BMP2 and GDF5 induce neuronal differentiation through a Smad dependant pathway in a model of human midbrain dopaminergic neurons.

Parkinson's disease is the second most common neurodegenerative disease, and is characterised by the progressive degeneration of the nigrostriatal dopaminergic (DA) system. Current treatments are symptomatic, and do not protect against the DA neuronal loss. One of the most promising treatment approaches is the application of neurotrophic factors to rescue the remaining population of nigrostriatal DA neurons. Therefore, the identification of new neurotrophic factors for midbrain DA neurons, and the subsequent elucidation of the molecular bases of their effects, are important. Two related members of the bone morphogenetic protein (BMP) family, BMP2 and growth differentiation factor 5 (GDF5), have been shown to have neurotrophic effects on midbrain DA neurons both in vitro and in vivo. However, the molecular (signalling pathway(s)) and cellular (direct neuronal or indirect via glial cells) mechanisms of their effects remain to be elucidated. Using the SH-SH5Y human neuronal cell line, as a model of human midbrain DA neurons, we have shown that GDF5 and BMP2 induce neurite outgrowth via a direct mechanism. Furthermore, we demonstrate that these effects are dependent on BMP type I receptor activation of canonical Smad 1/5/8 signalling.

Quantitative morphometric and cell-type-specific population analysis of microglia-enriched cultures subcloned to high purity from newborn rat brains.

Morphological and functional characterizations of cultured microglia are essential for the improved understanding of their roles in neuronal health and disease. Although some studies (phenotype analysis, phagocytosis) can be carried out in mixed or microglia-enriched cultures, in others (gene expression) pure microglia must be used. If the use of genetically modified microglial cells is not feasible, isolation of resident microglia from nervous tissue must be carried out. In this study, mixed primary cultures were established from the forebrains of newborn rats. Secondary microglia-enriched cultures were then prepared by shaking off these cells from the primary cultures, which were subsequently used to establish tertiary cultures by further shaking off the easily detachable microglia. The composition of these cultures was quantitatively analyzed by immunocytochemistry of microglia-, astrocyte-, oligodendrocyte- and neuron-specific markers to determine yield and purity. Microglia were quantitatively characterized regarding morphological and proliferation aspects. Secondary and tertiary cultures typically exhibited 73.3% ± 17.8% and 93.1% ± 6.0% purity for microglia, respectively, although the total number of microglia in the latter was much smaller. One in seven attempts of culturing the tertiary cultures had ~99% purity for microglia. The overall yield from the number of cells plated at DIV0 to the Iba1-positive microglia in tertiary cultures was ~1%. Astrocytic and neuronal contamination progressively decreased during subcloning, while oligodendrocytes were found sporadically throughout culturing. Although the tertiary microglia cultures had a low yield, they produced consistently high purity for microglia; after validation, such cultures are suitable for purity-sensitive functional screenings (gene/protein expression).

Endocytosis contributes to BMP2-induced Smad signalling and neuronal growth.

Bone morphogenetic protein 2 (BMP2) is a neurotrophic factor which induces the growth of midbrain dopaminergic (DA) neurons in vitro and in vivo, and its neurotrophic effects have been shown to be dependent on activation of BMP receptors (BMPRs) and Smad 1/5/8 signalling. However, the precise intracellular cascades that regulate BMP2-BMPR-Smad-signalling-induced neurite growth remain unknown. Endocytosis has been shown to regulate Smad 1/5/8 signalling and differentiation induced by BMPs. However, these studies were carried out in non-neural cells. Indeed, there are scant reports regarding the role of endocytosis in BMP-Smad signalling in neurons. To address this, and to further characterise the mechanisms regulating the neurotrophic effects of BMP2, the present study examined the role of dynamin-dependent endocytosis in BMP2-induced Smad signalling and neurite growth in the SH-SY5Y neuronal cell line. The activation, temporal kinetics and magnitude of Smad 1/5/8 signalling induced by BMP2 were significantly attenuated by dynasore-mediated inhibition of endocytosis in SH-SY5Y cells. Furthermore, BMP2-induced increases in neurite length and neurite branching in SH-SY5Y cells were significantly reduced following inhibition of dynamin-dependent endocytosis using dynasore. This study demonstrates that BMP2-induced Smad signalling and neurite growth is regulated by dynamin-dependent endocytosis in a model of human midbrain dopaminergic neurons.