Abstract
Bardet-Biedl syndrome (BBS), a ciliopathy, is a rare genetic condition characterised by retina degeneration, obesity, kidney failure and cognitive impairment. In spite of a progress made in general understanding of BBS aetiology, the molecular mechanism of cognitive impairment remains elusive. Here we report that loss of Bardet-Biedl syndrome proteins causes synaptic dysfunction in principal neurons providing possible explanation for cognitive impairment phenotype in BBS patients. Using synaptosomal proteomics and immunocytochemistry we demonstrate the presence of Bbs in postsynaptic density of hippocampal neurons. Loss of Bbs results in the significant reduction of dendritic spines in principal neurons of Bbs mice models. Furthermore, we demonstrate that spine deficiency correlates with events that destabilize spine architecture, such as, impaired spine membrane receptors signalling known to be involved in the maintenance of dendritic spines. Finally, we show that voluntary exercise rescues spine deficiency in the neurons. Based on our data, we propose a model in which Bbs proteins, similar to their function in primary cilia, regulate trafficking of signalling receptors into and out of the membrane of dendritic spines, thus providing the basis for synaptic plasticity.
Introduction
Dendritic spines are small protrusions that cover the dendrites of most principal neurons in the vertebrate central nervous system (CNS), where they typically serve as the postsynaptic part of excitatory synapses (1). Recent studies have revealed that alterations in dendritic spines are associated with a wide range of cognitive related conditions ranging from rare monogenic neurodevelopmental syndromes to common psychiatric diseases, including schizophrenia and bipolar disorder (2–4). Dendritic spine shape, size and number are regulated in a spatiotemporal manner that is tightly coordinated with synaptic function and plasticity (5, 6). Formation, maintenance, stability and pruning of spines is tightly coordinated by a wide range of surface receptors that, when activated by extracellular ligands, trigger diverse downstream signalling pathways. Membrane receptors such as Insulin-Like Growth Factor (IGF-1R), ephrin B (EphB), and TrkB have profound effects on neuroplasticity in CNS (7, 8). There are myriad of ways in which the activation of these receptors may mediate neuroplasticity including modulation of Akt/mTOR pathway, macroautophagy, small GTPases activity, and glutamate receptors (GluRs) membrane expression (9).
BBS proteins comprise of a group of unrelated ciliary proteins that, when mutated, cause a rare genetic disorder, Bardet-Biedl syndrome (BBS). BBS is a genetically heterogeneous, autosomal recessive disorder characterised by early-onset retinal degeneration, obesity, polydactyly, renal dysfunction, and cognitive impairment (10, 11). BBS was one of the first multi-system disorder ascribed to dysfunctional non-motile cilia, microtubule based membranous projections protruding from the cell surface of most mammalian cells, including neurons (12). It was shown that eight highly conserved BBS proteins form a coat like complex, BBSome that is responsible for the trafficking of signalling receptors into and out of cilia (13). Loss of BBS proteins affect entry and exit of signalling receptors such as somatostatin receptor type 3 (Sstr3), melanin-concentrating hormone receptor 1 (Mchr1), dopamine receptors (D1), Gpr161, and BDNF receptors (TrkB) (14, 15). One area of growing interest is the role of BBS proteins in cognition. The majority of individuals with BBS, experience developmental disabilities ranging from mild impairment or delayed emotional development to severe mental and psychiatric disorders (16). The frequency of neuro-psychiatric disorders and autism in BBS group exceeds the incidence rate of these disorders in the general population (17).
Here we show for the first time significant morphological aberrations of dendritic spines in the principal neurons of Bbs murine models which correlate with impaired contextual and cued fear conditioning test. While investigating the molecular mechanisms of dendritic spine loss in Bbs mice models, we excluded reduced synaptic activity and mitochondria dysfunction as a likely cause of spine loss. At the same time we found that spine deficiency correlates with impaired spine membrane receptors signalling known to be involved in the maintenance of dendritic spines among them IGF-1R and ephrin B receptor signalling. Moreover, our finding of BBS proteins localization to the postsynaptic densities (PSD) of hippocampal neurons prompted us to propose a model for BBS proteins function in synapses. Together, our data reveal that BBS proteins, their role so far being confined only to the functional maintenance of cilia, play an important role in synaptic structure and function and suggest that aberrant spine formation and maintenance may underlie the cognitive impairment in BBS patients.
Results
Loss of Bbs proteins affect principal neuron morphology
To investigate the effect of Bbs protein loss on the neuronal structure we analysed the dendritic morphology of principal neurons of Bbs mice models which closely mimic the human BBS phenotype. We measured dendritic length, spine count and spine density of dentate gyrus (DG), basolateral amygdala (BLA) and layer V pyramidal neurons of the frontal cortex using a Golgi-Cox impregnation method. We found that the total spine density was reduced by 55% in DG granule cells of 6 weeks old Bbs4−/− mice (Fig.1a,b and Supplementary Movie 1). Total basal and apical spine density of layer V neurons was reduced by 55% and 54%, (Fig.1a,e), respectively, and total basal and apical spine density of BLA neurons was reduced by 23% and 22%, respectively (Fig.1a,j). Sholl analysis revealed a significant reduction in spine density of all branches and per 30 μm interval in DG with the exception of the most distal branch and 300 μm circle in Bbs4−/− mice (Fig.1c,d). Similar Sholl analysis results were found in apical and basal dendrites of Layer V neurons where dendritic spine density per branch order and per 30 μm interval was affected (Fig.1f-i). Apical and basal BLA dendrites of Bbs4 knockout mice revealed unequal pattern in spine reduction affecting only few branches and concentric circles (Fig.1k-n). A number of dendritic intersections in DG, BLA and Layer V neurons were not affected when compared to control mice (Supplementary Fig.1 a-e). Total dendritic length was reduced by 48% in DG neurons and by 25% and 12% in basal and apical dendrites of layer V cortex neurons. BLA apical and basal dendrites showed a statistically significant length reduction of 14% and 19%, respectively (Supplementary Fig.1f-j). Overall, this data show significant aberrations in dendritic morphology.
In order to investigate when the dendritic architecture of Bbs4−/− DG neurons begins to change, we analysed dendritic length, spine density per branch order and per 30μm intervals at P21 mice. The results were similar to those obtained at 6 weeks of age: a significant reduction in dendritic length, spine density per branch order, and per 30μm interval (Supplementary Fig.2a, g-k). However, at E19.5, the density of filopodia in Bbs4−/− DG neurons was not affected (Supplementary Fig.2a-f). Taken together, the Bbs4−/− murine model show a progressive decrease in dendritic spine density at 3 weeks old mice (38%) and 6 weeks old mice (55%) but not during late embryonic stages (Supplementary Fig.2l).
To investigate whether similar dendritic abnormalities can be detected in the other Bbs models, we analysed DG dendrite morphology of 3 weeks old Bbs5 knockout and Bbs1 M390R knock-in models. Notably, loss of Bbs5 protein led to significant reduction in DG dendritic length (34%) and overall spine density (32%) in the dentate granule cells of knockout mice (Supplementary Fig.3a-c). Sholl analysis also revealed abnormal spine density in Bbs5−/−mice with a significant spine reduction from the 2nd to 5th branch order and from 60μm to 150μm interval, respectively (Supplementary Fig.3d-f). Interestingly, the Bbs1 M390R knock-in model was associated with consistent but moderate abnormalities in spino- and dendritogenesis of DG neurons showing only a 10% reduction in the total spine density (Supplementary Fig.3a, g-k). This finding is in agreement with our clinical observations that BBS1 M390R patients have the mildest cognitive phenotype.
Dendritic spines, especially on pyramidal cells, could be subdivided into categories based on their size and shape (Supplementary Fig.4a) (18). We next asked whether certain subtypes of spines were over-represented on DG neurons of Bbs4−/− mice. We observed that total spine count was reduced in all spine subtypes except ‘branched’ spines, however, when the reduction of dendritic length of Bbs4 neurons were taken into account, we found that only the density of ‘thin’ spines was significantly reduced (22%) (Supplementary Fig.4b-e).
Reduced contextual and cued fear memory but no impairment in anxiety-like behaviour in Bbs4−/− mice
Hippocampus, amygdala and prefrontal cortex are structures involved in learning, memory and social interaction. To investigate whether the loss of dendritic spines of DG, BLA and prefrontal cortex neurons correlates with behavioural changes of the Bbs4−/− mice, we performed analysis of data obtained from a battery of behavioural tests in 8 weeks old knockout and control mice. The contextual and cued fear conditioning tests are widely used to assess fear memory. In the conditioning session at Day 1, freezing behaviour and distance travelled during the first 150 s without introducing a conditioned stimulus (tone) and unconditioned stimulus (footshock) were used to evaluate baseline activity in the novel environment in the context fear experiment. There was no effect of Bbs4 loss on the percentage of freezing or distance travelled in baseline activity of Bbs4−/− males and female mice. However, after introduction of the paired tone-foot shock stimulus at Day 1, post-hoc analysis revealed a significant decrease in percentage of freezing and an increase in the distance travelled on Day 2 of Bbs4−/− female mice compared to female wild type mice. Male mice showed a significant decrease in percentage of freezing and an increase in distance travelled when the un-paired, two-tailed t test (p<0.05) was used (Fig.2a, b, d). Altered context at Day 2 before the introduction of the tone was used as a baseline for the cue data. There was no significant change in the percentage of freezing in altered context baseline activity (Fig.2c, d). During the cued conditioning session with the tone (Day 2, 180-360 s), the percentage of freezing was significantly reduced and distance travelled increased in Bbs4−/− males (Fig.2c, d). This set of results indicates that loss of Bbs4 protein affects contextual and cued fear memory.
Anxiety-like behaviour, locomotor activity and spatial memory were also assessed by using open field, light dark box and paddling Y-maze. No significant changes were obtained between Bbs4+/+ and Bbs4−/−mice (data not shown).
Miniature excitatory postsynaptic currents (mEPSCs) amplitude is increased in Bbs4−/− DG neurons
The morphology of dendritic spines is highly dynamic and their formation and maintenance depend on neuronal activity (19). As all existing Bbs mice models exhibit various age dependent sensory deficits such as loss of vision, obesity and thermo- and mechanosensory aberrations (11, 20) we measured intrinsic properties and synaptic function of hippocampal granule cells of 6 weeks old Bbs4−/− and Bbs4−/− mice in vitro. We found that intrinsic properties of Bbs4−/− neurons were unaffected (Fig.3a-c). To evaluate the synaptic properties of granule cells in these two groups, we measured mEPSCs. Notably, while the frequency of mEPSCs was not different between the two groups, mEPSC amplitudes were significantly larger in Bbs4−/− neurons (Fig.3d-f). These data show that there is no reduction in the brain activity in Bbs4−/− mice which makes it unlikely that reduced neuronal activity might be the cause of reduced spine density. It also suggests an activation of compensatory mechanisms at the presynaptic and/or postsynaptic sites in response to spine loss.
IGF-1R downstream signalling is dysregulated in Bbs4−/− synaptosomes
It has been established that a number of tyrosine kinase receptors (RTK), including IGF, RET, TrkB, PDGF and EphB enhance dendritic growth and promote formation and maintenance of dendritic spines (7, 21). To assess the signalling of RTK in the brain of Bbs4−/− and Bbs4+/+ mice we quantified the phosphorylation level of RTK receptors using a Phospho-RTK Array. Total brain extracts of P7 Bbs4−/− and Bbs4+/+ mice were incubated with the membrane containing immobilised RTK antibody followed by detection of RTK phosphorylation by a pan anti-phospho-tyrosine antibody. Interestingly, phosphorylation levels of a number of RTKs were altered including insulin and IGF1 receptors (Fig.4a). Annotation and the summary of phosphorylation levels of all analysed RTK are given in Supplementary figure 5. We continued this study with the focus on IGF-1R/Insulin receptor (IR) signalling as it is known to have a profound effect on neuroplasticity in the CNS (7–9). Pull down experiments confirmed that phosphorylation of IGF-IR/InsulinR was decreased in P7 enriched synaptosomal fraction of Bbs4−/− mice (Fig.4b). Additionally, phosphorylation levels of Akt, a downstream target of canonical IGF signalling, were significantly reduced (Fig.4b). Next we tested phosphorylation level of IRSp58 protein, an adaptor protein that is phosphorylated by IR and IGF-1R (22) and is highly enriched in the postsynaptic density (PSD) of glutamatergic synapses (23). We found that phosphorylation of IRSp58 was significantly reduced in synaptosomal fraction of P7 Bbs4−/− mice (Fig.4b). Furthermore, as the activities of IGF-1R and IRSp58 depend on interaction with Rho family GTPases (24, 25), we investigated the activities of Rac1 and RhoA GTPases. We observed that activity of RhoA was increased and, concurrently, Rac1 activity was decreased in the enriched synaptosomal fraction of P7 Bbs4−/− mice (Fig.4c, d). We next assessed whether dysregulation of IGF-1 signalling in Bbs−/− mice affects the levels of NMDA and AMPA receptors in the total and synaptosomal fractions of Bbs4−/− and Bbs4+/+ mice by western blotting. We observed a significant increase in the level of NMDA and AMPA receptors in synaptosomal fraction of P7 Bbs4−/− mice, whereas the changes in the receptors level in the total brain fraction were not detected (Fig.4e). These data are consistent with our previous findings of increased mEPSC amplitudes in Bbs4−/− neurons (Fig.3e).
One of the possible mechanisms of dendritic spine pruning is macroautophagy (18) process that is tightly regulated by IGF-1R signalling and small GTPases. To test whether autophagy is dysregulated in our Bbs model, we analysed the level of autophagy markers LC3-II and p62 in the enriched synaptosomal fractions isolated from Bbs4−/− and Bbs4+/+ mice brains at different postnatal stages. We observed a significant increase in LC3-II level at P1 and P7 of Bbs4−/− mice (Fig.4f). Given the widely recognised notion that the level of p62 correlates inversely with autophagy, it was unexpected to see p62 increase in P1 synaptosomes in our experiment. However, it is in line with the reports that p62 levels can be up-regulated during high autophagic flux due to a p62 protein multifunctional role (26). To exclude mitochondrial dysfunction and oxidative stress as triggers of autophagic induction (27) we assessed the activities of respiratory chain complexes I, II, III, IV and SCC (complex II and III combined) in total brain homogenates of P7 Bbs4−/− and Bbs4+/+ mice. Oxidative phosphorylation (OXPHOS) complex activities were determined and the results were normalized to the activity of citrate synthase (CS). We have found no significant differences in the activities of OXPHOS between Bbs4−/− and Bbs4+/+ mice (Fig.4g) thus ruling out mitochondrial dysfunction as a cause of autophagy in BBS.
Our finding that aberrant IGF-1 signalling leads to the dysregulation of various cellular pathways that tightly control actin re-arrangements provide a possible explanation for spine density reduction in the absence of functional Bbs proteins.
Synaptic localization of BBS proteins
To our knowledge, all previous studies confined the function of BBS proteins to structural and functional regulation of cilia. However, a reduction in dendritic spine density along with aberrant synaptic IGF receptor signalling and altered neurotransmitter receptor levels (NMDA and AMPA) suggested a potential synaptic role of Bbs proteins. Re-evaluation of our earlier mass spectrometric analyses of synaptosomal (28, 29) and crude synaptosomal fractions (30) of the rat cortex, dorsal striatum and dentate gyrus revealed the presence of Bbs1, Bbs2, Bbs4, Bbs5, Bbs7 and Bbs10 proteins in these fractions (Supplementary Table1).
To elaborate synaptic localization of BBS proteins biochemically, we enriched the cytosolic, detergent-soluble synaptosomal (DSS, pre-synapse enriched) and PSD fractions of synaptosomal preparations from the adult rat hippocampi using previously described method (31). Experimental procedure is summarised in the Supplementary Figure 6. Label-free MS1 intensity based LCMS quantitation revealed a high abundance of Bbs1, Bbs2, Bbs5, Bbs9 proteins in PSD fraction whereas Bbs7 was present mostly in the cytosolic fraction (Fig.5a, b). The low abundance Bbs4 protein was also unambiguously identified in the PSD fractions (Fig.5a,b). Immunofluorescence analysis of Bbs4 and Bbs5 protein localization confirmed the presence of Bbs punctae throughout the entire dendritic tree of mouse dissociated hippocampal neurons (Fig.5c and Supplementary Fig.7). Collectively, these data clearly indicate the presence of Bbs proteins in the neuronal processes and postsynaptic densities.
Voluntary exercise partly reverses dendritic spine loss in Bbs4−/− mice
Aerobic exercise is known to promote spinogenesis and, even though the exact mechanisms are unclear, a number of studies suggested that it might work through several signalling molecules including IGF-1 and BDNF (32, 33). In the light of our finding of aberrant IGF-1 signalling we investigated whether voluntary wheel exercise is able to increase spinogenesis in Bbs4−/− mice. We analysed the spine density of DG neurons in 6 weeks old Bbs4−/−, Bbs4−/− and Bbs4−/− mice after 2 weeks of wheel exercise. We found that remarkably only 2 weeks of wheel exercise significantly increased the total spine density of Bbs4−/− mice DG neurons (Fig.6a,b). Analysis of spine density per branch order revealed increased density of spines in the 1st, 5th and 6th branch orders. Notably, although statistically not significant, there was a trend in spine density increase in the other branch orders (Fig.6c).
Discussion
Our study reveals a previously unknown role for BBS proteins in neuronal function. We found significant morphological changes in dendritic spines and dendritic length in various brain regions of Bbs mouse models. Several studies have shown an association of neurodevelopmental and neuro-psychiatric disorders with morphological and physiological alterations in dendritic spines (2, 3) which prompted us to speculate that cognitive deficits in BBS patients are likely caused by the loss of dendritic spines. Our data provide several associative links between perturbed spine integrity and cognitive deficits in BBS: first, we show that Bbs4−/− mice display reduced contextual and cued fear memory. Secondly, loss of spines occurs in a spine sub-type-dependent manner, affecting mostly “thin” spines, which are thought to be specifically involved in learning processes, but not in memory maintenance (34). This finding is in good agreement with our clinical observation that learning difficulties in BBS patients are more prevalent than memory deficits (unpublished data). Finally, we found that the functional loss of different Bbs proteins appears to affect spines to different degree, for example, the Bbs1 M390R missense mutation has only a marginal effect on the spine loss. Together, these data support the idea that dendritic spine aberration might be an essential contributing factor to cognitive deficits in BBS.
Loss of dendritic spines may be attributed to a number of mechanisms including reduced synaptic activity, mitochondrial dysfunction etc. Our data indicate that as the intrinsic properties of Bbs4−/− neurons were unaffected and mEPSC amplitudes were significantly larger in Bbs4−/− neurons, it is it unlikely that reduced neuronal activity might be the cause of reduced spine density suggesting compensatory responses to the spine loss. Similarly, we did not obtain any mitochondrial dysfunction in Bbs4−/− brain. Strikingly, we found that several tyrosine kinase receptors signalling was affected including IGF-1R and ephrinB2 receptor. A number of studies reported the effect of IGF-1R and ephrin B2 receptor signalling on dendritic growth, formation, maintenance, and stabilization of dendritic spines (7, 35). Our present work shows that disruption in IGF-1 signalling initiates a cascade of downstream events such as increased autophagy, reduced phosphorylation of IRSp58 and aberrant activity of small GTPases, Rac1 and RhoA. All these events are known to have a diminishing effect on the spine density. Thus, our finding of aberrant cellular pathways that tightly control actin re-arrangements provide a plausible explanation for spine density reduction in the absence of functional Bbs proteins.
How ciliary Bbs proteins affect the signalling of the receptors integrated into spine membrane? Our study revealing the presence of BBS proteins in post synaptic density of the spines. Furthermore, comparison between primary cilia and dendritic spines highlights remarkable parallels between protein composition, membrane domain architecture and dynamic nature of primary cilia and dendritic spine assembly/ disassembly (36). Collectively, it is suggestive that BBS proteins might have similar function in both structures. Therefore, we propose a model in which BBS proteins are localised to the dendritic spines and stabilize microtubule polymerization (invasions) into dendritic spines thus facilitating the transport of the signalling receptors to the spine membrane (Fig.7). It would require further investigations to determine whether BBSome complex is present in the spines similar to primary cilia.
Finally, we show that that in Bbs4 mice model spine density can be increased by only two weeks of voluntarily wheel exercise. Accumulating evidences suggest that voluntary wheel running has an enhancing effect on the dendritic spine density and can even reverse spine loss in the mouse model of Parkinson’s disease (37, 38). Although the exact mechanisms of neuroplasticity enhancement during aerobic exercise remains unclear, several important signalling molecules including IGF-1 and BDNF were shown to be implicated (32, 33). In the light of this evidence, it is interesting that Bbs4 protein was found to be necessary for TrkB receptor localization to the cilia membrane and activation by BDNF(15). Considering structural similarity between primary cilia and dendritic spines it is plausible that Bbs4 proteins are involved in the localization of TrkB receptors on the spine membrane.
Even though it was not investigated in this work, our data may be pivotal in understanding already known interaction between BBS proteins and the Disrupted-in-schizophrenia 1 (DISC1) protein, the disruption of which can result in a wide a range of psychiatric problems including schizophrenia, bipolar disorder and major depression (39). As loss of spines is relevant to many brain disorders including neurodegeneration, probing the synaptic role of BBS proteins will contribute to a deeper understanding of the aetiology of these disorders.
Authors’ contributions
N.H. performed the experiments and generated the figures. C.S-H. performed the electrophysiology experiments and assisted with manuscript preparation. F.S. carried out proteomics experiments, analysed the data and prepared proteomics figures. L.C. isolated mouse hippocampal neurons and performed immunocytochemical experiments. D.M performed multiphoton microscope imaging. M.S., L.B., S.W., and P.N performed behavioural tests. R.R performed mitochondria function tests. E.F. assisted with manuscript preparation. M.C.W guided dendritic spine analysis and made a video of reconstructed neurons. P.S. guided in vitro experiments. G.L. guided proteomics experiments. M.H. guided electrophysiology experiments and assisted with manuscript preparation. P.L.B assisted with manuscript preparation. S.C-S conceived the study, performed experiments, generated the figures, and wrote the manuscript.
Competing interests
The authors declare no competing interests
Correspondence and requests for materials should be addressed to S.C-S
Methods
Mice and ethics statement
Animal maintenance, husbandry, and procedures are defined and controlled by the Animal (Scientific Procedures) Act 1986. All animal experimentations has been carried out under licences granted by the Home Secretary (PIL No. 70/7892) in compliance with Biological Services Management Group and the Biological Services Ethical Committee, UCL, London, UK. Mice were group housed in IVC cages and were kept on a 12-h light-dark cycle with ad libitum access to food and water.
Bbs1 M390R knock in model was purchased from Jackson Laboratory, Bbs4 gene trap model we received as a part of previous collaborative work (41), and targeted knockout C57BL/6NTac-Bbs5tm1b(EUCOMM)Wtsi/H strain model was received from MRC Harwell as part of the International Mouse Phenotyping Consortium. Bbs1 and Bbs4 mice were backcrossed with C57BL/6Ntac strain for 5 generations to keep the background consistent with Bbs5 mice obtained from MRC Harwell.
Statistical analysis
Analysis with two groups were performed using an unpaired, two-tailed Student’s t-test. Analysis with more than two groups and with one variable were performed using one-way analysis of variance (ANOVA) and Tukey’s post hoc tests. Kolmogorov-Smirnov test was used to determine the cumulative distribution function of a continuous random variables such as frequency and amplitudes of mEPSCs.
Data Availability
The authors confirm that the data supporting the findings of this study are available within the article and its Supplementary material. Raw data can be requested from the corresponding author: s.christou{at}ich.ucl.ac.uk.
Supplementary Table 1
Published and current proteomics data where BBS proteins were identified. LCMS-based proteomic analyses of rat synaptosomal and membrane preparations
Supplementary Video 1
Reconstructed hippocampal Bbs4−/− neurons show a significant reduction in total dendritic spines when compared to Bbs+/+ neurons. For 3D neuron reconstruction (Neurolucida, MBF), the image stacks of representative neurons per group were selected in order to align serial contoured objects, including the soma, apical and basal dendrites and associated spines. The module “3D visualization” of Neuroludica software was turned on in order to automatically generate 3D visualization of representative neurons.
Supplementary Fig.1
(a-e) Sholl analysis of DG, BLA and Layer V frontal cortex neurons of 6 weeks old Bbs4 mice. Frequency of intersections per 30 μm interval in DG (a), apical dendrites of layer V pyramidal neurons (b) and basal dendrites of layer V pyramidal neurons (c), apical dendrites of BLA (d), basal dendrites of BLA (e). (NWT=5; NKO=7, Mean ± SD, *P< 0.05). One-way ANOVA, Tukey post hoc test
(f-j) Dendritic length of DG, BLA and layer V neurons. (Biological samples: NWT=5; NKO=7; Total number of analysed cells: NWT=25; NKO=35, for DG; Biological samples: NWT=3; NKO=3; Total number of analysed cells: NWT=15; NKO=15 for BLA and LV) Mean ± SD, *P< 0.05. Unpaired t-test
Supplementary Fig.2
Defects in dendritic morphology of DG granule cells of 3 weeks old Bbs5 knockout and Bbs1 M390R knock in models
(a) Representative images of Golgi-impregnated dentate gyrus (DG) granule cells of Bbs5 and Bbs1 M390R models.
(b-f) Analysis of DG granule cells of Bbs5 mice. (b) total spine density, (c) dendritic length, (d) spine density per branch order, (e) spine density per 30 μm interval, (f) frequency of intersections per 30 μm interval
(g-k) Analysis of DG granule cells of Bbs1M390R knock-in mice. (g) total spine density, (h) dendritic length, (i) spine density per branch order, (j) spine density per 30μm interval, (k) frequency of intersections per 30 μm interval.
Biological samples: NWT=5; NKO=5; Total number of analysed cells: NWT=25; NKO=25, Mean ± SD, *P< 0.05, P***<0.01. One-way ANOVA, Tukey post hoc test. Scale bar 5 μm. # represents a significant level if un-paired, two-tailed t test is used (p<0.05).
Supplementary Fig. 3
‘Thin’ spines are abandoned on DG neurons of 6 weeks old Bbs4knockout mice
(a) Illustration of dendritic spine sub-classes. Adopted from Oliver von Bohlen und Halbach, Annals of Anatomy(18)
(b) Total count of ‘thin’, ‘stubby’, ‘mushroom’, ‘filopodia’ and ‘branched’ spines
(c) Total dendritic density of thin, stubby, mushroom, filopodia and branched spines. Density is calculated as the number of spines per micrometre of dendrite. Boxed panel shows the reduction in dendritic length.
(d) Sholl analysis of ‘thin’ and ‘mushroom’ spines (Nmice/WT=3; Nmice/KO=3, Ncells/WT=15, Ncells/KO=15, Mean ± SD, ***P<0.001; **P<0.01; *P< 0.05). One-way ANOVA, Tukey post hoc test
Supplementary Fig. 4
Dendritic spine density is reduced in DG neurons of Bbs4 knockout mice at P21 but not at E19.5
(a) Representative images of Golgi-Cox impregnated dentate granule (DG) of Bbs4 KO and WT mice at E19.5 and P21 (100x; scale bar 5 μm)
(b-f) Analysis of DG neuron morphology at E19.5. Total spine density (b), dendritic length (c), spine density per branch order (d), spine density per 30 μm interval (e), frequency of intersections per 30 μm interval (f)
(g-k) Analysis of DG neuron morphology at P21. Total spine density (h), dendritic length (i), spine density per branch order (j), spine density per 30 μm interval (k), frequency of intersections per 30 μm interval (l)
(Nmice/WT=3; Nmice/KO=3, Ncells/WT=24, Ncells /KO=24, Mean ± SD, ***P<0.001; **P<0.01; *P< 0.05). One-way ANOVA, Tukey post hoc test except for b, c, h, i where unpaired t-test was used
Supplementary Fig.5
RTK phosphorylation assay of enriched synaptosomal fractions of P7 Bbs4 knockout and control mice
(a) Representative image of Phospho-RTK array. Blue and red squares indicate the position of Ins-R and IGF-1R, respectively.
(b) Quantitative dot-blot analysis reveals significant decrease in phosphorylation of a number of RTK in P7 of Bbs4−/− mice brain(N=3, mean ± SD); unpaired t-test; ImageJ software.
Supplementary Fig.6
Experimental Workflow. Cytoplasmic and Synaptosomal fractions were isolated from the cortices of three Wistar rats. Synaptosomes were further separated into detergent soluble synaptosomal (DSS) and postsynaptic density (PSD) fractions. A FASP protocol adapted for synaptic membrane proteins is coupled to a gel-free LCMS to allow analyses of synaptosomal fractions. Database searching was performed with search engines against the rat SwissProt protein database. Quantitative information were determined using the software tools, Proteome Discoverer and Isobar.
Supplementary Fig.7
Validation of the specificity of Bbs4 and Bbs5 antibodies
(a) List of published validations of Bbs4 (12766-1-AP) and Bbs5 (14569-1-AP) ProteinTech antibodies used in this study
(b) Protein extracts from the brain of the wild-type and knockout Bbs4 and Bbs5 mice were immunoblotted with Bbs4 and Bbs5 antibodies as indicated. Approximate molecular weights are listed on the left side. Gapdh was used as the loading control. Wild type Bbs4 and Bbs5 mice showed a specific single band. Western blot with Bbs4 and Bbs5 antibodies did not detect any specific band in Bbs4 and Bbs5 knockout mice.
Acknowledgements
We thank International Mouse Phenotyping Consortium at Harwell for providing C57BL/6NTac-Bbs5tm1b(EUCOMM)Wtsi/H strain model and for assistance in experimental techniques. This study was supported by grants from Biotechnology and Biological Sciences Research Council (BBSRC) (BB/M020991/1) and New Life Foundation (14-15/20), UK.