The brain-derived neurotrophic factor enhances synthesis of Arc in synaptoneurosomes - PubMed (original) (raw)

The brain-derived neurotrophic factor enhances synthesis of Arc in synaptoneurosomes

Yong Yin et al. Proc Natl Acad Sci U S A. 2002.

Abstract

Protein synthesis in neurons is essential for the consolidation of memory and for the stabilization of activity-dependent forms of synaptic plasticity such as long-term potentiation (LTP). Activity-dependent translation of dendritically localized mRNAs has been proposed to be a critical source of new proteins necessary for synaptic change. mRNA for the activity-regulated cytoskeletal protein, Arc, is transcribed during LTP and learning, and disruption of its translation gives rise to deficits in both. We have found that selective translation of Arc in a synaptoneurosomal preparation is induced by the brain-derived neurotrophic factor, a neurotrophin that is released during high-frequency stimulation patterns used to elicit LTP. This effect involves signaling through the TrkB receptor and is blocked by the N-methyl-d-aspartate-type glutamate receptor antagonist, MK801. The results suggest there is a synergy between neurotrophic and ionotropic mechanisms that may influence the specificity and duration of changes in synaptic efficacy at glutamatergic synapses.

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Figures

Figure 1

Figure 1

Electrophoretic gel of RT-PCR-amplified mRNAs in SNS. Total RNA extracted from SNS samples was used for RT-PCR amplification. Primers for specific segments of Arc, CaMKIIα, and neural cell adhesion molecule 140 and -180 isoforms were used for PCR detection.

Figure 2

Figure 2

BDNF increases incorporation of [35S]methionine into a subset of proteins. Total synaptoneurosomal protein was extracted, and proteins were separated by 2D electrophoresis from both control and BDNF-treated samples after radioactive labeling. Proteins with increased 35S label in response to BDNF are marked with open arrowheads. As determined from several independent experiments, the protein at molecular mass 34 kDa (c) was increased by 34% ± 3.9% (n = 8, P < 0 01), at 46 kDa (b) by 43.4% ± 2.4% (n = 6, P < 0.01), and at 56 kDa (a) by 40.4% ± 2.3% (n = 7, P < 0.01). Stated values are means ± SE. Most proteins showed no change and some (marked with filled arrowheads in Inset) were actually reduced in samples treated with BDNF (Right). The intensity ratios (BDNF to control) of spots x, y, and z in the rectangle are 0.78, 0.89, and 0.96, respectively.

Figure 3

Figure 3

BDNF selectively enhances Arc label incorporation and expression. Arc protein incorporation and net protein expression are analyzed with autoradiography (A) followed by immunoblot (B) of the same membrane after 2D electrophoresis. Both 35S incorporation and overall Arc protein expression measured on blots, marked with circles, were significantly increased by BDNF treatment. However, BDNF treatment reduced the incorporation of 35S into spots a (0.86), b (0.64), c (0.82), and d (0.91) and increased spots e (1.25) and f (2.74), compared with the control. BDNF did not significantly increase β-tubulin levels overall, and in the experiment represented in B actually decreased β-tubulin by 37% of the control. (C) Summary (range, quartiles, median) of independent experimental results obtained from both autoradiography and immunoblots. The variance of basal Arc protein levels in SNSs was ± 2.1% (SEM) as determined with immunoblots (n = 3) normalized to synaptotagmin levels. On average, BDNF increased Arc 35S incorporation and overall Arc protein levels by 43% ± 4.8% (n = 8, P < 0.01) and 70% ± 9.13% (_n_ = 12, _P_ < 0.01), respectively, relative to paired controls. However, BDNF treatment did not change synaptotagmin (99% ± 1.4%, _n_ = 7, _P_ > 0.05) and β-tubulin (90% ± 7.7%, n = 8, P > 0.05) protein levels compared with controls.

Figure 4

Figure 4

The NMDA receptor antagonist MK801 prevents BDNF-induced increases in Arc protein synthesis. (A) The effect of the NMDA receptor antagonist MK801 on Arc protein incorporation was monitored with autoradiography (A) and immunoblot (B) after 2D electrophoresis. BDNF-induced Arc 35S incorporation (A) and overall Arc protein levels (B) were blocked by incubation with MK801 (filled arrowheads). Meanwhile, MK801 did not decrease the incorporation of radiolabeled amino acids into spots a (1.14), b (1.14), and c (1.15) marked with blank arrowheads. (C) Summary chart (range, quartiles, and median) displaying the results of six independent experiments performed as in A and analyzed for Arc and synaptotagmin protein levels. On average, MK801 suppressed BDNF-induced up-regulation of Arc 35S incorporation by 24% ± 4.7% (P < 0.01) and overall Arc protein levels by 34% ± 2.5% (_P_ < 0.01) relative to BDNF alone. MK801 treatment did not significantly affect synaptotagmin levels (101% ± 7.6%, _P_ > 0.05). MK801 alone (not shown) did not significantly affect the basal level of Arc protein (97% ± 10.26% compared with untreated controls). (D) A typical 1D SDS/PAGE Western blot in which BDNF and BDNF + MK801-treated samples were compared by using anti-Arc, anti-synaptotagmin, and anti-β-tubulin Abs. Preincubation with MK801 suppressed the BDNF-induced Arc protein increase by 35%; little or no change was evident for synaptotagmin (93% of BDNF alone) or β-tubulin (95% of BDNF alone).

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