Gene delivery to overcome astrocyte inhibition of axonal growth: An in vitro Model of the glial scar

Abstract

After injury to the central nervous system, a glial scar develops that physically and biochemically inhibits axon growth. In the scar, activated astrocytes secrete inhibitory extracellular matrix, of which chondroitin sulfate proteoglycans (CSPGs) are considered the major inhibitory component. An inhibitory interface of CSPGs forms around the lesion and prevents axons from traversing the injury, and decreasing CSPGs can enhance axon growth. In this report, we established an in vitro interface model of activated astrocytes and subsequently investigated gene delivery as a means to reduce CSPG levels and enhance axon growth. In the model, a continuous interface of CSPG producing astrocytes was created with neurons seeded opposite the astrocytes, and neurite crossing, stopping, and turning were evaluated as they approached the interface. We investigated the efficacy of lentiviral delivery to degrade or prevent the synthesis of CSPGs, thereby removing CSPG inhibition of neurite growth. Lentiviral delivery of RNAi targeting two key CSPG synthesis enzymes, chondroitin polymerizing factor and chondroitin synthase-1, decreased CSPGs, and reduced inhibition by the interface. Degradation of CSPGs by lentiviral delivery of chondroitinase also resulted in less inhibition and more neurites crossing the interface. These results indicate that the interface model provides a tool to investigate interventions that reduce inhibition by CSPGs, and that gene delivery can be effective in promoting neurite growth across an interface of CSPG producing astrocytes.

Targeting Beclin1 as an Adjunctive Therapy against HIV Using Mannosylated Polyethylenimine Nanoparticles

“…Since autophagy is an essential pathway for neuronal homeostasis and survival, we have modified our previously reported nanoformulation, which consisted of PEI-siBeclin1, with the addition of mannose molecules to specifically target mannose receptor-expressing glial cells. The mannose receptor is a transmembrane glycoprotein from the C-type lectin family that can be used as a potential surface target for drug delivery using nanocarriers [ 63 , 64 , 65 ]. This receptor is expressed in a wide range of cells, including macrophages, dendritic cells, and, as mentioned earlier, glial cells [ 66 , 67 ].…”

Astrocytes, therapeutic targets for neuroprotection and neurorestoration in ischemic stroke

“…Enzymatic disruption of neurocan with chondroitinase ABC, which cleaves glycosaminoglycan side chains, infused directly into the infarct cavity for 7 days, beginning 7 days after MCAo, improves gross anatomical, histological, and functional outcome in the chronic phase of experimental stroke in rats (Hill et al , 2012). In addition, using an in vitro interface model of activated astrocytes, lentiviral delivery of RNAi targeting two key CSPG synthesis enzymes, chondroitin polymerizing factor and chondroitin synthase-1, reduces CSPG levels and enhance axon growth (Tuinstra et al , 2013). Ephrin-A5 is expressed in reactive astrocytes in the peri-infarct region, and inhibits axonal sprouting; while inhibition of ephrin-A5 in the peri-infarct area 7 days after stroke promotes axonal outgrowth and behavioral recovery in mice (Overman et al , 2012).…”

Destination Brain: the Past, Present, and Future of Therapeutic Gene Delivery

“…Other RNAi-associated studies employed LVs to silence astrocyte marker molecules GFAP and vimentin (Desclaux et al, 2009), chondroitin polymerizing factor (CPF) and chondroitin synthase-1 (CS-1) (Tuinstra et al., 2013), as well as miR145 (Wang et al, 2015). Astrocyte intermediate filaments, GFAP and vimentin, are upregulated during inflammation adversely affecting axonal and neurite regeneration post-injury.…”

“…Thus, LV-shGFAP and shVimentin delivery decreased astrogliosis, astrocyte migration in scratch assay and improved neuronal survival in co-cultures (Desclaux et al, 2009). Astrocyte-secreted chondroitin sulfate proteoglycans were activated by LV-miCPF and -miCS-1 delivery during CNS injury, which increased the neurite outgrowth in neuroglial cultures (Tuinstra et al, 2013). A negative regulator of reactive astrogliosis, miR145, delivered with LV reduced astrocyte activation, proliferation, and migration in an in vitro SCI model (Wang et al, 2015).…”