Nek1 shares structural and functional similarities with NIMA kinase

AbstractThe Aspergillus NIMA serine/threonine kinase plays a pivotal role in controlling entrance into mitosis. A major function attributed to NIMA is the induction of chromatin condensation. We show here that the founder murine NIMA-related kinase, Nek1, is larger than previously reported, and that the full-length protein conserves the structural hallmarks of NIMA. Even though Nek1 bears two classical nuclear localization signals (NLS), the endogenous protein localizes to the cytoplasm. Ectopic overexpression of various Nek1 constructs suggests that the C-terminus of Nek1 bears cytoplasmic localization signal(s). Overexpression of nuclear constructs of Nek1 resulted in abnormal chromatin condensation, with the DNA mainly confined to the periphery of the nucleus. Advanced condensation phenotype was associated with nuclear pore complex dispersal. The condensation was not accompanied by up-regulation of mitotic or apoptotic markers. A similar phenotype has been described following NIMA overexpression, strengthening the notion that the mammalian Nek1 kinase has functional similarity to NIMA

Similar synapse elimination motifs at successive relays in the same efferent pathway during development in mice

In many parts of the nervous system, signals pass across multiple synaptic relays on their way to a destination, but little is known about how these relays form and the function they serve. To get some insight into this question we ask how the connectivity patterns are organized at two successive synaptic relays in a simple, cholinergic efferent pathway. We found that the organization at successive relays in the parasympathetic nervous system strongly resemble each other despite the different embryological origin and physiological properties of the pre- and postsynaptic cells. Additionally, we found a similar developmental synaptic pruning and elaboration strategy is used at both sites to generate their adult organizations. The striking parallels in adult innervation and developmental mechanisms at the relays argue that a general strategy is in operation. We discuss why from a functional standpoint this structural organization may amplify central signals while at the same time maintaining positional targeting. DOI: http://dx.doi.org/10.7554/eLife.23193.00

Multispectral labeling technique to map many neighboring axonal projections in the same tissue

We describe a method to map the location of axonal arbors of many individual neurons simultaneously based on the spectral properties of retrogradely transported dyelabeled vesicles. We inject overlapping regions of an axon target area with three or more different colored retrograde tracers. Based on the combinations and intensities of the colors in the individual vesicles transported to neuronal somata we calculate the projection sites of each neuron’s axon.This neuronal positioning system (NPS) enables mapping of many axons in a simple automated way. NPS combined with spectral (Brainbow) labeling of the input to autonomic ganglion cells show that the locations of ganglion cell projections to a mouse salivary gland relate to the identities of their preganglionic axonal innervation. We also show that NPS can delineate projections of many axons simultaneously in the mouse CNS.Molecular and Cellular Biolog

Perineural invasion detection in pancreatic ductal adenocarcinoma using artificial intelligence

Abstract Perineural invasion (PNI) refers to the presence of cancer cells around or within nerves, raising the risk of residual tumor. Linked to worse prognosis in pancreatic ductal adenocarcinoma (PDAC), PNI is also being explored as a therapeutic target. The purpose of this work was to build a PNI detection algorithm to enhance accuracy and efficiency in identifying PNI in PDAC specimens. Training used 260 manually segmented nerve and tumor HD images from 6 scanned PDAC cases; Analytical performance analysis used 168 additional images; clinical analysis used 59 PDAC cases. The algorithm pinpointed key areas of tumor-nerve proximity for pathologist confirmation. Analytical performance reached sensitivity of 88% and 54%, and specificity of 78% and 85% for the detection of nerve and tumor, respectively. Incorporating tumor-nerve distance in clinical evaluation raised PNI detection from 52 to 81% of all cases. Interestingly, pathologist analysis required an average of only 24 s per case. This time-efficient tool accurately identifies PNI in PDAC, even with a small training cohort, by imitating pathologist thought processes

Abnormal Reinnervation of Denervated Areas Following Nerve Injury Facilitates Neuropathic Pain

An injury to peripheral nerves leads to skin denervation, which often is followed by increased pain sensitivity of the denervated areas and the development of neuropathic pain. Changes in innervation patterns during the reinnervation process of the denervated skin could contribute to the development of neuropathic pain. Here, we examined the changes in the innervation pattern during reinnervation and correlated them with the symptoms of neuropathic pain. Using a multispectral labeling technique—PainBow, which we developed, we characterized dorsal root ganglion (DRG) neurons innervating distinct areas of the rats’ paw. We then used spared nerve injury, causing partial denervation of the paw, and examined the changes in innervation patterns of the denervated areas during the development of allodynia and hyperalgesia. We found that, differently from normal conditions, during the development of neuropathic pain, these areas were mainly innervated by large, non-nociceptive neurons. Moreover, we found that the development of neuropathic pain is correlated with an overall decrease in the number of DRG neurons innervating these areas. Importantly, treatment with ouabain facilitated reinnervation and alleviated neuropathic pain. Our results suggest that local changes in peripheral innervation following denervation contribute to neuropathic pain development. The reversal of these changes decreases neuropathic pain

Local sharing as a predominant determinant of synaptic matrix molecular dynamics.

Recent studies suggest that central nervous system synapses can persist for weeks, months, perhaps lifetimes, yet little is known as to how synapses maintain their structural and functional characteristics for so long. As a step toward a better understanding of synaptic maintenance we examined the loss, redistribution, reincorporation, and replenishment dynamics of Synapsin I and ProSAP2/Shank3, prominent presynaptic and postsynaptic matrix molecules, respectively. Fluorescence recovery after photobleaching and photoactivation experiments revealed that both molecules are continuously lost from, redistributed among, and reincorporated into synaptic structures at time-scales of minutes to hours. Exchange rates were not affected by inhibiting protein synthesis or proteasome-mediated protein degradation, were accelerated by stimulation, and greatly exceeded rates of replenishment from somatic sources. These findings indicate that the dynamics of key synaptic matrix molecules may be dominated by local protein exchange and redistribution, whereas protein synthesis and degradation serve to maintain and regulate the sizes of local, shared pools of these proteins

Mutated <i>TP53</i> in Circulating Tumor DNA as a Risk Level Biomarker in Head and Neck Squamous Cell Carcinoma Patients

Circulating tumor DNA (ctDNA) has been suggested as a surrogate biomarker for early detection of cancer recurrence. We aimed to explore the utility of ctDNA as a noninvasive prognostic biomarker in newly diagnosed head and neck squamous cell carcinoma (HNSCC) patients. Seventy HNSCC specimens were analysed for the detection of TP53 genetic alterations utilizing next-generation sequencing (NGS). TP53 mutations were revealed in 55 (79%). Upon detection of a significant TP53 mutation, circulating cell-free DNA was scrutinized for the presence of the tumor-specific mutation. ctDNA was identified at a minimal allele frequency of 0.08% in 21 out of 30 processed plasma samples. Detectable ctDNA correlated with regional spread (N stage ≥ 1, p = 0.011) and poorer 5-year progression-free survival (20%, 95% CI 10.9 to 28.9, p = 0.034). The high-risk worst pattern of invasion (WPOI grade 4–5) and deep invasion were frequently found in patients whose ctDNA was detected (p = 0.087 and p = 0.072, respectively). Detecting mutated TP53 ctDNA was associated with poor progression-free survival and regional metastases, indicating its potential role as a prognostic biomarker. However, ctDNA detectability in early-stage disease and the mechanisms modulating its release into the bloodstream must be further elucidated

ProSAP2 Lost from Dendritic Spine Heads Is Incorporated into PSDs of Adjacent Spines

<p>A dendritic segment expressing both CFP (A) and PA-GFP:ProSAP2 (B). At time <i>t</i> = 0, PA-GFP:ProSAP2 within the region enclosed in rectangles was photoactivated by selective illumination at 405 nm. With time from photoactivation, fluorescence at tips of remote spines increased, whereas spine head fluorescence within the photoactivated region diminished. The contrast in (B) was enhanced linearly to emphasize fluorescence changes in remote spines, resulting in the apparent saturation at photoactivated spines. Spatial relationships between spines and PA-GFP:ProSAP2 puncta before and 29 min after photoactivation are shown in (C) and (D), respectively. In these images, PA-GFP:ProSAP2 fluorescence data were overlaid onto the CFP images after rendering the latter with the “emboss” filter of Adobe Photoshop. Note that PA-GFP:ProSAP2 fluorescence is restricted to spine heads, with little fluorescence observed in spine necks or the dendrite shaft. This distribution indicates that the PA-GFP:ProSAP2 that migrated to adjacent spines had become integrated into the PSD at these sites. Bar, 10 μm. Quantification of fluorescence changes at photoactivated (E) and neighboring spine heads (F) reveals a gradual decrease of fluorescence in the photoactivation region concomitant with fluorescence increases at nearby spines, most prominent at spines nearest to the photoactivation site. Values are normalized to prephotoactivation fluorescence levels.</p