Objective-To report clinical and immunological investigations of contactin-associated proteinlike 2 (Caspr2), an autoantigen of encephalitis and peripheral nerve hyperexcitability (PNH) previously attributed to voltage-gated potassium channels (VGKC).Methods-Clinical analysis of patients with encephalitis, PNH, or both. Immunoprecipitation and mass spectrometry were used to identify the antigen and to develop an assay with Caspr2-expressing cells. Immunoabsorption with Caspr2 and comparative immunostaining of brain and peripheral nerve of wild-type and Caspr2-null mice were used to assess antibody specificity.
Paranodal junctions of myelinated nerve fibers are important for saltatory conduction and function as paracellular and membrane protein diffusion barriers flanking nodes of Ranvier. The formation of these specialized axoglial contacts depends on the presence of three cell adhesion molecules: neurofascin 155 on the glial membrane and a complex of Caspr and contactin on the axon. We isolated axonal and glial membranes highly enriched in these paranodal proteins and then used mass spectrometry to identify additional proteins associated with the paranodal axoglial junction. This strategy led to the identification of three novel components of the paranodal cytoskeleton: ankyrinB, ␣II spectrin, and II spectrin. Biochemical and immunohistochemical analyses revealed that these proteins associate with protein 4.1B in a macromolecular complex that is concentrated at central and peripheral paranodal junctions in the adult and during early myelination. Furthermore, we show that the paranodal localization of ankyrinB is disrupted in Caspr-null mice with aberrant paranodal junctions, demonstrating that paranodal neuron-glia interactions regulate the organization of the underlying cytoskeleton. In contrast, genetic disruption of the juxtaparanodal protein Caspr2 or the nodal cytoskeletal protein IV spectrin did not alter the paranodal cytoskeleton. Our results demonstrate that the paranodal junction contains specialized cytoskeletal components that may be important to stabilize axon-glia interactions and contribute to the membrane protein diffusion barrier found at paranodes.
Caspr and Caspr2 regulate the formation of distinct axonal domains around the nodes of Ranvier. Caspr is required for the generation of a membrane barrier at the paranodal junction (PNJ), whereas Caspr2 serves as a membrane scaffold that clusters Kv1 channels at the juxtaparanodal region (JXP). Both Caspr and Caspr2 interact with protein 4.1B, which may link the paranodal and juxtaparanodal adhesion complexes to the axonal cytoskeleton. To determine the role of protein 4.1B in the function of Caspr proteins, we examined the ability of transgenic Caspr and Caspr2 mutants lacking their 4.1-binding sequence (d4.1) to restore Kv1 channel clustering in Caspr-and Caspr2-null mice, respectively. We found that Caspr-d4.1 was localized to the PNJ and is able to recruit the paranodal adhesion complex components contactin and NF155 to this site. Nevertheless, in axons expressing Caspr-d4.1, Kv1 channels were often detected at paranodes, suggesting that the interaction of Caspr with protein 4.1B is necessary for the generation of an efficient membrane barrier at the PNJ. We also found that the Caspr2-d4.1 transgene did not accumulate at the JXP, even though it was targeted to the axon, demonstrating that the interaction with protein 4.1B is required for the accumulation of Caspr2 and Kv1 channels at the juxtaparanodal axonal membrane. In accordance, we show that Caspr2 and Kv1 channels are not clustered at the JXP in 4.1B-null mice. Our results thus underscore the functional importance of protein 4.1B in the organization of peripheral myelinated axons.
N-WASP–deficient Schwann cells sort and ensheath axons but arrest at the promyelinating stage.
SummaryInhibitory microcircuits are wired with a precision that underlies their complex regulatory roles in neural information processing. In the spinal cord, one specialized class of GABAergic interneurons (GABApre) mediates presynaptic inhibitory control of sensory-motor synapses. The synaptic targeting of these GABAergic neurons exhibits an absolute dependence on proprioceptive sensory terminals, yet the molecular underpinnings of this specialized axoaxonic organization remain unclear. Here, we show that sensory expression of an NB2 (Contactin5)/Caspr4 coreceptor complex, together with spinal interneuron expression of NrCAM/CHL1, directs the high-density accumulation of GABAergic boutons on sensory terminals. Moreover, genetic elimination of NB2 results in a disproportionate stripping of inhibitory boutons from high-density GABApre-sensory synapses, suggesting that the preterminal axons of GABApre neurons compete for access to individual sensory terminals. Our findings define a recognition complex that contributes to the assembly and organization of a specialized GABAergic microcircuit.
Background Postpartum depression is a widespread disorder, adversely affecting the well-being of mothers and their newborns. We aim to utilize machine learning for predicting risk of postpartum depression (PPD) using primary care electronic health records (EHR) data, and to evaluate the potential value of EHR-based prediction in improving the accuracy of PPD screening and in early identification of women at risk. Methods We analyzed EHR data of 266,544 women from the UK who gave first live birth between 2000 and 2017. We extracted a multitude of socio-demographic and medical variables and constructed a machine learning model that predicts the risk of PPD during the year following childbirth. We evaluated the model’s performance using multiple validation methodologies and measured its accuracy as a stand-alone tool and as an adjunct to the standard questionnaire-based screening by Edinburgh postnatal depression scale (EPDS). Results The prevalence of PPD in the analyzed cohort was 13.4%. Combing EHR-based prediction with EPDS score increased the area under the receiver operator characteristics curve (AUC) from 0.805 to 0.844 and the sensitivity from 0.72 to 0.76, at specificity of 0.80. The AUC of the EHR-based prediction model alone varied from 0.72 to 0.74 and decreased by only 0.01–0.02 when applied as early as before the beginning of pregnancy. Conclusions PPD risk prediction using EHR data may provide a complementary quantitative and objective tool for PPD screening, allowing earlier (pre-pregnancy) and more accurate identification of women at risk, timely interventions and potentially improved outcomes for the mother and child.
Germline BRCA–associated pancreatic ductal adenocarcinoma (glBRCA PDAC) tumors are susceptible to platinum and PARP inhibition. The clinical outcomes of 125 patients with glBRCA PDAC were stratified based on the spectrum of response to platinum/PARP inhibition: (i) refractory [overall survival (OS) <6 months], (ii) durable response followed by acquired resistance (OS <36 months), and (iii) long-term responders (OS >36 months). Patient-derived xenografts (PDX) were generated from 25 patients with glBRCA PDAC at different clinical time points. Response to platinum/PARP inhibition in vivo and ex vivo culture (EVOC) correlated with clinical response. We deciphered the mechanisms of resistance in glBRCA PDAC and identified homologous recombination (HR) proficiency and secondary mutations restoring partial functionality as the most dominant resistant mechanism. Yet, a subset of HR-deficient (HRD) patients demonstrated clinical resistance. Their tumors displayed basal-like molecular subtype and were more aneuploid. Tumor mutational burden was high in HRD PDAC and significantly higher in tumors with secondary mutations. Anti–PD-1 attenuated tumor growth in a novel humanized glBRCA PDAC PDX model. This work demonstrates the utility of preclinical models, including EVOC, to predict the response of glBRCA PDAC to treatment, which has the potential to inform time-sensitive medical decisions. Significance: glBRCA PDAC has a favorable response to platinum/PARP inhibition. However, most patients develop resistance. Additional treatment options for this unique subpopulation are needed. We generated model systems in PDXs and an ex vivo system (EVOC) that faithfully recapitulate these specific clinical scenarios as a platform to investigate the mechanisms of resistance for further drug development.
Background. Neoadjuvant chemotherapy (NACT) is increasingly administered for high-risk breast cancer patients. Yet, oncologists lack an effective method for assessing response to treatment beyond clinicpathological features. Organ tissue slices freshly obtained from the tumor and incubated in appropriate media, recapitulate the tumor’s heterogeneity and may provide a superior ex-vivo model for predicting response/resistance to treatment. While these models were tested in metastatic tumors, no studies up to date have been reported in early breast cancer or for NACT. These tests have short turnaround time (7-9 days), critical for the neoadjuvant setting. Here we aimed to prove the feasibility of performing cResponse assay, an ex vivo organ culture (EVOC), on freshly derived pre-treatment core needle biopsies and to evaluate it as a tool for predicting response to NACT in breast cancer. Methods. Pre-treatment tumor biopsies were obtained from breast cancer patients about to undergo NACT at the time of routine tumor clip marking. The fresh samples were immediately placed in cold medium, sliced into 250um sections and cultured in multiwell plates. The samples were treated with the clinically administered therapies or a vehicle control. The various therapies included Doxorubicin (A), Paclitaxel (T), Cytoxan (C) and in TN cases, Carboplatin (Cr), as single agents or in combination in clinically administered ratios. After 4 days of treatment, tumor slices were fixed and stained to allow morphological analysis. A trained pathologist examined the stained slides and scored the viable cancer cells. A response-score was generated and compared to the pathological result (pCR status and RCB class) of the patient at surgery.Results. Tumor samples from 15 breast cancer patients were examined. The cohort median age was 42 (32—82), LN involvement 9/15, subtypes status: 9 HR+, 2 TNBC, 3 HR+HER2+ and 1 HR-HER2+. Nine samples were either excluded due to insufficient viable cancer cells or necrosis and one sample failed technically. Five samples (2 TNBC, 2 HR+ and 1 HR+HER2+) were adequate for analysis and obtained cResponse scores. The HR+HER2+ case received a different protocol than actually tested, thus was not compared. For the four successful cases, the cResponse score was highly concordant with response to therapy (Table I). For the two TNBC samples, a maximal cResponse score (100) was demonstrated for AC-T or AC-TC combination and both patients achieved complete response (pCR, RCB-0) at surgery. Notably, in one patient, the assay predicted maximal response for the TC combination, suggesting that this protocol could be sufficient. For the HR+ patients, one patient demonstrated a strong cResponse to AC-T (85) and achieved near complete response (RCB-I), while the second patient demonstrated a moderate cResponse score (70) and achieved partial pathological response (RCB-II). Conclusions. We provide initial evidence for the feasibility and validity of an EVOC platform to predict response to NACT in breast cancer. Further optimizations are needed to increase the assay’s success rates. We expect the results to set the ground for a clinical trial, examining the utility of the cResponse test as predictive biomarker in determining NACT. This technology may provide a tool for the oncologist to select the most efficient therapies, maximizing pCR rates and minimizing toxicity from ineffective drugs, eventually improving prognosis of breast cancer patients. Table IPatientSubtypeBest cResponse scoreBest treatmentsPathological ResponseRCB classNAT-2HR+70AC-T/AC/CPartialRCB-IINAT-3TN100AC-TCr/TCrpCRRCB-0NAT-12TN100A/AC-TpCRRCB-0NAT-16HR+85AC-TPartialRCB-I Citation Format: Einav Nili Gal-Yam, Miri Sklair-Levi, Yaeli Vachnish, Nora Balint-Lahat, Dana Morzaev-Sulzbach, Michal Bakalenik-Gavry, Renata Fearmann, Osnat Halshtok, Anat Shalmon, Michael Gotlieb, Yael Yagil, Keren Levanon, Rinat Bernstein-Molho, Amit Itay, Tali Shapira-Rotenberg, Opher Globus, Iris Barshack, Seth Salpeter, Vered Bar, Sara Aharon, Lubov Turovsky, Adi Zundelevich, Giuseppe Mallel, Hamutal Shahar, Hagit Shapira, Maya Dadiani. Evaluating an ex vivo organ culture system for predicting response to neoadjuvant chemotherapy in breast cancer patients [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr P1-08-25.
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