Pαx6 Expression in Postmitotic Neurons Mediates the Growth of Axons in Response to SFRP1

During development, the mechanisms that specify neuronal subclasses are coupled to those that determine their axonal response to guidance cues. Pax6 is a homedomain transcription factor required for the specification of a variety of neural precursors. After cell cycle exit, Pax6 expression is often shut down in the precursor progeny and most postmitotic neurons no longer express detectable levels of the protein. There are however exceptions and high Pax6 protein levels are found, for example, in postmitotic retinal ganglion cells (RGCs), dopaminergic neurons of the olfactory bulb and the limbic system in the telencephalon. The function of Pax6 in these differentiating neurons remains mostly elusive. Here, we demonstrate that Pax6 mediates the response of growing axons to SFRP1, a secreted molecule expressed in several Pax6-positive forebrain territories. Forced expression of Pax6 in cultured postmitotic cortical neurons, which do not normally express Pax6, was sufficient to increment axonal length. Growth was blocked by the addition of anti-SFRP1 antibodies, whereas exogenously added SFRP1 increased axonal growth of Pax6-transfected neurons but not that of control or untransfected cortical neurons. In the reverse scenario, shRNA-mediated knock-down of Pax6 in mouse retinal explants specifically abolished RGCs axonal growth induced by SFRP1, but had no effect on RGCs differentiation and it did not modify the effect of Shh or Netrin on axon growth. Taken together these results demonstrate that expression of Pax6 is necessary and sufficient to render postmitotic neurons competent to respond to SFRP1. These results reveal a novel and unexpected function of Pax6 in postmitotic neurons and situate Pax6 and SFRP1 as pair regulators of axonal connectivity

Estudio de la regulación transcripcional de la guía axonal. Papel de los factores de transcripción Cux1, Cux2 y Pax6

Tesis doctoral inédita leída en la Universidad Autónoma de Madrid, Facultad de Ciencias, Departamento de Biología Molecular. Fecha de lectura: 27-10-201

Knock-down of <i>Pax6</i> blocks SFRP1 stimulated growth of retinal axons.

<p><b>a</b>) Low magnification images (a) and confocal micrographs (b) of explants from control (a) or electroporated retinal explants (b) seeded onto laminin coated coverslip and cultured in the absence or presence of recombinant SFRP1 as indicated in the panels. Explants in (a) were stained with β-tubulin III whereas those in (b) were co-electroporated with CAG-GFP and shRNA control or shRNA targeting <i>Pax6</i> and axons were visualized by GFP expression. Graph shows quantification of the proportion of total axon longer than 900 (a) or 200 (b) µm. Note that knocking-down <i>Pax6</i> inhibits the axonal response stimulated by SFRP1. Bar indicates 300 (a) and 150 µm (b). Data are expressed as the mean ± SD. (*) p<0.05 comparing stimulated populations; (**) p<0.01 and (***) p<0.001 compared with control.</p

Knock-down of <i>Pax6</i> in the embryonic retina does not interfere with the generation of RGCs.

<p>Frontal cryostat sections of E19.5 retinas after electroporation at E13.5 with shRNAs constructs control and targeting <i>Pax6</i> and immunostained (red) for Pax6 and Caspase 3 (<b>a</b>) or Islet-1/2 and Brn3 (<b>b</b>) as indicated in the panels. Electroporated cells are visualized with GFP (green). The graphs show the quantification of the proportion of the double positive cells for the indicated marker. Note that shRNA efficiently target Pax6 without inducing cell death (a) or changes in expression of markers for postmitotic RGCs (b). Bar indicates 10 in (a) and 40 µm in (b). Data are expressed as the mean ± SD. (*) p<0.05; (**) p<0.01. Number of axons per condition >60 (n = 3). INL, inner layer; VZ, ventricular zone.</p

Ectopic expression of <i>Pax6</i> stimulates axonal growth in cortical neurons.

<p>NSCs were nucleofected with CAG-empty vector or CAG-<i>Pax6</i> and co-electroporated with CAG-<i>GFP</i>. <b>a</b>) The graph shows the percentage of nucleofected neurons with respect to their axonal length after 9 days of differentiation. Over-expression of <i>Pax6</i> increments the axonal length compared with control neurons. <b>b</b>) The graph shows quantification of the axonal length of a cohort of BrdU positive neurons. <b>c</b>) Nucleofected neurons were identified by the specific expression of β-tubulin III and GFP. BrdU staining allowed us to compared neurons of similar birth dates. The effect in axonal growth is unrelated to an early exit from the cell cycle. Arrows point to BrdU stained nuclei. Bar indicates 50 µm. <b>d</b>) Cortical primary neurons were also transfected with CAG-empty vector or CAG<i>-Pax6</i>, and with CAG-<i>GFP</i>. Transfected primary axons were identified by GFP and MAP1b staining. Arrows indicate the distal part of the axon stained by MAP1b. Bar indicates 70 µm. <b>e</b>) The graph shows the percentage of primary neurons with respect to their axonal length. Data are expressed as the mean ± SD. (*) p<0.05; (**) p<0.01.</p

Effect of the COVID-19 pandemic on surgery for indeterminate thyroid nodules (THYCOVID): a retrospective, international, multicentre, cross-sectional study

Background: Since its outbreak in early 2020, the COVID-19 pandemic has diverted resources from non-urgent and elective procedures, leading to diagnosis and treatment delays, with an increased number of neoplasms at advanced stages worldwide. The aims of this study were to quantify the reduction in surgical activity for indeterminate thyroid nodules during the COVID-19 pandemic; and to evaluate whether delays in surgery led to an increased occurrence of aggressive tumours. Methods: In this retrospective, international, cross-sectional study, centres were invited to participate in June 22, 2022; each centre joining the study was asked to provide data from medical records on all surgical thyroidectomies consecutively performed from Jan 1, 2019, to Dec 31, 2021. Patients with indeterminate thyroid nodules were divided into three groups according to when they underwent surgery: from Jan 1, 2019, to Feb 29, 2020 (global prepandemic phase), from March 1, 2020, to May 31, 2021 (pandemic escalation phase), and from June 1 to Dec 31, 2021 (pandemic decrease phase). The main outcomes were, for each phase, the number of surgeries for indeterminate thyroid nodules, and in patients with a postoperative diagnosis of thyroid cancers, the occurrence of tumours larger than 10 mm, extrathyroidal extension, lymph node metastases, vascular invasion, distant metastases, and tumours at high risk of structural disease recurrence. Univariate analysis was used to compare the probability of aggressive thyroid features between the first and third study phases. The study was registered on ClinicalTrials.gov, NCT05178186. Findings: Data from 157 centres (n=49 countries) on 87 467 patients who underwent surgery for benign and malignant thyroid disease were collected, of whom 22 974 patients (18 052 [78·6%] female patients and 4922 [21·4%] male patients) received surgery for indeterminate thyroid nodules. We observed a significant reduction in surgery for indeterminate thyroid nodules during the pandemic escalation phase (median monthly surgeries per centre, 1·4 [IQR 0·6-3·4]) compared with the prepandemic phase (2·0 [0·9-3·7]; p&lt;0·0001) and pandemic decrease phase (2·3 [1·0-5·0]; p&lt;0·0001). Compared with the prepandemic phase, in the pandemic decrease phase we observed an increased occurrence of thyroid tumours larger than 10 mm (2554 [69·0%] of 3704 vs 1515 [71·5%] of 2119; OR 1·1 [95% CI 1·0-1·3]; p=0·042), lymph node metastases (343 [9·3%] vs 264 [12·5%]; OR 1·4 [1·2-1·7]; p=0·0001), and tumours at high risk of structural disease recurrence (203 [5·7%] of 3584 vs 155 [7·7%] of 2006; OR 1·4 [1·1-1·7]; p=0·0039). Interpretation: Our study suggests that the reduction in surgical activity for indeterminate thyroid nodules during the COVID-19 pandemic period could have led to an increased occurrence of aggressive thyroid tumours. However, other compelling hypotheses, including increased selection of patients with aggressive malignancies during this period, should be considered. We suggest that surgery for indeterminate thyroid nodules should no longer be postponed even in future instances of pandemic escalation. Funding: None

Evolution over Time of Ventilatory Management and Outcome of Patients with Neurologic Disease∗

OBJECTIVES: To describe the changes in ventilator management over time in patients with neurologic disease at ICU admission and to estimate factors associated with 28-day hospital mortality. DESIGN: Secondary analysis of three prospective, observational, multicenter studies. SETTING: Cohort studies conducted in 2004, 2010, and 2016. PATIENTS: Adult patients who received mechanical ventilation for more than 12 hours. INTERVENTIONS: None. MEASUREMENTS AND MAIN RESULTS: Among the 20,929 patients enrolled, we included 4,152 (20%) mechanically ventilated patients due to different neurologic diseases. Hemorrhagic stroke and brain trauma were the most common pathologies associated with the need for mechanical ventilation. Although volume-cycled ventilation remained the preferred ventilation mode, there was a significant (p &lt; 0.001) increment in the use of pressure support ventilation. The proportion of patients receiving a protective lung ventilation strategy was increased over time: 47% in 2004, 63% in 2010, and 65% in 2016 (p &lt; 0.001), as well as the duration of protective ventilation strategies: 406 days per 1,000 mechanical ventilation days in 2004, 523 days per 1,000 mechanical ventilation days in 2010, and 585 days per 1,000 mechanical ventilation days in 2016 (p &lt; 0.001). There were no differences in the length of stay in the ICU, mortality in the ICU, and mortality in hospital from 2004 to 2016. Independent risk factors for 28-day mortality were age greater than 75 years, Simplified Acute Physiology Score II greater than 50, the occurrence of organ dysfunction within first 48 hours after brain injury, and specific neurologic diseases such as hemorrhagic stroke, ischemic stroke, and brain trauma. CONCLUSIONS: More lung-protective ventilatory strategies have been implemented over years in neurologic patients with no effect on pulmonary complications or on survival. We found several prognostic factors on mortality such as advanced age, the severity of the disease, organ dysfunctions, and the etiology of neurologic disease