Presynaptic α2δ subunits are key organizers of glutamatergic synapses.

In nerve cells the genes encoding for α2δ subunits of voltage-gated calcium channels have been linked to synaptic functions and neurological disease. Here we show that α2δ subunits are essential for the formation and organization of glutamatergic synapses. Using a cellular α2δ subunit triple-knockout/knockdown model, we demonstrate a failure in presynaptic differentiation evidenced by defective presynaptic calcium channel clustering and calcium influx, smaller presynaptic active zones, and a strongly reduced accumulation of presynaptic vesicle-associated proteins (synapsin and vGLUT). The presynaptic defect is associated with the downscaling of postsynaptic AMPA receptors and the postsynaptic density. The role of α2δ isoforms as synaptic organizers is highly redundant, as each individual α2δ isoform can rescue presynaptic calcium channel trafficking and expression of synaptic proteins. Moreover, α2δ-2 and α2δ-3 with mutated metal ion-dependent adhesion sites can fully rescue presynaptic synapsin expression but only partially calcium channel trafficking, suggesting that the regulatory role of α2δ subunits is independent from its role as a calcium channel subunit. Our findings influence the current view on excitatory synapse formation. First, our study suggests that postsynaptic differentiation is secondary to presynaptic differentiation. Second, the dependence of presynaptic differentiation on α2δ implicates α2δ subunits as potential nucleation points for the organization of synapses. Finally, our results suggest that α2δ subunits act as transsynaptic organizers of glutamatergic synapses, thereby aligning the synaptic active zone with the postsynaptic density.

Cost-Utility of Partially Implantable Active Middle Ear Implants for Sensorineural Hearing Loss: A Decision Analysis.

BACKGROUND: Partially implantable active middle ear implants (aMEIs) offer a solution for individuals who have mild to severe sensorineural hearing loss and an outer ear medical condition that precludes the use of hearing aids. When otherwise left untreated, individuals report a lower quality of life, which may further decrease with increasing disability. In the lack of cost-effectiveness studies and long-term data, there is a need for decision modeling. OBJECTIVE: To explore individual-level variance in resource utilization patterns following aMEI implantation. METHODS: A Markov model was developed and analyzed as microsimulation to estimate the incremental cost utility ratio (ICUR) of partially implantable aMEIs compared with no (surgical) intervention in individuals with sensorineural hearing loss and an outer ear medical condition in Australia. Cost data were derived mostly from the Medicare Benefit Schedule and effectiveness data from published literature. A third-party payer perspective was adopted, and a 5% discount rate was applied over a 10-year time horizon. RESULTS: Compared with baseline strategy, aMEIs yielded an incremental cost of Australian dollars (AUD) 13,339.18, incremental quality-adjusted life-year (QALY) of 1.35, and an ICUR of AUD 9,913.72/QALY. Of the respective number of simulated patients who visited each health state, 75.73% never had a minor adverse event, 99.82% did not experience device failure, and 97.75% did not cease to use their aMEIs. Probabilistic sensitivity analyses showed the ICUR to differ by only 0.95%. CONCLUSIONS: In the Australian setting, partially implantable aMEIs offer a safe and cost-effective solution compared with no intervention and are also well accepted by users.

Serum Autoantibodies in Chronic Prostate Inflammation in Prostate Cancer Patients.

BACKGROUND: Chronic inflammation is frequently observed on histological analysis of malignant and non-malignant prostate specimens. It is a suspected supporting factor for prostate diseases and their progression and a main cause of false positive PSA tests in cancer screening. We hypothesized that inflammation induces autoantibodies, which may be useful biomarkers. We aimed to identify and validate prostate inflammation associated serum autoantibodies in prostate cancer patients and evaluate the expression of corresponding autoantigens. METHODS: Radical prostatectomy specimens of prostate cancer patients (N = 70) were classified into high and low inflammation groups according to the amount of tissue infiltrating lymphocytes. The corresponding pre-surgery blood serum samples were scrutinized for autoantibodies using a low-density protein array. Selected autoantigens were identified in prostate tissue and their expression pattern analyzed by immunohistochemistry and qPCR. The identified autoantibody profile was cross-checked in an independent sample set (N = 63) using the Luminex-bead protein array technology. RESULTS: Protein array screening identified 165 autoantibodies differentially abundant in the serum of high compared to low inflammation patients. The expression pattern of three corresponding antigens were established in benign and cancer tissue by immunohistochemistry and qPCR: SPAST (Spastin), STX18 (Syntaxin 18) and SPOP (speckle-type POZ protein). Of these, SPAST was significantly increased in prostate tissue with high inflammation. All three autoantigens were differentially expressed in primary and/or castration resistant prostate tumors when analyzed in an inflammation-independent tissue microarray. Cross-validation of the inflammation autoantibody profile on an independent sample set using a Luminex-bead protein array, retrieved 51 of the significantly discriminating autoantibodies. Three autoantibodies were significantly upregulated in both screens, MUT, RAB11B and CSRP2 (p>0.05), two, SPOP and ZNF671, close to statistical significance (p = 0.051 and 0.076). CONCLUSIONS: We provide evidence of an inflammation-specific autoantibody profile and confirm the expression of corresponding autoantigens in prostate tissue. This supports evaluation of autoantibodies as non-invasive markers for prostate inflammation.

Putative Prostate Cancer Risk SNP in an Androgen Receptor-Binding Site of the Melanophilin Gene Illustrates Enrichment of Risk SNPs in Androgen Receptor Target Sites.

Genome-wide association studies have identified genomic loci, whose single-nucleotide polymorphisms (SNPs) predispose to prostate cancer (PCa). However, the mechanisms of most of these variants are largely unknown. We integrated chromatin-immunoprecipitation-coupled sequencing and microarray expression profiling in TMPRSS2-ERG gene rearrangement positive DUCaP cells with the GWAS PCa risk SNPs catalog to identify disease susceptibility SNPs localized within functional androgen receptor-binding sites (ARBSs). Among the 48 GWAS index risk SNPs and 3,917 linked SNPs, 80 were found located in ARBSs. Of these, rs11891426:T>G in an intron of the melanophilin gene (MLPH) was within a novel putative auxiliary AR-binding motif, which is enriched in the neighborhood of canonical androgen-responsive elements. T→G exchange attenuated the transcriptional activity of the ARBS in an AR reporter gene assay. The expression of MLPH in primary prostate tumors was significantly lower in those with the G compared with the T allele and correlated significantly with AR protein. Higher melanophilin level in prostate tissue of patients with a favorable PCa risk profile points out a tumor-suppressive effect. These results unravel a hidden link between AR and a functional putative PCa risk SNP, whose allele alteration affects androgen regulation of its host gene MLPH.

miR-22 and miR-29a Are Members of the Androgen Receptor Cistrome Modulating LAMC1 and Mcl-1 in Prostate Cancer.

The normal prostate as well as early stages and advanced prostate cancer (PCa) require a functional androgen receptor (AR) for growth and survival. The recent discovery of microRNAs (miRNAs) as novel effector molecules of AR disclosed the existence of an intricate network between AR, miRNAs and downstream target genes. In this study DUCaP cells, characterized by high content of wild-type AR and robust AR transcriptional activity, were chosen as the main experimental model. By integrative analysis of chromatin immunoprecipitation-sequencing (ChIP-seq) and microarray expression profiling data, miRNAs putatively bound and significantly regulated by AR were identified. A direct AR regulation of miR-22, miR-29a, and miR-17-92 cluster along with their host genes was confirmed. Interestingly, endogenous levels of miR-22 and miR-29a were found to be reduced in PCa cells expressing AR. In primary tumor samples, miR-22 and miR-29a were less abundant in the cancerous tissue compared with the benign counterpart. This specific expression pattern was associated with a differential DNA methylation of the genomic AR binding sites. The identification of laminin gamma 1 (LAMC1) and myeloid cell leukemia 1 (MCL1) as direct targets of miR-22 and miR-29a, respectively, suggested a tumor-suppressive role of these miRNAs. Indeed, transfection of miRNA mimics in PCa cells induced apoptosis and diminished cell migration and viability. Collectively, these data provide additional information regarding the complex regulatory machinery that guides miRNAs activity in PCa, highlighting an important contribution of miRNAs in the AR signaling.

Differential upregulation in DRG neurons of an α2δ-1 splice variant with a lower affinity for gabapentin after peripheral sensory nerve injury.

The α2δ-1 protein is an auxiliary subunit of voltage-gated calcium channels, critical for neurotransmitter release. It is upregulated in dorsal root ganglion (DRG) neurons following sensory nerve injury, and is also the therapeutic target of the gabapentinoid drugs, which are efficacious in both experimental and human neuropathic pain conditions. α2δ-1 has 3 spliced regions: A, B, and C. A and C are cassette exons, whereas B is introduced via an alternative 3' splice acceptor site. Here we have examined the presence of α2δ-1 splice variants in DRG neurons, and have found that although the main α2δ-1 splice variant in DRG is the same as that in brain (α2δ-1 ΔA+B+C), there is also another α2δ-1 splice variant (ΔA+BΔC), which is expressed in DRG neurons and is differentially upregulated compared to the main DRG splice variant α2δ-1 ΔA+B+C following spinal nerve ligation. Furthermore, this differential upregulation occurs preferentially in a small nonmyelinated DRG neuron fraction, obtained by density gradient separation. The α2δ-1 ΔA+BΔC splice variant supports CaV2 calcium currents with unaltered properties compared to α2δ-1 ΔA+B+C, but shows a significantly reduced affinity for gabapentin. This variant could therefore play a role in determining the efficacy of gabapentin in neuropathic pain.

IL6 sensitizes prostate cancer to the antiproliferative effect of IFNα2 through IRF9.

Development and progression of prostate cancer (PCa) are associated with chronic inflammation. The cytokine interleukin 6 (IL6) can influence progression, differentiation, survival, and angiogenesis of PCa. To identify novel pathways that are triggered by IL6, we performed a gene expression profiling of two PCa cell lines, LNCaP and MDA PCa 2b, treated with 5 ng/ml IL6. Interferon (IFN) regulatory factor 9 (IRF9) was identified as one of the most prevalent IL6-regulated genes in both cell lines. IRF9 is a mediator of type I IFN signaling and acts together with STAT1 and 2 to activate transcription of IFN-responsive genes. The IL6 regulation of IRF9 was confirmed at mRNA and protein levels by quantitative real-time PCR and western blot respectively in both cell lines and could be blocked by the anti-IL6 antibody Siltuximab. Three PCa cell lines, PC3, Du-145, and LNCaP-IL6+, with an autocrine IL6 loop displayed high expression of IRF9. A tissue microarray with 36 PCa tissues showed that IRF9 protein expression is moderately elevated in malignant areas and positively correlates with the tissue expression of IL6. Downregulation and overexpression of IRF9 provided evidence for an IFN-independent role of IRF9 in cellular proliferation of different PCa cell lines. Furthermore, expression of IRF9 was essential to mediate the antiproliferative effects of IFNα2. We concluded that IL6 is an inducer of IRF9 expression in PCa and a sensitizer for the antiproliferative effects of IFNα2.

Sprouty2 and -4 regulate axon outgrowth by hippocampal neurons.

Sprouty proteins act as negative feedback inhibitors of fibroblast growth factor (FGF) signaling. FGFs belong to the neurotrophic factors and are involved in axonal growth during development and repair. We investigated the expression of Sprouty isoforms in hippocampal neurons as well as the regulation of Sprouty2 and -4 during development and their role in axon growth. Sprouty2 and -4 were located in the nucleus, the cytoplasm, in dendrites, and axons of hippocampal neurons concentrated in growth cones. During development in vivo and differentiation in vitro, expression of Sprouty2 and -4 was gradually downregulated in hippocampal neurons. Between 5 and 24 days in culture expression of both Sprouty isoforms was reduced by 70%. In vivo expression of Sprouty2 was reduced by 79% and of Sprouty4 by 93% on postnatal day 14 compared to embryonic day 16.5. Downregulation of Sprouty2 and -4 by shRNAs strongly promoted elongative axon growth by cultured hippocampal neurons, which was further increased by FGF-2 treatment. In addition, FGF-2 reduced expression of Sprouty2 by 33% and of Sprouty4 by 44%. Together, our results imply that Sprouty2 and -4 are downregulated in the hippocampus during postnatal brain development and that they can act as regulators of developmental axon growth.

Reciprocal interactions regulate targeting of calcium channel beta subunits and membrane expression of alpha1 subunits in cultured hippocampal neurons.

Auxiliary beta subunits modulate current properties and mediate the functional membrane expression of voltage-gated Ca(2+) channels in heterologous cells. In brain, all four beta isoforms are widely expressed, yet little is known about their specific roles in neuronal functions. Here, we investigated the expression and targeting properties of beta subunits and their role in membrane expression of Ca(V)1.2 alpha(1) subunits in cultured hippocampal neurons. Quantitative reverse transcription-PCR showed equal expression, and immunofluorescence showed a similar distribution of all endogenous beta subunits throughout dendrites and axons. High resolution microscopy of hippocampal neurons transfected with six different V5 epitope-tagged beta subunits demonstrated that all beta subunits were able to accumulate in synaptic terminals and to colocalize with postsynaptic Ca(V)1.2, thus indicating a great promiscuity in alpha(1)-beta interactions. In contrast, restricted axonal targeting of beta(1) and weak colocalization of beta(4b) with Ca(V)1.2 indicated isoform-specific differences in local channel complex formation. Membrane expression of external hemagglutinin epitope-tagged Ca(V)1.2 was strongly enhanced by all beta subunits in an isoform-specific manner. Conversely, mutating the alpha-interaction domain of Ca(V)1.2 (W440A) abolished membrane expression and targeting into dendritic spines. This demonstrates that in neurons the interaction of a beta subunit with the alpha-interaction domain is absolutely essential for membrane expression of alpha(1) subunits, as well as for the subcellular localization of beta subunits, which by themselves possess little or no targeting properties.

A CaV1.1 Ca2+ channel splice variant with high conductance and voltage-sensitivity alters EC coupling in developing skeletal muscle.

The Ca(2+) channel alpha(1S) subunit (Ca(V)1.1) is the voltage sensor in skeletal muscle excitation-contraction (EC) coupling. Upon membrane depolarization, this sensor rapidly triggers Ca(2+) release from internal stores and conducts a slowly activating Ca(2+) current. However, this Ca(2+) current is not essential for skeletal muscle EC coupling. Here, we identified a Ca(V)1.1 splice variant with greatly distinct current properties. The variant of the CACNA1S gene lacking exon 29 was expressed at low levels in differentiated human and mouse muscle, and up to 80% in myotubes. To test its biophysical properties, we deleted exon 29 in a green fluorescent protein (GFP)-tagged alpha(1S) subunit and expressed it in dysgenic (alpha(1S)-null) myotubes. GFP-alpha(1S)Delta 29 was correctly targeted into triads and supported skeletal muscle EC coupling. However, the Ca(2+) currents through GFP-alpha(1S)Delta 29 showed a 30-mV left-shifted voltage dependence of activation and a substantially increased open probability, giving rise to an eightfold increased current density. This robust Ca(2+) influx contributed substantially to the depolarization-induced Ca(2+) transient that triggers contraction. Moreover, deletion of exon 29 accelerated current kinetics independent of the auxiliary alpha(2)delta-1 subunit. Thus, characterizing the Ca(V)1.1 Delta 29 splice variant revealed the structural bases underlying the specific gating properties of skeletal muscle Ca(2+) channels, and it suggests the existence of a distinct mode of EC coupling in developing muscle.