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

Abstract

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.

Fig 1. Nuclear localization of Dp71s during neuronal differentiation process.

The temporal course of the nuclear expression of both Dp71s was performed, in primary cultures of hippocampal neurons. Hippocampal neurons were cultured for 0 (1h), 2, 4, 10, 15, and 21 days in vitro (DIV), and were double stained for Dp71d (Dys-2 Ab, green color, panel A) or Dp71f (5F3 Ab, green color, panel B) and GAD67 or GLUR1 Ab, respectively (red color, panel A and B). Both Dp71s were expressed in nuclei of neurons cultured for 0, 2, 4, 10, 15, and 21 DIV. Quantification of fluorescence intensity in the nucleus of each Dp71 is shown in panel C. The highest level of nuclear expression of both Dp71 proteins were at 10 DIV. Values are means ± SEM, ٭٭p˂0.01. Scale bar = 10 μm.

Fig 2. Localization of Dp71s in the nucleus of bipolar and multipolar hippocampal neurons.

Cultured hippocampal neurons at 21 DIV were used for immunostaining assays. Subsequently, were fixed and immunoreacted with Dys-2 (Dp71d) or 5F3 (Dp71f) Ab and DAPI, respectively. Dp71d colocalized with DAPI into the nucleus of bipolar hippocampal neurons (green color, panel A). Whereas, Dp71f colocalized with DAPI into the nucleus of the multipolar hippocampal neurons (green color, panel B). Quantification of fluorescence intensity between cytoplasm and nucleus of each Dp71 is shown in panel C. The localization of each Dp71 at bipolar or multipolar neurons is shown in the panel D. Values are means ± SEM, ٭p˂0.05, ٭٭p˂0.01. Scale bar = 10 μm.

Fig 3. Expression of Dp71s and DAP in subnuclear fractions from hippocampal neurons.

Protein samples from cultured hippocampal neurons at 21 DIV: total extract (Total), hippocampal nuclei (Nuclear), nucleoskeleton and chromatin, were obtained and analyzed by WB. In panel A, the specific protein markers of each subnuclear fraction are enriched in the corresponding sample: Calnexin for total extract, Emerin for inner nuclear membrane, Lamin A/C (Lam A/C), Matrin-3 and hnRNP C1/C2 for nucleoskeleton, and Histone H4 for chromatin. No contamination with Calnexin (endoplasmic reticulum marker) is observed in the nuclear fractions. In these conditions, using specific Abs (Table 1), the nuclear expression of Dp71s, and α-DG, β-DG, α1-, β-, α2-DB, α-SYN, nNOS and Utrophins (Up400 and Up71) is analyzed (panel B).

Fig 4. Dp71s-DAP complexes immunoprecipitated from nucleoskeleton fraction of hippocampal neurons.

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).

Fig 5. Colocalization of Dp71s with SC35 or DAP in bipolar and multipolar hippocampal neurons.

Cultured hippocampal neurons at 21 DIV were used for immunostaining assays. Hippocampal neurons were double stained with either Dp71d (Dys-2 Ab, green color, panel A) or Dp71f (5F3 Ab, green color, panel B) and SC35, α-DG (K8), β-DG (JAF), α1-, α2-DB (D124), β-DB (β-Brevin), and α-SYN (C4) Abs, respectively (red color, panel A and B). DAPI was used to stain the nuclei, and the fucsia color indicates the colocalization of Dp71s and each DAP in nuclear speckles. The colocalization rate of each Dp71 with SC35 or DAP is shown in the panel C. Values are means ± SEM, ٭٭p˂0.01. Scale bar = 5 μm.

Fig 6. Colocalization of Dp71s with SC35 or DAP in nucleoskeleton preparations from bipolar and multipolar hippocampal neurons.

Nucleoskeleton preparations of cultured hippocampal neurons at 21 DIV were used for immunostaining assays (see Materials and Methods section). Nucleoskeleton preparations of bipolar or multipolar hippocampal neurons were double stained with either Dp71d (Dys-2 Ab, green color, panel A) or Dp71f (5F3 Ab, green color, panel B); and SC35, α-DG (K8), β-DG (JAF), α1-, α2-DB (D124), β-DB (β-Brevin), and α-SYN (C4) Ab, respectively (red color, panel A and B). The colocalization between each Dp71 and SC35 or DAP was more evident in nuclear speckles of bipolar than multipolar hippocampal neurons, except for α1-, α2-DB (panel A and B). Yellow regions indicate the colocalization of Dp71s with SC35 or with each DAP in nuclear speckles. The colocalization rate of each Dp71 with SC35 or DAP is shown in the panel C. Values are means ± SEM, ٭٭p˂0.01. Scale bar = 5 μm.

Fig 7. Localization of Dp71s in nuclear speckles of cultured hippocampal neurons.

Cultured hippocampal neurons at 21 DIV were treated or non-treated with heat shock (see Materials and Methods section). Subsequently, were fixed and immunoreacted with Mandra-1 Ab (which detects both Dp71s isoforms, panel A and C) or anti-SC35 Ab that stains nuclear splicing speckles (panel B and D), respectively. 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.

Fig 8. (A) Hypothetical model showing the nuclear localization of Dp71s-DAPC in hippocampal neurons. This model shows, the localization of Dp71d/f-DAP complexes in the nucleus of cultured hippocampal neurons at 21 DIV. Dp71d, Dp71f, and DAP (β-DG, α1-DB, β-DB, α2-DB, and α-SYN), as well as, nNOS, integrate protein complexes. Dp71d/f-DAP complexes were mainly localized in splicing speckle structures, which are anchored to the nucleoskeleton, possibly through structural proteins such as: lamins A/C and actin (see discussion for more details). (B) Representation of the neuronal differentiation and the nuclear expression pattern of the Dp71 isoforms d and f along the curse of the primary culture. Here 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.