Introduction {#Sec1}
============

Long-term sickness absence is of great concern in the developed industry because of high productivity loss, and high compensation and treatment costs \[[@CR1]\]. While most workers return to work (RTW) within the first months of sickness absence, one-third of sick-listed workers remain absent for a much longer period of time \[[@CR1], [@CR2]\]. The leading causes for long-term sickness absences are chronic disorders, based on mental, musculoskeletal and cardio-vascular health complaints \[[@CR3]\]. Most of these health complaints can be explained by well-defined diseases; however, there are also persistent subjective health complaints (SHC) that cannot be fully explained by such well-defined diseases \[[@CR4]\]. SHC refer to symptoms (e.g. fatigue, pain, dizziness) and syndromes (e.g. fibromyalgia, irritable bowel syndrome), for which no clear organic cause is currently found after appropriate medical examination. SHC are identical to other common terms, such as medically unexplained physical symptoms (MUPS) or persistent physical symptoms (PPS), which also refer to complaints with an unknown underlying pathology.

Research has suggested that long-term sick-listed workers with SHC have an increased risk of permanent disability, a weakened financial position, social isolation and a compromised quality of life \[[@CR5], [@CR6]\]. RTW is an important factor in reducing these economic, societal and personal consequences. In most European countries, physicians have to support sick-listed workers in their RTW process \[[@CR7]\]. Physicians, however, have reported difficulties in supporting the RTW process of sick-listed workers with SHC in particular, due to the lack of objective medical findings and limited knowledge on relevant factors in long-term sickness absence and RTW for workers with SHC \[[@CR8]\].

Most studies on long-term sickness absence and RTW have been performed for workers with well-defined diseases, specific physical symptoms or across several health conditions \[[@CR9]--[@CR12]\]. These studies have revealed that health-related factors, such as the severity of the disease and the symptoms, seem to become less relevant for RTW in long-term sickness absence than for RTW in short-term sickness absence \[[@CR9]--[@CR12]\]. External and psychosocial factors, such as self-perceived health and disability, job demands and strain, claim-related aspects, age, self-efficacy and own expectations for RTW seem to become more important for RTW in the later phases of sickness absence \[[@CR9]--[@CR12]\]. This suggests that the RTW process after long-term sickness absence benefits from a more phase-specific and multifactorial approach across several health conditions.

To date, little attention has been devoted to determine RTW factors for long-term sick-listed workers with SHC, and the evidence that is available is conflicting and of low quality \[[@CR13]\]. More knowledge of factors on RTW after long-term sickness absence for workers with SHC is highly relevant for physicians to better identify sick-listed workers with SHC and to better support these workers in their RTW process. Medico-legal criteria on which disability systems are often based together with the lack of objective medical findings can make it difficult to use SHC to claim work disability benefits \[[@CR14], [@CR15]\]. The system in the Netherlands, in which a well-defined medical disease is not a prerequisite for a work disability benefit, provides an unique opportunity to analyse relevant prognostic factors for RTW for long-term sick-listed workers with SHC \[[@CR16]\].

This study was designed to evaluate the prognostic factors for RTW for workers with SHC after long-term sickness absence (\> 84 weeks) and to compare these factors with the prognostic factors for RTW for long-term sick-listed workers with other disorders as a reference group. We believe that understanding the most relevant factors for RTW for long-term sick-listed workers with SHC can help reduce sickness absence among these workers and optimise their rehabilitation and RTW process. Our results will give physicians more insight into whether they should give comparable advice and suggest comparable interventions for RTW for long-term sick-listed workers with SHC and for those with other disorders.

Materials and methods {#Sec2}
=====================

Study population and design {#Sec3}
---------------------------

This study used data from the Forward study, a Dutch longitudinal cohort of 2593 out of 44,379 long-term sick-listed workers aged 18--65 years, who had been registered as sick-listed for at least 84 weeks between June 2014 and May 2015 in the electronic database of The Dutch Social Security Institute (UWV). The participants of Forward did not meet the exclusion criteria of Forward (unable to fill in questionnaires; no longer sick-listed; hospitalised; involved in judicial procedures; pregnant in the three months before study entry; suffering from cancer, a psychotic disease or dementia in the twelve months before study entry; and a PHQ-15 score of ≤5) \[[@CR17]\]. The Forward study followed the included participants for 24 months after baseline, and measurements with questionnaires were taken at baseline (T0), after one year (T1) and after two years (T2). Further information about the study population of Forward has been described comprehensively elsewhere \[[@CR18]\].

Figure [1](#Fig1){ref-type="fig"} shows the flowchart of the study sample for the present study. The present study selected 1250 participants out of the 2593 Forward participants. Participants were included if they were not returned to work at baseline, their work status had been fully documented in the questionnaires during follow-up and if they were clearly diagnosed with SHC (subjective health complaints) or another disorder. Information about diagnoses was derived from medical work disability assessment data of UWV. In the Netherlands, workers who are sick listed for at least 84 weeks can apply for a medical work disability assessment at UWV. These assessment result in a diagnosis by an insurance physician (IP) based on the International Classification of Diseases (ICD classification) \[[@CR19]\]. IPs can report 10 functional somatic syndromes: Chronic Fatigue syndrome, Fibromyalgia, Irritable Bowel Syndrome, Pelvic Girdle Pain, Repetitive Strain Injury, Somatic (Pain) Syndrome, Somatization disorder, Tension Headache, Tietze Syndrome and Whiplash \[[@CR20]\]. IPs can also report one of the 25 functional somatic symptoms that match with the 23 (partially) unexplained physical complaints of the Robbins list \[[@CR21]\]. For this study, participants were defined as suffering from SHC if the IP reported a functional somatic syndrome or symptom. All other participants with a clear diagnosis were defined as the reference group. Fig. 1Flow chart of the present study population

Measures {#Sec4}
--------

### Dependent variable {#Sec5}

The primary outcome measure was RTW (return to work) during follow-up, with RTW defined as a partial or full return to a paid job for a duration of at least 28 days. This outcome measure was based on self-reported answers to the follow-up questionnaires at T1 and T2. The answer options in the questionnaire were: partial or full return to my usual job or another paid job for ≥28 days;partial or full return to my usual job or another paid job for \< 28 days;no return to a paid job;no return to a job at all.

Participants who had partially or fully returned to their usual or another paid job for \< 28 days or who had not returned to a paid job or any job at all were combined into one category.

### Independent variables {#Sec6}

The selection of independent variables was based on literature regarding predictors for RTW and work ability outcomes in general \[[@CR9]--[@CR12]\]. The selected variables were divided into domains based on the biopsychosocial model. This is a universal, well-known conceptual framework that focuses on health conditions and internal and external contextual factors. It was chosen for this study as it is useful for assessing all aspects of disability and functioning \[[@CR15], [@CR22]\]. The independent variables were classified into the following four domains: DemographicSocio-economic and work-relatedHealth-relatedSelf-perceived ability

All variables were collected at baseline via self-reported questionnaires, except for the variable work disability benefits in the socio-economic and work-related domain, which was derived from UWV data after the medical work disability assessment.

#### Demographic domain {#FPar1}

The demographic domain included answers to questions about age (years), gender (male/female), marital or partner status (yes/no), breadwinner of the family (yes/no), land of birth (The Netherlands or another country), and educational level (primary school/secondary school/high school/bachelor and master).

#### Socio-economic and work-related domain {#FPar2}

The socio-economic and work-related domain contained answers to questions about the participants job and work status: collar job (blue/white/pink), being employed (yes/no), usual working time (hours), regular work schedule (yes/no), managerial position (yes/no), job demands (physical/psychological/combination of both), and previous absenteeism for the same reason (yes/no). This domain also contained information about work disability benefits (no/partial/complete) and about stressors and support, based on answers to the following validated questionnaire: The Work and Well-Being Inventory (WBI) questionnaire. The stressors and support variables were based on two subscales of the WBI. The stressors subscale contains 16 questions, with a scoring range between 16 and 64 (higher scores indicate more stressors); the support subscale contains 21 questions, with a scoring range between 21 and 84 (higher scores indicate better or more support) \[[@CR23]\].

#### Health-related domain {#FPar3}

The health-related domain included answers to questions about the use of specialist or psychiatric care in the last two years (yes/no) and the use of medication (yes/no). It also included information on the presence of a depressive or anxiety disorder, the severity of complaints, the physical and mental health, the presence of hypochondria, and symptom scale and coping strategies, based on answers to the following validated questionnaires: The Hospital Anxiety and Depression Scale (HADS). The presence of a depressive or anxiety disorder was assessed by using two subscales of the HADS. Each subscale contains seven questions about the presence of a depressive or anxiety disorder, with a scoring range of 0--21 for each separately. Scores of ≤7 mean no disorder (no), scores between 8 and 10 mean a possible disorder (maybe), and scores of ≥11 mean a definite disorder (yes) \[[@CR24]\].The Patient Health Questionnaire (PHQ-15). The severity of complaints was based on the PHQ-15. This questionnaire contains 15 questions about the severity of complaints, with a scoring range of 5--30. Scores between 5 and 10 represent mild somatic complaints, scores between 10 and 15 represent moderate somatic complaints, and scores of ≥15 represent severe somatic complaints \[[@CR17]\].The Short Form Health Survey 36 (SF-36). The physical and mental health (PCS and MCS) and the health change (SF-2) were measured by using the SF-36. PCS and MCS were measured by using a validated formula on total scores of the SF-36. The scoring range lies between 0 and 100 for each score separately, with higher scores indicating better levels of mental and physical health and functioning. The SF-2 was compiled from the following question on the SF-36: "How is your health in general compared with a year ago?" We categorised the five answering options of SF-2 into three categories: one category with the answers 'much better' and 'somewhat better' (better), one category with the answer 'no difference' (same), and one category with the answers 'somewhat worse' and 'much worse' (worse) \[[@CR25], [@CR26]\].The Whitely Index questionnaire (WI). The presence of hypochondria was measured with the WI. This questionnaire contains 14 questions, with a scoring range between 0 and 14. Scores between 0 and 8 mean 'no hypochondria' (no) and scores of ≥8 mean 'definitely hypochondria' (yes) \[[@CR27]\].The WBI questionnaire. The symptom scale and coping strategies were based on two subscales of the WBI. The subscale about the symptom scale contains 20 questions, with a scoring range between 20 and 80 (a higher score means a higher risk for symptoms); the subscale about coping strategies contains 21 questions, with a scoring range between 17 and 68 (a higher score means less coping) \[[@CR23]\].

#### Self-perceived ability domain {#FPar4}

The self-perceived ability domain contained one answer to a question about RTW expectations (yes or maybe/no). It also contained answers to the following validated questionnaires about disability, work ability in general and in the context of work load, and possibilities for RTW. The WBI questionnaire. Self-perceived disability was based on the disability subscale of the WBI. The subscale contains seven questions, with a scoring range between 7 and 28. Higher scores on this subscale mean more self-perceived disability \[[@CR23]\].The Work Ability Index (WAI). Perception of work ability in general and in the context of work load were both derived from the WAI. The category about work ability in general, also called The Work Ability Score (WAS), contains one question, with a scoring range between 0 and 10. The category about work ability in the context of work load contains two questions, with a scoring range between 2 and 10. For both categories higher scores indicate higher self-perceived work ability \[[@CR28]\].The Obstacles to Return to Work Questionnaire (ORQ). The self-perceived possibilities for RTW were derived from the subscale "Perceived Prognosis of Work Return" of the ORQ. This subscale contains six questions, with a scoring range between 0 and 36. Higher scores mean higher self-perceived possibilities for RTW \[[@CR29]\].

Statistics {#Sec7}
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For the analyses, participants were divided into two subgroups: workers with SHC and those with other disorders as the reference group. All further analyses were performed for both groups separately. Firstly, descriptive analyses were used to describe both groups at baseline. Secondly, to obtain information about possible predictors for RTW, univariable logistic regression analyses were performed for all independent variables per domain separately (i.e. demographic, socio-economic and work-related, health-related, and self-perceived ability), with the dependent variable partial or full RTW to a paid job for ≥28 days.

A cut-off *p*-value ≤0.157 \[[@CR30]\] was used for the univariable analyses. Multicollinearity between the variables was checked. Multicollinearity was assumed if the analyses showed variance inflation factor (VIF) scores of ≥10 \[[@CR31]\]. Variables that had a *p*-value ≤0.157 in the univariable analyses and a VIF score of \< 10 in the correlation analyses were included in a combined multivariable logistic regression analysis with backward selection per domain separately. In the next step, all variables that had a *p*-value ≤0.157 in the combined models per domain were included in one multivariable model. Odds ratios (OR) and 95% confidence intervals (95% CI) were calculated to show associations with RTW in this multivariable model. Subsequently, variables with a p-value ≤0.05 were combined in a final model. The Hosmer and Lemeshow test was performed and the Nagelkerke's R2 was assessed to measure the overall fit and the overall predictive ability of the final model \[[@CR31]\].

The analyses were based on complete case analyses. In complete case analyses, missing data may give bias due to selective loss to follow-up. To explore the robustness of the complete case analyses, missing data sensitivity analyses were also performed by using a multiple imputation approach \[[@CR32]\]. The analyses were identical for both approaches. SPSS version 24.0 and R-studio were used for all statistical analyses.

Results {#Sec8}
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Baseline characteristics of the participants with SHC (subjective health complaints) (*n* = 213) and of the reference group (*n* = 1037) are shown separately in Table [1](#Tab1){ref-type="table"}. On average, the participants with SHC were more often women, less often the breadwinner of the family, usually worked fewer hours and received less complete work disability benefits than the reference group (Table [1](#Tab1){ref-type="table"}). Table 1Baseline characteristics of the study populationDomainsSHC^a^ (N = 213)Other disorders (N = 1037)Categories/RangesMean/N^b^SD^c^/%Mean/NSD/%***Demographic*** Age in years18--34168%545%35--443617%12612%45--547535%35034%55--658640%50749% GenderMale7636%56154% Marital statusMarried or partner15271%77775% Breadwinner of the familyYes12760%67665% Land of birthThe Netherlands19391%94791% Educational levelNone/Primary school2411%878%Secondary school7837%41941%High school6832%33532%Bachelor/Master4320%19419%***Socio-economic and work-related*** Collar jobBlue6029%33734%White8742%33634%Pink6129%31632% EmployerYes7736%36637% Usual working time in hours4--6031.6410.3432.6910.91 Work scheduleRegular14468%66464% Managerial positionYes4220%22722% Job demandsPsychological4823%24424%Physical7736%33032%Psychological and physical8841%45644% Stressors16--6438.569.4438.279.25 Support21--8458.2013.0258.9012.73 Previous absenteeism same reasonYes10951%47947% Work disability benefitNo5126%14414%Partial5025%25726%Complete9949%60060%***Health-related*** Use of specialist care last 2 yearsYes19190%87985% Use of psychiatric care last 2 yearsYes12056%48647% Use of medicationYes19692%92289% Depressive disorderNo6832%38337%Maybe5024%23323%Yes9444%41940% Anxiety disorderNo8239%41240%Maybe4421%24423%Yes8640%38137% Severity of complaintsMild3918%34433%Moderate5626%36235%Severe11856%33132% Physical Health0--10029.738.8931.579.79 Mental Health0--10033.3212.5834.6813.81 Health change comparing last yearWorse12458%55654%Same5124%30629%Better3718%17417% HypochondriaYes15573%70668% Symptom scale20--8046.6011.1644.9412.05 Coping strategies17--6843.409.7742.709.52***Self-perceived ability*** Return to work expectationYes or maybe14870%68866% Disability7--2825.313.5024.843.64 Work ability in general0--101.991.782.201.95 Work ability in context of work load0--104.131.504.281.62 Possibilities for returning to work0--369.837.819.538.15^a^SHC = Subjective Health Complaints^b^N = Number^c^SD = Standard Deviation

RTW (return to work) predictors for participants with SHC {#Sec9}
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Of the 213 participants with SHC, 47 participants (22%) returned to work. For RTW after two years of sickness absence we found significant univariable associations (*P* ≤ 0.157) in the domains demographic, socio-economic and work-related, health-related and self-perceived ability (Table [2](#Tab2){ref-type="table"}). We found no multicollinearity for any of the variables in the domains (data not shown). We used backward selection and further select one or two variables with a *P*-value ≤0.157 in all four domains, which we combined in a multivariable analysis. One variable in the socio-economic and work-related domain and one variable in the self-perceived ability domain remained statistically significant (*P* ≤ 0.05) (Table [3](#Tab3){ref-type="table"}), which we combined in a final multivariable model. In the final model, we found that the chance of RTW after two years of sickness absence decreased if participants obtained a partial (OR 0.62, 95% CI 0.26--1.47) or a complete (OR 0.24, 95% CI 0.10--0.58) work disability benefit after these two years. In addition, we found that a higher self-perceived possibility for RTW increased the chance for RTW after two years of sickness absence (OR 1.06, 95% CI 1.01--1.11). The Hosmer and Lemeshow test was not statistically significant (*P*-value 0.19), indicating that there was a good fit of the final model, and the Nagelkerke's R2 was 0.22. Table 2Univariable logistic regression analyses of potential predictors for participants with SHC^a^ and other disorders separatelyDomainsSHC^a^ (N = 213)Other disorders (N = 1037)Categories/RangesOR^b^95% CI^c^OR95% CI***Demographic*** Age in years18--34Reference**Reference**^d^35--440.480.13--1.72**0.710.36--1.37**45--540.320.10--1.04**0.520.29--0.95**55--650.540.18--1.66**0.230.12--0.42** GenderMaleReferenceReferenceFemale0.870.44--1.690.870.64--1.18 Married or partnerNoReferenceReferenceYes0.820.41--1.650.990.70--1.41 Breadwinner of the familyNoReferenceReferenceYes1.410.72--2.781.100.80--1.52 Land of birthThe Netherlands**ReferenceReference**Other country**2.631.01--6.881.480.90--2.42** Educational levelNone/Primary school**ReferenceReference**Secondary school**1.270.33--4.941.860.89--3.87**High school**2.710.73--10.172.761.33--5.76**Bachelor/Master**3.030.77--11.982.771.29--5.95*Socio-economic and work-related*** Collar jobBlueReferenceReferenceWhite1.010.46--2.240.990.68--1.43Pink1.080.46--2.540.990.68--1.44 EmployerNoReferenceReferenceYes1.120.57--2.201.170.86--1.60 Usual working time in hours4--601.000.97--1.03**1.011.00--1.03** Work scheduleIrregularReferenceReferenceRegular0.810.41--1.590.940.69--1.28 Managerial positionNoReference**Reference**Yes1.130.51--2.51**1.721.22--2.41** Job demandsPsychologicalReferenceReferencePhysical0.670.28--1.600.810.54--1.21Psychological and physical0.940.42--2.130.880.60--1.28 Stressors16--640.990.95--1.021.010.99--1.03 Support21--841.000.98--1.03**0.990.98--1.00** Previous absenteeism same reasonNoReferenceReferenceYes1.100.57--2.100.920.68--1.25 Work disability benefitNo**ReferenceReference**Partial**0.590.25--1.380.620.41--0.94**Complete**0.190.08--0.450.080.05--0.12*Health-related*** Use of specialist care last 2 yearsNoReference**Reference**Yes0.960.33--2.75**0.540.37--0.79** Use of psychiatric care last 2 yearsNoReferenceReferenceYes1.060.55--2.041.130.84--1.53 Use of medicationNoReference**Reference**Yes2.240.49--10.14**0.440.29--0.67** Depressive disorderNoReferenceReferenceMaybe0.420.16--1.101.450.98--2.15Yes0.740.36--1.521.120.79--1.60 Anxiety disorderNoReferenceReferenceMaybe1.190.50--2.801.050.70--1.57Yes0.940.45--1.971.230.87--1.74 Severity of complaintsMildReference**Reference**Moderate1.290.48--3.46**0.830.58--1.19**Severe1.040.43--2.55**0.660.45--0.97** Physical Health0--100**1.030.99--1.061.051.03--1.06** Mental Health0--1001.010.99--1.041.000.98--1.01 Health Change comparing last yearWorse**ReferenceReference**Same**0.780.32--1.861.801.26--5.57**Better**2.841.29--6.283.132.12--4.64** HypochondriaNo**Reference**ReferenceYes**0.570.28--1.13**0.920.66--1.26 Symptom scale20--800.980.95--1.011.000.99--1.02 Coping strategies17--681.000.97--1.041.000.98--1.01***Self-perceived ability*** Return to work expectationNo**ReferenceReference**Yes or maybe**2.421.06--5.533.472.32--5.19** Disability7--28**0.910.84--0.990.860.83--0.89** Work ability in general0--10**1.301.09--1.561.351.25--1.46** Work ability in the context of work load0--10**1.261.01--1.571.341.22--1.47** Possibilities for returning to work0--36**1.081.04--1.121.131.11--1.15**^a^SHC = Subjective Health Complaints^b^OR = Odds ratio^c^95% CI = 95% confidence intervals^d^Numbers in bold had a p-value of ≤0.157Table 3Multivariable logistic regression analysis of all predictors for participants with SHC^a^ and other disorders separatelyDomainsSHC^a^ (N = 213)Other disorders (N = 1037)Categories/RangesOR^b^95% CI^c^OR95% CI***Demographic*** Age in years18--34**Reference**^d^35--44**0.910.39--2.16**45--54**0.720.32--1.61**55--65**0.390.17--0.88** Land of birthThe NetherlandsReferenceReferenceAnother country2.610.88--7.771.140.61--2.15 Educational levelNone/Primary schoolReferenceReferenceSecondary school1.130.26--4.951.250.52--2.99High school1.950.45--8.401.710.71--4.07Bachelor/Master2.220.49--10.121.520.60--3.82***Socio-economic and work-related*** Usual working time in hours4--601.010.99--1.03 Manegerial positionNo**Reference**Yes**1.561.00--2.45** Work disability benefitNo**ReferenceReference**Partial**0.710.29--1.780.660.40--1.08**Complete**0.260.10--0.660.120.07--0.20*Health-related*** Use of medicationNoReferenceYes0.880.51--1.50 Physical Health0--1001.000.98--1.03 Health Change comparing last yearWorseReferenceReferenceSame0.510.18--1.450.980.63--1.54Better1.310.49--3.510.840.48--1.48***Self-perceived ability*** Work ability in general0--10**1.111.00--1.24** Possibilities for returning to work0--36**1.051.00--1.111.081.05--1.11**^a^SHC = Subjective Health Complaints^b^OR = Odds ratio^c^95% CI = 95% confidence intervals^d^Numbers in bold had a p-value of ≤0.05 and were combined the final model

RTW predictors for the reference group (participants with other disorders than SHC) {#Sec10}
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In the reference group (*n* = 1037), 211 participants (20%) returned to work. We found significant univariable associations (*P* ≤ 0.157) in all four domains for RTW (Table [2](#Tab2){ref-type="table"}), and no multicollinearity in any of the domains (data not shown). After the backward selection, all four domains contained two or more significant variables (P ≤ 0.157), which were combined in a multivariable model (Table [3](#Tab3){ref-type="table"}). We analysed the five remaining significant variables (*P* ≤ 0.05) in a final multivariable model. In the final model, the demographic domain showed that older participants were less likely to RTW (OR 0.37, 95% CI 0.16--0.81). For the socio-economic and work-related domain, we found that participants who previously worked in a managerial position were more likely to return to work (OR 1.64, 95% CI 1.06--2.53). If participants received a partial or complete work disability benefit, they returned to work less often (OR 0.69, 95% CI 0.43--1.12 and OR 0.12, 95% CI 0.07--0.20). Within the domain of self-perceived ability, those who reported a good self-perceived work ability (OR 1.11, 95% CI 1.00--1.23) and a high possibility to RTW (OR 1.08, 95% CI 1.05--1.11) more often returned to work. The Hosmer and Lemeshow test was not statistically significant (*P*-value 0.82) in the final model, indicating that there was a good fit of the model. The Nagelkerke's R2 was 0.37.

Missing data {#Sec11}
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Missing data analyses showed that participants with an unknown RTW outcome differed significantly from the participants with a known RTW outcome. Participants with an unknown RTW outcome reported less good health, more complaints, less socio-economic status and less support (Additional file [1](#MOESM1){ref-type="media"}). Although the sensitivity analyses did not show any differences on regression coefficients in the multivariable and final model (Additional file [2](#MOESM2){ref-type="media"}), this meant that we could not completely rule out that the missing data was not merely a coincidence \[[@CR32]\]. Therefore, we included only the results of the complete case analyses in this study; however, the results of the missing data analyses are presented in the supplementary materials for comparison (Additional files [1](#MOESM1){ref-type="media"} and [2](#MOESM2){ref-type="media"}).

Discussion {#Sec12}
==========

The main purpose of this study was to evaluate prognostic factors for RTW (return to work) after long-term sickness absence for workers with SHC (subjective health complaints). In our Dutch population, we found that receiving a work disability benefit after two years of sickness absence significantly predicted less RTW, and that high self-perceived possibilities for RTW resulted in more RTW after those two years for workers with SHC. These prognostic factors for RTW, as well as the number of workers that returned to work, were comparable for the reference group with other disorders; however, we found three additional factors that predicted RTW for the reference group: a lower age, a previous managerial position and a high self-perceived work ability. Our results suggest that non-health-related factors are more important than health-related factors in predicting RTW after long-term sickness absence.

Our results reveal that receiving a work disability benefit after two years of sickness absence is negatively related to the chances of returning to work successfully for workers with SHC. While some previous studies have supported that claim-related factors and compensation status are indeed associated with poorer health, longer sickness absence and less RTW \[[@CR6], [@CR33], [@CR34]\] the literature in general has not paid much attention to this topic \[[@CR35]\]. It is therefore difficult to determine whether it is poorer health status that leads to compensation and less RTW, or whether receiving compensation is a factor in RTW in and of itself. The literature that is available on this topic seems divided \[[@CR36]--[@CR39]\].

Our results seem to show an anti-therapeutic effect of disability compensation, as not the severity of the complaints but receiving work disability benefits had a negative influence on RTW for workers with SHC. The exact underlying mechanisms of this anti-therapeutic effect, however, are still difficult to determine \[[@CR34]\]. Cassidy et al. \[[@CR36]\] have argued that it could in part be explained by the theory of financial incentives, or secondary gain, as they found that removing the compensation increased health in workers with SHC. The explanation behind this hypothesis is that workers with SHC focus more on proving that their health complaints are real in the claim process at the expense of their RTW options because they are reluctant to RTW (i.e. less RTW willingness) for fear of losing their compensation and the validation of their being disabled \[[@CR1], [@CR37]\].

In contrast to the anti-therapeutic effect suggested by Cassidy et al. and others \[[@CR36], [@CR37]\], it is important to take into account that workers, regardless of their own feelings of recovery status, may be forced to RTW or to seek for another compensation because of financial necessity if they are not eligible for a work disability benefit. Although information about the course of those workers is scarce, the limited evidence on this topic revealed a high mental impact \[[@CR38]\]. In addition, some studies have suggested that the process of applying for compensation can in fact make people more ill \[[@CR39]--[@CR41]\]. This is explained in terms of the distress caused by these claim settlement processes outweighing the possible positive effect of the expectation of gain \[[@CR39], [@CR40]\]. Importantly, this is irrespective of the underlying cause of the injury or the underlying pathology of the disease \[[@CR41]\]. This is in line with the results of this study where the effect of a work disability benefit was not only valid for workers with SHC: we found comparable results for workers with other disorders. The results of this study corroborates the view that the process of applying for or receiving a disability compensation in and of itself may be a greater risk factor for permanent disability and less RTW than the severity and underlying pathology of the complaints and the health status in and of itself.

We also found that workers' self-perceived possibilities for RTW was one of the most important factors for workers with SHC as well as for workers without SHC after long-term sickness absence. Young et al. \[[@CR42]\] state that researchers have assumed that health-related factors, which were found as important factors for RTW in short-term sickness absence, remain the most relevant predictors for RTW after long-term sickness absence. However, a body of evidence supports the theory that for several chronic disorders, including persistent SHC, the importance of precipitating factors for RTW shifts during the sickness absence process \[[@CR9], [@CR10], [@CR12], [@CR43]\]. In fact, some studies on RTW after long-term sickness absence have indeed highlighted the workers' own expectations for RTW as an important factor \[[@CR9]--[@CR12]\] and have shown that health-related factors become less important during sickness absence \[[@CR13]\]. The present study indicates that this effect on RTW is indeed true for all workers: health-related factors, such as the underlying pathology and the severity of the disorder, became less relevant, and the non-health-related factors, such as the self-perceived expectations, became more relevant, for RTW after long-term sickness absence. In addition, factors that seem to be especially important for RTW for workers with other disorders than SHC, can also be classified as non-health-related factors. Contrasting to our expectations based on the literature beforehand \[[@CR5]--[@CR8]\], we found similar rates in RTW for workers with SHC and those with other disorders, which also corroborates the comparable results between these two groups in the present study.

Strengths and limitations {#Sec13}
-------------------------

The main strengths of this study were the use of broad data from participants from all regions of the Netherlands, which increases generalisability, and its prospective design. We asked workers to participate in the study when they were already sick-listed for two years, but just before their medical work disability assessment. We followed them for another two years, even if they were not granted a work disability benefit. This provided a unique opportunity to follow workers on RTW after long-term sickness absence, and to include the effect of a work disability benefit.

There are also some weaknesses in the present study. The first is the small response rate, due to the manner in which we included the participants. Because of stringent privacy regulations, we were not able to make a selection of workers beforehand. Therefore, we asked all 44,379 workers in the electronic database of UWV (The Dutch Social Security Institute) who were registered as sick listed for ≥84 weeks to participate in the study. They were asked to fill in a checklist without assistance and to respond only if they did not meet one of the criteria on the checklist and wanted to participate in the study. Out of the approached workers 9% responded, which is lower than average \[[@CR44]\].

A second weakness follows from the first: we could not obtain more information about the non-responders as their data was not available. While it is certain that many workers who received a participation letter would normally not have been contacted, we may still assume -- based on the high rate of non-response -- that the characteristics of the study population may have caused some selection bias. It is possible that the non-responders were unhealthier than the responders, with possibly as result more positive outcomes in the present study. We did find, however, that the study sample was quite comparable with earlier studies on RTW for workers with other chronic diseases \[[@CR13]\].

A further conceivable weakness is that as an outcome measure in research, data on sickness absence gathered from data files is preferable to data based on questionnaires \[[@CR45]\]. However, questionnaires may still be considered a valuable source of information on overall sickness absence, and we had to use the questionnaires for the outcome measure due to the fact that this data on RTW after long-term sickness absence was not available in the UWV records.

In addition, missing follow-up questionnaires and missing answers in submitted questionnaires led to the exclusion of one-third of the respondents. However, the sensitivity analyses between the complete case analyses and the multiple imputation analyses for all participants showed comparable results on the regression coefficients in the final models. We take this to mean that there is missing at random (MAR), and that the data in the complete case analyses is robust, unselective and also representative for other workers.

Implications for practice and future research {#Sec14}
---------------------------------------------

Based on the present study, support of RTW after long-term sickness absence has to be based especially on modifiable non-health-related factors, irrespective of the underlying pathology of the disorder. Previous studies have reported that delayed recovery could be improved by the implementation of more assistance, less medical assessments that have no therapeutic value, more personalized assessments, and more clarity in decision making in order to reduce the stressfulness for workers in the claim management process \[[@CR39], [@CR46], [@CR47]\]. In addition, previous studies have reported that behaviour change interventions and interventions on self-efficacy may have the potential of optimizing the RTW process \[[@CR48], [@CR49]\]. However, more research is required to better examine the important underlying factors for positive RTW expectations and which interventions can help to change negative expectations for RTW into positive ones.

Conclusion {#Sec15}
==========

Not receiving a work disability benefit and having positive expectations for RTW are the most important factors in RTW successfully after long-term sickness absence, both for workers with SHC as for those with other disorders. This suggests that non health-related factors are more important than health-related factors to predict RTW after long-term sickness absence.

Supplementary information
=========================

 {#Sec16}

######

**Additional file 1.** Missing data analyses of the baseline characteristics of the present study population.

######

**Additional file 2.** Multivariable logistic regression multiple imputation analysis (pooled data) of all potential predictors for participants with SHC^a^ and other disorders separately.

CI

:   95% confidence intervals

HADS

:   Hospital anxiety and depression scale

MUPS

:   Medically Unexplained Physical Symptoms

OR

:   Odds ratio

ORQ

:   Obstacles to return to work questionnaire

PHQ-15

:   Patient health questionnaire-15

PPS

:   Persistent Physical Symptoms

RTW

:   Return to work

SF-36

:   Short form health survey-36

SHC

:   Subjective health complaints

UWV

:   Dutch social security institute

VIF

:   Variance inflation factor

WAI

:   Work ability index

WAS

:   Work ability score

WBI

:   Work and well-being inventory

WI

:   Whitely index

**Publisher's Note**

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Supplementary information
=========================

**Supplementary information** accompanies this paper at 10.1186/s12889-020-09203-5.

Ms. M. Maaker-Berkhof, employee of Amsterdam UMC location Vrije Universiteit Amsterdam, assisted in preparing and processing the data of the Forward study.

KW and KB prepared, processed, analysed and interpreted the data of the Forward study. MH was also a major contributor in analysing and interpreting the data. KW, FS, JA and AB were major contributors in writing the manuscript. All authors have seen, read and approved the first and revised version of the manuscript being submitted. The article is the authors' original work, hasn't received prior publication and isn't under consideration for publication elsewhere.

This study was supported by The Dutch Social Security Institute (UWV) (funding number 2002045). The funding organisation had no further role in the analysis and interpretation of the data, nor in writing the paper or in the decision to submit the paper for publication.

The datasets generated and/or analysed during the current study are not publicly available due to possible individual privacy compromising but are available from the corresponding author on reasonable request.

The Medical Ethics Committee of Amsterdam UMC, Vrije Universiteit Amsterdam, gave Ethical approval for the study (reference number 2014--197). They declared that no comprehensive ethical review was needed for this study. All procedures performed in this study were in accordance with the ethical standards of this institutional research committee and with the 1964 Helsinki declaration and its later amendments or comparable ethical standards.

All patients have given consent to the inclusion of material pertaining to themselves and publication. They were informed that we have fully anonymised all data so that their identity cannot be identified via the paper.

Authors K. Weerdesteijn and K. Bonefaas-Groenewoud are employees of The Dutch Social Security Institute (UWV). Author J.R. Anema is shareholder of the VUmc spin-off company Evalua Nederland B.V. ([www.evalua.nl](http://www.evalua.nl)) and holds a chair in Insurance Medicine on behalf of UWV. Author A.J. van der Beek is shareholder of the VUmc spin-off company Evalua Nederland B.V. ([www.evalua.nl](http://www.evalua.nl)). All authors declare that they have no competing interests in the collection, analyses and interpretation of the data, nor in writing the paper or in the decision to submit the paper for publication.


Introduction
============

The release of neurotransmitters by Ca^2+^-triggered synaptic vesicle exocytosis is a key event in interneuronal communication. Release involves a series of steps that include vesicle docking to the plasma membrane, priming to a release-ready state, and Ca^2+^-triggered membrane fusion ([@bib45]). The protein machinery that governs these steps contains components that have homologues in most types of intracellular membrane traffic and are thus believed to underlie a conserved mechanism of membrane fusion. Particularly important for fusion are proteins from the Sec1/Munc18 (SM) and SNARE families, which, in neuronal synapses, are represented by Munc18-1 and the SNAREs syntaxin-1, SNAP-25, and synaptobrevin/vesicle-associated membrane protein ([@bib38]; [@bib49]; [@bib26]). The SNAREs function by forming tight four-helix bundles called SNARE complexes through sequences known as SNARE motifs ([@bib42]; [@bib35]; [@bib46]); assembly of these complexes brings the two membranes together ([@bib24]) and is key for membrane fusion ([@bib4]; [@bib37]). The function of SM proteins is less clear.

Munc18-1 was identified and linked to synaptic vesicle fusion by virtue of its tight binding to syntaxin-1 ([@bib25]). In addition to a SNARE motif preceding a C-terminal transmembrane region, syntaxin-1 contains a flexible N-terminal sequence, a three-helix bundle called the H~abc~ domain, and a flexible linker ([Fig. 1 A](#fig1){ref-type="fig"}; [@bib18]). The H~abc~ domain and SNARE motif bind intramolecularly, forming a "closed conformation" that is crucial for tight binding to Munc18-1 ([Fig. 1 B](#fig1){ref-type="fig"}; [@bib12]; [@bib30]). Sso1p, the yeast plasma membrane syntaxin, also adopts a closed conformation ([@bib31]; [@bib19]), but this feature is not generally conserved in syntaxins ([@bib13], [@bib14]), and the yeast SM protein Sec1p binds to assembled SNARE complexes rather than to isolated Sso1p ([@bib7]). Moreover, the syntaxins from the ER, Golgi, TGN, and early endosomes of yeast and mammals bind tightly to their cognate SM proteins through the short N-terminal sequence ([@bib3]; [@bib14], [@bib15]; [@bib53]). The apparently diverging picture emerging from these early findings was partially unified by evidence showing that SM proteins generally bind to their cognate SNARE complexes and that most of these interactions involve syntaxin N-terminal sequences ([@bib33]; [@bib6]; [@bib28]; [@bib44]; [@bib48]; [@bib17]; [@bib41]). These data suggest that Munc18-1 interacts with the SNAREs in at least two different modes: a binary interaction with the syntaxin-1 closed conformation that is not universal and likely meets specific requirements of regulated exocytosis ([@bib20]) and a multifaceted interaction with the SNARE complex that likely underlies the general function of SM proteins ([Fig. 1 B](#fig1){ref-type="fig"}; [@bib17]; [@bib41]).

![**Design of mutations to disrupt Munc18-1--SNARE interactions.** (A) Domain diagram of syntaxin-1. NTS, N-terminal sequence; TM, transmembrane region. (B) Diagrams of the binary complex between Munc18-1 and closed syntaxin-1 (left) and the Munc18-1--SNARE complex assembly (right). Munc18-1 is purple, synaptobrevin is red, SNAP-25 is green, and syntaxin-1 is orange (H~abc~ domain) and yellow (SNARE motif and transmembrane region). The model of the binary complex is based on its crystal structure ([@bib30]; [@bib5]). The model of the Munc18-1--SNARE complex assemblies is based on NMR data suggesting a multifaceted interaction and illustrates the overall notion that these assemblies are critical for membrane fusion ([@bib17]; and for a concrete physical model of how these assemblies can induce membrane fusion, see [@bib39]). (C) Models of potential interactions between Munc18-1 and open syntaxin-1 within syntaxin-1--SNAP-25 heterodimers. The model on the left is based on the finding that Munc18-1 can bind to the isolated syntaxin-1 N-terminal region ([@bib27]; [@bib5]), whereas the model on the right also incorporates interactions with the SNARE motifs ([@bib51]). (D) Ribbon diagram of the binary Munc18-1--syntaxin-1 complex with Munc18-1 colored in purple except for the N-terminal domain, which is in cyan, and syntaxin-1, which is in orange (H~abc~ domain) and yellow (linker and SNARE motif). The red asterisk indicates the position where cerulean was inserted for the rescue experiments. A close-up of the interface showing the mutated residues is shown on the right. The diagrams were prepared with Pymol (DeLano Scientific).](JCB_200812026_RGB_Fig1){#fig1}

The physiological relevance of the binary Munc18-1--syntaxin-1 complex was suggested by diverse evidence ([@bib52]; [@bib50]; [@bib38]) and has been demonstrated through analysis of knockin mice bearing an LE (L165A, E166A) mutation that destabilizes the syntaxin-1 closed conformation and impairs Munc18-1 binding, leading to an increase in vesicle release probability ([@bib20]). This phenotype likely arises because the LE mutation facilitates SNARE complex formation, as the SNARE complex is incompatible with the closed conformation ([@bib12]; [@bib30]), and Munc18-1 binding stabilizes this conformation ([@bib9]), thus hindering SNARE complex assembly ([@bib54]).

Munc18-1--SNARE complex assemblies were proposed to form the core of the membrane fusion machinery ([Fig. 1 B](#fig1){ref-type="fig"}; [@bib17]), and their importance for exocytosis was supported by transfection assays and peptide injection experiments in the calyx of Held synapse ([@bib27]). Moreover, reconstitution assays suggested that Munc18-1 stimulates the rate of lipid mixing between SNARE proteoliposomes, interacting with both syntaxin-1 and synaptobrevin ([@bib41]; [@bib40]). However, a recent study concluded that Munc18-1--SNARE complex binding is mediated only by the syntaxin-1 H~abc~ domain and N-terminal sequence ([@bib5]), and Munc18-1 was shown to also bind to syntaxin-1--SNAP-25 heterodimers ([@bib22]; [@bib51]). Although the results of these latter studies do not truly contradict the model of [Fig. 1 B](#fig1){ref-type="fig"}, they do suggest that Munc18-1 can interact with open syntaxin-1 within more than one type of complex ([Fig. 1, B and C](#fig1){ref-type="fig"}) and emphasize that we are still far from understanding how these interactions control release.

To reach such an understanding, it is crucial to determine which of the steps leading to release depends on the binding of Munc18-1 to open syntaxin-1. Moreover, because several components of the release machinery also bind to SNARE complexes ([@bib45]; [@bib4]), elucidating whether such interactions are compatible with Munc18-1 binding is critical to dissect the order of the molecular events leading to release. Particularly important in this context is to unravel whether Munc18-1 competes for SNARE complex binding with complexins because it is well established that these small soluble proteins function in the Ca^2+^-triggering step of release ([@bib36]; [@bib47]), and they are generally believed to be bound to the SNARE complex before Ca^2+^ influx ([@bib37]). In this study, we have addressed these questions using a combination of biophysical experiments and a Munc18-1 knockout (KO) rescue approach. This approach has been hindered because Munc18-1--deficient neurons die early ([@bib50]) and are thus difficult to analyze. We have overcome this problem through lentiviral expression of Munc18-1 in neurons from Munc18-1 KO mice. We have designed mutations in Munc18-1 that impair binding to SNARE complexes containing open syntaxin-1 while still retaining tight binding to closed syntaxin-1 and the ability to rescue survival in Munc18-1 KO neurons. Importantly, these mutations cause a selective disruption of synaptic vesicle priming without altering the efficiency of release of primed vesicles. Moreover, we show that Munc18-1 and complexin-1 bind simultaneously to the SNARE complex. Our data show that interactions of Munc18-1 with open syntaxin-1 are critical for priming vesicles to a release-ready state that likely involves macromolecular assemblies comprising Munc18-1, SNAREs, and complexins.

Results
=======

Design of mutations to distinguish Munc18-1--SNARE interactions
---------------------------------------------------------------

The x-ray structure of the syntaxin-1--Munc18-1 complex revealed that Munc18-1 has an arch shape with a cavity where the syntaxin-1 closed conformation binds ([Fig. 1 D](#fig1){ref-type="fig"}; [@bib30]). The N-terminal domain of Munc18-1 ([Fig. 1 D](#fig1){ref-type="fig"}, cyan) plays a key role in the interaction, making extensive contacts with the H~abc~ domain and the SNARE motif of syntaxin-1 ([Fig. 1 D](#fig1){ref-type="fig"}, orange and yellow, respectively). The syntaxin-1 N-terminal sequence was not observable in the initial crystal structure but also participates in binding ([@bib27]; [@bib5]). Although no high resolution structure of the Munc18-1--SNARE complex is available, nuclear magnetic resonance (NMR) data showed that formation of the complex involves the syntaxin-1 N-terminal sequence and H~abc~ domain as well as the four-helix bundle formed by the SNARE motifs ([Fig. 1 B](#fig1){ref-type="fig"}, right; [@bib17]). These observations suggest that the interactions of Munc18-1 with the syntaxin-1 C terminus must change drastically in the transition between the two complexes, whereas interactions with the N-terminal sequence and H~abc~ domain may involve similar residues in both complexes. However, the energetic contributions of individual interactions to binding are likely to change during the transition between the two complexes because of the syntaxin-1 C-terminal rearrangements, particularly if the interactions involve residues near the interface between the H~abc~ domain and SNARE motif in the closed conformation. Thus, replacing residues near this interface is likely to have differential disruptive effects on binding of Munc18-1 to syntaxin-1 or the SNARE complex. Based on these considerations, three residues of the Munc18-1 N-terminal domain that contact the syntaxin-1 H~abc~ domain (E59), the SNARE motif (K63), or both (E66; [Fig. 1 D](#fig1){ref-type="fig"}) were selected for mutagenesis, and three point mutants of Munc18-1 bearing substitutions in one of these three residues were prepared. Two of the substitutions (E59K and K63E) were charge reversals to try to enhance their disruptive effects, whereas the third (E66A) only neutralized the charge to aim for more moderate effects.

The effects of the three mutations on the binary Munc18-1--syntaxin-1 interaction were investigated by isothermal titration calorimetry (ITC; [Fig. 2](#fig2){ref-type="fig"}). For this purpose, we used a syntaxin-1 fragment encompassing residues 2--243, which include all of the sequences that make contact with Munc18-1 in the crystal structure of the binary complex (ITC experiments using a slightly longer fragment, syntaxin-1\[2--253\], yielded comparable but less consistent results because of the tendency of this fragment to oligomerize; [@bib9]). Triplicate experiments with syntaxin-1(2--243) and the wild-type (WT) or mutant Munc18-1s yielded the following mean *K*~d~ and standard deviations: WT, 7.5 ± 2.7 nM; E59K, 12.0 ± 5.6 nM; K63E, 20.5 ± 11.7 nM; and E66A, 11.3 ± 4.1 nM. The *K*~d~ measured for WT Munc18-1 is comparable with the 10--20-nM *K*~d~ values measured previously by other methods ([@bib34]; [@bib27]) and the 2.7-nM *K*~d~ reported in a recent ITC study ([@bib5]). The differences between these values can be attributed to differences in experimental conditions and/or the protein fragments used as well as to intrinsic difficulties in obtaining accurate measurements for such high affinities, which also underlie the relatively large standard deviations in our measurements. Despite these difficulties, it is clear from our ITC data that the three Munc18-1 mutants retain tight binding to syntaxin-1. Each of the mutations do appear to impair the binary interaction slightly, particularly the K63E mutation, but none of the differences between the *K*~d~ measured for WT Munc18-1 and the mutants is statistically significant.

![**ITC analysis of binding of WT and mutant Munc18-1 to syntaxin-1.** (A--D) Illustrative examples of the ITC data obtained for binding of WT Munc18-1 (A) and E59K (B), E66A (C), and K63E (D) Munc18-1 mutants to syntaxin-1(2--243) are shown. A polynomial baseline correction was applied to remove a slight drift in the initial points of each titration before fitting the data to a single-site binding model. This correction did not substantially alter the *K*~d~ values obtained.](JCB_200812026_GS_Fig2){#fig2}

ITC experiments with WT Munc18-1 and the SNARE complex yielded very small binding enthalpies ([@bib5]; and unpublished data), which suggests that binding is entropically driven and hinders quantitative comparisons by ITC. To measure the effects of the mutations in Munc18-1 on its interaction with open syntaxin-1 within the SNARE complex, we turned to an NMR method that we used previously to demonstrate this interaction ([@bib17]). The method is based on the observation of a decrease in the intensity of the strongest methyl resonance (SMR) in 1D ^13^C-edited ^1^H-NMR spectra of a ^13^C-labeled protein (or complex) upon binding to an unlabeled protein as a result of the broadening caused by formation of a larger species ([@bib1]). To apply this method to study Munc18-1--SNARE complex binding, we prepared SNARE complexes containing ^13^C-labeled syntaxin-1 (hereafter referred to as ^13^C-labeled SNARE complex for simplicity) and added WT or mutant Munc18-1s. Addition of 2.5 µM WT Munc18-1 induced a moderate but reproducible decrease in the SMR intensity of 2 µM ^13^C-labeled SNARE complex ([Fig. 3 A](#fig3){ref-type="fig"}), which reflects formation of the Munc18-1--SNARE complex assembly. Interestingly, the K63E Munc18-1 mutant had a similar effect as WT Munc18-1, whereas the E59K mutant did not significantly decrease the SMR intensity of the ^13^C-labeled SNARE complex, and the E66A mutant had an intermediate effect ([Fig. 3 A](#fig3){ref-type="fig"}). Multiple titrations in which we measured the decrease in the SMR intensity of the ^13^C-labeled SNARE complex as a function of WT Munc18-1 concentration ([Fig. 3 B](#fig3){ref-type="fig"}) yielded a *K*~d~ of 266 ± 41 nM, which is consistent with the values of 100--300 nM that we estimated previously ([@bib17]). Titrations with the Munc18-1 mutants yielded a *K*~d~ of 310 ± 82 nM for K63E, which is not significantly different from WT, and a *K*~d~ of 1.61 ± 0.35 µM for E66A, which reveals a considerable disruption of Munc18-1 binding to the SNARE complex. Titrations with the E59K mutant consistently showed that this mutation strongly impairs SNARE complex binding, although there appeared to be some residual binding at the higher concentrations ([Fig. 3 B](#fig3){ref-type="fig"}). Based on the sensitivity of the method, we estimate a *K*~d~ of \>30 µM for this mutant. Thus, these results show that the three mutations in Munc18-1 have markedly different effects on SNARE complex binding and that two of them (E66A and E59K) disrupt this interaction much more strongly than the binary interaction with the syntaxin-1 closed conformation.

![**Differential disruption of Munc18-1 binding to the SNARE complex by Munc18-1 point mutations.** (A) Sample traces of the methyl regions of 1D ^13^C-edited ^1^H-NMR spectra of 2 µM SNARE complex containing uniformly ^13^C-labeled syntaxin-1(2--243) in the absence or presence of 2.5 µM of unlabeled WT or mutant Munc18-1s. ppm, parts per million. (B) Binding curves obtained from the SMR intensities observed in 1D ^13^C-edited ^1^H-NMR spectra of 2 µM SNARE complex containing uniformly ^13^C-labeled syntaxin-1(2--243) in the presence of increasing amounts of unlabeled WT or mutant Munc18-1s. The data were fit to a standard single-site binding model and normalized to the percentage of binding using as limit values the initial intensity in the absence of Munc18-1 (0% binding) and the intensity extrapolated to infinite Munc18-1 concentration (100% binding).](JCB_200812026_RGB_Fig3){#fig3}

Rescue of survival and neurotransmitter release in Munc18-1 KO neurons
----------------------------------------------------------------------

Munc18-1 KO mice die immediately at birth and exhibit a total abrogation of spontaneous, hypertonic sucrose-induced and Ca^2+^-triggered neurotransmitter release ([@bib50]). To explore whether we could rescue release in neurons from these mice by overexpression of WT Munc18-1, we used primary cortical cultures from mouse embryos at embryonic day (E) 16.5. During the first week in vitro, Munc18-1--deficient neurons exhibited apparently normal neurite outgrowth and synapse formation as judged by immunocytochemistry and electron microscopy (unpublished data). Subsequently, neurons from Munc18-1 KO mice degenerated rapidly, and cultures did not survive \>10 d in vitro (DIV; [Fig. 4](#fig4){ref-type="fig"}). To overcome this problem, we used lentiviral expression of Munc18-1. In these experiments, we aimed to visualize the expressed Munc18-1 to make it easier to monitor the levels of WT and mutant Munc18-1. Thus, we tagged Munc18-1 with the cerulean variant of GFP. We initially explored a C-terminal cerulean fusion protein and three fusion proteins in which cerulean was inserted into loops of Munc18-1. The three loops were chosen in exposed surface locations of Munc18-1 that, based on the crystal structure of the Munc18-1--syntaxin-1 complex ([@bib30]), were close to syntaxin-1 (for future fluorescence resonance energy transfer studies in vivo) and were predicted to be able to harbor the insertion of cerulean without disrupting folding and/or binding. Although no systematic experiments were performed, preliminary experiments indicated that the insertion of cerulean between residues 24 and 25 ([Fig. 1 D](#fig1){ref-type="fig"}, red asterisk) allowed efficient rescue of the survival and the neurotransmitter release phenotypes in Munc18-1--deficient neurons (Fig. S1, A and B, available at <http://www.jcb.org/cgi/content/full/jcb.200812026/DC1>) and that the rescue was better than that observed with the other fusion proteins. Therefore, we performed all of our functional experiments with this cerulean fusion protein of Munc18-1.

![**Rescue of neuronal survival in cortical cultures by Munc18-1 expression.** Representative images of cortical synapses from littermate heterozygotes (top), homozygote KOs for Munc18-1 (middle), or Munc18-1 KOs infected with Munc18-1-containing lentivirus (bottom). Cells were maintained in culture for 11 d before being labeled with antibodies against the presynaptic marker synapsin (first column), the neurofilament marker MAP2 (second column), and the nuclear DAPI marker (third column). The last column shows the combined image of the three labeling procedures with colors that match the relevant labels in the other columns. Bars, 20 µm.](JCB_200812026_RGB_Fig4){#fig4}

We infected cultured Munc18-1--deficient neurons at 1 DIV with lentiviruses expressing WT or E59K, E66A, or K63E mutant Munc18-1 that was fused to cerulean and monitored the survival and morphology of the neurons and expression levels using fluorescence microscopy at 11 DIV. All Munc18-1 proteins rescued neuronal survival, and no significant difference was observed between the number of synapses in WT neurons and in neurons rescued with WT or mutant Munc18-1 proteins (Fig. S1 C). Similar expression levels were observed for the WT and mutant Munc18-1 proteins as monitored from cerulean fluorescence intensities (Fig. S2 A, available at <http://www.jcb.org/cgi/content/full/jcb.200812026/DC1>), although Western blots indicated lower levels of E59K mutant (25--37% compared with WT; Fig. S2 B). The reason for this discrepancy is unclear, but these data suggest that the electrophysiological results described below for this mutant need to be interpreted with caution.

Next, we used patch-clamp recordings to test whether the cerulean-tagged WT Munc18-1 rescues neurotransmitter release in Munc18-1 KO neurons. As expected, no evoked or spontaneous activity was observed in untreated KO cultures at 6--7 DIV, when there are still some surviving neurons (unpublished data), which confirmed previous findings ([@bib50]). Lentiviral expression of WT Munc18-1 rescued spontaneous neurotransmitter release ("minis") and also restored release evoked by field stimulation at low frequency (0.4 Hz), as monitored by recordings at 12--18 DIV ([Fig. 5](#fig5){ref-type="fig"}). Similarly, release stimulated at higher frequencies (10 Hz; Fig. S3, available at <http://www.jcb.org/cgi/content/full/jcb.200812026/DC1>) or by hyperosmotic sucrose ([Fig. 6](#fig6){ref-type="fig"}) was rescued by WT Munc18-1. These results show that lentiviral expression of WT Munc18-1 is efficient enough to confer WT electrophysiological responses on Munc18-1--deficient neurons.

![**Synaptic release depends on Munc18-1 binding to the SNARE complex.** (A) Analysis of spontaneous synaptic release upon rescue with WT and mutant Munc18-1s. Representative 10-s segments from 10-min-long traces of spontaneous excitatory synaptic activity, which was recorded at a holding potential of −70 mV in the presence of 1 µM tetrodotoxin and 50 µM picrotoxin. (B) Bar diagram describing the frequency (top) and amplitude (bottom) of spontaneous release (WT, *n* = 13; Munc18, *n* = 19; E59K, *n* = 10; K63E and E66A, *n* = 9). (C) Representative traces of field stimulation (at 0.4 Hz) evoked excitatory responses from neurons of WT (*n* = 14) or Munc18-1 KO infected with WT (*n* = 9), E59K (*n* = 7), E66A (*n* = 16), or K63E (*n* = 11) Munc18-1--24-cerulean. Note that only the first 400 ms of the traces are shown for clarity. (D) Bar diagram summarizing the amplitudes of evoked responses for cultures rescued with the WT Munc18-1 and different Munc18-1 mutants. (E) Synaptic responses characterized as the amount of transferred charge. Asterisks in the bar diagrams mark statistical significance of the difference between the WT and mutant rescues (\*, P \< 0.05; \*\*\*, P \< 0.005). (B, D, and E) Data are shown as means ± SEMs. Dashed lines indicate WT values.](JCB_200812026_GS_Fig5){#fig5}

![**Munc18-1 binding to the SNARE complex is critical for release readiness of synaptic vesicles.** (A) Representative traces of synaptic excitatory responses to hypertonic solution (+500 mM sucrose to the bath) in WT neurons from littermate controls and Munc18-1 KO neurons rescued with lentivirus-expressing WT Munc18-1--24-cerulean or Munc18-1--24-cerulean with the E59K, K63E, or E66A mutations. (B) Bar diagram depicting the amplitudes of responses to hypertonic sucrose solution for WT cultures or Munc18-1 KO cultures rescued with the WT and mutant Munc18-1s (WT, *n* = 5; Munc18-1, K63E, and E66A, *n* = 8; E59K, *n* = 5). (C) Readily releasable synaptic excitatory transmission characterized as the amount of charge transfer induced by hypertonic sucrose. Asterisks in the bar diagrams mark statistical significance of the difference between the WT and mutant rescues (\*, P \< 0.05; \*\*, P \< 0.01). (B and C) Data are shown as means ± SEMs. Dashed lines indicate WT values.](JCB_200812026_GS_Fig6){#fig6}

Mutations in Munc18-1 impair vesicle priming
--------------------------------------------

We next investigated the effects of the three mutations in Munc18-1 on its ability to rescue neurotransmitter release. We first examined spontaneous release (minis) and found that the K63E mutation had no significant effect, but the E66A mutation strongly decreased the mini frequency (\>60%) while leaving the mini amplitude unaltered ([Fig. 5, A and B](#fig5){ref-type="fig"}). The E59K mutation decreased the mini frequency even more strongly (90%). We then measured the effects of the Munc18-1 mutations on evoked neurotransmitter release ([Fig. 5, C--E](#fig5){ref-type="fig"}). The K63E mutation had no statistically significant effect, although there was a trend for less release. The E66A mutation again caused a considerable impairment of release (50%), which was even more pronounced for the E59K mutant (80%). Note that the finding that the E66A mutation markedly impairs spontaneous and evoked release, whereas the K63E mutation has little or no effect, clearly correlates with the effects of these mutations on SNARE complex binding. The E59K mutant data further extends this correlation, although we cannot rule out the possibility that the disruption of release caused by this mutation arises in part from decreased protein levels.

The decrease in evoked release caused by the mutations could in principle arise from a reduction in the size of the readily releasable pool (RRP) of vesicles and/or in the vesicular release probability. To distinguish between these possibilities, we analyzed evoked responses at 10-Hz stimulation frequency. In these experiments, a reduced release probability is expected to lead to synaptic facilitation. The effects of the mutations on the amplitude of the first response of the train paralleled those observed at low frequency stimulation, but, importantly, all mutants exhibited strong synaptic depression during the stimulus train (Fig. S3). We then determined the size of the RRP by measuring synaptic response to 0.5 M hypertonic sucrose. We found that the E66A mutation led to a marked decrease in the size of the RRP (∼50% decrease), whereas the E59K mutation decreased the size of the RRP even more strongly (76%), and the K63E mutation caused no significant effect ([Fig. 6](#fig6){ref-type="fig"}). Thus, the effects of the mutations on the RRP parallel those observed in the spontaneous and evoked responses ([Fig. 5](#fig5){ref-type="fig"}) as well as in the Munc18-1--SNARE complex binding assays ([Fig. 3](#fig3){ref-type="fig"}). Correspondingly, the ratio between the synaptic charge transfer in evoked release ([Fig. 5 E](#fig5){ref-type="fig"}) and the sucrose-induced charge transfer ([Fig. 6 C](#fig6){ref-type="fig"}) was very similar for the WT and mutant Munc18-1 rescues (Fig. S4, available at <http://www.jcb.org/cgi/content/full/jcb.200812026/DC1>), suggesting that, for the vesicles that are primed, Ca^2+^ triggering of fusion is normal. These results show that the impairment in release caused by the Munc18-1 mutations occurs at the vesicle-priming step and suggest that the interaction of Munc18-1 with the H~abc~ domain in open syntaxin-1 is critical for this step but not for the downstream events that lead to release.

A Munc18-1--SNARE--complexin macromolecular assembly
----------------------------------------------------

A fundamental question to understand the mechanism of action of Munc18-1 in neurotransmitter release is to characterize the relation between its binding to the SNARE complex and other interactions that have been described for this complex ([@bib45]; [@bib4]). It is particularly crucial to determine whether Munc18-1 and complexins can bind simultaneously to the SNARE complex because complexins function at the Ca^2+^-triggering step of release ([@bib36]) and they are generally believed to be bound to the SNARE complex before Ca^2+^ influx ([@bib37]). Thus, such simultaneous binding would suggest that Munc18-1 is bound to the SNAREs within the release-ready state that results after vesicle priming.

To address this question, we first used 1D isotope-edited ^1^H-NMR assays ([@bib1]; [@bib16]) that rely on the same principles as the experiments of [Fig. 3](#fig3){ref-type="fig"}. Thus, the SMR intensity of 1D ^13^C-edited ^1^H-NMR spectra of 2 µM Munc18-1 (65 kD) was considerably reduced upon addition of unlabeled SNARE complex (55 kD), reflecting the formation of the Munc18-1--SNARE complex assembly ([Fig. 7 A](#fig7){ref-type="fig"}). Addition of ^15^N-labeled complexin-1 (16 kD) led to a further decrease in the SMR intensity of Munc18-1, which can be attributed to binding of complexin-1 to the Munc18-1--SNARE complex assembly (note that if complexin-1 displaced Munc18-1 from the SNARE complex, the SMR intensity would have been restored to that of isolated Munc18-1). The formation of a Munc18-1--SNARE--complexin macromolecular assembly was confirmed by 1D ^15^N-edited ^1^H-NMR spectra of 2 µM ^15^N-labeled complexin-1 ([Fig. 7 B](#fig7){ref-type="fig"}). Many complexin-1 signals in these spectra are broadened beyond detection upon SNARE complex binding and are still not observable upon addition of Munc18-1, showing that Munc18-1 does not displace complexin-1 from the SNARE complex (signals that remain observable correspond to complexin-1 regions that are flexible regardless of the presence or absence of Munc18-1). In particular, note that the spectra containing ^13^C-labeled Munc18-1, unlabeled SNARE complex, and ^15^N-labeled complexin-1 ([Fig. 7, A and B](#fig7){ref-type="fig"}, right) were acquired on the same sample and unambiguously demonstrate that Munc18-1 and complexin-1 can bind simultaneously to the SNARE complex.

![**Munc18-1 and complexin-1 can bind simultaneously to the SNARE complex.** (A) Sample traces of the methyl regions of 1D ^13^C-edited ^1^H-NMR spectra of 2 µM Munc18-1 in the absence or presence of 2 µM of unlabeled SNARE complex or 2 µM of unlabeled SNARE complex plus 2 µM ^15^N-labeled complexin-1. (B) Sample traces of 1D ^15^N-edited ^1^H-NMR spectra of 2 µM ^15^N-labeled complexin-1 in the absence or presence of 2 µM of unlabeled SNARE complex or 2 µM of unlabeled SNARE complex plus 2 µM ^13^C-labeled Munc18-1. (A and B) The spectra on the right were acquired with the same sample. ppm, parts per million. (C) Gel filtration on a Superdex S200 (10/300GL) column of Munc18-1, complexin-1, SNARE complex (SC), and mixtures of the SNARE complex with Munc18-1, complexin (Cpx), or both. The SNARE complexes used for all of these experiments contained syntaxin-1(2--253), synaptobrevin-2(29--93), SNAP-25(11--82), and SNAP-25(141--203).](JCB_200812026_RGB_Fig7){#fig7}

To confirm this conclusion by a different method, we used gel filtration experiments ([Fig. 7 C](#fig7){ref-type="fig"}). A mixture of complexin-1 and SNARE complex coeluted at smaller volumes than those of separate samples of complexin-1 and SNARE complex, as expected from the high affinity of their interaction ([@bib29]). Similar results were obtained with samples of Munc18-1 and SNARE complex, as described previously ([@bib17]). Importantly, addition of complexin-1 further decreased the elution volume of the Munc18-1--SNARE complex assembly ([Fig. 7 C](#fig7){ref-type="fig"}), demonstrating that complexin-1 binds to this assembly. It is worth noting in these profiles that the elution of the SNARE complex is shifted to smaller volumes by complexin-1 than by Munc18-1 despite the much smaller size of complexin-1. We attribute this difference to the formation of a more compact structure in the SNARE complex upon Munc18-1 binding as the result of interactions of Munc18-1 with the four-helix bundle and the N-terminal region of syntaxin-1 ([@bib17]) and to the fact that complexin-1 contains large unfolded regions even after SNARE complex binding ([@bib32]). To rule out the possibility that the coelution of Munc18-1 with complexin-1 and the SNARE complex might arise from the binding of Munc18-1 to these unfolded regions of complexin-1, we performed additional gel filtration experiments with a shorter complexin-1 fragment (residues 26--83), which spans the region that becomes structured upon SNARE binding ([@bib32]; [@bib8]). Analogous results were obtained with this fragment (Fig. S5, available at <http://www.jcb.org/cgi/content/full/jcb.200812026/DC1>), again confirming the formation of a Munc18-1--SNARE--complexin assembly.

Discussion
==========

The importance of Munc18-1 and the SNAREs for neurotransmitter release is well established, but it is still unclear how their functions are coupled. The binary interaction initially identified between Munc18-1 and the closed conformation of syntaxin-1 ([@bib25]; [@bib12]), which stabilizes both proteins and gates the entry of syntaxin-1 into SNARE complexes ([@bib50]; [@bib20]), does not appear to be general and may have emerged to meet specific requirements of regulated secretion. Munc18-1 binding to SNARE complexes containing open syntaxin-1 does seem to be universal ([Fig. 1 B](#fig1){ref-type="fig"}; [@bib17]; [@bib41]; [@bib40]), and functional data provided evidence for the physiological relevance of SM protein--SNARE complex interactions in diverse systems ([@bib7]; [@bib21]; [@bib53]; [@bib10]; [@bib27]; [@bib41]). However, the point of action of Munc18-1--open syntaxin-1 interactions in release was unknown, and the relationship between these interactions and those of the SNAREs with other key proteins such as complexins was unclear. Our data now provide fundamental insights into these questions, showing that binding of Munc18-1 to the H~abc~ domain of open syntaxin-1 is critical for synaptic vesicle priming but not for the release step and that Munc18-1 and complexin-1 can bind simultaneously to the SNARE complex.

The development of a strategy to rescue survival and neurotransmitter release in neurons from Munc18-1 KO mice, which is strongly hindered by the deleterious effects arising from deletion of this protein, was key for this study. In addition, we wanted to perform the rescue with fluorescently tagged Munc18-1 to ensure that we could monitor the proper localization of the expressed protein. After extensive efforts, we overcame these difficulties by identifying a Munc18-1--cerulean fusion that rescues the Munc18-1 deficiency phenotype when expressed with a lentivirus, which allowed us to study the functional effects of the three Munc18-1 mutations with the precision of electrophysiology and correlate them with our in vitro binding data.

Although the energetic contributions of individual residues to protein--protein interactions are difficult to predict from 3D structures, the observed biochemical effects of the Munc18-1 mutations can be rationalized according to general knowledge on the energetics of protein--protein interactions and to the model used to design these mutations. In this model, the syntaxin-1 SNARE motif contributes strongly to the binary interaction with Munc18-1 but needs to be released upon formation of the Munc18-1--SNARE complex assembly, leading to a different interaction of Munc18-1 with the four-helix bundle ([Fig. 1 B](#fig1){ref-type="fig"}). In contrast, the H~abc~ domain--Munc18-1 interface is likely similar in both complexes ([@bib27]). The latter assumption implies that, in the Munc18-1--SNARE complex assembly, E59 contacts the H~abc~ domain extensively, E66 makes fewer contacts, and K63 does not interact with the H~abc~ domain (in the binary complex, E66 is between the H~abc~ domain and the SNARE motif, whereas K63 only contacts the SNARE motif; [Fig. 1 D](#fig1){ref-type="fig"}); this prediction correlates very well with the relative impairments in Munc18-1--SNARE complex binding caused by the E59K and E66A mutations and the lack of an effect by the K63E mutation. A key prediction of the model was that, because of the proximity of E59, K63, and E66 to the H~abc~ domain--SNARE motif interface in the binary Munc18-1--syntaxin-1 complex and because of the large rearrangement of the syntaxin-1 SNARE motif upon formation of the Munc18-1--SNARE complex assembly, the energetic contributions of these residues to binding would be different in the two complexes. The different effects of the mutations on the binary Munc18-1--syntaxin-1 interaction compared with those caused on Munc18-1--SNARE complex binding agree with this prediction. It is not surprising that none of the mutations strongly reduced Munc18-1 binding to syntaxin-1 given the adaptability of protein--protein interfaces upon introduction of point mutations ([@bib2]), particularly for complexes of high affinity and involving large interfaces such as that in the Munc18-1--syntaxin-1 complex (\>4,000 Å of buried surface area; [@bib30]; [@bib5]). Although not reaching statistical significance, the K63E mutation did appear to have an effect on Munc18-1--syntaxin-1 binding, which is consistent with the notion that interactions with the SNARE motif contribute strongly to the affinity of the binary complex; such a contribution likely facilitates toleration of the E59K and E66A mutations.

The considerable impairment of Ca^2+^-evoked, sucrose-induced, and spontaneous release caused by the E66A mutation and the little functional consequences of the K63E mutation ([Figs. 5](#fig5){ref-type="fig"} and [6](#fig6){ref-type="fig"}) correlate very well with the effects of these mutations on binding of Munc18-1 to SNARE complexes containing open syntaxin-1 ([Fig. 3](#fig3){ref-type="fig"}) but not with their effects on binding of Munc18-1 to closed syntaxin-1 ([Fig. 2](#fig2){ref-type="fig"}). Although the functional data obtained for the E59K mutant needs to be interpreted with caution, the data appear to extend the correlation between impairment of release and disruption of Munc18-1--SNARE complex binding but not Munc18-1--syntaxin-1 binding. Note that the expression of this mutant is sufficient to rescue survival in Munc18-1 KO neurons and that decreased Munc18-1 levels in syntaxin-1B LE mutant mice lead to a much more moderate decrease in the RRP ([@bib20]) than that caused by the E59K mutation ([Fig. 6 C](#fig6){ref-type="fig"}). Thus, it seems likely that the strong impairment of release caused by this mutation arises at least in part from disruption of the interaction between Munc18-1 and open syntaxin-1. Importantly, the decreases in spontaneous and Ca^2+^-evoked release caused by the E66A and E59K mutations mirror the corresponding RRP reductions. This finding is in sharp contrast with the phenotype observed in the syntaxin-1 LE mutant mice, in which the RRP decreased (likely because of the lower protein levels), but spontaneous release and release of primed vesicles were enhanced ([@bib20]). Because the LE mutation impairs Munc18-1 binding to closed syntaxin-1 but not to SNARE complexes ([@bib20]), this contrast further supports the conclusion that the functional effects of the E66A and E59K mutations arise from the disruption of interactions of Munc18-1 with open syntaxin-1 rather than closed syntaxin-1.

Synaptic vesicle docking was not affected in Munc18-1 KO mice ([@bib50]). Thus, it is unlikely that the E66A and E59K mutations alter docking. This observation together with the finding that these mutations cause parallel decreases in spontaneous, sucrose-induced, and Ca^2+^-triggered release, resulting in evoked release/RRP ratios that are similar to WT (Fig. S4), strongly suggest that the mutations selectively disrupt synaptic vesicle priming but not the downstream events that lead to evoked release. Immediate questions that arise are do Munc18-1--SNARE interactions play any role after priming, and does Munc18-1 form part of the macromolecular complex that results after priming and is poised for Ca^2+^-triggered release? As a first step to address these questions, we examined whether complexin-1 and Munc18-1 compete for SNARE complex binding. Complexins play a role in the Ca^2+^-triggering step of release ([@bib36]) and bind tightly to SNARE complexes ([@bib29]), interacting with the SNARE four-helix bundle ([@bib8]). These observations strongly suggest that complexins are key components of the primed macromolecular assembly that is ready for release ([@bib37]), and our demonstration that Munc18-1 and complexin-1 can bind simultaneously to the SNARE complex suggests that Munc18-1 likely forms part of this assembly as well. Structural studies will be required to characterize in detail the resulting Munc18-1--SNARE--complexin assembly, but it is noteworthy that Munc18-1 binding barely shifts the elution profile of the complexin-1--SNARE complex despite doubling its molecular mass ([Fig. 7 C](#fig7){ref-type="fig"}). This observation suggests that such binding leads to a more compact shape caused by interactions of Munc18-1 with both the N-terminal region of syntaxin-1 and the four-helix bundle, as proposed for the Munc18-1--SNARE complex assembly ([Fig. 1 B](#fig1){ref-type="fig"}, right; [@bib17]).

In contrast, a recent study concluded that binding of Munc18-1 to the SNARE complex involves interactions with only the syntaxin-1 N-terminal region (residues 1--179) based on the similar affinities of Munc18-1 for the SNARE complex and syntaxin-1(1--179) ([@bib5]). However, this conclusion ignores the possibility that the energy gained from Munc18-1--four-helix bundle interactions may be offset by release of interactions contributing to the affinity of Munc18-1 for syntaxin-1(1--179) (e.g., involving the syntaxin-1 linker region), and abundant evidence has demonstrated interactions of Munc18-1 with the SNARE motifs ([@bib17]; [@bib41]; [@bib40]; [@bib51]). Nevertheless, the similar affinities suggest that there is little cooperativity between interactions of Munc18-1 with the four-helix bundle and the syntaxin-1 N-terminal region. Conversely, Munc18-1--membrane interactions seem to cooperate with binding to the four-helix bundle, as the syntaxin-1 N-terminal region is required for binding of Munc18-1 to soluble SNARE complexes but not for binding to membrane-anchored SNARE complexes ([@bib41]).

These observations, together with our data, suggest a model whereby synaptic vesicle priming involves the opening of syntaxin-1, and binding of Munc18-1 to the syntaxin-1 H~abc~ domain is critical for the opening reaction but not for downstream events leading to release. In this model, transition from the Munc18-1--closed syntaxin-1 complex to the Munc18-1--SNARE complex assembly ([Fig. 1 B](#fig1){ref-type="fig"}) involves a series of intermediate states. Thus, release of the SNARE motif from closed syntaxin-1 to bind to SNAP-25 (likely assisted by Munc13-1; [@bib22]; [@bib51]) may involve a transient state in which Munc18-1 is only interacting with the syntaxin-1 N-terminal region ([Fig. 1 C](#fig1){ref-type="fig"}, left). This interaction might keep Munc18-1 near the site of action to facilitate the establishment of new interactions with the syntaxin-1/SNAP-25 SNARE motifs ([Fig. 1 C](#fig1){ref-type="fig"}, right), forming an acceptor complex for synaptobrevin binding. The resulting Munc18-1--SNARE complex assembly ([Fig. 1 B](#fig1){ref-type="fig"}, right) may involve cooperative interactions of Munc18-1 with the four-helix bundle and one or both membranes, which might be key for membrane fusion but might shift the energetic balance so that the interactions of Munc18-1 with the syntaxin-1 N-terminal region become dispensable. These features can explain why binding of Munc18-1 to the H~abc~ domain of open syntaxin-1 is crucial for priming but not for the downstream events leading to fusion. The existence of the proposed intermediate states is supported by the finding that Munc18-1 can bind to isolated syntaxin-1 N-terminal fragments ([@bib27]; [@bib5]) and to syntaxin-1--SNAP-25 heterodimers ([@bib22]; [@bib51]). The hypothesis that Munc18-1 binds to the four-helix bundle and the two apposed membranes correlates with the role proposed for the homotypic fusion and vacuole protein-sorting complex (which includes the Munc18-1 homologue Vps33p) in discriminating trans- from cis-SNARE complexes in yeast vacuolar fusion ([@bib43]). Our model is also consistent with evidence suggesting that Munc18-1 plays multiple roles in the different steps that lead to release ([@bib23]), but, clearly, further research will be required to test this model and to elucidate how the function of Munc18-1 is coupled to those of other SNARE-binding proteins such as complexins, Munc13s, and synaptotagmin-1.

Materials and methods
=====================

Constructs
----------

Bacterial expression vectors to express full-length rat Munc18-1 and full-length rat complexin-1 and fragments corresponding to the cytoplasmic region of rat syntaxin-1A (residues 2--243 or 2--253), the SNARE motifs of rat synaptobrevin-2(29--93) and human SNAP-25B (11--82 and 141--203), or residues 26--83 of rat complexin-1 as GST fusion proteins were described previously ([@bib29]; [@bib8]; [@bib17]). Analogous vectors to express full-length rat Munc18-1 point mutants (E59K, K63E, and E66A) were generated from the WT construct using the QuikChange Site-Directed Mutagenesis kit (Agilent Technologies) and custom-designed primers and were verified by sequencing. For the construction of Munc18-1--24-cerulean constructs, a Munc18-1 (aa 1--24) fragment was first generated by PCR to introduce EcoRI and SseI cloning sites at the N and C termini, and then a Munc18-1 (aa 25--601) fragment was produced by PCR to introduce BsrGI and XbaI cloning sites at the N and C termini. These two PCR products were then inserted to the N terminus and C terminus of cerulean, respectively, on a PCMV5-cerulean vector. The cDNAs of WT and mutant Munc18-1--24-cerulean versions were subcloned between the EcoRI and BamHI sites into the pFUGW shuttle vector for virus production.

Preparation of recombinant proteins and SNARE complexes
-------------------------------------------------------

Syntaxin-1A(2--243), syntaxin-1A(2--253), synaptobrevin-2(29--93), SNAP-25(11--82), SNAP-25(141--203), complexin-1, complexin-1(26--83), and WT and mutant full-length Munc18-1 were expressed in bacteria as GST fusion proteins, isolated by affinity chromatography, cleaved with thrombin, and purified by ion exchange or gel filtration chromatography as described previously ([@bib12]; [@bib8]; [@bib17]). Mass spectrometry analysis showed that the proteolysis of the syntaxin-1A N terminus reported in a recent study ([@bib5]) does not occur using our protocols. Uniform ^13^C or ^15^N labeling was accomplished by expression in bacteria using ^13^C~6~-glucose as the sole carbon source or ^15^NH~4~Cl as the sole nitrogen source, respectively. SNARE complexes were prepared by overnight incubation of the four purified SNARE fragments (with either syntaxin-1A\[2--243\] or syntaxin-1A\[2--253\] plus synaptobrevin-2\[29--93\], SNAP-25\[11--82\], and SNAP-25\[141--203\]), and further purification was performed by gel filtration on a column (Superdex 200; GE Healthcare) in PBS, pH 7.4, containing 2 mM Tris(2-carboxyethyl)phosphine ([@bib17]).

ITC
---

ITC experiments were performed using a VP-ITC system (GE Healthcare) at 20°C in PBS, pH 7.4, containing 2 mM Tris(2-carboxyethyl)phosphine with samples of 5--10 µM WT or mutant Munc18-1s in the sample cell and successive injections of 100--150 µM syntaxin-1A(2--243). All proteins were purified by gel filtration on a Superdex 200 column in the same buffer before the experiments. After polynomial baseline correction to remove a slight drift of the initial data points, the data were fitted with a nonlinear least squares routine using a single-site binding model with Origin for ITC version 5.0 (GE Healthcare). The baseline correction did not substantially alter the *K*~d~ values obtained.

NMR experiments
---------------

1D ^13^C-edited or ^15^N-edited ^1^H-NMR spectra were acquired on a spectrometer (INOVA600; Varian) at 25°C in 20 mM sodium phosphate, pH 7.1, 150 mM NaCl, and 2 mM DTT, acquiring the first trace of ^1^H-^13^C or ^1^H-^15^N heteronuclear single quantum correlation spectra (1,500 scans and 30-min total acquisition time). For the titrations of [Fig. 3 B](#fig3){ref-type="fig"}, samples contained 2 µM SNARE complex (with uniformly ^13^C-labeled syntaxin-1A\[2--243\]) and the desired concentration of unlabeled WT or mutant Munc18-1s; a separate sample was prepared for each concentration. The data were fit to a single-site binding model using SigmaPlot (SPSS, Inc.; [@bib1]).

Cortical cultures
-----------------

Homozygote Munc18-1 KO mice were bred by crossing heterozygous mutant Munc18-1 KO mice ([@bib50]). Cortical neurons from littermate mice at E16 were dissociated by trypsin (5 mg/ml for 5 min at 37°C), triturated with a siliconized Pasteur pipette, and plated onto 12-mm coverslips coated with Matrigel (∼12 coverslips/cortex). Neurons were cultured at 37°C in a humidified incubator with 95% air and 5% CO~2~ in minimal essential media containing 5 g/liter glucose, 0.1 g/liter transferrin, 0.25 g/liter insulin, 0.3 g/liter glutamine, 5--10% heat-inactivated FCS, 2% B-27 supplement, and 1 µM cytosine arabinoside and were used after 5--22 DIV.

Lentiviral infection
--------------------

Constructs were cotransfected with plasmids for viral enzymes and envelope proteins into HEK 293 cells using a transfection system (FuGENE6; Roche) according to the manufacturer\'s specifications, and lentivirus-containing culture medium was harvested 2 d later, filtered through 0.45-µm pores, and immediately used for infection or frozen in liquid nitrogen and stored at −80°C. Cortical cultures were infected at 1 DIV by adding 300 µl of viral suspension to each well.

Immunocytochemistry
-------------------

Neurons attached to the glass coverslips were rinsed once in PBS and fixed for 15 min on ice in 4% formaldehyde and 4% sucrose in PBS. After fixation, the neurons were washed with PBS twice and then incubated for 30 min in blocking solution, PBS containing 3% milk, 0.1% saponin, and in PBS followed by 1-h incubation with primary and rhodamine- and FITC-conjugated secondary antibodies diluted in blocking solution. The coverslips were then mounted on glass slides with Aqua-Poly/Mount medium (Polysciences, Inc.) and analyzed at room temperature (22--24°C) with a confocal microscope (DMIRE2; Leica) equipped with a confocal system (TCS SPX; Leica) using a 63× NA 1.32 oil immersion objective. The images were collected using confocal software (Leica) and processed using Photoshop software (Adobe). All digital manipulations were equally applied to the entire image.

Electrophysiology
-----------------

Synaptic responses were recorded from pyramidal cells in modified Tyrode bath solution in the whole-cell patch configuration. The solution routinely contained 50--100 µM picrotoxin to block inhibitory synaptic currents via γ-aminobutyric acid receptors. For spontaneous release experiments ([@bib11]), 1 µM tetrodotoxin was added to inhibit voltage-gated sodium channels and action potential propagation. For evoked responses, tetrodotoxin was omitted from the bath. Data were acquired with an amplifier (Axopatch 200B; MDS Analytical Technologies) and Clampex 8.0 software (MDS Analytical Technologies), filtered at 2 kHz, and sampled at 200 µs. The internal pipette solution was set to 300 mosM and included 115 mM Cs-MeSO~3~, 10 mM CsCl, 5 mM NaCl, 0.1 mM CaCl~2~, 10 mM Hepes, 4 mM Cs-BAPTA, 20 mM triethylamine-Cl, 4 mM Mg-ATP, 0.3 mM Na~2~-GTP, and 10 mM lidocaine *N*-ethyl-bromide, pH 7.35. A hypertonic solution, which was prepared by the addition of 500 mM sucrose to the nominally Ca^2+^-free Tyrode solution, was perfused directly into the close vicinity of the cell from which the recording was made. Field stimulations (24-mA pulses of 0.6 ms) were applied with parallel platinum electrodes immersed into the perfusion chamber.

Data analysis
-------------

Evoked responses were adjusted with baseline subtraction for each stimulus. Synchronized responses were determined as those within 100 ms of the stimulus, and transferred charge was also calculated for this period. Normal distribution of data was analyzed with the Shapiro-Wilk\'s W test. Paired Student\'s *t* test or variance analysis followed with Tukey\'s test was used to determine significance, which is marked on the figures as asterisks (\*, P \< 0.05; \*\*, P \< 0.01; and \*\*\*, P \< 0.005 levels of significance).

Gel filtration binding assays
-----------------------------

Samples contained 5 µM Munc18-1, 7.5 µM complexin-1, 5 µM SNARE complex (formed with syntaxin-1A\[2--253\], synaptobrevin-2\[29--93\], SNAP-25\[11--82\], and SNAP-25\[141--203\]), or different combinations of these proteins at the same concentrations and were dissolved in 400 µl of 20 mM Tris-HCl, pH 7.4, and 120 mM NaCl. The samples were incubated for 30 min, injected in a column (Superdex S200 10/300 GL; GE Healthcare), and eluted with the same buffer.

Online supplemental material
----------------------------

Fig. S1 shows a comparison of Ca-dependent synaptic release from WT neurons and Munc18-1 KO neurons rescued with WT Munc18-1 or Munc18-1--24-cerulean, as monitored by FM2-10 destaining, and the quantification of synaptic densities in the rescue experiments with WT and mutant Munc18-1s. Fig. S2 shows the protein expression levels in the rescue experiments with WT and mutant Munc18-1s as assessed by fluorescence and Western blotting. Fig. S3 shows the synaptic depression observed for WT Munc18-1 and Munc18-1 mutants at 10-Hz field stimulation. Fig. S4 shows the ratios between charge transfer induced by an action potential and by hypertonic sucrose in the rescue experiments with WT and mutant Munc18-1s. Fig. S5 shows gel filtration profiles of Munc18-1, complexin-1(26--83), SNARE complex, and mixtures of them. Online supplemental material is available at <http://www.jcb.org/cgi/content/full/jcb.200812026/DC1>.

We would like to thank Siqi Liu for preparing bacterial expression vectors for Munc18-1 mutants, Andrea Roth, Iza Kornblum, and Ewa Borowicz for excellent technical assistance, and Lin Fan and Jason Mitchell for help with mouse husbandry.

This work was supported by grant I-1304 from the Welch Foundation and by grant NS37200 (to J. Rizo) from the National Institutes of Health.

Abbreviations used in this paper: DIV, day in vitro; ITC, isothermal titration calorimetry; KO, knockout; NMR, nuclear magnetic resonance; RRP, readily releasable pool; SM, Sec1/Munc18; SMR, strongest methyl resonance; WT, wild type.

[^1]: F. Deák and Y. Xu contributed equally to this paper.

[^2]: F. Deák\'s present address is Dept. of Neuroscience, Mayo Clinic, Jacksonville, FL 32224.

[^3]: I. Dulubova\'s present address is Reata Pharmaceuticals, Inc., Irving, TX 75063.

[^4]: T.C. Südhof\'s present address is Dept. of Molecular and Cellular Physiology and Stanford Institute for Neuro-Innovation and Translational Neuroscience, Stanford University School of Medicine, Stanford, CA 94305.


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Monday, August 15, 2011

7 and a half years, 3 children and it finally happened

There are those rites of passage that all parents go through. Being puked on, pooped on, blow-outs, accidents, green beans being raspberried all over the place, and the list goes on.

All parents realize, at some point, your child will lock themselves in a vehicle.

It took longer than I would have expected, but we finally went through that rite of passage.

Miss Rose & Gracie were headed to spend the night with their Aunt who lives about 90 miles away. We met halfway to do the exchange. Lily wasn't quite ready for a sleepover (although mama would have gladly let her go!). While I was moving carseats over, Lily climbed in the front of the van (which was off) and was playing.

The transitions were made and I grabbed Lily and put her in the back to climb into her seat, which is on the driver's side. Went around to open the door to buckle her in and...all the doors were locked!

I quickly ran back around to my sister's car before she could pull away. I needed some moral support and perhaps her phone, since all my stuff was in the van. I was nervously laughing, knowing things would be okay, but still panicked!

Thankfully, the keys were sitting on top of my purse on the passenger seat. I had to yell to get Lily's attention. I am so glad she wasn't buckled in then! It took a few minutes to communicate to her to get reach up front and get my keys. Then it took a few more minutes of telling her to hit the buttons on the alarm for her to finally hit the "unlock"button, but it worked!

I'm sure it took no more than five minutes, but it did feel like an eternity. Lily was quite proud of herself proclaiming "I did it" several times, although I don't think she realized that she was locked in the van.

And I'm thankful we didn't have the experience my friend shared on Facebook with me, where his 7 year old son locked himself in the car in their garage, and remembered about the "emergency hammer" in the glove compartment to break windows. He broke two windows out of the vehicle. I'm thinking a 7 year old would have been capable of unlocking a door, but the hammer was much more exciting.

1 comment:

I know it was probably really nerve racking when it happened. When I accidently locked my son in the car he was less than a year old and in no position to help. Also he thought mommy running around the car trying doors was hysterically funny. Luckily one window was cracked open and I was able to fit something through it to unlock the door. Crazy.

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Acute compartment syndrome of the forearm caused by calcific tendinitis of the distal biceps.
Acute compartment syndrome of the forearm requires immediate treatment to avoid damage of the soft tissues and a poor functional outcome for the forearm. Muscular and bone lesions are the main causes of acute compartment syndromes. We report a case of acute compartment syndrome of the forearm caused by a calcific tendinitis of the distal biceps.


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