Novel Nuclear Protein Complexes of Dystrophin 71 Isoforms in Rat Cultured Hippocampal GABAergic and Glutamatergic Neurons

The precise functional role of the dystrophin 71 in neurons is still elusive. Previously, we reported that dystrophin 71d and dystrophin 71f are present in nuclei from cultured neurons. In the present work, we performed a detailed analysis of the intranuclear distribution of dystrophin 71 isoforms (Dp71d and Dp71f), during the temporal course of 7-day postnatal rats hippocampal neurons culture for 1h, 2, 4, 10, 15 and 21 days in vitro (DIV). By immunofluorescence assays, we detected the highest level of nuclear expression of both dystrophin Dp71 isoforms at 10 DIV, during the temporal course of primary culture. Dp71d and Dp71f were detected mainly in bipolar GABAergic (≥60%) and multipolar Glutamatergic (≤40%) neurons, respectively. We also characterized the existence of two nuclear dystrophin-associated protein complexes (DAPC): dystrophin 71d or dystrophin 71f bound to β-dystroglycan, α1-, β-, α2-dystrobrevins, α-syntrophin, and syntrophin-associated protein nNOS (Dp71d-DAPC or Dp71f-DAPC, respectively), in the hippocampal neurons. Furthermore, both complexes were localized in interchromatin granule cluster structures (nuclear speckles) of neuronal nucleoskeleton preparations. The present study evinces that each Dp71’s complexes differ slightly in dystrobrevins composition. The results demonstrated that Dp71d-DAPC was mainly localized in bipolar GABAergic and Dp71f-DAPC in multipolar Glutamatergic hippocampal neurons. Taken together, our results show that dystrophin 71d, dystrophin 71f and DAP integrate protein complexes, and both complexes were associated to nuclear speckles structures.

Introduction protein complex in the nuclei; (iii) Dp71-DAP complexes were differentially localized in bipolar GABAergic or multipolar Glutamatergic hippocampal neurons; (iv) the Dp71-DAP complexes are localized in nuclear speckles associated to the nuclear skeleton; and (v) the nuclear localization or composition of these complexes probably determine specific characteristics of each neuronal phenotype. These nuclear protein complexes that appears to be structurally similar to those present in the plasma membrane, offer a new functional alternative to Dp71 isoforms, not only involved in the plasma membrane and cytoskeletal architecture but probably also, in a nuclear process that determine and regulate the peripheral function of these complexes.

Materials and Methods
All procedures were in accordance with the current Mexican legislation, NOM-062- ZOO-1999 (SAGARPA), based on the Guide for the Care and Use of Laboratory Animals. The Center for Research and Advanced Studies of the National Polytechnic School (CINVESTAV-IPN) Institutional Animal Care and Use Committee (IACUC), Mexico City, approved our procedures for animal use (protocol # 091-02).

Ethics Statement
All efforts were made to minimize animal suffering.

Animals
All animals were maintained under constant room temperature (RT; 23°C), 12 h-12 h lightdark cycle, with food and water ad libitum (S1 ARRIVE Checklist). Female Postnatal day 7 Wistar rats (10-15 g of body weight), bred in our facilities, were sacrificed according to the Mexican official norm NOM-062-ZOO-1999 for the production, care, use and sacrifice of laboratory animals. Briefly, 5 female rat pups of 7 days postnatal, were anesthetized (at noon) via intraperitoneal Pentobarbital injection (30 to 40 mg/kg, i.p.) and after that were decapitated, and the brains were dissected to obtain the hippocampal neurons for cell culture. See more details in the cell culture section of this Material and Methods.

Antibodies
The properties and dilution of the antibodies used during this study are shown in Table 1.

Cell treatments
For heat shock experiments, Cells cultured in Petri dishes containing coverslips were switched to pre-warmed medium (45°C) and incubated for 15 min in a water bath preheated at 45°C, prior to fixation for immunofluorescence microscopy. Control cells were also seeded in the same type of Petri dishes with cover slips, and their culture medium was changed to pre-warmed 37°C medium and incubated for the same time at 37°C prior fixation.

Nuclei isolation
Isolation of nuclei was essentially carried out as previously reported [28].

Subnuclear fractions
Briefly, isolated nuclei were stripped of outer and inner nuclear membranes, by treatment in 200 μl of CSK-1 buffer (10 mM PIPES pH 6.8, 100 mM NaCl, 0.3 M Sucrose, 1.5 mM MgCl 2 , 1.0 mM EGTA and 0.5% [v/v] Triton X-100), followed by incubation on ice for 5 min. Membrane-depleted nuclei were collected by centrifugation at 5,000 × g for 5 min at 4°C, the supernatant contained the nuclear membranes fraction. Chromatin and soluble proteins were

Immunoprecipitation
Immunoprecipitation of Dp71d (20 μl of Dys-2 Ab) and Dp71f (20 μl of 5F3 Ab) or both dystrophin isoforms (20 μl of Mandra-1 Ab) was performed with the nucleoskeleton fraction from cultured hippocampal neurons. Briefly, nucleoskeleton fraction (1 mg) was precleared with 20 μl of recombinant protein G-agarose beads overnight at 4°C. The beads were removed by centrifugation at 16,000 rpm for 5 min, and precleared extract was incubated overnight at 4°C with the immunoprecipitating Ab, previously bound to recombinant protein G-Agarose (Invitrogen, Carlsbad, CA, USA). As negative control, parallel incubation with a non-related Ab (monoclonal anti-CD4 Ab, as nonspecific immunoprecipitation) was performed. The immune complexes were collected by centrifugation at 16,000 g for 5 min, and washed three times for 10 min with 1 ml of RIPA buffer and then eluted by boiling in Laemmli buffer. Immunoprecipitated proteins were then analyzed by western blot as described above. with Fluorogel (Electron Microscopy Sciences). Mounted coverslips were examined using a Leica laser scanning model SP8 confocal microscope (TCP-SP8, Leica, Heidelberg, Germany) and analyzed with LAS AF Image software, Ver. 4. For the colocalization analyze using the LAS AF software we selected the nuclear area or the cytoskeletal area and obtained the colocalization rate for that specific area of the cell. We measured 10 cells per each immunostaining condition, and calculate the average ± the SEM. Data was plotted using Graph Pad Prism 5.

Nucleoskeleton Preparations
Nucleoskeleton preparations were obtained as previously reported [30], with some modifications. Briefly, cultured hippocampal neurons grown on coverslips at 21 DIV, were washed twice with ice cold PBS followed by incubation with CSK buffer (10 mM Pipes pH 6.8, 100 mM NaCl, 30 mM sucrose, 3 mM MgCl 2 , 1 mM EGTA, 0.5% (v/v) Triton X-100), to remove cytosolic soluble proteins. After 10 min at 4°C, the chromatin was eliminated by incubating with DNase I digestion buffer (CSK buffer containing 500 U/ml of RNase free DNase I) for 2h at 37°C. Next, to remove the remaining DNA and soluble nuclear proteins, the preparations were incubated with extraction buffer (CSK buffer containing 650 mM [NH 4 ] 2 SO 4 ) for 20 min at 4°C. Samples were washed with the CSK buffer and then, fixed with CSK and 4% (w/v) paraformaldehyde for 40 min in CSK. No staining of nucleoskeleton with DAPI was observed, demonstrating the effective removal of DNA by this procedure. Fixed nucleoskeleton was blocked for 20 min with 1.0% (w/v) IgG-free albumin (1331-A, Research Organics, Cleveland, OH, USA) in PBS, and incubated overnight at 4°C with primary Ab, diluted in PBS (see Table 1). Following incubation with the primary Ab, the samples were washed three times with PBS. Secondary Ab Alexa Fluor 488 donkey anti-mouse, Alexa Fluor 594 donkey anti-rabbit and Alexa Fluor 647 donkey anti-goat or Alexa Fluor 594 donkey anti-goat (Invitrogen, Carlsbad, CA, USA) were used; Secondary Abs were diluted 1:500 in PBS. Immunofluorescence was evaluated using a confocal inverted microscope (TCP-SP8, Leica, Heidelberg, Germany) and analyzed with the LAS AF Confocal software of Leica version 4.0. Immunofluorescence experiments were performed at least three times from three different experiments. Nonspecific labeling was assessed by exclusion of the primary Ab.

Data analysis
Statistical analyze was performed using the Graph Pad Prism software, version 5.0. Results are expressed as mean ± standard error of the mean (SEM). Differences between means from two groups were evaluated by the Student's t-test. One way analysis of variance (ANOVA) was used for multiple comparisons among groups. Bonferroni post hoc test was performed. ‫٭‬P<0.05 and ‫٭‬ ‫٭‬ P<0.01 were considered to be statistically significant.

Nuclear expression of Dp71 isoforms during the temporal course of cultured hippocampal neurons
To assess if the subcellular distribution of Dp71 isoforms (Dp71s) change during the neuronal differentiation processes, we studied the temporal course of cultured hippocampal neurons, and evaluate the expression of both Dp71s in neuronal nuclei. By immunofluorescence assays with specific antibodies (Abs; see Table 1), we found that Dp71s were expressed in nuclei from neurons cultured for 0 (1h), 2, 4, 10, 15, and 21 DIV, but its level of expression varied along the time of culture (Fig 1). We detected a weak expression of nuclear Dp71s in fresh isolated primary hippocampal neurons seeded for only 1h (0 DIV; Fig 1C). After this time, we observed that the nuclear expression of both Dp71s increased over time of culture. It was found that the highest level of nuclear expression of both Dp71 proteins was reached at 10 DIV (Fig 1C). This level of expression of nuclear Dp71d was maintained through the 15 DIV, but at 21 DIV, a decrease of this isoform was observed; while the level of nuclear expression of Dp71f drops at 15 and 21 DIV, to a similar level reached at 2 DIV (Fig 1C).
To analyze the nuclear distribution of Dp71s in primary cultures of hippocampal neurons, we identified the GABAergic or Glutamatergic phenotype of these cultured neurons by double immunostaining of Dp71s, with anti-glutamic acid decarboxylase 67 (GAD67, a marker of GABA neurons) or anti-glutamatergic receptor 1 (GLUR1, a marker of glutamate neurons) Abs, respectively (Fig 1A and 1B), and the Abs for the Dp71s (see methods). Dp71d was mainly expressed in bipolar GABAergic neurons (60%), whereas Dp71f was found in multipolar glutamatergic neurons (40%; Fig 2D). Additionally, both Dp71s were localized into the nucleus, as tiny spots (nuclear speckles-like granules) frequently present (Fig 1A and 1B). This nuclear  speckles-like immunostaining was more evident in neurons cultured for 21 DIV; and the nuclear immunofluorescence of Dp71d was more intense than Dp71f (Fig 2). Collectively, these results show that Dp71d or Dp71f were localized in the nuclei of bipolar GABAergic or multipolar glutamatergic hippocampal neurons, respectively. Moreover, Dp71s were localized in nuclear speckle-like granules; and the Dp71d nuclear immunostaining was more intense than Dp71f in these primary cultures of hippocampal neurons at 21 DIV (Fig 2C). Furthermore we measured the colocalization of Dp71s with DAPI in the nucleus of primary hippocampal neuronal cells, to verify its nuclear localization (S1 Fig).
These results suggest that the presence of Dp71s in the nucleus of hippocampal neurons might be associated to the process of differentiation and the acquisition of morphological and physiological characteristics of the GABAergic or Glutamatergic cells. However, further investigation is required to explain this phenomenon.

Subnuclear distribution of Dp71s and DAP in hippocampal neurons
To gain more insight into the intranuclear distribution of Dp71s and DAP, we purified nuclei and obtained the nucleoskeleton and chromatin fractions from hippocampal neuron cultures at 21 DIV. The purity of each fraction was validated by Western blot (WB), and specific protein markers were detected: Calnexin for endoplasmic reticulum, Emerin for inner nuclear membrane, Lamins A/C (Lam A/C), Matrin-3 and hnRNP C1/C2 for nucleoskeleton, as well as Histone H4 for chromatin (Fig 3A). The subnuclear localization of each protein marker was observed in its respective fraction: nuclear, nucleoskeleton and chromatin, compared with the total cell extract (Total).
To verify the nuclear expression of other dystrophin isoforms (Dp427) in hippocampal neurons cultured for 21 DIV, we compared total cell and nuclear extracts by WB, using Mandra-1 Ab (which detects Dp427 and both Dp71s). Dp71d and Dp71f were the only DMD gene products, expressed in nuclear extracts, as compared with the total extract (S2A Fig). Dp71d expression was more robust than Dp71f in the nuclear fraction (S2B Fig), and these data are consistent with the intensity of the immunofluorescence for each dystrophin shown in the Fig 2C. These data showed that the Dp71s and DAP are co-expressed in nucleoskeleton fractions from primary hippocampal neurons cultured from 1h to 21 DIV.

Dp71 isoforms and DAP integrate specific nuclear complexes in hippocampal neurons
To further analyze, if Dp71d or Dp71f interact with DAP forming the respective complexes in the nucleoskeleton fraction; we carried out immunoprecipitation assays (IP). Each Dp71 precipitated was analyzed by WB, for the presence of Dp71d or Dp71f coupled to β-DG, α1-DB, β-DB, α2-DB, α-SYN and nNOS (Fig 4). We found that either Dp71s were associated to these proteins, forming the respective complexes (Dp71d-DAPC or Dp71f-DAPC). Additionally, we Nucleoskeleton fractions were immunoprecipitated (IP) with either Dp71d or Dp71f (Dys-2 or 5F3 Ab, respectively). Samples of the input nucleoskeleton fraction (Input), unbound and bound (IP-Dp71d or IP-Dp71f) proteins were analyzed by WB. The Dp71s, β-DG, α1-DB, β-DB, α2-DB, α-SYN and nNOS were detected as indicated in the right side. The immunoprecipitate from the nucleoskeleton fraction with Dp71d, contain a similar pattern of proteins than Dp71f. The only difference observed among Dp71s-DAP complexes was the pattern of associated dystrobrevins: Dp71d was preferentially bound to β-DB and Dp71f to α1-DB, α2-DB. As control for non-specific interactions, immunoprecipitation with CD4 (see Table 1), a non-related protein Ab was performed (IP-Unrelated Ab). found that the pattern of associated dystrobrevins to each Dp71s-DAPC was different: Dp71d was preferentially bound to β-DB and Dp71f to α1-DB, α2-DB (Fig 4). These results are consistent with the nuclear distribution of both Dp71s and DAP in the nucleoskeleton fraction (Fig 3B). Together, the immunofluorescence assays and IP data confirm that the Dp71s and DAP integrate two different complexes in the nucleoskeleton fraction, from bipolar GABAergic (expressing nuclear Dp71d) and multipolar Glutamatergic (expressing nuclear Dp71f) hippocampal neurons, respectively.
Dp71s-DAP complexes localized in nuclear speckles are associated to nucleoskeleton preparations of hippocampal neurons In eukaryotic cells, using immunofluorescence assays, the pre-messenger RNA splicing factors, small nuclear ribonucleoprotein and arginine/serine proteins have been localized in granule nuclear structures, termed SC35 domains, splicing-factor compartments or nuclear speckles [31][32][33]. Dp71d and Dp71f were observed, as tiny spots, into the nucleus, during the temporal course of the neuronal culture. This nuclear granule immunostaining was more evident in neurons cultured at 21 DIV. Accordingly, we analyzed the colocalization of Dp71s with DAP in these nuclear structures, by immunofluorescence and confocal microscopy assays. As shown in Fig 5, we found that the colocalization of Dp71d or Dp71f with SC35 (a nuclear speckle marker), α-DG, β-DG, α1-, α2-DB, β-DB and α-SYN was clearly observed in nuclear speckles of bipolar and multipolar neurons, respectively (Fig 5A and 5B), except for nNOS that was distributed in a more diffused way (data not shown).
Previously, it has been described that the nuclear speckles are tightly associated to the nucleoskeleton [34,35], also known as nuclear scaffold or nuclear matrix [36,37]. To further demonstrate if Dp71d or Dp71f nuclear colocalization with DAP detected in nuclear speckles, is associated to the nucleoskeleton, we obtained nucleoskeleton preparations from these neuronal cultures (Fig 6). Again, both Dp71s colocalized with SC35, α-DG, β-DG, α1-, α2-DB, β-DB and α-SYN, in nucleoskeleton preparations of bipolar and multipolar hippocampal neurons, respectively (Fig 6A and 6B). Furthermore, we measured the colocalization rate of Dp71 isoforms with DAP, and found that it was more evident in nucleoskeleton preparations, as compared to intact cells (Figs 6C and 5C, respectively). It is important to note, that Dp71d was better colocalized with β-DB and Dp71f with α1-DB, α2-DB in the nucleoskeleton preparations ( Fig 6C); and these data are consistent with the IP assays (Fig 4).
These results demonstrated that the Dp71d-DAPC and Dp71f-DAPC were localized in nuclear speckles, and associated to nucleoskeleton, from bipolar GABAergic and multipolar Glutamatergic hippocampal neurons, respectively.

Heat shock treatment effects the distribution of Dp71s in nuclear speckles
To further corroborate whether Dp71s staining patters indeed are localized to nuclear speckles, we studied the pattern distribution of Dp71s under heat shock stress conditions, at 45°C for 15 min, to characterize if this condition, differentially alter the distribution of Dp71s as has been shown for SC35 protein [38]. The nuclear granules stained with anti-Dp71d/f (Mandra-1) or anti-SC35 Ab were observed as grains or pellets in control neurons (no heat shock treated, Fig  7A and 7B), while in heat shock treated neurons (H-shock), the granules were diffusely stained with both Ab (Fig 7C and 7D, respectively).
Taken together, these findings demonstrate that nuclear Dp71s are mainly localized in nuclear speckles from hippocampal neurons.

Discussion
We have previously reported the intranuclear localization of Dp71d and Dp71f, in granular structures of cultured neurons from hippocampus. Both isoforms were colocalized with neurofilaments and with protein kinase C β II (PKCβII) in nuclear speckles-like structures [28]. It has been described the presence of Dp71d-DAP complexes in the nuclei of non-neuronal cells In non-treated neurons (Control), Dp71d, Dp71f and SC35 were clearly localized in nuclear speckles structures or nuclear rod structures (white arrowheads, Z-stacks). In contrast, when neurons were heat shocked at 45°C for 15 min (H-shock), the localization of Dp71d, Dp71f and SC35 were dispersed, and speckles appear to be poorly stained or empty (panel C and D, respectively; Z-stacks). Scale bar = 10 μm.
doi:10.1371/journal.pone.0137328.g007 [39][40][41], however, Dp71d-DAP and Dp71f-DAP complexes have not been detected in the nucleoskeleton of primary cultures of hippocampal neurons. Herein, we demonstrated that Dp71d or Dp71f and DAP were expressed in the nucleus of primary hippocampal neurons cultured from 1h to 21 DIV, where Dp71d was more abundant in the nuclei of bipolar cells while Dp71f was more abundant in the nuclei of multipolar cells (Figs 1, 2 and 5). It is known that bipolar cells are mainly GABAergic neurons, and multipolar cells are predominantly Glutamatergic neurons, and has been suggested that nuclear Dp71s-DAPC may be linked to the regulation of the neurotransmitter function, at their respective synapses [17]. These groups of proteins integrate specific Dp71s-DAP complexes in nucleoskeleton from the cultured hippocampal neurons (Fig 4). Nuclear expression of both Dp71s varied along the temporal course of hippocampal neuronal culture; but, the highest level of nuclear expression of both Dp71s was at 10 DIV. After this time, the level of expression of nuclear Dp71d was maintained through the 15 DIV, but at 21 DIV, a decrease of this isoform was observed; while the level of nuclear expression of Dp71f drops at 15 and 21 DIV, to a similar level reached at 2 DIV (Fig 1C). Therefore, Dp71s may participate in the neuronal differentiation process, during the time course of the culture of hippocampal neuronal cells, as has been suggested for Dp71d in N1E-115 and PC12 cell lines [42,43]. Here we report for the first time the nuclear localization of both Dp71s isoforms d and f in primary hippocampal neuronal cells, since it was only been described the nuclear localization of Dp71d in neuroblastome cell lines, but not Dp71f [42,43]. Therefore we were able to characterize the nuclear localization pattern of both Dp71s in different neuronal populations: Dp71d was mainly localized to the nucleus of GABAergic neuronal cells, while Dp71f was mainly localized to the nucleus of Glutamatergic neurons (Fig 1).
Of note, is the fact that the protein complexes, Dp71d-DAPC and Dp71f-DAPC, colocalized in nuclear speckles of primary hippocampal cells (Figs 5 and 7). Moreover, the localization of Dp71s-DAPC, in nuclear speckles suggests, its participation in the regulation of alternative splicing and the co-activation of transcription [44], as it has been proposed for other nuclear proteins containing WW domains [45,46].
Nuclear localization signal in Dp71 has been characterized in the ZZ domain in non-neuronal cells [47]. The specific nuclear localization of the each Dp71-DAP complex, observed here in GABAergic or Glutamatergic hippocampal neurons, appears to be related to specific synaptic functions of each type of neuron. It has been shown that highly mobile protein nuclear factors (HMG) are ubiquitous non-histone proteins that bind to DNA and regulate the expression of neuron-specific genes [48]. It was also demonstrated that β-DB interacts with some nuclear factors (iBRAF/HMG20a and BRAF35/HMG20b) [49]. iBRAF and BRAF35 have opposite roles: iBRAF activates REST-responsive genes through the modulation of histone methylation and BRAF35 mediates the repression of neuron-specific genes through remodeling chromatin structure [50,51]. Accordingly, Dp71-DAP complexes may be regulating the co-activation or co-repression of neural specific genes [49], through the specific dystrobrevin isoform associated to each complex. In addition, β-DB binds to and represses the promoter of synapsin I, a marker for neuronal differentiation [49]. Synapsin I, is a neuronal phosphoprotein implicated in synaptogenesis and the modulation of neurotransmitter release [52,53].
The main structural nuclear protein components are lamins A/C and actin, which are linked to some cytoskeleton proteins [54][55][56]. The findings reported here, indicate that Dp71s are bound to lamins A/C and actin (S3 Fig), therefore to the nucleoskeleton, thus Dp71s, may regulate the interactions cytoskeleton/nucleoskeleton [57]. It is possible that nuclear Dp71 isoforms may auto-modulate their function at the plasma and synaptic membranes [58][59][60]. As structural proteins, Dp71s may also participate in maintaining the nuclear structure and spatial compartmentalization required for specific nuclear functions. It has been shown that Dp71d is necessary to maintain the integrity of the nuclear architecture in C2C12 cell line [47]. Hence, nuclear alterations that affect their localization or the signaling of these protein complexes, may impact the function and viability of neurons.
Our data show that nNOS colocalized with Dp71d and Dp71f, showing a non-granular pattern, suggesting a different nuclear localization. nNOS is recruited to the nucleus, through its association with α-SYN, where it interacts with the Sp1 nuclear factor, and decreases the superoxide dismutase expression [61,62]. Therefore, nNOS could participate in other signaling processes in the neuronal nucleus, such as the protection of neuronal cells, against oxidative stress [63].
β-DG is a key protein with versatile functions [64], that participates in cell signaling during cell polarity or cancer progression [65,66]. Its signaling functions are associated to the presence of over 40 protein interactive domains, and 19 functional motifs, present at the β-DG Cterminal region rich in proline residues [67]. β-DG is an HMG protein and is associated to emerin, lamins A/C and B1, SC35, p80-coilin, and Nopp140 proteins in nucleus of HeLa cells and C2C12 cells, respectively [68,69], suggesting that it may have a dynamic regulatory function. Thus, it is not surprising that we found that β-DG is associated to Dp71s-DAPC in the nucleoskeleton fraction by co-immunoprecipitation studies of primary cultures of hippocampal neuronal cells.
In prostate cancer, β-DG is tyrosine phosphorylated and translocated to the nucleus, where changes the gene transcriptional activity during the progression of cancer [70]. Consequently, β-DG is a multifunctional protein that participates in signaling from the plasma membrane to the cytoskeleton, that may also participates in the signaling processes from cytoskeleton/ nucleoskeleton [71]. Thus, based on the data presented herein, we suggest that β-DG might participate in the transcription and regulation of genes involved in nuclear signaling processes and neuronal differentiation. The role of β-DG in the regulation of Dp71s complexes with DAP in neurons, remains to be elucidated.
Taken together our results, we propose a hypothetical model (Fig 8), in which, either Dp71 isoform could enter the nucleus throw the nuclear pore, and locate to the nuclear speckles, where it could form complexes with DAP, and interact with SC35, possibly modulating transcription and splicing (Fig 8A). Additionally we proposed that the differential nuclear localization of the Dp71 isoforms between the two principal neuronal phenotypes could be related to its role in neuronal differentiation (Fig 8B). In this model we show the progression of neuronal differentiation throw the days of culture from 1h to 21 days, the nuclear localization of the Dp71 isoforms, and the expression phenotypic markers of GABAergic or Glutamatergic neurons. Thus Dp71d is primarily expressed in the nucleus of GABAergic neuronal cells, while Dp71f is preferentially expressed in the nucleus of Glutamatergic neurons. Mouse lacking full length Dystrophin Dp427, or the short C-terminal isoforms Dp71s, show altered presynaptic ultrastructure in excitatory (Glutamatergic) hippocampal synapses, which may contribute to the intellectual disability characteristic of the X-linked DMD [59], and supports our observation about the possible contribution of Dp71s expression in the acquisition of the neurotransmitter phenotype (GABAergic vs Glutamatergic) during hippocampal neuronal differentiation.
In conclusion, in this study we demonstrate that: (i) Dp71d, Dp71f and DAP are localized in the nuclei of hippocampal neurons cultured at 21 DIV, (ii) Dp71d and Dp71f form complexes with DAP in neuronal nucleoskeleton, (iii) Dp71d-DAPC and Dp71f-DAPC are localized in speckles of nucleoskeleton from cultured hippocampal neurons (see hypothetical model in Fig  8), (iv) the complexes of Dp71d-DAPC and Dp71f-DAPC were detected in nuclear speckles of different neuronal phenotypes: GABAergic and Glutamatergic, respectively. The critical question about the functions of these sophisticated nuclear multi-protein complexes need to be Here we show the progression of neuronal differentiation throw the days of culture from 1h to 21 days, the nuclear localization of the Dp71 understood in the total cellular context, therefore this work opens an interesting avenue concerning the function of these multi-protein complexes. Nucleoskeleton fractions were IP with Mandra-1 Ab (that detects both Dp71s), and shows that Lamins A/C (Lam A/C) and Actin interact with both Dp71 isoforms. As control for non-specific interactions, immunoprecipitation with CD4 (see Table 1), a non-related protein Ab was performed (IP-Unrelated Ab). (TIF)