Combined extrinsic and intrinsic manipulations exert complementary neuronal enrichment in embryonic rat neural precursor cultures: An in vitro and in vivo analysis

Abstract

Numerous CNS disorders share a common pathology in dysregulation of γ-amino butyric acid inhibitory signaling. Transplantation of GABA-releasing cells at the site of disinhibition holds promise for alleviating disease symptoms with fewer side effects than traditional drug therapies. We manipulated FGF-2 deprivation and MASH1 transcription factor levels in an attempt to amplify the default GABAergic neuronal fate in cultured rat embryonic neural precursor cells (NPCs) for use in transplantation studies. Naïve and MASH1 lentivirus-transduced NPCs were maintained in FGF-2 or deprived of FGF-2 for varying lengths of time. Immunostaining and quantitative analysis showed that GABA- and β-III-tubulin-immunoreactive cells generally decreased through successive passages, suggesting a loss of neurogenic potential in rat neurospheres expanded in vitro. However, FGF-2 deprivation resulted in a small, but significantly increased population of GABAergic cells derived from passaged neurospheres. In contrast to naïve and GFP lentivirus-transduced clones, MASH1 transduction resulted in increased BrdU incorporation and clonal colony size. Western blotting showed that MASH1 overexpression and FGF-2 deprivation additively increased β-III-tubulin and decreased CNPase expression, while FGF-2 deprivation alone attenuated GFAP expression. These results suggest that low FGF-2 signaling and MASH1 activity can operate in concert to enrich NPC cultures for a GABA neuronal phenotype. When transplanted into the adult rat spinal cord, this combination also yielded GABAergic neurons. These findings indicate that, even for successful utilization of the default GABAergic neuronal precursor fate, a combination of both extrinsic and intrinsic manipulations will likely be necessary to realize the full potential of NSC grafts in restoring function.

Characterization of NPY Y2 receptor protein expression in the mouse brain. II. Coexistence with NPY, the Y1 receptor, and other neurotransmitter‐related molecules

“…Guinea pig anti-GABA polyclonal antibody, raised against GABA conjugated to keyhole limpet hemocyanin via glutaraldehyde (Chemicon/Millipore, Billerica, MA; Code No., Ab 175). Previously, preadsorption of this antiserum with 500 lM glutaraldehyde-treated GABA eliminated all tissue staining, while preadsorption of the antiserum with 500 lM glutaraldehyde-treated taurine or glutamate failed to block staining (McDonald and Pearson, 1989;Roettger et al, 1989; see also Furmanski et al, 2009). For preadsorption control in a subsequent study, equal volumes of GABA-BSA conjugate (2.5 mg/mL) and GABA antibody were combined before the primary incubation step, which completely blocked immunoreactivity to GABA (Hantman et al, 2004).…”

“…A 2052). According to Sigma, this antibody shows positive binding with GABA and GABA-KLH in a dotblot assay, and negative binding with BSA (see also Furmanski et al, 2009). Staining patterns of this antibody are consistent with the distribution of GABAergic neurons and terminals in the rat CNS revealed by GAD immunohistochemistry (e.g., Mugnaini and Oertel, 1985) and those patterns obtained using GADgreen fluorescent protein (GFP) heterozygous (Dneo) knockin (GAD67 gfp/þ ) mice (Tamamaki et al, 2003).…”

Crosstalk among electrical activity, trophic factors and morphogenetic proteins in the regulation of neurotransmitter phenotype specification

“…Differentiation of GABAergic phenotypes is thought to be a default fate of differentiating neuronal precursors (Furmanski et al, 2009), which depends on the expression of particular transcription factors. For instance, the Dlx transcription factors promote differentiation of olfactory GABAergic interneurons in mice by regulating the expression of Wnt5a (Paina et al, 2011).…”

Neuropathic Pain Following Nerve Injury

“…Enhanced delivery of GABA via gene therapy approaches have shown promise in preclinical models, notably the administration of GAD65-expressing rAAV2 to sciatic nerve or DRG (Kim et al, 2009) and peripherally delivered HSV-based vectors engineered to produce either GAD65 or GAD67 in DRG (Hao et al, 2005) can reduce peripheral neuropathic or SCI pain. Previous findings in our laboratory have shown that the transplantation of neural progenitor cells expressing GABA into the dorsal horn of animals with excitotoxic spinal cord injury can reduce symptoms of spontaneous pain, and can reduce spinal hyperexcitability (wind-up) and hyperalgesia in animals with chronic constriction injury (Jergova et al, 2009;Lee et al, 2001). To enhance the efficiency of GABAergic cell therapy, several approaches are investigated.…”

“…Recent study demonstrated increased yield of GABAergic precursor cells under a low concentration of the fibroblast growth factor in a cell culture. Also, genetic modification where specific transcriptional factor was blocked promoted GABAergic and reduced glial differentiation (Furmanski et al, 2009). …”