Fig 1.
Dendritic spine morphology of DG, BLA, and frontal cortex neurons is affected by the loss of Bbs4 protein.
(A) Representative images of Golgi-impregnated DG, BLA, and Layer V pyramidal neurons of P42 Bbs4−/− and Bbs4+/+ mice (100x; scale bar, 5 μm). (B-D) Spine density of DG neurons. (B) Total spine density. (C) Spine density per branch order. (D) Spine density per 30-μm interval. (E-I) Spine density of layer V pyramidal neurons. (E) Total spine density. (F) Spine density in apical dendrites per branch order. (G) Spine density in basal dendrites per branch order. (H) Spine density in apical dendrites per 30-μm interval. (I) Spine density in basal dendrites per 30-μm interval. (J-N) Total spine density of BLA. (J) Total spine density. (K) Spine density in apical dendrites per branch order. (l) Spine density in basal dendrites per branch order. (M) Spine density in apical dendrites per 30-μm interval. (N) Spine density in basal dendrites per 30-μm interval (Nmice/WT = 5; Nmice/KO = 7, Ncells/WT = 25, Ncells /KO = 35, mean ± SD, ***P < 0.001; **P < 0.01; *P < 0.05). One-way ANOVA, Tukey post hoc test except for B, E, J, where unpaired t test was used. Underlying data are available in S1 Data. Bbs4, Bardet-Biedl syndrome 4; BLA, basolateral amygdala; DG, dentate gyrus; KO, knockout; ns, not significant; WT, wild type.
Fig 2.
Aberrant fear conditioning behaviour in Bbs4 knockout mice.
(A) Schematic presentation of the contextual and cued fear conditioning test. At Day 1, mice were placed in the fear conditioning chamber for 616.6 seconds. After 150 seconds, a 5-second tone is played, followed by a 0.5-second, 0.5-mA shock. The tone and shock are repeated two more times at 150-second intervals. At Day 2 mice were placed in exactly the same chamber for 300 seconds without tones or shocks. After 4 hours (Day 2), mice are placed in the altered context and left for 180 seconds. At 180 seconds, a 5-second tone is played, which is repeated twice at 60-second intervals. The first 150 seconds of the conditioning trial were used as a baseline for the context data. The first 180 seconds in the altered context were used as the baseline for the cue data. (B) Freezing (%) in the contextual memory test. (C) Freezing (%) in the cue memory test. (D) Distance travelled (cm) in the conditioning test, context test, and cued test (females: NWT = 11, NKO = 11; males: NWT = 13, NKO = 12; mean ± SD, ***P < 0.001; **P < 0.01; *P < 0.05). One-way ANOVA, Tukey post hoc test. #It was noted that there was a significant level of reduction of percent time freezing and distance travelled in Bbs4−/− mice when unpaired, two-tailed t test (P < 0.05) was used. Underlying data are available in S3 Data. Bbs4, Bardet-Biedl syndrome 4; KO, knockout; WT, wild type.
Fig 3.
Comparison of electrophysiological properties of hippocampal neurons between Bbs4−/− and Bbs4+/+ mice in vitro using acute hippocampal slices.
(A) Comparison of firing patterns in response to current injections during current clamp recordings from hippocampal granule cells. (B) Bar graphs summarising passive membrane properties. No significant differences were found between Bbs4−/− and Bbs4+/+ mice in input resistance (left), membrane time constant (middle), and resting membrane potential (right). (C) Firing frequency was plotted against current injection amplitudes. No significant differences were found between Bbs4−/− and Bbs4+/+ mice. (D) mEPSCs were recorded from hippocampal granule cells in Bbs4−/− and Bbs4+/+ mice (N = 6). (E) Cumulative probability plot comparing mEPSC amplitudes between Bbs4−/− and Bbs4+/+ mice. mEPSC amplitudes in granule cells of Bbs4−/− mice are significantly larger (N = 6, P < 0.05, Kolmogorov-Smirnov test). (F) Cumulative probability plot comparing inter-event intervals (IEIs) of mEPSCs between Bbs4−/− and Bbs4+/+ mice (N = 6; P = 0.27, Kolmogorov-Smirnov test). Underlying data are available in S3 Data. Bbs4, Bardet-Biedl syndrome 4; IEI, inter-event interval; KS, Kolmogorov-Smirnov; mEPSC, miniature excitatory postsynaptic current.
Fig 4.
InsulinR/IGF1-R signalling is dysregulated in the synaptosomal fraction of P7 Bbs4−/− mice.
(A) Phospho-RTK array reveals significant decrease in phosphorylation of insulin and IGF1 receptors in Bbs4−/− (N = 2, mean ± SD). (B) Pull-down analysis shows aberrant IGF-1R downstream signalling. Bbs4−/− and Bbs4+/+ enriched synaptosomal fractions were incubated with mouse anti-phosphotyrosine antibody overnight, followed by incubation with Dynabeads M-280 for 2 hours. Immunoblotting analysis of the proteins eluted from the beads was performed using anti-IGFR/InsR, anti-Akt, and anti-IRS p58 antibodies. Input: the total brain protein fraction before the incubation with anti-phosphotyrosine antibody, which indicates the total level of IGFR/InsR, Akt, and IRS p58 in Bbs4−/− and Bbs4+/+ mice. (C, D) RhoA and Rac1 G-LISA Activation Assays. Levels of activated RhoA (c) and Rac1 (d) were measured in the total brain extracts and enriched synaptosomal fraction of Bbs4−/− and Bbs4+/+ mice (N = 3, mean ± SD; unpaired t test). (E) Representative image of western blot analysis of NMDA and AMPA receptors levels in the total brain extract and enriched synaptosomal fraction of Bbs4−/− and Bbs4+/+ mice. (F) Representative western blots of autophagy markers LC3-II and p62 in the synaptosomal fractions of Bbs4−/− and Bbs4+/+ mice at P1, P7, P14, and P21 (N = 3, mean ± SD). LC3-I is a cytosolic form of LC3. LC3-II is a LC3-phosphatidylethanolamine conjugate (LC3-II), which is recruited to autophagosomal membranes. LC3-II and p62 levels were quantified by measuring western blot band intensities using the Image J programme (N = 3, mean ± SD, unpaired t test). Housekeeping genes (actin, GAPDH, etc.) could not be used as normalisation controls due to the changes in their gene expression levels in Bbs4−/− mice (our unpublished observations). (G) Measurement of oxidative phosphorylation (OXPHOS) complex activities in the whole brain homogenates of Bbs4−/− and Bbs4+/+ mice. Units: mU:U CS; raw data were normalised to citrate synthase; N = 4, mean ± SD; ns, not significant; unpaired t test. Underlying data are available in S2 Data. AMPAR, alpha-Amino-3-Hydroxy-5-Methyl-4-Isoxazole Propionic Acid receptor; NMDAR, N-methyl-D-aspartate receptor; Bbs4, Bardet-Biedl syndrome 4; CI, mitochondria complex I; CII, mitochondria complex II; CIII, mitochondria complex III; CS, citrate synthase, GAPDH, Glyceraldehyde 3-phosphate dehydrogenase; GluR, glutamate receptor; IGF-1R, insulin-like growth factor receptor; InsR, insulin receptor; LC3, microtubule-associated protein 1A/1B-light chain 3; LC3-I, cytosolic form of LC3; LC3-II, LC3-phosphatidylethanolamine conjugate recruited to autophagosomal membranes; OXPHOS, oxidative phosphorylation; RTK, tyrosine kinase receptor; SCC, succinate:cytochrome c oxidoreductase (= complex II + III combined); WB, western blot.
Fig 5.
Biochemical and immunocytochemical profiling of BBS proteins.
(A) Proteomic profile of the BBS proteins in biochemical fractions using nano-LC-MS/MS analysis. Protein levels of Bbs proteins and synaptic markers were estimated by label-free LC-MS analyses from following biochemical fractions of the rat hippocampi: cytosolic, detergent-soluble synaptosomal preparation (DSS, pre-synapse enriched), and postsynaptic density preparation (PSD). Protein levels of the presynaptic (VGLU1, SYP) and postsynaptic (NMDAR1, PSD95) protein markers are enriched in DSS and PSD preparations, respectively. (B) The protein abundances illustrated in the heat map are obtained from the total MS1 peptide intensities scaled to the mean of all the samples. Protein levels of the presynaptic (VGLU1, SYP) and postsynaptic (NMDAR1, PSD95) protein markers are enriched in DSS and PSD preparations, respectively. (C) Representative image of immunolabelling of Bbs proteins. Cultured mouse hippocampal neurons at low density were immunolabelled with Bbs4 and Bbs5 antibodies (red), phalloidin (green), and beta-III tubulin (green) after 6 days in vitro (DIV6). Scale bar, 20 μm (top panel) and 5 μm (bottom panels). Underlying data are available in S4 Data. BBS, Bardet-Biedl syndrome; DIV6, six days in vitro hippocampal culture; DSS, detergent-soluble synaptosomal; LC-MS/MS, liquid chromatography- tandem mass spectrometry; MS, mass spectrometry; NMDAR1, N-methyl-D-aspartate receptor 1; PSD, postsynaptic density; SYP, synaptophysin; TUBB3, β-tubulin III encoded by TIBB3 gene; VGLU1, vesicular glutamate transporter 1.
Fig 6.
Working model of the role of Bbs proteins in synapses.
This simplified schematic is based on (i) the data shown in this paper, (ii) the previously described role of Bbs proteins in primary cilium, and (iii) known dendritic spine homeostasis. It illustrates possible mechanisms by which Bbs proteins may impact synapse plasticity. (A) In the presence of Bbs proteins at the dendritic shafts, microtubules are anchored, facilitating receptor transport to the plasma membrane of the spines. Bbs proteins may also be involved in the transport of the receptors in and out of the spine membrane. (B) In the absence of Bbs proteins the microtubule anchoring and receptor sorting may be destabilised. The receptor transport to the spines becomes dependent on other transport mechanisms, e.g., exocytosis of vesicles in the dendritic shafts or a myosin-based transport [46]. This may result in reduction of the spine membrane receptor abundance (e.g., IGF-1R). This may trigger a number of downstream events, leading to destabilisation of actin filaments and eventually dendritic spines. AMPAR, alpha-Amino-3-Hydroxy-5-Methyl-4-Isoxazole Propionic Acid receptor; Bbs, Bardet-Biedl syndrome; EphB2, ephrin B 2; IGF-1R, insulin-like growth factor; NMDAR, N-methyl-D-aspartate receptor.