The Shortest Isoform of Dystrophin (Dp40) Interacts with a Group of Presynaptic Proteins to Form a Presumptive Novel Complex in the Mouse Brain

Abstract

Duchenne muscular dystrophy (DMD) causes cognitive impairment in one third of the patients, although the underlying mechanisms remain to be elucidated. Recent studies showed that mutations in the distal part of the dystrophin gene correlate well with the cognitive impairment in DMD patients, which is attributed to Dp71. The study on the expression of the shortest isoform, Dp40, has not been possible due to the lack of an isoform specific antibody. Dp40 has the same promoter as that found in Dp71 and lacks the normal C-terminal end of Dp427. In the present study, we have raised polyclonal antibody against the N-terminal sequence common to short isoforms of dystrophin, including Dp40, and investigated the expression pattern of Dp40 in the mouse brain. Affinity chromatography with this antibody and the consecutive LC-MS/MS analysis on the interacting proteins revealed that Dp40 was abundantly expressed in synaptic vesicles and interacted with a group of presynaptic proteins, including syntaxin1A and SNAP25, which are involved in exocytosis of synaptic vesicles in neurons. We thus suggest that Dp40 may form a novel protein complex and play a crucial role in presynaptic function. Further studies on these aspects of Dp40 function might provide more insight into the molecular mechanisms of cognitive impairment found in patients with DMD.Electronic supplementary materialThe online version of this article (doi:10.1007/s12035-012-8233-5) contains supplementary material, which is available to authorized users.

Somatodendritic and excitatory postsynaptic distribution of neuron-type dystrophin isoform, Dp40, in hippocampal neurons

“…Considering the fact that Dp427 and the other Dp isoforms, including Dp71, are localized beneath the plasma membranes of muscle and/or neuronal cells by interacting with dystroglycan-related molecules [5][6][7][8]15], it is possible that common molecular mechanisms will be involved in the Dp40 localization. However, we previously showed that Dp40 is not likely associated with membrane-spanning glycoproteins [14], suggesting that a particular molecular mechanism different from that related to dystroglycan-related molecules might be responsible for the Dp40 localization. Thus, we must consider the question, ''What kinds of molecules are responsible for the Dp40 localization?''…”

“…The amino acid sequence used for the antigen is totally included in full-length Dp (also known as Dp427) and a short isoform of Dp, Dp71 (Fig. 1A) [6,14]. To validate the reactivity of the anti-Dp40 antibody, anti-Dp40, antiDp71, or control IgG immunoprecipitations were performed using adult mouse cerebrum followed by Western blotting with the anti-Dp40 antibody, showing that the anti-Dp40 antibody was able to immunoprecipitate and detect both Dp40 and Dp71 proteins (Fig.…”

“…Dp71 is an abundant product of the DMD gene that is expressed in the brain and found in both neurons and glia [5]. Most recently, it was found that the shortest dystrophin isoform, Dp40, the NH 2 -terminal partial product of Dp71 produced by alternative splicing [13], is also expressed in the brain [14], although little is known about Dp40.…”

EF-hand domains are involved in the differential cellular distribution of dystrophin Dp40

“…The tissue distribution of the Dp40 transcript is similar to that of Dp71 and it is also expressed in early development and in murine ES cells [4]. Previously, the expression of Dp40 protein in the mouse brain and its interaction with a group of presynaptic proteins was reported [5]. Recently, somatodendritic and nuclear localization of Dp40 wild type, in cultured mouse hippocampal neurons, has been shown and function for this protein in neuronal maturation and postsynaptic processes has been proposed [6].…”

Acetylcholine, GABA and neuronal networks: A working hypothesis for compensations in the dystrophic brain

“…Some results suggest that most of the alterations associated with cognitive impairment are related to mutations of the specific dystrophin isoforms Dp140 and Dp71 (Moizard et al, 1998(Moizard et al, , 2000. Further studies suggest the possible involvement of other isoforms (Giliberto et al, 2004;Taylor et al, 2010;D'Angelo et al, 2011;Tozawa et al, 2012). A variety of studies have focused their attention mainly on the disruption of neuronal function, yet there is evidence that some brain functions (such as working and reference memory) are not compromised despite the lack of dystrophin (Sesay et al, 1996;Vaillend et al, 1998;Hinton et al, 2007).…”