AB3202 Sigma-AldrichAnti-LIM-3 Antibody

An unresolved debate in Alzheimer's disease (AD) is whether amyloid plaques are pathogenic, causing overt physical disruption of neural circuits, or protective, sequestering soluble forms of amyloid-β (Aβ) that initiate synaptic damage and cognitive decline. Few animal models of AD have been capable of isolating the relative contribution made by soluble and insoluble forms of Aβ to the behavioral symptoms and biochemical consequences of the disease. Here we use a controllable transgenic mouse model expressing a mutant form of amyloid precursor protein (APP) to distinguish the impact of soluble Aβ from that of deposited amyloid on cognitive function and synaptic structure. Rapid inhibition of transgenic APP modulated the production of Aβ without affecting pre-existing amyloid deposits and restored cognitive performance to the level of healthy controls in Morris water maze, radial arm water maze, and fear conditioning. Selective reduction of Aβ with a γ-secretase inhibitor provided similar improvement, suggesting that transgene suppression restored cognition, at least in part by lowering Aβ. Cognitive improvement coincided with reduced levels of synaptotoxic Aβ oligomers, greater synaptic density surrounding amyloid plaques, and increased expression of presynaptic and postsynaptic markers. Together these findings indicate that transient Aβ species underlie much of the cognitive and synaptic deficits observed in this model and demonstrate that significant functional and structural recovery can be attained without removing deposited amyloid.

Regeneration of auditory hair cells (HCs) is a promising approach to restore hearing. Recent studies have demonstrated that induced pluripotent stem cells/embryonic stem cells or supporting cells (SCs) adjacent to HCs can be converted to adopt the HC fate. However, little is known about whether new HCs are characteristic of outer or inner HCs. Here, we showed in vivo conversion of 2 subtypes of SCs, inner border cells (IBs) and inner phalangeal cells (IPhs), to the inner HC (IHC) fate. This was achieved by ectopically activating Atoh1, a transcription factor necessary for HC fate, in IBs/IPhs at birth. Atoh1+ IBs/IPhs first turned on Pou4f3, another HC transcription factor, before expressing 8 HC markers. The conversion rate gradually increased from ∼ 2.4% at 1 week of age to ∼ 17.8% in adult. Interestingly, new HCs exhibited IHC characteristics such as straight line-shaped stereociliary bundles, expression of Fgf8 and otoferlin, and presence of larger outward currents than those of outer HCs. However, new HCs lacked the terminal differentiation IHC marker vGlut3, exhibited reduced density of presynaptic Cbtp2 puncta that had little postsynaptic GluR2 specialization, and displayed immature IHC outward currents. Our results demonstrate that the conversion rate of IBs/IPhs in vivo by Atoh1 ectopic expression into the IHC fate was higher and faster and the conversion was more complete than that of the 2 other SC subtypes underneath the outer HCs; however, these new IHCs are arrested before terminal differentiation. Thus, IBs/IPhs are good candidates to regenerate IHCs in vivo.

Unlike nonmammalian vertebrates, mammals cannot convert inner ear cochlear supporting cells (SCs) into sensory hair cells (HCs) after damage, thus causing permanent deafness. Here, we achieved in vivo conversion of two SC subtypes, pillar cells (PCs) and Deiters' cells (DCs), into HCs by inducing targeted expression of Atoh1 at neonatal and juvenile ages using novel mouse models. The conversion only occurred in ∼10% of PCs and DCs with ectopic Atoh1 expression and started with reactivation of endogenous Atoh1 followed by expression of 11 HC and synaptic markers, a process that took approximately 3 weeks in vivo. These new HCs resided in the outer HC region, formed stereocilia, contained mechanoelectrical transduction channels, and survived for greater than 2 months in vivo; however, they surprisingly lacked prestin and oncomodulin expression and mature HC morphology. In contrast, adult PCs and DCs no longer responded to ectopic Atoh1 expression, even after outer HC damage. Finally, permanent Atoh1 expression in endogenous HCs did not affect prestin expression but caused cell loss of mature HCs. Together, our results demonstrate that in vivo conversion of PCs and DCs into immature HCs by Atoh1 is age dependent and resembles normal HC development. Therefore, combined expression of Atoh1 with additional factors holds therapeutic promise to convert PCs and DCs into functional HCs in vivo for regenerative purposes.

The GnRH receptor (GnRH-R) plays a central role in mammalian reproductive function throughout adulthood. It also appears as an early marker gene of the presumptive gonadotrope lineage in developing pituitary. Here, using transient transfections combined with DNA/protein interaction assays, we have delineated cis-acting elements within the rat GnRH-R gene promoter that represent targets for the LIM-homeodomain (LIM-HD) proteins, Isl-1 and Lhx3. These factors, critical in early pituitary development, are thus also crucial for gonadotrope-specific expression of the GnRH-R gene. In heterologous cells, the expression of Isl-1 and Lhx3, together with steroidogenic factor 1 (SF-1), culminates in the activation of both the rat as well as human GnRH-R promoter, suggesting that this combination is evolutionarily conserved among mammals. The specificity of these LIM-HD factors is attested by the inefficiency of related proteins, including Lhx5 and Lhx9, to activate the GnRH-R gene promoter, as well as by the repressive capacity of a dominant-negative derivative of Lhx3. Accordingly, targeted deletion of the LIM response element decreases promoter activity. In addition, experiments with Gal4-SF-1 fusion proteins suggest that LIM-HD protein activity in gonadotrope cells is dependent upon SF-1 binding. Finally, using a transgenic model that allows monitoring of in vivo promoter activity, we show that the overlapping expression of Isl-1 and Lhx3 in the developing pituitary correlates with promoter activity. Collectively, these data suggest the occurrence of a specific LIM-HD pituitary code and designate the GnRH-R gene as the first identified transcriptional target of Isl-1 in the anterior pituitary.