The nucleolar δ isoform of adapter protein SH2B1 enhances morphological complexity and function of cultured neurons

Abstract

The adapter protein SH2B1 is recruited to neurotrophin receptors, including TrkB (also known as NTRK2), the receptor for brain-derived neurotrophic factor (BDNF). Herein, we demonstrate that the four alternatively spliced isoforms of SH2B1 (SH2B1α-SH2B1δ) are important determinants of neuronal architecture and neurotrophin-induced gene expression. Primary hippocampal neurons from Sh2b1-/- [knockout (KO)] mice exhibit decreased neurite complexity and length, and BDNF-induced expression of the synapse-related immediate early genes Egr1 and Arc. Reintroduction of each SH2B1 isoform into KO neurons increases neurite complexity; the brain-specific δ isoform also increases total neurite length. Human obesity-associated variants, when expressed in SH2B1δ, alter neurite complexity, suggesting that a decrease or increase in neurite branching may have deleterious effects that contribute to the severe childhood obesity and neurobehavioral abnormalities associated with these variants. Surprisingly, in contrast to SH2B1α, SH2B1β and SH2B1γ, which localize primarily in the cytoplasm and plasma membrane, SH2B1δ resides primarily in nucleoli. Some SH2B1δ is also present in the plasma membrane and nucleus. Nucleolar localization, driven by two highly basic regions unique to SH2B1δ, is required for SH2B1δ to maximally increase neurite complexity and BDNF-induced expression of Egr1, Arc and FosL1.

Keywords: Brain-derived neurotrophic factor; Hippocampal neurons; Immediate early genes; Obesity; Protein isoforms; Signal-transducing adapter proteins.

Fig. 1.

Sh2b1 KO neurons exhibit decreased neurite complexity and length. (A) Schematic of SH2B1 isoforms modified from Cote et al. (2021). Different colored C-terminal tails denote isoform-specific amino acids. Numbers indicate amino acids in mouse and human sequences. P, proline-rich region; DD, dimerization domain; NLS, nuclear localization sequence; NES, nuclear export sequence; PH, pleckstrin homology domain; SH2, Src homology 2 domain. (B,C) mRNA levels of (B) total Sh2b1 or (C) Sh2b1 isoforms were measured in WT hippocampal neurons by qPCR. The number (n) of biological replicates was: DIV2,5,8, n=2; DIV14, n=3. (D) Representative WT and KO hippocampal neurons transiently expressing GFP (DIV4) and imaged (DIV5) using live-cell fluorescence microscopy. Images inverted. Scale bars: 20 µm. (E–G) Parameters measured using Simple Neurite Tracer on neuron images prepared as in D. The number (n) of neurons from seven distinct experiments was: WT, n=92; KO, n=89. (H–J) Parameters were obtained via Sholl analysis of images used in E–G. H inset, subset of data in H. (K) Proteins in hippocampal neuron lysates were immunoblotted with antibodies to PSD-95 (αPSD-95) or ERK1/2 (αERK1/2). Migration of molecular mass standards is on the right. PSD-95 signals were normalized to ERK1/2 signals and then to WT values. Data are means±s.e.m. Statistics: B,C,E–G,I,J, one-tailed unpaired t-test compared to DIV2 (B,C) and WT (E–G,I,J); H, inset, two-way repeated measures ANOVA with Dunnett's multiple comparisons test, thick line indicates significance; K, one-tailed paired t-test. *P<0.05.

Fig. 2.

SH2B1 isoforms increase neurite complexity and/or length. (A–C) Parameters measured using Simple Neurite Tracer on images of Sh2b1 KO hippocampal neurons transiently expressing GFP (−) or GFP–SH2B1 isoforms. Neurons were transfected (DIV4) and imaged (DIV5) using fluorescence microscopy. The number (n) of neurons from three distinct experiments was: GFP, n=44; GFP–SH2B1α, n=45; GFP–SH2B1β, n=48; GFP–SH2B1γ, n=46; GFP–SH2B1δ, n=44. (D–F) Parameters obtained from Sholl analysis of images used in A–C. D inset, subset of data in D. Data are means±s.e.m. Statistics: A–C,E,F, one-tailed unpaired t-test, WT versus KO neurons expressing GFP (^#P<0.05); one-way ANOVA with Dunnett's multiple comparisons test, KO neurons expressing GFP versus each GFP–SH2B1 isoform (*P<0.05); D, inset, two-way repeated measures ANOVA with Dunnett's multiple comparisons test, KO neurons expressing GFP versus each GFP–SH2B1 isoform, thick lines indicate significance (*P<0.05).

Fig. 3.

The ability of SH2B1δ to increase neurite complexity is altered by some human obesity-associated SH2B1 variants. (A) Schematic of SH2B1δ showing human obesity-associated variants. (B–D) Parameters measured using Simple Neurite Tracer on images of Sh2b1 KO hippocampal neurons transiently expressing GFP (−) or the indicated GFP–SH2B1δ construct. Neurons were transfected (DIV4) and imaged (DIV5) using fluorescence microscopy. The number (n) of neurons from three distinct experiments was: GFP, n=34; GFP–SH2B1δ(WT), n=45; GFP–SH2B1δ(P90H), n=38; GFP–SH2B1δ(P322S), n=39; GFP–SH2B1δ(A484T), n=38; GFP–SH2B1δ(T546A), n=43; GFP–SH2B1δ(R680C), n=39. (E) Sholl analysis of images used in B-D. Inset: subset of data in E. Data are means±s.e.m. Statistics: B–D, one-tailed unpaired t-test, GFP versus GFP–SH2B1δ(WT) (^#P<0.05); one-way ANOVA with Dunnett's multiple comparisons test, GFP–SH2B1δ(WT) versus each GFP–SH2B1δ variant (no significant differences detected); E, inset, two-way repeated measures ANOVA with Dunnett's multiple comparisons test, GFP–SH2B1δ(WT) versus each GFP–SH2B1δ variant, thick lines indicate significance (*P<0.05).

Fig. 4.

SH2B1δ localizes to nucleoli and the plasma membrane. (A) PC12 cells transiently co-expressing GFP–SH2B1 isoforms and mCherry–nucleolin were stained with Alexa Fluor 488–conjugated wheat germ agglutinin (WGA) and imaged using live-cell confocal microscopy. Left five panels: low photomultiplier gain. Right two panels: high photomultiplier gain. Images representative of ≥20 cells/isoform. DIC, differential interference contrast. (B) Sh2b1 KO hippocampal neurons were transiently transfected with the indicated GFP–SH2B1 isoforms (DIV5) and imaged (DIV6) using fluorescence (top panels) or confocal microscopy of fixed neurons stained with antibody to GFP (αGFP) (middle panels) or live-cell confocal microscopy (bottom panels). Images inverted in top and middle panels. Arrowheads (middle panels) denote microstructures protruding from dendritic shafts. Scale bars: 10 µm.

Fig. 5.

SH2B1δ promotes NGF-stimulated signaling activities. (A) Serum-starved PC12 cells stably expressing GFP, GFP–SH2B1β or GFP–SH2B1δ were stimulated with 25 ng/ml NGF as indicated. Proteins in cell lysates were immunoblotted (IB) with the indicated antibodies. Migration of molecular mass standards is on the left. (B–D) Quantification of immunoblots in A plus two additional sets of immunoblots, each performed using a distinct biological sample. Relative signals of phosphorylated proteins were normalized first to the signal for the total amount of that protein and then to the signal for GFP-expressing cells stimulated with NGF for 10 min. Data are means±s.e.m. Statistics: B–D, one-way ANOVA at 10 and 60 min with Tukey's multiple comparisons test to compare GFP versus GFP–SH2B1β (^#P<0.05) and GFP versus GFP–SH2B1δ (*P<0.05).

Fig. 6.

Bipartite NLS in C-terminal tail is required for SH2B1δ to localize in nucleoli. (A) Schematic of SH2B1δ with mutations and truncation. Magenta font and/or underline indicates mutation or removal of amino acids. mNLS, mutated NLS. (B) PC12 cells transiently co-expressing the indicated GFP–SH2B1δ mutant and mCherry–nucleolin were stained with Alexa Fluor 467-conjugated WGA and imaged using live-cell confocal microscopy. Left five panels: low photomultiplier gain. Right two panels: high photomultiplier gain. DIC, differential interference contrast. Scale bars: 10 µm. (C) Subcellular localization of the indicated SH2B1δ mutants. Bright, dim and absent refer to the signal intensity in indicated cellular compartment [bright, seen with low gain; dim, seen only with high gain; absent, not seen even with high gain]. The number (n) of cells from two or three distinct experiments were: SH2B1δ(WT), n=28; δ(mNLS2), n=24; δ(mNLS3), n=19; δ(mNLS2+3), n=25; δ(W712X), n=20; δ(mNLS1), n=25. PM, plasma membrane.

Fig. 7.

SH2B1δ must localize to nucleoli and the plasma membrane to maximally increase neurite complexity. (A) Schematic of SH2B1δ showing mutations. (B) Sh2b1 KO hippocampal neurons were transiently transfected (DIV5) with cDNA encoding the indicated GFP–SH2B1δ constructs and imaged (DIV6) using live-cell confocal microscopy. Left three panels: low photomultiplier gain. Right panel: high photomultiplier gain. Scale bars: 10 µm. (C–E) Parameters measured using Simple Neurite Tracer on images of KO neurons transiently expressing the indicated GFP–SH2B1δ construct. Neurons were transfected (DIV4) and imaged (DIV5) using fluorescence microscopy. The number (n) of neurons from three distinct experiments was: GFP, n=49; GFP–SH2B1δ(WT), n=58; GFP–SH2B1δ(mNLS2+3), n=41; GFP–SH2B1δ(W712X), n=28; GFP–SH2B1δ(mNLS1), n=27; GFP–SH2B1δ(R555E), n=34. (F) Sholl analysis of neuron images used in C–E. Inset: subset of data in F. Data are means±s.e.m. Statistics: C–E, one-tailed unpaired t-test, GFP versus GFP–SH2B1δ(WT) (^#P<0.05); one-way ANOVA with Dunnett's multiple comparisons test, GFP–SH2B1δ(WT) versus each GFP–SH2B1δ mutant (*P<0.05); F, inset, two-way repeated measures ANOVA with Dunnett's multiple comparisons test, GFP–SH2B1δ(WT) versus each GFP–SH2B1δ mutant, thick lines indicate significance (*P<0.05).

Fig. 8.

SH2B1δ enhances neurotrophin-induced expression of neuronal immediate early genes; enhancement is partially dependent on nucleolar SH2B1δ. (A–D) PC12 cells stably expressing GFP (−), GFP–SH2B1β or GFP–SH2B1δ were treated with or without NGF (100 ng/ml, 6 h). Relative mRNA levels of (A) Plaur, (B) Mmp3, (C) Mmp10 and (D) FosL1 were determined using qPCR. Data were normalized to NGF-treated GFP–SH2B1β values. n=3 biological replicates. (E,F) WT or KO hippocampal neurons (DIV5) were treated with or without BDNF (50 ng/ml, 1 h). Relative mRNA levels of (E) Egr1 and (F) Arc were determined using qPCR. n=3 biological replicates. (G–I) WT or KO neurons (DIV8) were infected with lentivirus encoding GFP, GFP–SH2B1β or GFP–SH2B1δ and treated with or without BDNF (50 ng/ml, 2 h) (DIV14). Relative mRNA levels of (G) FosL1, (H) Egr1 and (I) Arc were determined using qPCR. n=3−5 biological replicates; one set of biological replicates of GFP–SH2B1β (with or without BDNF) was averaged from duplicate samples. Data are means±s.e.m. Statistics: A–D, one-way ANOVA (randomized block experiment) on NGF-treated samples with Holm–Sidak's multiple comparisons test (*P<0.05); one-way ANOVA (randomized block experiment) on untreated samples (no significant differences detected); E,F, one-tailed paired t-test on BDNF-treated samples (*P<0.05); one-tailed paired t-test on untreated samples (no significant differences detected); G–I, one-way ANOVA (randomized block experiment with mixed effects analysis) with Holm–Sidak's multiple comparisons test on BDNF-treated samples, GFP versus GFP–SH2B1β versus GFP–SH2B1δ (*P<0.05); one-tailed paired t-test on BDNF-treated samples, GFP–SH2B1δ versus GFP–SH2B1δ(mNLS2+3) (^#P<0.05); one-way ANOVA (randomized block experiment with mixed effects analysis) with Holm–Sidak's multiple comparisons test on untreated samples (no significant differences detected).