SD-208

We previously showed the importance of TGFβ signaling in development of the mouse axial skeleton. Here, we provide the first direct evidence that TGFβ signaling is required for resegmentation of the sclerotome using chick embryos. Lipophilic fluorescent tracers, DiO and DiD, were microinjected into adjacent somites of embryos treated with or without TGFβRI inhibitors, SB431542, SB525334 or SD208, at developmental day E2.5 (HH16). Lineage tracing of labeled cells was observed over the course of 4 days until the completion of resegmentation at E6.5 (HH32). Vertebrae were malformed and intervertebral discs were small and misshapen in inhibitor injected embryos. Hypaxial myofibers were also increased in thickness after treatment with the inhibitor. Inhibition of TGFβ signaling resulted in alterations in resegmentation that ranged between full, partial, and slanted shifts in distribution of DiO or DiD labeled cells within vertebrae. Patterning of rostro-caudal markers within sclerotome was disrupted at E3.5 after treatment with TGFβRI inhibitor with rostral domains expressing both rostral and caudal markers. We propose that TGFβ signaling regulates rostro-caudal polarity and subsequent resegmentation in sclerotome during spinal column development.

HIV-1–associated neurocognitive disorders (HAND) progression is related to continued inflammation despite undetectable viral loads and may be caused by early viral proteins expressed by latently infected cells. Astrocytes represent an HIV reservoir in the brain where the early viral neurotoxin negative factor (Nef) is produced. We previously demonstrated that astrocytic expression of Nef in the hippocampus of rats causes inflammation, macrophage infiltration, and memory impairment. Since these processes are affected by TGFβ signaling pathways, and TGFβ-1 is found at higher levels in the central nervous system of HIV-1+ individuals and is released by astrocytes, we hypothesized a role for TGFβ-1 in our model of Nef neurotoxicity.

To test this hypothesis, we compared cytokine gene expression by cultured astrocytes expressing Nef or green fluorescent protein. To determine the role of Nef and a TGFβRI inhibitor on memory and learning, we infused astrocytes expressing Nef into the hippocampus of rats and then treated them daily with an oral dose of SD208 (10 mg/kg) or placebo for 7 days. During this time, locomotor activity was recorded in an open field and spatial learning tested in the novel location recognition paradigm. Postmortem tissue analyses of inflammatory and signaling molecules were conducted using immunohistochemistry and immunofluorescence.

TGFβ-1 was induced in cultures expressing Nef at 24 h followed by CCL2 induction which was prevented by blocking TGFβRI with SD208 (competitive inhibitor). Interestingly, Nef seems to change the TGFβRI localization as suggested by the distribution of the immunoreactivity. Nef caused a deficit in spatial learning that was recovered upon co-administration of SD208. Brain tissue from Nef-treated rats given SD208 showed reduced CCL2, phospho-SMAD2, cluster of differentiation 163 (CD163), and GFAP immunoreactivity compared to the placebo group.

Consistent with our previous findings, rats treated with Nef showed deficits in spatial learning and memory in the novel location recognition task. In contrast, rats treated with Nef + SD208 showed better spatial learning suggesting that Nef disrupts memory formation in a TGFβ-1-dependent manner. The TGFβRI inhibitor further reduced the induction of inflammation by Nef which was concomitant with decreased TGFβ signaling. Our findings suggest that TGFβ-1 signaling is an intriguing target to reduce neuroHIV.