Dysregulation of brain-derived neurotrophic factor expression and neurosecretory function in Mecp2 null mice - PubMed (original) (raw)

Comparative Study

Dysregulation of brain-derived neurotrophic factor expression and neurosecretory function in Mecp2 null mice

Hong Wang et al. J Neurosci. 2006.

Abstract

Disruptions in brain-derived neurotrophic factor (BDNF) expression are proposed to contribute to the molecular pathogenesis of Rett syndrome (RTT), a severe neurological disorder caused by loss-of-function mutations in methyl-CpG-binding protein-2 (MeCP2). Although MeCP2 is a transcriptional regulator of BDNF, it is unknown how MeCP2 mutations affect transsynaptic BDNF signaling. Our findings demonstrate an early, abnormal neurosecretory phenotype in MeCP2-deficient neurons characterized by significant increases in the percentage of cellular BDNF content available for release. However, loss of MeCP2 also results in deficits in total cell BDNF content that are developmentally regulated in a cell-type-specific manner. Thus, the net effect of MeCP2 loss on absolute BDNF secretion changes with age and is determined by both the amount of BDNF available for release and progressive declines in total cellular BDNF. We propose, therefore, that loss of MeCP2 function disrupts transsynaptic BDNF signaling by perturbing the normal balance between BDNF protein levels and secretion. However, mutant neurons are capable of secreting wild-type levels of BDNF in response to high-frequency electrical stimulation. In addition, we found elevated exocytic function in Mecp2(-/y) adrenal chromaffin cells, indicating that the Mecp2 null mutation is associated with alterations of neurosecretion that are not restricted to BDNF. These findings are the first examples of abnormal neuropeptide and catecholamine secretion in a mouse model of RTT.

PubMed Disclaimer

Figures

Figure 1.

Figure 1.

Mecp2 −/y mice exhibit progressive deficits in BDNF in vivo. A, BDNF protein levels were compared in P0 and P35 wild-type and Mecp2 −/y NGs, brainstem (b.stem), hippocampus (hipp.), and cortex by ELISA. Mutant values are expressed as a fraction of the corresponding wild-type control (wild type = 1). Each bar represents the mean ± SEM from at least three animals. *p < 0.05, **p < 0.01 (Student’s t test). B, BDNF immunostaining is depleted in the terminal field of NG neurons in the brainstem nTS in P35 Mecp2 −/y mice (−/y) compared with Mecp2 +/y controls (+/y). CC, Central canal; DMnX, dorsal motor nucleus of the vagus nerve; tS, solitary tract. Scale bar, 100 μm.

Figure 2.

Figure 2.

Mecp2 −/y neurons exhibit age-dependent defects in spontaneous BDNF release. Spontaneous BDNF release from dissociated cultures of wild-type and Mecp2 −/y NGs and cortical neurons was measured during 3 d in culture using ELISA in situ. A, The amount of BDNF released as a percentage of total BDNF content. B, C, The absolute amount of BDNF released. Each bar represents the mean ± SEM from at least three independent experiments. *p < 0.05, **p < 0.01 (Student’s t test).

Figure 3.

Figure 3.

Evoked BDNF release in Mecp2 −/y NG neurons. BDNF release evoked by patterned electrical field stimulation was compared in dissociate cultures of P35 wild-type and Mecp2 −/y NG neurons. Cultures were stimulated once every 20 s, with 2 s trains of biphasic pulses at 100 Hz, and assayed for released BDNF by ELISA in situ and for cellular BDNF content by standard ELISA. A, Wild-type and Mecp2 −/y neurons released the same absolute amount of BDNF during the period of stimulation (stimulated release = total release after stimulation − unstimulated release as measured in control cultures). However, as shown in B, cellular BDNF content was significantly lower in mutant neurons compared with wild-type cells, as in vivo. Therefore, as shown in C, mutant neurons released a significantly greater percentage of total BDNF content during stimulation than did wild-type cells. Each bar represents the mean ± SEM from at least three independent experiments. *p < 0.05 (ANOVA).

Figure 4.

Figure 4.

Mecp2 −/y adrenal chromaffin cells exhibit an increase in the catecholamine RRP and evoked release. The RRP was quantified in wild-type and Mecp2 −/y chromaffin cells as described previously (Gillis et al., 1996). Ai, Cells were voltage clamped at −80 mV and stimulated with a pair of 100 ms depolarizations, first to 0 mV and then to +5 mV, to balance Ca2+ influx. The top trace shows evoked current influx (IMon.), and the bottom trace shows cell capacitance (Cm). In this example, the first pulse resulted in a greater capacitance increase than the second pulse (Cm1 and Cm2, respectively), reflecting a consumption of release-ready granules by the first pulse, leaving few for the second pulse to access. Formally, the RRP can thus be quantified as follows: RRP = S/(1 − R 2), where S is the sum of Cm1 and Cm2 and R is the ratio of Cm2 to Cm1. Aii, This dual-pulse protocol was repeated on MeCP2+/y and MeCP2−/y cells. The quantified RRP sizes from Mecp2 +/y and Mecp2 −/y records (n = 36 and 65, respectively) are summarized and show that the RRP was significantly larger in the Mecp2 −/y (*p < 0.02, paired Student’s t test). Bi, To correlate the increased RRP to evoked catecholamine release, the amperometric (I Amp.) current was measured from chromaffin cells in control Ringer’s solution and during nicotine stimulation. A representative record from Mecp2 +/+ chromaffin tissue is plotted. Bii, Amperometric currents (ΣAmp.) recorded under this protocol from Mecp2 +/y and Mecp2 −/y cells were integrated to allow relative comparison of catecholamine release (n = 8 and 10 for Mecp2 +/y and Mecp2 −/y cells, respectively). These data show significant increases in both spontaneous and evoked catecholamine release from Mecp2 −/y cells compared with Mecp2 +/y (*p < 0.02, paired Student’s t test).

Similar articles

Cited by

References

    1. Amir RE, Van den Veyver IB, Wan M, Tran CQ, Francke U, Zoghbi HY. Rett syndrome is caused by mutations in X-linked MECP2, encoding methyl-CpG-binding protein 2. Nat Genet. 1999;23:185–188. - PubMed
    1. Balkowiec A, Katz DM. Activity-dependent release of endogenous brain-derived neurotrophic factor from primary sensory neurons detected by ELISA in situ. J Neurosci. 2000;20:7417–7423. - PMC - PubMed
    1. Balkowiec A, Kunze DL, Katz DM. Brain-derived neurotrophic factor acutely inhibits AMPA-mediated currents in developing sensory relay neurons. J Neurosci. 2000;20:1904–1911. - PMC - PubMed
    1. Brady R, Zaidi SI, Mayer C, Katz DM. BDNF is a target-derived survival factor for arterial baroreceptor and chemoafferent primary sensory neurons. J Neurosci. 1999;19:2131–2142. - PMC - PubMed
    1. Chan SA, Polo-Parada L, Landmesser LT, Smith C. Adrenal chromaffin cells exhibit impaired granule trafficking in NCAM knockout mice. J Neurophysiol. 2005;94:1037–1047. - PubMed

Publication types

MeSH terms

Substances

LinkOut - more resources