2PMPA was preferentially taken-up by microglial cells (A-D and E-H). Microglial uptake of Cy5-D-2PMPA continued at 12 hours post-treatment (I-L, white arrows). At 12 hours post treatment, astrocytes also demonstrated some signs of uptake (I-L, white arrowheads). Scale bar 20 µm.

Figure 5

Effect of D-2PMPA on LPS-treated glial cultures. D-2PMPA treatment resulted in a dose-dependent upregulation of NAAG (A), TGFβ mRNA expression (B), and mGluR3 mRNA expression (C). D-2PMPA had no effect on cell viability as assessed by the MTT assay (D). Significantly different from control at *P<0.05, **P<0.01. Significantly different from LPS at ⁺P<0.05, ⁺⁺P<0.01, ⁺⁺⁺P<0.001.

Figure 6

Cy5-D-2PMPA is selectively taken up by activated microglia. EAE mice were administered a single dose of Cy5-D-2PMPA at 14 days post-immunization and sacrificed 24 hours later. Representative brain images illustrate selective uptake Cy5-D-2PMPA (C, violet cells) in Iba1 positive activated microglia (B, red cells) along the edge of the dentate gyrus. Merged image of Iba1 and Cy5-D-2PMPA positive cells (D, pink cells). GFAP positive astrocytes (A, green cells) have no Cy5-D-2PMPA signal (D, arrowheads). Nuclei are stained blue in the merged image (D). Scale bars, 50 µm.

Figure 7

D-2PMPA improves cognitive function and inhibits hippocampal GCPII activity in EAE mice. Biweekly D-2PMPA (20mg/kg) treatment initiated at the onset of physical disease had no impact on EAE physical disease severity (A). At 5 weeks post-immunization, EAE mice were tested in the Barnes maze. EAE mice exhibited impaired cognition function compared to non-EAE control mice that was reversed with D-2PMPA treatment as measured by the change between the first and last trial in latency to find the target (B) and path efficiency (C). Representative track plot from the final Barnes maze trial illustrate the impaired performance in EAE mice versus controls that is improved with D-2PMPA treatment (D-F). Experiments were performed in duplicate and data shown are mean ± SD of a representative experiment, n=7-8/group. G. Following Barnes maze testing, EAE mice received D-Veh or D-2PMPA and sacrificed 24 hours later and CD11b+ cells were isolated from hippocampi. GCPII enzymatic activity increased 24.7-fold in EAE mice treated with D-Veh versus Control mice. D-2PMPA treatment in EAE mice robustly attenuated the increased GCPII activity. Significantly different from EAE+D-Veh at P<0.001(***); Data are mean ± SD, n=5-8/group.