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Role of Nesprin-2 and RanBP2 in BICD2-associated brain developmental disorders [1]

['Julie Yi', 'Department Of Pathology', 'Cell Biology', 'Columbia University Medical Center', 'New York', 'United States Of America', 'Xiaoxin Zhao', 'Department Of Chemistry', 'Binghamton University', 'Binghamton']

Date: 2023-03

Bicaudal D2 (BICD2) is responsible for recruiting cytoplasmic dynein to diverse forms of subcellular cargo for their intracellular transport. Mutations in the human BICD2 gene have been found to cause an autosomal dominant form of spinal muscular atrophy (SMA-LED2), and brain developmental defects. Whether and how the latter mutations are related to roles we and others have identified for BICD2 in brain development remains little understood. BICD2 interacts with the nucleoporin RanBP2 to recruit dynein to the nuclear envelope (NE) of Radial Glial Progenitor cells (RGPs) to mediate their well-known but mysterious cell-cycle-regulated interkinetic nuclear migration (INM) behavior, and their subsequent differentiation to form cortical neurons. We more recently found that BICD2 also mediates NE dynein recruitment in migrating post-mitotic neurons, though via a different interactor, Nesprin-2. Here, we report that Nesprin-2 and RanBP2 compete for BICD2-binding in vitro. To test the physiological implications of this behavior, we examined the effects of known BICD2 mutations using in vitro biochemical and in vivo electroporation-mediated brain developmental assays. We find a clear relationship between the ability of BICD2 to bind RanBP2 vs. Nesprin-2 in controlling of nuclear migration and neuronal migration behavior. We propose that mutually exclusive RanBP2-BICD2 vs. Nesprin-2-BICD2 interactions at the NE play successive, critical roles in INM behavior in RGPs and in post-mitotic neuronal migration and errors in these processes contribute to specific human brain malformations.

Mutations in BICD2 gene have been found to cause brain developmental defects, as well as other disorders, like Spinal Muscular Atrophy with Lower Extremity Dominance 2 (SMA-LED2). Despite its impact on human disorders, the underlying cause of BICD2 associated disease conditions remains incompletely understood. During brain development, BICD2 recruits dynein to the nuclear envelope of pre- and post-mitotic neuronal cells via successive interactions with two different nuclear envelope proteins, RanBP2 and Nesprin-2. How the switch in BICD2-dynein cargo interactions is achieved and its physiological necessity have emerged as important questions for understanding normal and abnormal brain development. To answer the questions, we first performed biochemical assays to compare RanBP2 and Nesprin-2 bindings to BICD2. We find that RanBP2 and Nesprin-2 bind to distinct sites within BICD2 region, but their interactions are mutually exclusive. Second, we test the relative physiological roles of the two mechanisms by expressing disease-causing BICD2 variants in embryonic rat brains. We find that BICD2’s ability to bind RanBP2 vs. Nesprin-2 controls specific brain developmental steps, and defects in the binding to these NE proteins contribute to the specific brain deformities associated with BICD2 mutations.

As yet, only a small number of known BICD2 mutations have been implicated in brain developmental disorders, and there is yet relatively little evidence linking genotype and phenotype. In this study, we address this issue by focusing on the effect of BICD2 mutations on the interaction between BICD2 and its two NE associated interactors. We provide the first evidence identifying the Nesprin-2 binding site within BICD2, which proves to be distinct from, though partially overlapping with that for RanBP2. We also show a clear correlation between BICD2 mutations that preferentially bind Nesprin-2- vs. RanBP2- with specific developmental defects in the embryonic rat brain, providing important new guidance linking phenotype with genotype. We also sought to identify BICD2 mutations that might selectively interfere with the BICD2-RanBP2 vs. BICD2-Nesprin-2 interactions in the developing brain, to help distinguish the specific developmental contributions of altered INM vs. post-mitotic neuronal migration in the developing brain. Our findings have potentially important implications for understanding BICD2 pathophysiology and the mechanisms responsible for normal cortical development.

The physiological roles of BICD2 In the developing brain have also been assessed in a knock-out mouse model, which displayed hydrocephalus and cerebellar malformation, the latter was proposed to result from impaired secretion of Tenascin-C by the cerebellar Bergman Glial cells [ 38 ]. Also, cortical expression of the polymicrogyria-causing BICD2 R694C mutation caused delayed migration of postmitotic neurons within the cortical plate [ 39 ], but the underlying molecular defect causing the delay remains unknown. More recently, a C-terminally truncated Lissencephaly-associated form of BICD2 (K775X) was found to disrupt Nesprin-2 binding and to cause a severe defect in post-mitotic neuronal migration in mouse cerebral cortex [ 35 ]. But its effect on RanBP2 mediated function or interaction has not been studied.

During brain development, the nucleus in Radial Glial Progenitor (RGP) has long been known to oscillate away from and back to the ventricular surface of the embryonic brain as these cells multiply and begin to differentiate to form postmitotic neurons ( Fig 1C ; Left). The latter then travel to the cortical plate (CP), where they become mature neurons. We found that BICD2 plays a central role in dynein recruitment to the nuclear envelope (NE) in both RGPs and postmitotic neurons. In particular, knockdown of BICD2 in embryonic rat brain impaired both dynein-mediated apical nuclear migration in the RGP cells, as well as migration of post-mitotic neurons to the CP [ 5 ]. We found BICD2 itself to be recruited to the NE in these cells via two distinct mechanisms: BICD2 binds to the RGP NE via the nucleoporin RanBP2 [ 5 , 14 ], but to the NE of post-mitotic neurons via the LINC complex component Nesprin-2 [ 3 ] ( Fig 1C ; Right).

Our lab had identified multiple roles for cytoplasmic dynein, BICD2 and other dynein regulators and accessory factors in rat brain development. We found clear, important roles for BICD2 in neurogenesis in the radial glial progenitor cells, as well as in subsequent neuronal migration during specific developmental stages [ 3 , 5 , 14 ]. Consistent with these findings, so far, three different BICD2 mutations have been reported to cause developmental brain diseases [ 22 , 28 , 35 ].

To date, 22 independent disease-causing mutations have been reported at sites throughout the human BICD2 coding region [ 22 – 36 ], revealing a range of clinical phenotypes. Most of BICD2 mutations have been found to cause Spinal Muscular Atrophy with Lower Extremity Dominance 2 (SMA-LED2), characterized by slowly progressive muscle wasting, and weakness in the lower extremities [ 25 – 27 , 33 ]. An impaired Rab6A mediated secretion has been shown in the SMA-LED2 patient fibroblasts [ 37 ]. But, the underlying molecular mechanisms leading to the disease phenotype still remain elusive, particularly owing to an incomplete understanding of BICD2’s roles in the neuromuscular system.

BICD2 is one of four vertebrate BICD orthologues, which also include BICD1, BICDR-1, and BICDR-2. The BICD2 gene encodes a 95 kDa polypeptide with a substantial coiled-coil α-helical structure ( Fig 1A , top). The coiled-coil 1 (CC1) and coiled-coil 2 regions (CC2) interact, respectively, with cytoplasmic dynein/dynactin [ 15 ] and kinesin [ 13 ]. The coiled-coil 3 region of BICD2 (CC3) serves in cargo binding [ 4 – 7 , 9 ]. When not bound to cargo, BICD2 adopts an autoinhibited conformation involving an intramolecular interaction between CC1 and CC3 ( Fig 1B ) that masks the dynein/dynactin binding site, thus preventing dynein recruitment [ 4 , 9 , 16 – 18 ]. Autoinhibition, in turn, has been proposed to be regulated by another unusual BICD2 feature, a coiled-coil registry-shift within CC3, which involves a vertical displacement of the two α-helices against each other by ~one helical turn [ 16 , 19 ] to form another distinct coil-coil structure (see further in discussion ). Additionally, BICD2 and dynein interaction was also found to be under the control of the Cyclin-dependent kinase 1 (CDK1) and the Polo-family kinase 1 (PLK1) phosphorylation of BICD2 [ 20 ].

(A) Schematic representation of BicD2 (top), RanBP2 (middle), and Nesprin-2 (bottom). BicD2 (top): The colored boxes represent the three coiled-coil domains (CC1, CC2, and CC3) of the BICD2 protein. Interactors for each domain are noted on top. RanBP2 (Middle): A drawing of RanBP2 (modified from [ 14 ]), showing leucine-rich region (LRR), an E3 Sumo ligase domain (E3), and four Ran-binding domains (R), two of which flanking the BicD2 binding domain (BBD). “BBD” (highlighted in blue) of RanBP2 is used in the biochemical assays of this study. Nesprin-2 (bottom): A drawing of Nesprin-2 [ 3 , 21 ] with “N2G 52–56” highlighted in green. The BicD2 binding “N2G 52–56” fragment [ 3 ] is used in the biochemical assays of this study. (B) Schematic depiction of BICD2 in an autoinhibited state. (C) Drawing of the cerebral cortex during development (Left). Both the apical nuclear migration of Radial Glial Progenitors and the basal migration of post-mitotic neurons are mediated by dynein, which is recruited to the nuclear envelope via two distinct pathways: (2) RanBP2- or (1) Nesprin-2-BICD2 interactions (Right). (D) Left panel: BICD2 fragments used in the GST pull-down (Top left). A Coomassie-stained SDS-PAGE of the purified BICD2 fragments used in the GST pull-down assay is shown below. Middle panel: An SDS-PAGE of the elution fractions of the GST-pulldown assay is shown. The GST-tagged RanBP2 fragment (BICD2 binding domain, aa 2148–2240) pulls down the BICD2 fragments aa 630–800 and aa 711–800, but not the fragment aa 750–800. The GST-tagged Nesprin-2 fragment (BICD2/Dynein binding domain, for the sequence see S1A Fig ) pulls down each of the three BICD2 fragments. A pull-down assay with GST is shown as a negative control. The molar masses of standards are indicated on the left of each SDS-PAGE. Right panel: The intensities of the BICD2 gel bands from the pull-down assays with Nesprin-2 were quantified from 3 sets of experiments, a representative dataset is shown in S1B Fig . Discrete amounts of the three BICD2 fragments were analyzed on the SDS-PAGE and used as calibration standards to calculate the amounts of BICD2 bound to Nesprin-2 (μg) from the pull-down assays. Since the staining intensities are directly proportional to the molar masses of each fragment, in order to compare the number of bound BICD2 molecules, the BICD2 amounts were converted to the molar concentration divided by the molar concentration of Nesprin-2 and normalized to 1 for the longest fragment. The error was calculated as the standard deviation. Student’s t-test was performed against the BICD2 (aa 630–800) condition and the statistical significance is shown. (**** p<0.0001 and ns = not significant).

D. melanogaster BICD was initially identified as a polarity factor, which was later found to play an essential role in mRNA localization, facilitated by BICD-mediated interactions with Egalitarian [ 11 ] and Fragile X Mental retardation protein (FMRP) [ 12 ]. The mammalian homolog BICD2 was subsequently found to interact with a number of “cargo” proteins, including Rab6A [ 6 ], which links dynein [ 4 ] and kinesin to exocytic vesicles in cultured mammalian cells [ 13 ]. BICD2 ( Fig 1A , top) was more recently found to recruit dynein to the nucleoporin RanBP2 ( Fig 1A , middle) [ 7 ] and, by our lab, to the LINC complex protein Nesprin-2 at the nuclear envelope (NE) surface ( Fig 1A , bottom) [ 3 ], consistent with important roles for BICD2 in vertebrate brain development. We found that RanBP2 and Nesprin-2 play key roles in mediating nuclear migration in proliferating neuronal precursors (RGPs) [ 5 , 14 ] and migrating neurons [ 3 ], respectively.

Results

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[1] Url: https://journals.plos.org/plosgenetics/article?id=10.1371/journal.pgen.1010642

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