The patellofemoral joint alignment in patients with symptomatic accessory navicular bone

Quadriceps angle (Q angle) provides useful information about the alignment of the patellofemoral joint. The aim of the present study was to assess a possible link between malalignment of the patellofemoral joint and symptomatic accessory navicular (AN) bone as an underlying cause in early adolescence using Q angle measurements. This study was performed on patients presenting to the Foot and Ankle Clinic at the Jordanian Royal Medical Services because of pain on the medial side of the foot that worsened with activities or shoe wearing, with no history of knee pain, between September 2013 and April 2015. The Q angle was measured using a goniometer in 27 early adolescents aged 10-18 years diagnosed clinically and radiologically with symptomatic AN bone, only seven patients had associated pes planus deformity; the data were compared with age appropriate normal arched feet without AN. Navicular drop test (NDT) was used to assess the amount of foot pronation. The mean Q angle value among male and female patients with symptomatic AN with/without pes planus was significantly higher than in controls with normal arched feet without AN (p<0.05). Symptomatic AN feet were also associated with higher NDT values (p<0.001). The present findings suggest an early change in patellofemoral joint alignment in patients with symptomatic AN bone with/without arch collapse. Therefore, it is recommended that Q angle assessment should be an essential component of the examination in patients with symptomatic AN bone

Cyclin-dependent kinase 5 is involved in the phosphorylation of gephyrin and clustering of GABAA receptors at inhibitory synapses of hippocampal neurons.

CDK5 has been implicated in neural functions including growth, neuronal migration, synaptic transmission and plasticity of excitatory chemical synapses. Here we report robust effects of CDK5 on phosphorylation of the postsynaptic scaffold protein gephyrin and clustering of inhibitory GABAA receptors in hippocampal neurons. shRNA-mediated knockdown of CDK5 and pharmacological inhibition of cyclin-dependent kinases reduced phosphorylated gephyrin clusters and postsynaptic γ2-containing GABAA receptors. Phosphorylation of S270 is antagonized by PP1/PP2a phosphatase and site-directed mutagenesis and in vitro phosphorylation experiments indicate that S270 is a putative CDK5 phosphorylation site of gephyrin. Our data suggest that CDK5 plays an essential role for the stability of gephyrin-dependent GABAA receptor clusters in hippocampal neurons

CDK5 knockdown correlates with reduced numbers of GABA_A receptor clusters containing γ2-subunits.

<p>Hippocampal neurons (div14) were immunolabeled with anti GFP antibodies to detect infected neurons (upper panel), with phosphospecific anti gephyrin mAb7a antibody (middle panel), and with anti-GABA_A receptors γ2-subunit of (lower panel). (A) Non-infected cells; (A') CDK5-kd2 knockdown; (A'') control shRNA (CDK5-mismatch). Neurons were infected with the indicated viruses at div6. The scale bar represents 15 µm. (B) Quantification of the number of GABA_A receptor γ2 puncta on three proximal dendritic segments of 30 µm. 20 or 18 cells from n = 3 independent cultures of control or mismatch neurons and 25 cells (69 dendrites) from n = 4 independent cultures of kd2-infected neurons, mean ± SE. ANOVA with post-hoc test, *** P<0.001. (C) Quantification of the relative fluorescence intensities of GABA_A receptor γ2 puncta. Mean ± SE. Each value was calculated from data from the same cell numbers as in B from n = 3 independent cultures. ANOVA with post-hoc test: *** P<0.001, ** P<0.01.</p

CDK5 knockdown virus infection results in reduced CDK5 expression and reduced numbers of phospho-gephyrin clusters in cultured hippocampal neurons.

<p>Hippocampal neurons (div14) were stained with anti-GFP antibodies to detect infected neurons (upper panel), with anti-CDK5 antibody to quantify CDK5 expression levels (middle panel) and with the phosphospecific anti-gephyrin mAb7a antibody (lower panel). (A) Non-infected cells; (A') CDK5-knockdown; (A'') control shRNA (CDK5-mismatch). Neurons were infected with the indicated viruses at div6. The scale bar represents 15 µm. (B) Quantification of CDK5 fluorescence intensities of neurons infected with three different CDK5 knockdown viruses (kd1, kd2 and kd3). CDK5 knockdown cells were compared to non-infected neurons. n = 3, mean ± SD (C) Quantification of mAb7a cluster numbers of hippocampal neurons infected with three different CDK5 knockdown viruses (kd1, kd2, kd3) compared to non-infected and control shRNA (mismatch). 30 cells from n = 4 independent cultures (for each of non-infected, mismatch, kd2, kd1) or 30 cells from n = 3 independent cultures (kd3), mean ± SE, ANOVA with post-hoc test, ***P<0.001.</p

Synaptic mAb7a-specific gephyrin clusters are reduced upon CDK5 knockdown whereas VIAAT puncta were not reduced in perisomatic dendritic segments.

<p>Hippocampal neurons (div14) were immunolabeled with anti-GFP antibodies to detect infected neurons (upper panel), with phosphospecific anti-gephyrin mAb7a antibody (middle panel), and with anti-VIAAT antibody (lower panel). (A) Non-infected cells; (A') CDK5-kd2 knockdown; (A'') control shRNA (CDK5-mismatch). Neurons were infected with the indicated viruses at div6. The scale bar represents 15 µm. (B) Quantification of the number of VIAAT puncta, mAb7a puncta and overlapping mAb7a/VIAAT puncta (synaptic mAb7a clusters). Mean ± SE. Each value was calculated from analysis of 15 cells from n = 3 independent cultures, ANOVA with post-hoc test: *** P< 0.001, * P<0.05.</p

Gephyrin and GABA_A receptor γ2 puncta are reduced upon CDK5/2/1 inhibition.

<p>(A) Hippocampal neurons were double-immunolabeled for gephyrin with antibody mAb7a (red, upper panel) and with anti-VIAAT antibody (green, middle panel). Superposition of both immunolabelings (lower panel, merge). Neurons were fixed and immunolabeled at div16. (A) Control cells, non-treated; (A') cultures treated with aminopurvalanol A (5 µM) for 2 days; (A'') cultures treated with aminopurvalanol A (5 µM) for 3 days. Note a higher number of VIAAT-opposed gephyrin puncta (yellow, merge) under A, compared to A' and A''. The scale bar represents 15 µm. (B) Hippocampal neurons were double-immunolabeled for GABA_A receptors with anti-γ2-subunit antibody (red, upper panel) and with anti-VIAAT antibodies (green, middle panel). Superposition of both immunolabelings (lower panel, merge). Neurons were fixed and immunolabeled at div16. (A) Control cells, non-treated; (A') cultures treated with aminopurvalanol A (5 µM) for 2 days. (A'') cultures treated with aminopurvalanol A (5 µM) for 3 days. Note a higher number of VIAAT-opposed γ2-subunit puncta (yellow, merge) under B, compared to B' and B''. (C) Quantification of the number of gephyrin (mAb7a) puncta. Quantification was done with about 10 cells from 4 independent cultures. Mean ± S.E.; ANOVA with post-hoc test: * P<0.05. (D) Quantification of the number of GABA_A receptors with anti-γ2-subunit antibody. Mean ± SE. Quantification was done with 49 cells from n = 4 independent cultures (control), 34 cells from n = 3 independent cultures (2 days) and 18 cells from n = 3 independent cultures (3 days). ANOVA with post-hoc test: ** P<0.01.</p