Biosynthesis and release of brain-derived neurotrophic factor : a study using neurons derived from embryonic stem cells

Brain-derived neurotrophic factor (BDNF) is a secreted growth factor widely expressed in all major areas of the CNS where it regulates a number of different functions. In animal models of diseases, reduced levels of BDNF have been associated with several conditions, like Rett syndrome, HuntingtonÕs disease and depression. Moreover, reduced BDNF expression in humans has been recently linked with metabolic and neurocognitive impairments, including obesity, episodic memory loss and depression. Like other members of the neurotrophin family, BDNF is synthetized as a N-glycosylated precursor (pro-BDNF) that is post-translationally converted to mature protein. In CNS neurons, mature BDNF is subsequently sorted in large dense core vesicles, transported anterogradely to the synapses and released upon stimulation. As endogenous BDNF is expressed at extremely low levels, most previous studies on BDNF processing and release were performed using acute overexpression, an approach that can lead to the intracellular accumulation and secretion of unprocessed pro-BDNF. To test this possibility, an engineered ES cell line was generated in our laboratory by targeting Bdnf cDNA on Mapt locus. Since the expression of Mapt gene starts when progenitors exit from cell cycle, neurons derived from Mapt::Bdnf ES cells overexpress Bdnf in a controlled fashion throughout their maturation in culture.
My results indicate that BDNF overexpression is incompatible with complete processing and leads to a progressive accumulation and constitutive secretion of pro-BDNF. By contrast, in wild-type neuronal cultures pro-BDNF is fully converted to mature BDNF that is released in an activity dependent fashion.
Regarding the important question of the release of endogenous BDNF from neurons, I observed that basal BDNF release is fully dependent on extracellular calcium influx through specific voltage gate calcium channels. By contrast, calcium efflux from sarcoplasmic reticulum, which triggers BDNF release during specific stimulation patterns, does not affect the basal BDNF secretion.
In order to identify the mechanisms underlying BDNF release during elevated neuronal activity, I derived a new ES cell line from Bassoon mutant mice (Bsn m/m), which develop epileptic seizures and exhibit higher BDNF protein levels in various brain areas. Neurons derived from the Bsn m/m ES cell lines show significantly higher levels of BDNF secretion. In addition, the release of BDNF observed in Bassoon mutant ES cell-derived neurons activates TrKB in these cells and down-regulates the expression of KCC2, a gene encoding for the major neuronal Cl-/K+ co-transporter.
My results thus shed new light on physiological mechanisms of endogenous BDNF biosynthesis and release and invite a critical re-consideration of data obtained using overexpression paradigms.