Intramuscular injections of botulinum toxin block pre-synaptic cholinergic release at neuromuscular junctions producing a temporary paralysis of affected motor units. There is increasing evidence, however, that the effects are not restricted to the periphery and can alter the central excitability of the motoneurones at the spinal level. This includes increases in input resistance, decreases in rheobase currents for action potentials and prolongations of the post-spike after-hyperpolarization. The aim of our experiments was to investigate possible anatomical explanations for these changes. Unilateral injections of Botulinum toxin A mixed with a tracer were made into the gastrocnemius muscle of adult rats and contralateral tracer only injections provided controls. Immunohistochemistry for Ankyrin G and the vesicular acetylcholine transporter labelled axon initial segments and cholinergic C-boutons on traced motoneurones at 2 weeks post-injection. Soma size was not affected by the toxin; however, axon initial segments were 5.1% longer and 13.6% further from the soma which could explain reductions in rheobase. Finally, there was a reduction in surface area (18.6%) and volume (12.8%) but not frequency of C-boutons on treated motoneurones potentially explaining prolongations of the after-hyperpolarization. Botulinum Toxin A therefore affects central anatomical structures controlling or modulating motoneurone excitability explaining previously observed excitability changes. There is increasing evidence, however, that the effects may not be restricted to the periphery. Studies show that BoNT/A, injected intramuscularly, is transported both anterogradely along sensory axons and retrogradely along motoneurone axons to the motoneurone soma in the spinal cord5. Furthermore, there is now also evidence that BoNT/A can spread between networks of cells6, although this claim has been questioned by other studies and would appear to be dose dependent8. This raises the question as to what central effects BoNT/A may have on the motoneurone itself or on central cholinergic synapses contacting the motoneurone.Botulinum neurotoxin type-A (BoNT/A) is one of the most dangerous toxins known to man, yet it is being increasingly used, both cosmetically and clinically, to produce long lasting paralysis of specific muscles. After intramuscular injection, the toxin enters the synaptic vesicles of motor axon terminals by endocytosis. From here it silences synaptic transmission via specific proteolytic cleavage of SNAP-25 which prevents the release of acetylcholine from the motoneurone terminal resulting in flaccid paralysis9. This increased reflex was also seen when tested as simple monosynaptic responses to dorsal root stimulation suggesting the enhanced reflexes may be due to an increase in motoneurone excitability10. Consistent with this, intracellular recordings of gastrocnemius motoneurones show a reduction in rheobase currents for action potentials already by 5 days post-injection11 which is further reduced by 2–3 weeks12. There are a number of potential anatomical factors that could account for these changes. The first is a possible reduction in cell size. This is plausible given that increases in input resistance have been observed following BoNT/A treatment12. Plasticity at the site of action potential generation; the axon initial segment (AIS), is another possible explanation. The size and location of the AIS determines the excitability of a neurone and its response to inputs13. This structure is highly plastic, and can dynamically change its size and location to alter neuronal excitably in response to perturbations in inputs or activity levels16. Thus, AIS plasticity would also appear to be a likely candidate for reducing the rheobase after peripheral BoNT/A injection.Electrophysiological studies suggest changes in spinal motoneurone excitability after intramuscular BoNT/A injections of the gastrocnemius muscle. Increased stretch reflexes are seen at two weeks post-injection in rats12. A certain type of cholinergic synapse on spinal motoneurones; the C-bouton, plays a crucial role in the modulation of the motoneurone AHP of lumbar spinal neurones via their interaction with muscarinic (m2) receptors and the small conductance calcium-activated potassium (SK) channels mediating the AHP18. A reduction in the number and size of C-boutons on spinal motoneurones has been shown on axotomised motoneurones21 which may explain the prolongation of the AHP observed after axotomy23. As BoNT/A injections induce similar  changes in the AHP12 this suggests that a loss of functional connectivity at the neuromuscular junction may be enough to drive such changes in C-boutons. Alternatively, if BoNT/A is transported retrogradely and transsynaptically this could also influence the C-boutons.A prolongation of the post-spike after-hyperpolarisation (AHP) of gastrocnemius motoneurones has also been observed at 2 weeks post BoNT/A injectionWe therefore investigated possible intramuscular BoNT/A injection-induced anatomical changes in cell size, axon initial segment size/location and size/frequency of C-boutons on gastrocnemius motoneurones that could explain the altered excitability. BoNT/A was mixed with a tracer, Cholera toxin subunit B (CTB) before being injected unilaterally into the gastrocnemius muscle and CTB only injections on the contralateral side provided an internal control Fig. .Figure 1To determine that mixing the BoNT/A with tracer did not impair its effects, in 3 additional rats we recorded the maximal Compound Muscle Action Potential (CMAP) from the gastrocnemius muscles before and after bilateral injections of BoNT/A with CTB mixed with the BoNT/A on one side only. In all rats, by 4 days post-injection a drastic reduction in CMAP amplitude was observed on both sides (>75%) regardless of whether the BoNT/A had been mixed with tracer or not Fig.  confirmiExamples of traced gastrocnemius motoneurones for both the BoNT/A injected and contralateral control side can be seen in Fig. As motoneurone soma shapes varied considerably Fig.  we confi24. Examples of AISs from both BoNT/A treated and control gastrocnemius motoneurones can be seen in Fig. Immunohistochemistry for Ankyrin G was used to label AISs on traced gastrocnemius motoneurones as this scaffolding protein spans the entire length of the AISTo determine whether the observed differences were consistent across rats, means were calculated per animal and these compared using repeated-measures tests. This showed that the increase in length was consistent in 6 out of 7 rats  has the advantage of being an internal control, eliminating the effects of inter-rat variation, it is possible that the BoNT/A could have contralateral effects (see discussion). To test this, we compared the mean AIS length on the CTB only side with the mean AIS length measured in an additional 5 control rats in which only CTB was injected. The mean AIS length in these extra control rats was 23.5 µm which was remarkably close to the mean of 23.6 µm measured in the control side of the BoNT/A treated rats and was not significantly different . In fact, no significant differences were observed between the two different control groups with respect to any of the AIS parameters. A significant increase in AIS distance was still observed on the BoNT/A treated side when compared to these additional controls .26. Given that the number of proximal dendrites visible in a single 50 µm thick tissue section differed between cells we confined our analysis of C-boutons to the soma.C-boutons on the soma and proximal dendrites of spinal motoneurones were labelled using antibodies against vesicular acetylcholine transporter (VAChT). Examples of C-boutons labelled on BoNT/A treated and control motoneurones are shown in Fig. The mean number of C-boutons per cells was not significantly different between the BoNT/A treated and control sides . For cells with very large dendritic trees  modelling suggests that excitability is increased by lengthening the AIS and moving it further away from the soma28. In the context of these results, we can speculate that both of our observed changes of a 5.1% increase in length and 13.6% distal relocation following BoNT/A are likely to have a functional consequence.An important question remains as to whether the magnitude of the AIS changes would be sufficient to have a functional consequence. Here modelling studies, based on AIS plasticity following blast induced traumatic brain injury have shown that reductions in AIS length of just 4.5% result in small increases in action potential threshold as well as decreased amplitude and rates of rise of action potentials. Importantly, it also reduced the firing frequency in response to prolonged depolarizing currents29. The reduction in size we observed would consequently reduce the effectiveness of the C-boutons. Given the role that C-boutons play in the reduction of the AHP via their modulation of SK channels18, this could explain the prolongation of the AHP observed in gastrocnemius motoneurones. One caveat here is that the role of C-boutons is hypothesised to be task dependent modulation, having the most effect under states of moderate to strong physiological drive30 and all recordings of AHP following BoNT/A treatment, however, have been performed in immobile anaesthetised animals.Changes in the size but not frequency of C-boutons were also observed on spinal motoneurones. Bouton size has been shown to systematically correlate with both total and individual active zone area, the number of active zones and the number of synaptic vesiclesIt could be hypothesised that the AIS changes may result in a decrease in rheobase making the motoneurones easier to recruit and then once recruited the motoneurones would show increased firing rates. A prolongation of the AHP would therefore compensate to decrease firing rates, normalising the input-output function. From this perspective, the changes in C-boutons may potentially represent a homeostatic response to the AIS plasticity which may be particularly important under periods of intense drive to the motoneurones.14. One of the largest sources of excitatory sensory input to motoneurones are the Ia afferent synapses which mediate the stretch reflex. BoNT/A would alter this sensory input as the gamma motoneurone-intrafusal fiber connection also requires acetylcholine and is also blocked by intramuscular BoNT/A injections34. Such a block would result in a decreased sensitivity of the muscle spindles and consequently a reduced excitatory Ia input to the motoneurone which may trigger a homeostatic increase in AIS length in order to reduce rheobase to facilitate the afferent input reaching threshold. Ia afferent input may not be the only synaptic inputs to motoneurones that are affected. There are also experiments suggesting that after intramuscular injection, BoNT/A is not only retrogradely transported to the spinal cord35, but also travels transsynaptically and consequently may potentially be blocking other central synapses on the motoneurones36. Recent work extended on these findings showing that cleaved SNAP-25 can be visualised preferentially at cholinergic terminals on the soma of facial motoneurones following intramuscular BoNT/A injections37 suggesting that it is specifically blocking excitatory rather than inhibitory synaptic inputs to motoneurones. However, the question of whether, at clinically relevant doses, BoNT/A is actually transcytosed and the effects of this is a contentious issue. A recent study showed only sparse labelling of cleaved SNAP-25 in the spinal cord of rats following doses of BoNT/A at dosages close to those used in our experiments and suggested that the labelling seen in neighbouring neurones at higher dosages may be due to systemic spread7. In addition an in vitro study, using rat sympathetic neurones, suggested that while BoNT/A does migrate to the soma, no significant decrease in the amplitude of EPSPs recorded from the soma was seen at lower doses (but with significant decreases observed at much higher doses8). Further evidence to suggest that the reduction in C-bouton size observed in our experiments represents a homeostatic change rather than a toxic effect of the BoNT/A at the central cholinergic synapses are observations that the few central cholinergic synapses that had been shown previously to express cleaved SNAP-25 tend to increase in size due to vacuolisation in contrast to our observed general decrease in size37.The increases in AIS length following BoNT/A treatment may itself represent a homeostatic response either to the loss of functional connectivity at the neuromuscular synapse or changes in inputs to the motoneurones, rather than a direct toxic effect from the BoNT/A itself as injury to motoneurones tends to result in AIS shortening (unpublished data). Reductions in excitatory sensory input to neurones have been shown to induce homeostatic increases in AIS length in auditory neurones7) evidence that the BoNT/A once transported to the spinal cord, can spread to the contralateral spinal cord36, or can enter the blood system and go systemic introducing a possible caveat to our work in using the contralateral motoneurones as internal controls. Our measurements of C-bouton size for another study exploring the effects of axotomy38, performed at the same time using all the same reagents and antibodies produced similar measurements of C-bouton size on the control non-injured side (11.3 µm3 compared with the 11.7 µm3 observed on the control side on our BoNT/A experiments- unpublished observations) suggesting that contralateral C-boutons were not affected by the ipsilateral BoNT/A injections at this dosage. Extra control experiments performed later with a newer lot number of the VAChT antibody produced significantly less intense labelling in general making it invalid to apply the same threshold-based analysis with the same parameters to measure the C-boutons. Given that our AIS measurements are not affected by intensity but location of labelling and the same lot number of Ankyrin G was still available allowed us to confirm there were no effects of the BoNT/A injections on the AISs of contralateral motoneurones at this dose. This is consistent with findings that changes in excitability following BoNT/A are dependent on the degree of block12.There is also limited  and even for conditions such as migraine44 and chronic pain45. BoNT/A is also currently being used in younger patients, most notably in children with cerebral palsy46. It will therefore be crucial to determine if the changes we have observed (and the corresponding excitability changes) are irreversible.Importantly, all the work to date on excitability changes following BoNT/A have focused on the initial time period  after injection when the block is maximal. Whether the central excitability changes along with the anatomical changes that we have observed would return to normal upon full restoration of functional synaptic activity will be important to determine. Whilst a full restoration of central excitability cannot be discounted, neither can it be assumed. Similar, but more pronounced excitability changes are observed following axotomy and while most are returned to the pre-axotomy values after reinnervation of the muscle, not all are, including lasting impairments in proprioceptive inputs to the motoneuronesThe experimental procedures were approved by the Danish Animal Experiments Inspectorate (2014-15-0201-00442 and 2015-15-0201-00689) and were in accordance with the EU Directive 2010/63/EU.In 7 adult male rats  under isoflurane anaesthesia and aseptic conditions, the retrograde tracer, Cholera toxin subunit B , conjugated to Alexa Fluor 488, was injected bilaterally into gastrocnemius muscle . On one side, however, the CTB was mixed with 4IU BoNT/A  before injection. The side injected with CTB only served as an internal control. In one half of the rats the BoNT/A was injected on the right side and on the left side in the remainder.47. This went up to 76% at 6IU but with little additional change between this and 12IU units. Increasing dosages from 6 to 12IU, however, did result in an increasing effect on the contralateral limb (which in the current experiment we wanted to use as a control). Most importantly, the dosage we selected has previously been shown to result in enhanced stretch reflexes involving gastrocnemius motoneurones in rats at two weeks post injection10.The specific dosage of BoNT/A was selected to achieve a significant block whilst reducing confounds related to higher doses. Dose-response curves from another study using injections of BoNT/A into the whole rat triceps surae from 3IU up to 18IU show a 70% reduction in muscle force with dosages of only 3IUTM for oral intake). Two weeks after the injections the rats were transcardially perfused with 4% paraformaldehyde in phosphate buffer (pH 7.4).For pain management buprenorphine  was administered subcutaneously at the start of the procedure along with antibiotics  and buprenorphine was then administered post operatively for 3 days  BoNT/A was injected bilaterally into the gastrocnemius muscle but only on one side was the BoNT/A mixed with the tracer. The maximal CMAP was obtained before and 4 days after injection on both sides (under isoflurane anaesthesia). To do this, the sciatic nerve was stimulated using bipolar stimulating electrodes, increasing the intensity until the maximal CMAP was measured. This was measured using custom made silver ball electrodes that could be placed on the surface of the skin directly over the muscle. The signals were amplified and filtered using custom-made amplifiers (University of Copenhagen) and sampled and analysed using a 1401 digital to analogue converter run by Signal software . The maximum amplitude of the CMAP was measured peak to peak. In all rats a drop of at least 75% amplitude was observed on both sides confirming that the BoNT/A did not impair the functional ability of the BoNT/A.After perfusion, a laminectomy was performed and the lumbar enlargement of the spinal cord was removed. The lumbar cord was sectioned into 50 µm thick horizontal sections and the sections containing traced motoneurones were selected for immunohistochemistry. For AIS labelling an antibody against Ankyrin G  was used and for C-bouton labelling an antibody against the vesicular acetylcholine transporter was used . Immunohistochemistry was performed on free-floating horizontal sections. The tissue was first incubated in a blocking solution  for 4 hours followed by overnight incubation with the primary antibodies diluted in blocking solution. The tissue was then sequentially incubated in the dark for 2 hours for each secondary antibody - donkey anti-rabbit Alexa Fluor 568  and donkey anti-goat DyLight 650 . Sections were then rinsed, mounted and cover-slipped with fluorescent mounting medium (Dako Denmark).2 and with no C-boutons on the soma were excluded as they are presumed to be γ-motoneurones52. AISs originating from dendrites were also excluded.Tissue sections were imaged in Z-stacks with a Zeiss LSM700 confocal microscope, using a 20×/0.8 Plan Apochromat Air objective. For the AIS, image analysis was performed using ZEN 2.3 Black Software, and the following measurements were made: length of the AIS, distal and proximal width of the AIS, distance from the base of the axon hillock to the proximal end of the AIS and the 2D soma size calculated as an ellipse using the maximal and minimal diameter. Data from cells with an area of less than 600 µmC-bouton measurements were performed using a semi-automated script on Image J (NIH Software). All images were pre-processed using Background substraction and Gaussian blur (sigma = 1). Number and size (volume and surface area) of C-boutons was measured using the 3-D Object counter function. C-boutons were measured from cells which had somas entirely contained in the image and not overlapping with other cells. The number of boutons counted was then confirmed manually, excluding C-boutons that were not on the soma and correcting for adjoining boutons that were measured as one. Furthermore, the middle section of each cell was determined manually and the perimeter and the area of the soma in the respective Z-stack were measured using Freehand selection tool in Image J. C-boutons around the centre perimeter were then identified using the script described above and counted manually.All measurements were performed blinded and from the original raw data Z-stack files. Images for illustration were prepared using Image J (NIH Software) and Adobe Illustrator. Brightness and contrast were uniformly adjusted for image presentation using Image J.Statistical tests were performed using the GraphPad Prism software. Precise details of the individual statistical tests are provided in the results and figure legends. All tests were two-tailed. D’Agostino & Pearson omnibus normality tests were used to test for normality and parametric statistics or non-parametric tests were then applied appropriately. Statistical significance was accepted at the P ≤ 0.05 level. On all graphs, stars are used to indicate the following significant differences: *(P ≤ 0.05), **(P ≤ 0.01), ***. On the graphs, unless indicated, no significant difference was found. Initial analysis compared individual cells from the 2 groups. Further analysis compared the means between the two sides by animal to confirm if any differences were consistent across animals, although caution must be used with the interpretation of these particular statistics due to low statistical power.


IntroductionWe report our initial series of patients treated with radiosurgery to the Centromedian (CM) and Parafascicular (Pfc) Complex (CM-Pf) of the contralateral thalamus mainly for trigeminal neuralgia that had failed most known forms of conventional treatments. The coordinates were co-registered to a three-dimensional atlas of the thalamus in order to have a better comprehension of isodose curves distribution.MethodsA fully automated rotating gamma ray unit was used to deliver a high dose of radiation (140 Gy) using a 4-mm collimator to the CM-Pf of the contralateral thalamus in 14 patients suffering from refractory trigeminal pain and other complex pain syndromes. The best stereotactic coordinates were plotted in a thalamic three-dimensional atlas space along with isodose curves corresponding to 50% of the dose prescription and the dose gradient.ResultsFrom November 2016 to July 2019, 14 patients experiencing severe forms of different pain syndromes were treated, and 10 were eligible for follow-up evaluation. Pain deriving from trigeminal neuralgia was present in the majority (80%) of patients and from other complex pain syndromes in the rest (20%). Median follow-up was 384 days (range: 30-994). The Visual Analogue Scale (VAS) score before treatment was 9 (range: 7-10) and standardized to 10. Before treatment, all the patients had a Barrow Neurological Institute Pain Scale (BNI) of 5 (V). The median years suffering from pain was 4.5 years (range: 1-15), the number of procedures including radiosurgery to the trigeminal nerve before thalamotomy was four (range: 1-10). Most patients (90%) reported some form of relief, the average VAS at the time of response was 3.5 (range: 0-9), and the average time to response was 67.3 days (range: 2-210). The neuromodulation effect of radiation was seen in 60% of patients. The average BNI score at response was 2.7 (range: 1-5). The final VAS score at last follow-up was 5.5 (range: 0-10) in six patients. In four patients (40%), the procedure had failed with a final BNI of IV, and V, three patients (30%) had excellent response (BNI of I), and three patients (30%) had worthwhile results with BNI of IIIa and IIIb. The total success rate (BNI of I to IIIb) was 60%, and the number of patients experiencing more than 50% of pain reduction at final follow-up was five (50%). Excluding both patients that were treated for pain outside of trigeminal neuralgia, 75% of the patients responded. The best coordinates on average were X: 5.5 mm from the thalamic border, Y: 3.7 mm anterior to the posterior commissure, and Z: 3.7 mm from the intercomissural line. There were no complications to report.ConclusionRadiosurgery to the CM-Pf of the thalamus was demonstrated to be a safe and relatively effective alternative to treat refractory trigeminal neuralgia. Further studies are needed to optimize target dimensions based on the three-dimensional studies of isodose curves as well as coordinates. Longer follow-up is necessary to evaluate recurrence rates that could not be reached. Trigeminal neuralgia can constitute a true challenge for all modalities of treatments; the best results emerging from microvascular decompression, radiosurgery or percutaneous treatments still leave behind a small number of patients who do not obtain relief, particularly those suffering from secondary trigeminal neuralgia -8. Most What is more popularly known as “Medial thalamotomy” using radiosurgery for the management of pain dates to the origins of Gamma Knife® radiosurgery in the hands of Leksell . The funWe describe our initial experience with radiosurgical medial thalamotomy mainly for refractory trigeminal neuralgia using a rotating gamma ray system and describe the isodose curves coverage in relation to the CM-Pf with regards to the best clinical results that were obtained.Patient selectionWe conducted a retrospective study regarding radiosurgery to the CM-Pf for patients referred by pain and palliative medicine suffering mainly from anesthesia dolorosa in the trigeminal region or refractory trigeminal neuralgia that had completely failed radiosurgery to usual targets within the intracranial posterior fossa segments of the nerve at habitual doses of radiation (90 Gy). A complete failure of radiosurgery was defined as no perceived beneficial effect  six months after treatment. Other complex pain was included such as anesthesia dolorosa of the lesser occipital nerve and complex regional pain syndrome (CRPS) of the superior limb after failed cervical surgery. All patients were declared refractory to medical treatment including the addition of opioid therapy in whom no reasonable alternative intervention such as surgery was feasible. The trial was approved by the hospital’s ethical committee in November 2016. The inclusion criteria were patients with refractory trigeminal neuralgia with “complete failure” after radiosurgery to the nerve, patients that presented with anesthesia dolorosa secondary to surgical procedures, nerve sectioning or one case where there were no identifiable nerve portions due to tumor on stereotactic magnetic resonance imaging (MRI) studies. Also, the patients’ pain had to be categorized as severe (scoring 7 to 10) on the Visual Analog Scale (VAS) despite best medical algology practice and optimal pharmacological management. In addition, they had to be 18 years or older to enroll and needed to possess adequate family support to understand the expectations from treatment, the standardization of VAS to 10 before treatment, and how follow-up would be conducted. Interviews with patients and family members were routinely performed by the leading neurosurgeon and pain specialist. The cases were reviewed and approved by an ad-hoc committee that included a pain and palliative care specialist, neurosurgeon, and radiation therapy staff. Our main endpoints were the reduction of the VAS score by at least 50% and a reduction in pain medicine consumption as well as a monthly emergency room attendance of at least 25%.Radiosurgical techniqueOn the day of the procedure, patients fasted six hours and were prepared for sedation by the anesthesiologist. Under local anesthesia, an Infini™ stereotactic frame  was placed by a neurosurgeon, and the MR acquisition was performed with a 1.5-Tesla Avanto™ . Normally only one volumetric T2 of a 6-mm slice thickness with no spacing of the head (apex to foramen magnum) was acquired for defining the skin; T1 and T2 constructive interference in steady state of 1-mm slice thickness with no spacing from the corpus callosum covering the thalamus below the trigeminal nerve were also acquired. Images were transferred to the treatment planning station (Superplan®). For planning, the anterior commissure and posterior commissure (PC) were identified in the axial plane, then the images were transformed into the sagittal plane using fusion tools and the intercomissural line (ICL) was drawn between T1 and T2 sequences. The distance from the PC was taken anteriorly along the ICL  and confirmed and adjusted by taking 8 mm posterior to the midpoint of the ICL; this was identified as Y. A 90° angle was traced from the PC to the Y along the ICL, and Z was determined 3 to 4 mm above the ICL. Finally, images were reoriented to axial views, and X coordinates were 4 to 6 mm lateral from the contralateral thalamic border to the side of the pain. Using the 4-mm collimator, a single shot was placed and adjusted based on anatomy similitudes to the initial cases that provided the best response. Due to safety mechanisms specific to the Superplan® and the use of the prescribed high dose, we needed to place two shots in the same coordinates. The plan was reviewed and approved by radiation oncology and neurosurgery. The prescribed dose of 140 Gy to Dmax was administered to all patients with a gamma angle regularly fixed at 90°  was defined as a prompt relief of at least 25% of pain, usually occurring immediately after treatment or in a brief time span of fewer than 30 days. NER was usually categorized by being transient as pain relief was short, and pain recurs until definite sustained pain relief was obtained or failure of treatment was documented.Thalamic three-dimensional atlas plottingThe thalamic reconstruction is part of the São Paulo-Würzburg Atlas of the Human Brain, details on the histological processing and image registration pipelines can be found elsewhere . BrieflyFrom November 2016 to July 2019, 14 patients were treated, and 10 were eligible for follow-up evaluation, eight (80%) were female, and the mean age at treatment was 45 years (range: 22-72 years). The pain duration was 4.5 years (range: 1-15 years); the most affected side was the left side in seven patients (70%). Pain deriving from refractory or anesthesia dolorosa of the trigeminal neuralgia was present in the majority of the studied population (80%). One patient in the trigeminal pain group had anesthesia dolorosa secondary to a left maxillary sinus carcinoma resection and was the only patient categorized as a palliative oncological patient expected to survive less than six months. As for the other two patients eligible for follow-up who did not have trigeminal neuralgia pain, one experienced an anesthesia dolorosa of the left lesser occipital nerve do to multiple nerve blocks, radiofrequency, and subsequent surgical nerve sectioning and the other patient suffered from CRPS to the right superior limb after multiple cervical spine interventions and refusal to evaluate conventional neuromodulation therapy. The mean number of surgical or percutaneous procedures (either nerve blocks or in combination with radiofrequency and radiosurgery) to the trigeminal nerve was 4.6 (range: 1-10). The VAS score was 9 (range: 8-10) despite best medical treatment by pain and palliative medicine, and the VAS was standardized to 10. Before treatment, all the patients had a BNI of five (V). Of the patients suffering from trigeminal neuralgia, four experienced “complete failure” of radiosurgery to the trigeminal nerve at a habitual dose of 90 Gy. Median follow-up was 384 days  reported some form of relief. Six patients (60%) achieved the 50% or more pain relief threshold defined by the study characteristics as success. For four patients (40%), the procedure had failed as final BNI was IV and V, and pain never improved (VAS score less than 7). The NER was seen in 60% of the patients at a mean time of 8.5 days (range: 2-15 days) with a VAS score of 3 (range: 0-7). The duration of the NER effect lasted 15 days. In one patient, in whom thalamotomy was performed due to an inability to visualize the trigeminal nerve due to tumor, the pain had not recurred after 808 days. Another patient, in whom thalamotomy was used as a palliative alternative for anesthesia dolorosa secondary to a left maxillary sinus carcinoma resection, responded at 48 hours and died at 31 days after the procedure without pain.The average VAS score at the time of sustained response was 3.5 (range: 0-9); at 67.3 days (range: 2-210 days), the BNI score was 2.7 (range: 1-5). The final VAS score at last follow-up was 5.5 (range: 0-10). Three patients (30%) had an excellent response (BNI I), and three patients (30%) had worthwhile results . The total success rate by BNI I to IIIb was 60%, and the success rate by more than 50% of pain relief at last follow-up was 50%. One patient within the success group recurred after 798 days. Excluding both patients that were treated for pain different than trigeminal neuralgia, 75% of the patients responded. At last follow-up, one patient had died, and the rest had reduced their monthly visits to the emergency room at least 25% (range: 0%-100%), and 90% of the patients had a reduction of at least one of their pain medications, even in those with failed treatment failure (BNI IV and V).The best coordinates, on average, were X: 5 mm from the thalamic border, Y: 3.7 mm anterior to the posterior commissure, and Z: 3.7 mm from the intercommisural line. There were no statistical differences in coordinates to those whose treatment was considered a failure Table .There were no complications to report.Lesions and stimulation to the CM-Pf of the thalamus in order to alleviate intractable pain have a long neurosurgical tradition -13,15-17This protocol was implemented in our center as an alternative for those patients for whom radiosurgery to the nerve completely failed. This number in our initial series hovered between 14% and 16% of the population . Early rPossible neuromodulation effects by radiation, with sub-necrotic doses of radiation have been proposed by several authors. Ill-defined and poorly understood clinical results in epilepsy and trigeminal neuralgia patients who report immediate relief of symptoms or pain at the end of the radiosurgical procedure have been witnessed by most who practice functional radiosurgery and have been reported by some authors -21. OhyeThe areas that were irradiated regarding the isocenter and 50% of the Dmax (70 Gy) isodose curve in the present series correspond to the coordinates of the CM-Pf region adjacent and definitely intertwined with the ventroposteromedial (VPM) nucleus that, regardless of the steep dose gradients that characterize Gamma-ray (Knife) use, still cover an area with a diameter of 10 mm from the border of the thalamus to the farthest aspect of the isodose curve corresponding to 24 Gy Figure .A dose of 24 Gy is known to have a therapeutic effect in epilepsy trials, although this is by no means comparable to the effect of a thalamotomy . The lowInterestingly enough, all the patients in whom the procedure failed reduced their attendance to emergency units for pain management by at least 25%. We noticed that even though the treatment effect was not present at the beginning, family members reported fewer emergency unit visits prior to the feeling of pain relief. Habitual pain medication use was reduced in all but one patient. Due to the characteristics of this investigation and patients’ geographical origins and diverse healthcare systems, we could not adequately quantify these aspects that could potentially be of value.This is our first series that describes the clinical results of radiosurgery to the CM-Pf. The results as described seem useful as an alternative technique where most conventional alternatives to control pain have failed or are not viable in trigeminal neuralgia. This procedure seems safe, and three-dimensional atlas imaging provides potentially useful information regarding isodose line distribution in this area that can prompt further studies to refine the best areas to enhance clinical result. Recurrence rates are yet unknown if given enough time after treatment.


Trypsin-like serine proteases are essential in physiological processes. Studies have shown that N-glycans are important for serine protease expression and secretion, but the underlying mechanisms are poorly understood. Here, we report a common mechanism of N-glycosylation in the protease domains of corin, enteropeptidase and prothrombin in calnexin-mediated glycoprotein folding and extracellular expression. This mechanism, which is independent of calreticulin and operates in a domain-autonomous manner, involves two steps: direct calnexin binding to target proteins and subsequent calnexin binding to monoglucosylated N-glycans. Elimination of N-glycosylation sites in the protease domains of corin, enteropeptidase and prothrombin inhibits corin and enteropeptidase cell surface expression and prothrombin secretion in transfected HEK293 cells. Similarly, knocking down calnexin expression in cultured cardiomyocytes and hepatocytes reduced corin cell surface expression and prothrombin secretion, respectively. Our results suggest that this may be a general mechanism in the trypsin-like serine proteases with N-glycosylation sites in their protease domains. In the human genome, ~2% of the genes encode proteases, among which trypsin-like serine proteases are a major group . Most ofN-glycosylation is a common post-translational modification in proteins . About tCORIN and PCSK6 variants that impair corin cell surface expression and zymogen activation have been identified in patients with hypertensive diseases , a transmembrane serine protease, and prothrombin, a secreted serine protease. We found that N-glycosylation in the protease domain of corin, EK and prothrombin has a common role in regulating the extracellular expression of these proteases, which involves calnexin-assisted protein folding and ER exiting.N1022 is a conserved glycosylation site in the corin protease domain . AbolishThe cytoplasmic tail was shown to regulate corin intracellular trafficking . To testvs. resistant corin bands was higher in the N1022Q mutant than WT after CHX treatment for 4 hr (In Western blotting of lysates from cycloheximide (CHX)-treated cells, levels of WT and the N1022Q mutant decreased over time . After 8for 4 hr , indicatWe then co-stained corin and protein disulfide isomerase (PDI) in the cells. Without CHX treatment, WT or N1022Q corin and PDI staining mostly overlapped  with simTo identify proteins that interact differentially with corin WT and the N1022Q mutant, we treated the cells with dithiobis succinimidyl propionate (DSP), a protein cross-linker, and did proteomic analysis in samples co-immunoprecipitated with corin. A total of 387 proteins were detected . Among tTo show direct interactions between corin and calnexin or BiP, we immunoprecipitated corin in WT- and N1022Q-expressing cells and analyzed co-precipitated proteins by western blotting. Calnexin and BiP levels from N1022Q-expressing cells were 137 ± 9 and 562 ± 82%, respectively, of those from WT . In contIn calnexin-assisted glycoprotein folding , triglucWe next made a chimeric protein (CorinEK4N), in which the corin protease domain was replaced by the EK protease domain with four N-glycosylation sites , and addWe next studied EK , a transIn co-immunoprecipitation and western blotting, calnexin levels in EK-4Q- and PT-N416Q-expressing cells were 165 ± 12 and 171 ± 8%, respectively, of those in respective WT controls . BiP levWe verified our findings in murine HL-1 cardiomyocytes and human HepG2 hepatocytes expressing endogenous corin and prothrombin, respectively. In DNJ-treated HL-1 cells, cell surface corin levels were 26 ± 10% of untreated controls, as estimated by western blotting and densitometry . In DNJ-N-glycosylation is important in protein expression and function . PreviouCalnexin acts in glycoprotein folding . Unlike Human corin contains 19 N-glycosylation sites . Among tCalreticulin is a soluble calnexin homologous in the ER and acts as a key partner in the calnexin-calreticulin cycle . Like caThe importance of N-glycans in glycoprotein folding varies depending on proteins and cell types . In the In summary, we identify a common mechanism of N-glycosylation in the protease domains of corin, EK and prothrombin in calnexin-mediated folding and ER exiting. This process is calreticulin-independent, operates in a domain-autonomous manner, and involves two steps: direct calnexin binding to the target protein and subsequent calnexin binding to monoglucosylated N-glycans. Our findings suggest that this may be a general mechanism in the trypsin-like proteases with N-glycosylation sites in their protease domains. Naturally-occurring mutations disrupting such N-glycosylation sites may impair the expression and function of the trypsin-like serine proteases.The plasmids expressing corin WT and the mutants N1022Q and R801A were described . Human cHEK293 cells  were grown in DMEM with 10% fetal bovine serum at 37°C in humidified incubators. At 70–80% of confluency, the cells in six-well plates were transfected with the plasmids using Fugene reagents (Promega). To make stable cells expressing recombinant proteins, the transfected cells were cultured with G418 . After ~2 w, G418-resistant cells were selected and analyzed by western blotting.Recombinant proteins on the cell surface or in the conditioned media and lysates from the transfected cells were immunoprecipitated with an anti-V5 antibody  and protein A-Sepharose (Thermo Fisher) for western blotting, as described previously . AntibodHEK293 cells expressing corin WT or the N1022Q mutant in six-well plates were incubated with or without CHX . The cells were lysed at different time points for western blotting, as described above.Glycosidase Endo H was used to analyze N-glycans on corin in HEK293 cells. The cell lysates were incubated with Endo H  in 50 mM sodium acetate at 37°C for 1–2 hr. The Endo H-treated proteins were analyzed by Western blotting.HEK293 cells expressing corin were treated with CHX for 4 hr, fixed with 3% paraformaldehyde, permeabilized with 0.2% Triton X-100, and imminostained with antibodies against V5 (1:1000), PDI  or TGN46  and Alexa Fluor-594 or 488-labeled secondary antibody . In controls, the primary antibody was replaced by mouse  or rabbit  IgG. The stained cells were examined under a confocal microscope (Leica DM2500) and images were analyzed by ImageJ software.HEK293 cells expressing corin were incubated with dithiobis succinimidyl propionate (DSP)  at 4°C for 30 min. The reaction was stopped with 0.2 M glycine. Cell lysates were analyzed by immunoprecipitation and SDS-PAGE. Proteins on silver-stained gels were analyzed by liquid chromatography-mass spectrum at the Cleveland Clinic Proteomics Core to identify proteins interacting differentially with corin WT and the N1022Q mutant.Murine HL-1 cardiomyocytes were a generous gift from Dr. William Claycomb , as described previously . Human HTo digest cell surface proteins, HEK293 cells expressing corin or EK were incubated with trypsin  at 37°C for 10 min. After washing, cell lysates were prepared for western blotting.To examine effects of calnexin knockdown on corin expression in HL-1 and prothrombin expression in HepG2 cells, siRNAs targeting murine and human calnexin genes  and corresponding scrambled control siRNAs (Origene) were transfected using Lipofectamine reagents (Thermo Fisher). After 24–48 hr, the cells were collected. Calnexin, corin and prothrombin proteins were analyzed, as described above.t test was used to compare two groups with Prism (Graphpad). ANOVA followed by Tukey’s post hoc analysis was used to compare three or more groups. A p-value of < 0.05 was considered to be statistically significant.The sample size estimation was based on previous studies and pilot experiments. The Student’s  In the interests of transparency, eLife includes the editorial decision letter and accompanying author responses. A lightly edited version of the letter sent to the authors after peer review is shown, indicating the most substantive concerns; minor comments are not usually included.eLife. Your article has been reviewed by three peer reviewers, and the evaluation has been overseen by a Reviewing Editor, Charles Craik and a Senior Editor, Michael Marletta. The reviewers have discussed the reviews with one another and Charles Craik has drafted this decision to help you prepare a revised submission.Thank you for choosing to send your work entitled "N-glycosylation in the protease domain of trypsin-like serine proteases mediates calnexin-assisted protein folding" for consideration at The current work provides new evidence that protease domain N-glycosylation in the trypsin-like serine proteases corin, enteropeptidase and prothrombin is essential for extracellular protease expression and that elimination of protease domain N-glycosylation sites results in endoplasmic reticulum retention through the protein-protein interactions with the chaperone calnexin. The N-glycosylation-mediated folding and trafficking mechanism findings for this important class of enzymes are novel and interesting, experiments are carried out in a thorough, careful manner and the manuscript is well written with helpful, informative schematics that guide the reader through the experimental section.eLife's interest/mission. The reviewers raise valid points and a summary of our assessment follows:The manuscript is in principle within the scope of Appropriate controls for the immunoblots and the pull down experiments:Many of the conclusions in the article are based on subtle differences in protein expression levels as determined by semi-quantitative methods such as immunoblotting and immunocytochemistry. It is therefore important to include loading controls for all immunoblots as well as controls for the amount of protease immunoprecipitated for the V5 pull-downs as outlined by reviewer 1.Endogenous expression experiments:The experimental results presented in the manuscript are in heterologous expression systems using recombinantly expressed enzymes. Showing physiological relevance with endogenous protein would increase the biological significance of the findings. As described by reviewer 2, knock-down experiments with siRNA silencing to specifically target calnexin and/or BiP in cell lines that express one or two of the endogenously proteases under study is one approach that could address this concern. A complementary experiment would be DNJ treatment of cell lines expressing endogenous enzymes.Editorial comments:The authors are urged to provide greater clarity, particularly for the non-expert on why there is increased binding to N1022Q in the presence of DNJ and why PDI A3 and A4, which also showed enrichment in the N1022Q group proteomics studies were not pursued as pointed out by reviewer 3.Furthermore, does DNJ increase calnexin  binding of proteins other than trypsin fold serine proteases or is this is an effect specific to trypsin-fold serine proteases?Reviewer 1:This study reports new evidence that protease domain N-glycosylation in the serine proteases corin, EK and prothrombin is essential for extracellular protease expression and that elimination of protease domain N-glycosylation sites results in ER retention through the protein-protein interactions with the chaperone calnexin. The manuscript is well written and points towards new important functions for glycosylation of serine protease domains. Publication of the study is recommended with some corrections and additions.In general, loading controls for Western blots showing the total amount of protein loaded for each sample in is missing in most of the figures , which makes it difficult to conclude anything with certainty. A Western blot showing the expression level for a housekeeping gene  is needed for all blots. Furthermore, when using densiometry tools for quantification, the correct method is normalizing the amount of protein of interest (e.g. corin) to the loading control (e.g. GAPDH), to ensure that the difference in intensity is not due to variation in the amount of total protein loaded.In Figure 2A and B, the authors conclude that N1022 glycosylation promotes ER trafficking. These data are obtained by quantifying the protein expression levels as determined by the semi-quantitative method Western blotting followed by quantification using densiometric analysis on what appears to be two separate membranes, one for WT and one for N1022Q, which makes it difficult to compare. Although these are commonly used techniques in cell biology it is not a quantitative method and the intensity of the band is not necessarily linear for the protein concentrations detected in Figure 2A. Furthermore, a loading control is needed, as mentioned above. As impaired trafficking of the N1022Q mutant is one of the main conclusions from this study it would be nice to see this observation supported by other more quantitative techniques such as ELISA or similar.In Figure 2C, the authors conclude that N1022Q mutant is retained in the ER or early Golgi as compared to the WT based on Endo H sensitivity. Again Western blot analysis is used to quantify the amount of protein. The baseline protein expression of the N1022Q mutant appears higher than for the WT . Upon deglycosylation at timepoint 0 we see a down-switch in molecular weight for both WT and N1022Q, and the band intensity is comparable for + Endo H and –Endo H. In contrast, at timepoint 4 hours after CHX treatment, there is an inconsistency between the band intensity for WT -Endo H and WT +Endo H? The Endo H treatment should only affect the size of the protein, not the expression level ? Again, a loading control for the blot is needed as mentioned in the first point.In Figure 3, the authors write that corin N1022Q staining was "strong" compared to "little corin staining" in WT corin expressing cells after 4 h CHX treatment based on ICC analysis. This observation is not evident from the images shown in Figure 4A and B? Looking at Figure 3 there is still  corin in the WT  as compared to N1022Q ? It is also difficult to see the difference in overlap with the ER marker PDI and the WT versus the N1022Q as suggested by the authors.In Figure 4 the authors immunoprecipitate corin by a C-terminal V5 tag and find an increased level of the ER proteins calnexin and BIP in the N1022Q pull down as compared to WT corin pull down. In all previous figures comparing expression levels of N1022Q to WT it looks like there is more N1022Q in the lysate. It would be nice to see the amount of corin precipitated by the V5 tag for both the WT and the N1022Q mutant to rule out that the increased level of calnexin and BiP interaction in the N1022Q pull down is not due to more of the N1022Q protein precipitated as compared to WT.In Figure 5B a similar issue is found, as described above. It is not evident that the total amount of precipitated corin for the V5 pull down is comparable for WT and N1022Q. A Western blot of V5 immunoprecipitated corin is needed.In Figure 8 it is similarly important to show the amount of precipitated protease for both EK and prothrombin (PT) in the respective V5 pulldowns to exclude that the difference in calnexin and BiP levels is not due to varying levels of the protease in the samples.In summary, the study is extensive, systematic and reveals a potential new role for serine protease domain glycosylation in calnexin assisted protein folding and extracellular expression. However, most of the conclusions in the article are based on subtle differences in protein expression levels as determined by semi-quantitative methods such as Western blotting and immunocytochemistry. It is therefore crucial for publication to include loading controls for all Western blots as well as controls for the amount of protease immunoprecipitated for the V5 pull-downs.Reviewer 2:In the manuscript by Wang et al., with the title "N-glycosylation in the protease domain of trypsin-like serine proteases mediates calnexinassisted protein folding and extracellular expression" the authors set out to determine how N-glycosylation regulates the extracellular expression, secretion and activation of trypsin-like serine proteases. They report the identification of a common mechanism of N-glycosylation in the protease domains of corin, enteropeptidase and prothrombin in calnexin-mediated glycoprotein folding and extracellular expression. A substantial amount of data is included in the study and experiments are carried out in a thorough manner. The findings are novel and interesting to a wide audience; including scientists interested in basic biology of proteases as well as scientists focusing on post-translational modification of proteins and cellular trafficking.It is convincingly demonstrated that glycosylation at N1022 promotes corin exiting from the ER. Furthermore, increased N1022Q mutant corin binding to calnexin and BiP is shown. To demonstrate a functional role of calnexin and BiP for corin trafficking, cells were treated with DNJ, a glucosidase inhibitor. This experiment was performed based on the knowledge that in calnexin-assisted glycoprotein folding, triglucosylated oligosaccharides on nascent proteins are trimmed by α-glucosidases I and II to monoglucosylated oligosaccharides, allowing calnexin binding to N-glycans to assist protein folding. DNJ treatment indeed blocked calnexin binding to N-glycans on corin and impaired calnexin-assisted folding, resulting in increased direct corin binding to calnexin and BiP.1) While the DNJ experiments are informative, it cannot be conclusively established that calnexin and BiP are the essential players in corin glycoprotein folding and transport. The inclusion of selective silencing of calnexin and/or BiP by RNAi would provide valuable data and further substantiate their importance.2) All experiments are carried out in HEK293 cells using recombinant versions of serine proteases. The biological relevance is therefore unclear. Inclusion of data using cells expression endogenous protease(s) in combination with DNJ treatment and/or RNAi-mediated silencing of calnexin and BiP would greatly enhance the impact of the findings described here.In conclusion, the manuscript by Wang et al. is thorough and experiments are carried out in a careful manner. The findings are novel and interesting. A knock-down experiment to specifically target calnexin and/or BiP in cell lines that express one or two of the endogenously proteases under study (one or two different cell lines) would significantly enhance the biological relevance of this new exciting N-glycosylation-mediated folding and trafficking mechanism. These experiments would be expected to be manageable, relatively straight forward, and possible to carry out within a reasonable time-frame.Reviewer 3:This is well designed and executed study that adds to our understanding of the role of protease domain glycosylation of trypsin fold serine proteases. The results are clean and well interpreted. The manuscript has a good structure and the cartoons in the figures are very helpful to follow the experimental section. There are a couple of questions that need to be properly addressed and are also listed below.1) Figure 5B shows that DNJ increases calnexin and BiP binding to corin WT and to N1022Q (similar results for EK and prothrombin in later figures). I don't quite understand why there is increased binding to N1022Q in the presence of DNJ, since the N1022-attached glycan (which is absent in the mutant) seems to be the main driver for folding and export. Since the N1022Q mutant has no glycan attached at this position and, therefore, no glucose residues to begin with, it is not clearly understood (by me) why glucosidase I/II inhibition of the N1022Q mutant by DNJ would lead to even more unfolded (and retained) corin in the ER. Since in this case (N1022Q mutant) DNJ inhibits glycosylation at other sites (possibly 18 if I'm correct) the increased calnexin binding seems to be related to impaired trimming of some of these other glycan attachments. How then is the N1022 so important by itself? Maybe I have misunderstood this, but I urge the authors to work on improving the clarity on this section. In addition, the authors may comment in the Discussion as to whether DNJ increases calnexin  binding of proteins other than trypsin fold serine proteases or whether this is an effect specific to trypsin fold serine proteases (any literature on this?).2) Based on the proteomics results the authors have focused on calnexin and BiP, even though there are many other hits that came out of this experiment. A potentially relevant binding hit seems to be the PDI A3 and A4, which also showed enrichment in the N1022Q group (Table 1). Any reason why this was not pursued? […] The manuscript is in principle within the scope of eLife's interest/mission. The reviewers raise valid points and a summary of our assessment follows:Appropriate controls for the immunoblots and the pull down experiments:Many of the conclusions in the article are based on subtle differences in protein expression levels as determined by semi-quantitative methods such as immunoblotting and immunocytochemistry. It is therefore important to include loading controls for all immunoblots as well as controls for the amount of protease immunoprecipitated for the V5 pull-downs as outlined by reviewer 1.Loading controls in all immunoblots and controls for the V5 pull-downs have been included. Please see our responses to reviewer 1.Endogenous expression experiments:The experimental results presented in the manuscript are in heterologous expression systems using recombinantly expressed enzymes. Showing physiological relevance with endogenous protein would increase the biological significance of the findings. As described by reviewer 2, knock-down experiments with siRNA silencing to specifically target calnexin and/or BiP in cell lines that express one or two of the endogenously proteases under study is one approach that could address this concern. A complementary experiment would be DNJ treatment of cell lines expressing endogenous enzymes.The DNJ and siRNA silencing experiments have been performed in HL-1 cardiomyocytes and HepG2 hepatocytes, in which endogenous corin and prothrombin are expressed, respectively. The data, which are consistent with the results from HEK293 cells, have been included in new Figure 9. Please see our responses to reviewer 2.Editorial comments:The authors are urged to provide greater clarity, particularly for the non-expert on why there is increased binding to N1022Q in the presence of DNJ and why PDI A3 and A4, which also showed enrichment in the N1022Q group proteomics studies were not pursued as pointed out by reviewer 3.Human corin has 19 N-glycosylation sites. In the absence of DNJ, increased binding to calnexin and BiP in the N1022Q mutant, compared to WT corin, indicates the importance of N-glycans at N1022. In the presence of DNJ, all N-glycan-calnexin interactions are expected to be inhibited. Increased binding of N1022Q to calnexin, in the presence of DNJ, indicates that N-glycans at other N-glycosylation sites on corin are also involved in the calnexin interaction. We have revised the Discussion to clarify this point (second paragraph).In our proteomic studies, we chose 2-fold as the cut-off value for differential binding proteins. We provided this information in the footnote of Supplementary file 2. The N1022Q vs. WT ratios for PDI A3 and A4 were 1.24- and 1.67-fold, respectively, which were below the cut-off value.However, we do appreciate the Editor and reviewer’s comments. In this revision, we have included the cut-off information in the Results . We also performed new pull-down experiments using an anti-total PDI antibody , which showed similar PDI binding between WT corin and N1022Q. The results are included in the revised Figure 4A and D, and the text .Furthermore, does DNJ increase calnexin  binding of proteins other than trypsin fold serine proteases or is this is an effect specific to trypsin-fold serine proteases?DNJ is a glucosidase inhibitor. As calnexin and calreticulin are involved in the folding process of many glycoproteins, we do not expect the effect of DNJ to be specific to trypsin-like serine proteases. Surprisingly, only limited studies have been published in this area. In our PubMed search, we found only one report, in which DNJ treatment increased calreticulin binding to cruzipain, a protozoa cysteine protease . We did not find any papers reporting increased calnexin binding to trypsin-like serine proteases or other proteins in the presence of DNJ. In the revised Discussion, we have cited the Labriola paper (fourth paragraph).Reviewer 1:[…] In general, loading controls for Western blots showing the total amount of protein loaded for each sample in is missing in most of the figures , which makes it difficult to conclude anything with certainty. A Western blot showing the expression level for a housekeeping gene  is needed for all blots.The GAPDH controls were done in all our original Western blotting experiments. Some of them were not included in the original figures. These controls now have been added to Figure 1B and D, Figure 2A and C, Figure 6B and Figure 7C and D. For Western blots with cell membrane and secreted proteins, we have included Coomassie Blue-stained non-specific protein bands as controls for protein loading .Furthermore, when using densiometry tools for quantification, the correct method is normalizing the amount of protein of interest (e.g. corin) to the loading control (e.g. GAPDH), to ensure that the difference in intensity is not due to variation in the amount of total protein loaded.In our analyses to estimate protein cell surface expression and secretion, levels of recombinant cell surface proteins or secreted proteins were normalized to the total recombinant protein in cell lysates. Loading controls were also verified to ensure proper equal loading.In Figure 2A and B, the authors conclude that N1022 glycosylation promotes ER trafficking. These data are obtained by quantifying the protein expression levels as determined by the semi-quantitative method Western blotting followed by quantification using densiometric analysis on what appears to be two separate membranes, one for WT and one for N1022Q, which makes it difficult to compare.In this study, protein levels were normalized to the control sample at 0h from the same experiment and on the same blot. We have revised Figure 2 legend to clarify this.Although these are commonly used techniques in cell biology it is not a quantitative method and the intensity of the band is not necessarily linear for the protein concentrations detected in Figure 2A. Furthermore, a loading control is needed, as mentioned above. As impaired trafficking of the N1022Q mutant is one of the main conclusions from this study it would be nice to see this observation supported by other more quantitative techniques such as ELISA or similar.The GAPDH controls have been included in new Figure 2A. The conclusion of impaired trafficking is also supported by immunostaining  and increased calnexin and BiP binding . We have revised the Results to reflect this point .In Figure 2C, the authors conclude that N1022Q mutant is retained in the ER or early Golgi as compared to the WT based on Endo H sensitivity. Again Western blot analysis is used to quantify the amount of protein. The baseline protein expression of the N1022Q mutant appears higher than for the WT . Upon deglycosylation at timepoint 0 we see a down-switch in molecular weight for both WT and N1022Q, and the band intensity is comparable for + Endo H and -Endo H. In contrast, at timepoint 4 hours after CHX treatment, there is an inconsistency between the band intensity for WT -Endo H and WT +Endo H? The Endo H treatment should only affect the size of the protein, not the expression level ? Again, a loading control for the blot is needed as mentioned in the first point.The GAPDH controls have been added to Figure 2C. Indeed, comparing bands in WT and N1022Q with and without Endo H treatment is less desired. A better way is to compare the ratio of Endo H-sensitive vs. resistant bands within the same lane. Such analysis led to the same conclusion. We have revised the description in the Results .In Figure 3, the authors write that corin N1022Q staining was "strong" compared to "little corin staining" in WT corin expressing cells after 4 h CHX treatment based on ICC analysis. This observation is not evident from the images shown in Figure 4A and B? Looking at Figure 3 there is still  corin in the WT  as compared to N1022Q ? It is also difficult to see the difference in overlap with the ER marker PDI and the WT versus the N1022Q as suggested by the authors.In Figure 3, images in difference columns were from different cells. To ensure the visibility, photo exposure times may not be the same. Thus, images in different columns are not meant to be compared each other. In our analysis, we compared the intensity of corin (red) vs. PDI (green) staining in the same images, i.e. in the same individual cells. This should be clear when one looks at two bottom right panels in Figure 3A. We have revised the Results  and Figure 3 legend, to reflect this point.In Figure 4 the authors immunoprecipitate corin by a C-terminal V5 tag and find an increased level of the ER proteins calnexin and BiP in the N1022Q pull down as compared to WT corin pull down. In all previous figures comparing expression levels of N1022Q to WT it looks like there is more N1022Q in the lysate. It would be nice to see the amount of corin precipitated by the V5 tag for both the WT and the N1022Q mutant to rule out that the increased level of calnexin and BiP interaction in the N1022Q pull down is not due to more of the N1022Q protein precipitated as compared to wt.In Figure 5B a similar issue is found, as described above. It is not evident that the total amount of precipitated corin for the V5 pull down is comparable for WT and N1022Q. A Western blot of V5 immunoprecipitated corin is needed.We value the reviewer’s suggestion. In revised Figures 4A and 5B, corin proteins in V5-pull down samples are shown.In Figure 8 it is similarly important to show the amount of precipitated protease for both EK and prothrombin (PT) in the respective V5 pulldowns to exclude that the difference in calnexin and BiP levels is not due to varying levels of the protease in the samples.EK and PT were verified in the V5 pull-down samples and have been included in revised Figure 8.Reviewer 2:[…] 1) While the DNJ experiments are informative, it cannot be conclusively established that calnexin and BiP are the essential players in corin glycoprotein folding and transport. The inclusion of selective silencing of calnexin and/or BiP by RNAi would provide valuable data and further substantiate their importance.We have performed new DNJ and siRNA experiments in HL-1 cardiomyocytes and HepG2 hepatocytes. The results are consistent with the findings from HEK293 cells. The new data are included in new Figure 9.2) All experiments are carried out in HEK293 cells using recombinant versions of serine proteases. The biological relevance is therefore unclear. Inclusion of data using cells expression endogenous protease(s) in combination with DNJ treatment and/or RNAi-mediated silencing of calnexin and BiP would greatly enhance the impact of the findings described here.As indicated above, the suggested experiments have been done in HL-1 and HepG2 cells and the results are shown in new Figure 9.Reviewer 3:…1) Figure 5B shows that DNJ increases calnexin and BiP binding to corin WT and to N1022Q (similar results for EK and prothrombin in later figures). I don't quite understand why there is increased binding to N1022Q in the presence of DNJ, since the N1022-attached glycan (which is absent in the mutant) seems to be the main driver for folding and export. Since the N1022Q mutant has no glycan attached at this position and, therefore, no glucose residues to begin with, it is not clearly understood (by me) why glucosidase I/II inhibition of the N1022Q mutant by DNJ would lead to even more unfolded (and retained) corin in the ER. Since in this case (N1022Q mutant) DNJ inhibits glycosylation at other sites (possibly 18 if I'm correct) the increased calnexin binding seems to be related to impaired trimming of some of these other glycan attachments. How then is the N1022 so important by itself? Maybe I have misunderstood this, but I urge the authors to work on improving the clarity on this section. In addition, the authors may comment in the Discussion as to whether DNJ increases calnexin  binding of proteins other than trypsin fold serine proteases or whether this is an effect specific to trypsin-fold serine proteases (any literature on this?).As described above in our replies to the Editorial comments, human corin has 19 N-glycosylation sites. In the absence of DNJ, increased binding to calnexin and BiP in the N1022Q mutant, compared to WT corin, indicates the importance of N-glycans at N1022. In the presence of DNJ, all N-glycan-calnexin interactions are expected to be inhibited. Increased binding of N1022Q to calnexin, in the presence of DNJ, indicates that N-glycans at other N-glycosylation sites on corin are also involved in the calnexin interaction. We have revised the Discussion to clarify this point (second paragraph).Also described above, in our PubMed search we found only one report, in which DNJ treatment increased calreticulin binding to cruzipain, a protozoa cysteine protease . We did not find any papers reporting increased calnexin binding to trypsin-like serine proteases or other proteins in the presence of DNJ. In the revised Discussion, we have cited the Labriola paper (fourth paragraph).2) Based on the proteomics results the authors have focused on calnexin and BiP, even though there are many other hits that came out of this experiment. A potentially relevant binding hit seems to be the PDI A3 and A4, which also showed enrichment in the N1022Q group (Table 1). Any reason why this was not pursued?As indicated above in our replies to Editorial comments, we chose 2-fold as the cutoff value for differential binding proteins in our proteomic studies. We provided this info in the footnote of Supplementary file 2. The N1022Q vs. WT ratios for PDI A3 and A4 were 1.24- and 1.67-fold, respectively, which were below the cut-off value. However, we do appreciate the Editor and reviewer’s comments. In this revision, we have included the cut-off information in the Results . We also performed new pull-down experiments using an anti-total PDI antibody , which showed similar PDI binding between WT corin and N1022Q. The results are included in the revised Figure 4A and D.


Developmental dysgraphia is a disorder of writing/spelling skills, closely related to developmental dyslexia. For developmental dyslexia, profiles with a focus on phonological, attentional, visual or auditory deficits have recently been established. Unlike for developmental dyslexia, however, there are only few studies about dysgraphia, in particular about the variability of its causes. Research has demonstrated high similarity between developmental dyslexia and dysgraphia. Thus, the aim of the study was to investigate cognitive deficits as potential predictors of dysgraphia, analogously to those for dyslexia, in order to identify dysgraphia profiles, depending on the particular underlying disorder. Different tests were carried out with 3rd and 4th grade school children to assess their spelling abilities, tapping into phonological processing, auditory sound discrimination, visual attention and visual magnocellular functions as well as reading. A group of 45 children with developmental dysgraphia was compared to a control group. The results showed that besides phonological processing abilities, auditory skills and visual magnocellular functions affected spelling ability, too. Consequently, by means of a two-step cluster analysis, the group of dysgraphic children could be split into two distinct clusters, one with auditory deficits and the other with deficits in visual magnocellular functions. Visual attention was also related to spelling disabilities, but had no characteristic distinguishing effect for the two clusters. Together, these findings demonstrate that a more fine-grained diagnostic view on developmental dysgraphia, which takes the underlying cognitive profiles into account, might be advantageous for optimizing the outcome of individuum-centered intervention programs. Developmental dysgraphia is a disorder characterized by difficulties in the acquisition of writing/spelling skills despite adequate schooling, visus and normal IQ. It is closely related to developmental dyslexia, a disorder of the acquisition of reading skills, which has been more in the focus of investigation for the past years. As defined by the phonological processing abilities. For instance, automatization of linguistic, motor and cognitive skills is supported by the cerebellum  MorBecause of the heterogeneity of diverse underlying deficits, a lot of research has been conducted to identify profiles of developmental dyslexia e.g., . Heim etAs indicated in T-score < 37/percentile < 10 in a standardized German spelling assessment (see below) were assigned to the group of dysgraphic children. Children with a T-score ≥ 43  were assigned to the group of normally spelling children. The T-scores of 37 and 43 were chosen because of non-perfect reliability of the test: The 90% confidence interval for the T-score 40 denoting the lower boundary of the normal value range is 40 ± 3.2. Out of the 132 children who volunteered, 25 did not fit the inclusion criteria. Moreover, data sets of nine children were not complete and thus could not be included in the analysis, yielding a total of 98 (65 boys and 33 girls) valid and complete data sets. Fifty-four children were in 3rd grade, 44 in 4th grade. An overview of the sample of participants is shown in Table One hundred and thirty-two children and their parents agreed to participate. Out of these, those 98 children, who had a non-verbal IQ ≥ 70 and thus did not suffer from a general learning disorder according to the ICD-10; The volunteers for this study had been recruited from six different German primary schools and special education schools from Cologne and Mönchengladbach, which agreed to take part, furthermore from one practice for speech therapy in Aachen, in the period between March 2014 and April 2015. Parents were provided detailed information about the content of the study according to the Declaration of Helsinki . WrittenThere were two test sessions. Spelling abilities, IQ and reading abilities were tested in a group setting on the 1st day. On the 2nd day, dysgraphic and non-dysgraphic children who fulfilled the inclusion criteria stated above were tested individually for their performance in phonological processing, auditory sound discrimination, visual magnocellular functions, and visual attention. The order of tests on the 2nd day was counterbalanced over participants to avoid order effects. All tests were administered in a quiet room in the schools or in the practice for speech therapy.The tests for spelling ability and IQ were administered to check inclusion criteria and are therefore described first. The other tests served as the dependent variables when investigating for clusters in the dysgraphic sample. An overview of the different tests and their settings is presented in Table 1 (“Bert buys the _______.” [“book”]). This test provides T-scores for spelling accuracy.Spelling skills were tested with the German DRT-3  and one for reading performance (consisting of subtest 2–4), the latter entering the analysis. The test differentiates between monolingual and multicultural class norms (T-scores and percentile ranks) for grades 1 to 4, each in the middle or at the end of the school year. The tested classes hosted a variety of nationalities, so multicultural class norms were chosen.Reading competence of participants was assessed with the KNUSPEL-L3 . Childre5 (Rα:s] – [dak] – [lα:t]”: [dak] is pronounced with a short vowel in contrast to the other three words). Test 4 “Phoneme exchange” requires children to change the first two phonemes of words and pseudowords which are presented auditorily, . Separate norms for grades 1 to 4 were given for both tests. Because the scores of both tests were positively correlated in the previous study of r = 0.41; p < 0.001) and also in the present study , the average T-score was calculated for each child and used for further analyses as the measure for phonological awareness as in the The ability to work with the phonological structure of words like recognizing, segmenting, synthesizing and manipulating phonemes, syllables and onsets and rhymes was tested with the German test BAKO 1–45 . Two subT-scores constituted the basis for the analysis.Phonological working memory was tested with the “Mottier-Test” . Childre6  run on an Acer Travelmate 5744 laptop under Windows 7.All computerized tests were programmed and administered with Presentationn = 98) using SPSS 22 for Mac IOS . To ensure comparability, analysis in the present study was very similar to the analysis of the previous study about cognitive profiles of dyslexia  allow for the best separation of the whole dysgraphic group and the group of normally spelling children. We chose the discriminant analyses instead of a series of separate two-sample Next, a two-step cluster analysis was conducted to identify the optimum number of profiles in the dysgraphic sample. As previously done by p ≤ 0.05, exclusion criterion: p ≥ 0.10. Priors were set equal. Wilks’ lambda was calculated for each step. For the assignment of children to a particular group the leaving-one-out method was used to prevent biased (under-) estimates of misclassification rates.A series of linear discriminant analyses followed. The clusters were compared directly with each other and also with the control group. For all discriminant analyses the following settings were selected  was revalidated including only those dysgraphic children with no diagnosed reading deficits. To this end, the same parameter settings were used for a pre-defined 2-cluster solution. The coincidence in assignment of children with pure dysgraphia to the original clusters and the newly established clusters in that second analysis is reported in a 2 × 2 contingency table.p < 0.001) as well as in phonological awareness . Thirty-eight of the 45 dysgraphic children (84.4%) and 41 of the 53 normally spelling children (77.4%) were correctly assigned to their spelling skill groups on the basis of the set of selected cognitive variables using the leaving-one-out method, resulting in a total positive classification rate of 80.6% on the basis of the two variables for phonological processing. Table T-Scores and raw scores of the participants’ performance in the cognitive variables.The first discriminant analysis was employed to compare the whole group of dysgraphic children with the normally spelling children with respect to six dependent variables using a stepwise forward selection approach to find the best discriminating variables. The two groups differed significantly in phonological processing: in phonological working memory . The average T-Scores for spelling competence of the clusters and of the normally spelling group are displayed in Figure The cluster analysis of the dysgraphic group based on the set of six cognitive variables  yielded two clusters (Cluster 1: p < 0.001) and visual magnocellular functions  out of the profile of six variables contributed significantly to discrimination among both clusters. Cluster 1 could be identified as being significantly worse in visual magnocellular functions, Cluster 2 scored significantly worse in auditory sound discrimination – separated clusters with 16/17 (94.1%) correctly identified dysgraphic children in Cluster 1 and 27/28 (96.4%) in Cluster 2 with an overall correct assignment of 95.6%.In the subsequent discriminant analyses, the comparison of Clusters 1 and 2 revealed that auditory sound discrimination , and visual magnocellular functions . The overall rate of correct classifications was 91.4%, with 50/53 (94.3%) of the normally spelling children and 14/17 (82.4%) of the dysgraphic children in Cluster 1 correctly assigned.In a next step, Cluster 1 was compared to the normally spelling children. Cluster 1 differed from the normally spelling children in phonological working memory , auditory sound discrimination  and phonological awareness  with an overall classification rate of 87.7% .Children in Cluster 2 and control children differed in phonological working memory  for visual magnocellular functions and visual attention separately for each cluster and the control group.Figure p = 0.114; CVE: Wilks’ λ = 0.32; p = 0.341) and the normally spelling children .The two variables for visual attention revealed no significant differences between the clusters  and Cluster 2  showed significant differences in comparison to normally spelling children after Bonferroni-correction. The comparison of Clusters 1 and 2 revealed no mean difference  and thus confirmed and extended the previous results: phonological awareness deficits are a common factor for both dysgraphia clusters (as expressed in the significant t-tests) but explain independent variance to a different degree .Since the discriminant analysis revealed significant differences from controls in phonological awareness only for Cluster 2 but not for Cluster 1, an additional chi12 = 0.37, p = 0.546, grade: Pearson’s chi12 = 0.95, p = 0.758). The comparison of the whole group of dysgraphic vs. normally spelling children also revealed no sex differences  and no age differences .The chi-square analyses revealed no sex and grade differences across the clusters . In contrast, however, comparison of each cluster against normally spelling children revealed significant mean differences also after Bonferroni-correction: Normally spelling children vs. Cluster 1  and normally spelling children vs. Cluster 2 .In an additional analysis, we tested whether the actual degree of reading competence differed across the clusters and the normally spelling children. Mean and standard deviation of the reading skills of the two dysgraphic clusters and the normally writing children are displayed in Figure d = -1.75) and Cluster 2 vs. normally spelling children (Cohen’s d = -1.47).In a next step, the average scores for the variable reading competence of the two clusters versus normally spelling children were compared and revealed very high effect sizes : Clustern = 27 instead of n = 45) to the two clusters revealed that most children were assigned to the same clusters . The direct comparison of the two clusters revealed the differential impairment pattern in these profiles, with Cluster 1 demonstrating a visual impairment and Cluster 2 an auditory impairment.Even after excluding dyslexic children, the two clusters remained similar although the Cluster 1 group was reduced more extensively with 7 dysgraphic children left in contrast to the Cluster 2 group with 20 dysgraphic children remaining. After excluding dyslexic children 6 children fall in Cluster 1 and show a visual magnocellular deficit and 21 in cluster 2 with an auditory deficit. This leads to the conclusion that even if visual magnocellular functions play a role for spelling, auditory functions seem to influence dysgraphic children more often. The exact role auditory and visual functions play throughout the course of literacy development needs to be addressed further in longitudinal studies.In conclusion, the present study revealed new evidence that children with developmental dysgraphia are not homogeneous and that diverse cognitive variables, i.e., phonological awareness, phonological working memory, auditory processing and visual magnocellular function  are important for developmental dysgraphia. These findings show that spelling is a complex process, influenced by diverse cognitive variables, which should be taken into account for diagnosis and remediation. Usually only whole word and letter-by-letter reading/spelling are in the focus during the process of diagnosing dyslexics/dysgraphics. On the basis of the present data, inclusion of additional variables equivalent to those chosen for the study could be included in prediction and prevention programs. The question about the connection of developmental dysgraphia and dyslexia as well as their communalities and differences remains still unanswered and will be discussed below.The distributions of dysgraphic participants with vs. without accompanying reading difficulties across the clusters did not differ significantly in the present sample. These data can be taken to reflect either that the type of cognitive profile of a dysgraphic child is not influenced by his/her reading ability – or that the observed cognitive profiles have rather comparable impact on reading ability, at least in the sample studied here.In order to reflect the communalities of developmental dysgraphia and dyslexia, the new evidence about dysgraphic children will now be compared to the existing knowledge about dyslexia. In a first step, the underlying skills of the spelling and reading deficit are compared and in a second step, the clusters obtained in the present study are compared to those previously found by The present study corroborated the assumption that dyslexia and dysgraphia share several commonly underlying disorders : ProblemApart from phonological and auditory processing deficits, visual deficits have also been discussed as relevant for reading and dyslexia. Finally, visual attention requires additional consideration. In contrast to dyslexics e.g., , signifiIn summary, the present study revealed communalities with respect to deficits in underlying cognitive abilities of developmental dyslexia and dysgraphia, i.e., in phonological, auditory and visual magnocellular processing. The whole group of dysgraphic children in our study and dyslexics  share the fact that they have diverse underlying deficits and they (2) also share almost all of those deficits; they (3) moreover share the fact that impaired children can be subdivided into profiles and they (4) furthermore have in common that either impaired visual magnocellular functions or impaired auditory processing differentiate between dyslexic and dysgraphic children. With a closer look they also show differences in (5) the combination of those underlying deficits and finally (6) might differ with respect to possible profile groups. Thus, developmental dysgraphia and developmental dyslexia might be regarded as similar but not homologous with respect to their underlying cognitive profiles.Future research should include a broader diagnostic assessment approach, comprising a large pool of different functions assessed to investigate possible further dysgraphic profiles. Furthermore it would be inspiring to substantiate the new evidence with imaging techniques and thus get more precise information especially of the magnocellular system and the cerebellum with respect to visual and auditory processing. A more fine-grained analysis by contrasting exclusively dyslexic and exclusively dysgraphic children in comparison to children with a combined disorder of reading and spelling skills may reveal interesting information with respect to underlying additional deficits and eventually question the actually supposed communalities of the two disorders.The present study revealed new insights about underlying deficits and possible performance profiles of developmental dysgraphia and communalities of them with developmental dyslexia. In comparison to normally spelling children, dysgraphic children score worse in phonological processing skills, i.e., phonological awareness and phonological working memory. Based on six variables, measuring cognitive abilities and acquired skills, dysgraphic children could be subdivided into two profiles, one with an auditory and phonological processing  deficit and another with an impairment in visual magnocellular functions and phonological working memory. In summary, the present study revealed evidence for underlying deficits of developmental dysgraphia, i.e., phonological and auditory processing impairments as well as deficits in visual magnocellular functions. Finally, a comparison revealed that developmental dysgraphia and dyslexia are similar but not homologous. They share a common basis with different individual characteristics. As a consequence, it is reasonable to transfer this new evidence about impaired underlying functions to therapeutic everyday practice and conduct a more fine-graded diagnosis of dysgraphic children and consequently tailor remediation on the basis of the patient’s individual resources or barriers.DD: data acquisition, first manuscript draft, data analysis. DD, SH, and KW: concept, data analysis strategy, interpretation of results, revision of the manuscript drafts, and visualization of results.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.