Women with a history of gestational diabetes mellitus present an accumulation of cardiovascular risk factors at age 46-A birth cohort study.

INTRODUCTION: The incidence of gestational diabetes mellitus (GDM) is globally increasing, and it has been associated with later type 2 diabetes, metabolic syndrome (MetS), and cardiovascular disease (CVD). However, long-term population-based studies investigating common CVD risk factors years after pregnancy are lacking. To evaluate the future mortality and morbidity in cardiovascular and metabolic diseases, we conducted a thorough investigation of midlife risk factors in women with and without previous GDM. MATERIAL AND METHODS: A prospective population-based cohort study was conducted of 3173 parous women from the Northern Finland Birth Cohort, 1966. Study participants were obtained from the national register or patient records. Those with a GDM diagnosis formed the GDM cohort (n = 271), and those without a previous GDM diagnosis formed the control cohort (n = 2902). Clinical examinations were performed on participants at the age of 46 and included anthropometric measurements, oral glucose tolerance test (OGTT), biochemical measurements, and cardiovascular assessment. RESULTS: At the age of 46, women in the GDM cohort had a higher body mass index (BMI, 29.0 kg/m2 vs 26.3 kg/m2, p < 0.001) and greater waist circumference (94.1 cm vs 86.5 cm, p < 0.001) than the control cohort. In the GDM cohort, a higher incidence of impaired glucose tolerance (12.6% vs 7.3%, p = 0.002), more previously diagnosed and OGTT-detected type 2 diabetes (23.3% vs 3.9%, p < 0.001), lower high-density lipoprotein (1.53 mmol/L vs 1.67 mmol/L, p = 0.011), higher triglycerides (1.26 mmol/L vs 1.05 mmol/L, p = 0.002) and a higher fatty liver index (6.82 vs 2.47, p < 0.001), were observed even after adjusting for BMI, polycystic ovary syndrome, parity, level of education, physical activity, smoking, and alcohol consumption. The women in the GDM cohort also had more MetS (42.6% vs 21.9%, p < 0.001) and higher risk scores for CVD and fatal events (Framingham 4.95 vs 3.60, p < 0.001; FINRISK 1.71 vs 1.08, p < 0.001). CONCLUSIONS: Women with a previous diagnosis of GDM exhibit more risk factors for CVD in midlife and are at a higher risk for cardiovascular events later in life.

Longitudinal analysis of vaginal microbiota during IVF fresh embryo transfer and in early pregnancy.

IMPORTANCE: Infertility is a global public health issue which leads many couples to seek fertility treatments, of which in vitro fertilization (IVF) is considered to be the most effective. Still, only about one-third of the women achieve live birth after the first IVF embryo transfer (IVF-ET). Factors affecting embryo implantation are poorly known, but the female reproductive tract microbiota may play a key role. Our study confirms the beneficial role of vaginal lactobacilli, especially Lactobacillus crispatus, in the probability of achieving clinical pregnancy and live birth following IVF-ET. Our findings regarding the intra-individual shift of vaginal microbiota between non-pregnancy and pregnancy states are novel and provide new information about the dynamics of microbiota in the early steps of human reproduction. These findings may help clinicians in their attempts to optimize the conditions for ET by microbiota screening or modulation and timing the ET when the microbiota is the most favorable.

Autoantibodies predict type 1 diabetes after gestational diabetes - a 23-year cohort study.

Objective: To study the predictive value of autoantibodies for type 1 (T1DM) and type 2 (T2DM) diabetes morbidity after gestational diabetes (GDM) in a 23-year follow-up study. Design: Prospective population-based cohort study. Methods: We studied 391 women with GDM, and 391 age- and parity-matched controls, who delivered in 1984-1994. Four autoantibodies were analysed in first-trimester blood samples: islet cell autoantibodies (ICAs), glutamic acid decarboxylase autoantibodies (GADAs), insulin autoantibodies (IAAs) and insulinoma-associated antigen-2 autoantibodies (IA-2As). Two follow-up questionnaires (1995-1996, 2012-2013) were sent to assess development of T1DM and T2DM. Predictive value of autoantibodies and clinical factors were analysed by conditional linear regression and ROC analyses. Results: Single autoantibody positivity was detected in 12% (41/342) of the GDM cohort and in 2.3% (8/353) of the control cohort. In the GDM cohort, 2.6% (9/342) tested positive for two autoantibodies and 2.3% (8/342) for three autoantibodies, whereas only one subject in the control cohort had two autoantibodies. ICA positivity was found in 12.5% of the cases, followed by GADA (6.0%), IA-2A (4.9%) and IAA (1.2%). In the control cohort, GADA positivity was found in 1.4%, IA-2A in 0.8%, IAA in 0.6%, and ICA in 0.3% of the subjects. Detection of ICA, GADA and/or IA-2A autoantibodies decreased T1DM-free survival time and time to diagnosis. All subjects with three positive autoantibodies developed T1DM within seven years from the GDM pregnancy. Development of T2DM after GDM occurred independent of autoantibody positivity. Conclusion: Development of T1DM can be reliably predicted with GADA and ICA autoantibodies during early pregnancy.

Effects of estradiol- and ethinylestradiol-based contraceptives on adrenal steroids: A randomized trial.

OBJECTIVES: Ethinylestradiol (EE)-based combined oral contraceptives (COC) affect adrenal function by altering steroid and corticosteroid-binding globulin (CBG) synthesis that may contribute to adverse effects related to these drugs. The effects of COCs containing natural estrogens remain unclear. We compared the effects of COCs containing estradiol valerate (EV) and EE on cortisol and other adrenal steroid hormones. STUDY DESIGN: A spin-off study of a randomized, open-label trial. Fifty-nine healthy women were allocated to groups that engaged in the continuous use of EV+dienogest (DNG), EE+DNG, or DNG only for 9 weeks. We measured changes in adrenal steroids, CBG, and the free cortisol index (FCI). RESULTS: Treatment with EE+DNG increased total cortisol (mean increment 668 nmol/L, p < 0.001) and cortisone (10 nmol/L, p= 0.001) levels, whereas the change from the baseline was insignificant for the EV+DNG and DNG-only groups. Dehydroepiandrosterone sulfate decreased by 24% in the EE+DNG group but remained unchanged in the EV+DNG and DNG-only groups. Aldosterone and 17-hydroxyprogesterone levels did not differ between the groups. All preparations increased CBG, but the increase in the EE+DNG group (median increment 42 µg/mL, p < 0.001) was 9- and 49-fold higher than that in the EV+DNG and DNG-only groups, respectively. The FCI remained unchanged in all study groups, indicating that cortisol and CBG mainly increased in parallel, although some individuals demonstrated larger alterations in the cortisol-CBG balance. CONCLUSION: In COCs, EV had a milder effect on circulating CBG and adrenal steroid levels than EE; however, further research is necessary to determine the long-term effects. TRIAL REGISTRATION: ClinicalTrials.gov NCT02352090 IMPLICATIONS: EV-based COC had reduced effects on circulating CBG and adrenal steroids compared to EE, probably due to a lower hepatic impact. Whether the sensitization of the adrenals to ACTH varies according to COC contents and whether it relates to experienced side effects needs to be investigated. These results encourage further research and development of contraceptives containing natural estrogens.

Combined oral contraceptives containing estradiol valerate vs ethinylestradiol on coagulation: A randomized clinical trial.

INTRODUCTION: Contraceptives containing ethinylestradiol (EE) induce changes in the coagulation system and are associated with a risk of venous thromboembolism. However, studies comparing the effects of combined oral contraceptives containing EE and low-potency estrogens (ie, estradiol [E2 ] and estradiol valerate [EV]) on coagulation biomarkers are limited. This study represents secondary outcomes of a randomized trial comparing combined oral contraceptives containing EV + dienogest (DNG), EE + DNG, and DNG alone on selected coagulation biomarkers. We could compare the specific effects of the different estrogen components owing to the inclusion of preparations containing the same progestin. MATERIAL AND METHODS: We enrolled 59 healthy, 18- to 35-year-old, non-smoking women, of whom three discontinued. The participants were randomly allocated to 9 weeks of continuous treatment with EV 2 mg + DNG 2-3 mg (n = 20), EE 0.03 mg + DNG 2 mg (n = 20), or DNG 2 mg (n = 19). Blood samples were collected at baseline and after 9 weeks. We assessed coagulation in vitro by thrombin generation using the Calibrated Automated Thrombogram. Thrombin generation was evaluated by lag time, time to thrombin peak, thrombin peak, and endogenous thrombin potential in response to tissue factor (1 pm). In vivo coagulation assessment was based on levels of prothrombin fragment 1 + 2 (F1 + 2) (thrombin generation) and D-dimer (fibrin turnover). CLINICAL TRIAL REGISTRATION: NCT02352090. RESULTS: Lag time and time to thrombin peak remained unaltered after exposure to EV + DNG, whereas EE + DNG shortened both lag time (mean percentage change -24%, 95% confidence interval [CI] -32% to -15%; p < 0.01) and time to thrombin peak (-26%, 95% CI -37% to -16%; p < 0.01). EV + DNG induced lower thrombin peak and endogenous thrombin potential than EE + DNG (peak; +45%, 95% CI 22%-67% vs +147%,95% CI 96%-198%; p < 0.01, and endogenous thrombin potential; +26%, 95% CI 15%-38% vs +64%, 95% CI 51%-76%; p < 0.01). Median F1 + 2 levels remained unchanged with EV + DNG (p = 0.22) but increased within normal ranges with EE + DNG (from 152 pmol/L, 95% CI 127-206] pmol/L to 194 pmol/L, 95% CI 149-250 pmol/L, p = 0.04). The within-group change in D-dimer levels was not significant in any of the groups. DNG alone did not affect these biomarkers. CONCLUSIONS: Both in vitro and in vivo thrombin generation was lower after exposure to EV + DNG compared with EE + DNG. The lower thrombin generation measures after treatment with EV + DNG indicate less enhancement of coagulation potential and suggest that EV may be favorable to EE as a component of combined oral contraceptives.

Estradiol Valerate vs Ethinylestradiol in Combined Oral Contraceptives: Effects on the Pituitary-Ovarian Axis.

CONTEXT: Limited studies have compared the effects of combined oral contraceptives (COCs) containing natural estrogens and synthetic ethinylestradiol (EE) on reproductive hormones. OBJECTIVE: To compare estradiol valerate (EV) + dienogest (DNG), EE + DNG, and DNG alone (active control) on levels of follicle stimulating hormone (FSH), luteinizing hormone, anti-Müllerian hormone (AMH), ovarian steroids, sex hormone binding globulin (SHBG), and the free androgen index (FAI). METHODS: This spin-off study from a randomized trial enrolled 59 healthy, 18 to 35-year-old ovulatory women, outpatients at Helsinki and Oulu University Hospitals, Finland, who were randomized to EV 2 mg + DNG 2-3 mg (n = 20); EE 0.03 mg + DNG 2 mg (n = 20); and DNG 2 mg (n = 19) for 9 weeks. Blood samples were drawn at baseline, and at 5 and 9 weeks. Age and BMI were comparable between groups; 3 women discontinued. RESULTS: EV + DNG suppressed FSH by -27% (-51% to -3%) (median [95% CI]) vs EE + DNG, -64% (-78 to -51), P = 0.04, but AMH levels decreased similarly by -9% (-18 to -0.1) vs -13% (-28 to 0.2), P = 0.38, respectively. EV + DNG increased SHBG levels by 56% (30% to 82%) and EE + DNG by 385% (313% to 423%), P < 0.001. Total testosterone decreased by 16% (-27% to -5%) in the EV + DNG group but it did not decrease in the EE + DNG group, whereas the FAI decreased by -39% (-54% to -25%) vs -72% (-78% to -67%), P < 0.001. DNG alone did not induce changes in any of these parameters. CONCLUSION: Compared with EE + DNG, treatment with EV + DNG resulted in milder pituitary downregulation and reduced induction of hepatic SHBG synthesis-potentially carrying more beneficial health effects.

Heavy Menstrual Bleeding in Adolescent: Normal or a Sign of an Underlying Disease?

Heavy, and often irregular, menstrual bleeding (HMB) is a common gynecologic complaint among adolescents. During the first few post-menarcheal years, anovulatory cycles related to immaturity of the hypothalamic-pituitary-ovarian axis are the most common etiology for abnormal uterine bleeding and should be considered as a part of normal pubertal development rather than a disease. If an already regular menstrual cycle becomes irregular, secondary causes of anovulation should be ruled out. Inherited and acquired bleeding disorders, such as von Willebrand disease, and quantitative and qualitative abnormalities of platelets are relatively common findings in adolescents with HMB from menarche. History of excessive bleeding or a diagnosed bleeding disorder in the family supports this etiology, warranting specialized laboratory testing. First-line treatment of HMB among adolescents is medical management with hormonal therapy or nonhormonal options. Levonorgestrel-releasing intrauterine device is an effective tool also for all adolescents with menstrual needs.

Ethinyl estradiol vs estradiol valerate in combined oral contraceptives - Effect on glucose tolerance: A randomized, controlled clinical trial.

OBJECTIVE: To compare the effects of two formulations of combined oral contraceptives (COCs), estradiol valerate (EV) and ethinyl estradiol (EE) combined with dienogest (DNG), and DNG-only, on glucose tolerance. STUDY DESIGN: We performed a randomized, controlled 9-week clinical trial. Inclusion criteria were: age 18-35 years, regular menstrual cycle (28 ± 7 days), no polycystic ovaries, non-smoking, no contraindications for COC use and a 2-month wash-out from hormonal contraceptive use. The women were randomized to EV + DNG (n = 20), EE + DNG (n = 20), and DNG-only (n = 19), and evaluated at baseline, at 4-5 weeks and 8-9 weeks of treatment. Study medications were used continuously for 63 days. Primary outcome measure was change in the whole-body insulin sensitivity index (Matsuda index) derived from the oral glucose tolerance test (OGTT) over the treatment period. Secondary outcome measures were area under curves (AUC) of glucose and insulin, homeostatic model assessment - insulin resistance (HOMA-IR) and Insulin Sensitivity Index (ISI). RESULTS: Fifty-nine women enrolled, and 56 women completed the study. The Matsuda index changed from baseline as follows (mean percentage change, mean change [95%CI]): DNG-only -12%, -1.45 [95%CI -3.22-0.325] P = 0.10; EV + DNG + 2.7%, -0.10 [-1.34 to 1.14] P = 0.86; EE + DNG -5.5%, -1.02 [-2.51 to 0.46] P = 0.16, comparing the groups P = 0.27. There were no clinically significant differences in glucose tolerance between the COC groups, but the DNG-only group showed an improvement in the 2-h glucose levels (5.5 [95%CI 5.0-6.0] to 4.7 mmol/l [4.2-5.2], P = 0.001). CONCLUSION: We found no clinically significant differences between EV and EE combined with DNG and DNG-only on glucose tolerance in healthy, young, normal-weight women, indicating that these preparations appear close to neutral regarding glucose metabolism when used continuously for nine weeks. IMPLICATIONS: Combinations of both ethinyl estradiol and natural estradiol (estradiol valerate) with dienogest (DNG), as well as DNG-only, seem metabolically safe in young and healthy women in short-term continuous use.

Type 1 and type 2 diabetes after gestational diabetes: a 23 year cohort study.

AIMS/HYPOTHESIS: The aim of this work was to examine the progression to type 1 and type 2 diabetes after gestational diabetes mellitus (GDM) in a 23 year follow-up study. METHODS: We carried out a cohort study of 391 women with GDM diagnosed by an OGTT or the use of insulin treatment during pregnancy, and 391 age- and parity-matched control participants, who delivered in 1984-1994 at the Oulu University Hospital, Finland. Diagnostic cut-off levels for glucose were as follows: fasting, ≥4.8 mmol/l; 1 h, ≥10.0 mmol/l; and 2 h, ≥8.7 mmol/l. Two follow-up questionnaires were sent (in 1995-1996 and 2012-2013) to assess the progression to type 1 and type 2 diabetes. Mean follow-up time was 23.1 (range 18.7-28.8) years. RESULTS: Type 1 diabetes developed (5.7%) during the first 7 years after GDM pregnancy and was predictable at a 2 h OGTT value of 11.9 mmol/l during pregnancy (receiver operating characteristic analysis: AUC 0.91, sensitivity 76.5%, specificity 96.0%). Type 2 diabetes increased linearly to 50.4% by the end of the follow-up period and was moderately predictable with fasting glucose (AUC 0.69, sensitivity 63.5%, specificity 68.2%) at a level of 5.1 mmol/l (identical to the fasting glucose cut-off recommended by the International Association of Diabetes and Pregnancy Study Groups [IADPSG) and WHO]). CONCLUSIONS/INTERPRETATION: All women with GDM should be intensively monitored for a decade, after which the risk for type 1 diabetes is minimal. However, the incidence of type 2 diabetes remains linear, and therefore individualised lifelong follow-up is recommended.

Estradiol Valerate in COC Has More Favorable Inflammatory Profile Than Synthetic Ethinyl Estradiol: A Randomized Trial.

CONTEXT: Combined oral contraceptives (COCs) alter inflammatory status and lipid metabolism. Whether different estrogens have different effects is poorly understood. OBJECTIVE: We compared the effects of COCs containing ethinyl estradiol (EE) or estradiol valerate (EV) and dienogest (DNG) with those containing DNG only on inflammation and lipid metabolism. DESIGN: Randomized, controlled, open-label clinical trial. SETTING: Two-center study in Helsinki and Oulu University Hospitals. PARTICIPANTS: Fifty-nine healthy, young, nonsmoking women with regular menstrual cycles. Age, body mass index, and waist-to-hip ratio were comparable in all study groups at the beginning. Fifty-six women completed the study (EV + DNG, n = 20; EE + DNG, n = 19; DNG only, n = 17). INTERVENTIONS: Nine-week continuous use of COCs containing either EV + DNG or EE + DNG, or DNG only as control. MAIN OUTCOME MEASURES: Parameters of chronic inflammation (high-sensitivity C-reactive protein [hs-CRP], and pentraxin 3 [PTX-3]) and lipid profile (high-density lipoprotein [HDL], low-density lipoprotein [LDL], triglycerides, and total cholesterol). RESULTS: Serum hs-CRP increased after 9-week use of EE + DNG (mean change ± standard deviation 1.10 ± 2.11 mg/L) compared with EV + DNG (-0.06 ± 0.97 mg/L, P = 0.001) or DNG only (0.13 ± 0.68 mg/L, P = 0.021). Also, PTX-3 increased in the EE + DNG group compared with EV + DNG and DNG-only groups (P = 0.017 and P = 0.003, respectively). In the EE + DNG group, HDL and triglycerides increased compared with other groups (HDL: EE + DNG 0.20 ± 0.24 mmol/L vs EV + DNG 0.02 ± 0.20 mmol/L [P = 0.002] vs DNG 0.02 ± 0.18 mmol/L [P = 0.002]; triglycerides: EE + DNG 0.45 ± 0.21 mmol/L vs EV + DNG 0.18 ± 0.36 mmol/L [P = 0.003] vs DNG 0.06 ± 0.18 mmol/L [P < 0.001]). CONCLUSIONS: EV + DNG and DNG only had a neutral effect on inflammation and lipids, while EE + DNG increased both hs-CRP and PTX-3 levels as well as triglycerides and HDL. TRIAL REGISTRATION: ClinicalTrials.gov NCT02352090.

Long-term health of women with genetic POI due to FSH-resistant ovaries.

OBJECTIVE: To study the use of hormone therapy (HT), morbidity and reproductive outcomes of women with primary ovarian insufficiency (POI) due to FSH-resistant ovaries (FSHRO). DESIGN: A prospective follow-up study in a university-based tertiary clinic setting. METHODS: Twenty-six women with an inactivating A189V FSH receptor mutation were investigated by means of a health questionnaire and clinical examination. Twenty-two returned the health questionnaire and 14 were clinically examined. Main outcome measures in the health questionnaire were reported as HT, morbidity, medication and infertility treatment outcomes. In the clinical study, risk factors for cardiovascular disease (CVD) and metabolic syndrome (MetS) were compared to age-matched controls from a national population survey (FINRISK). Average number of controls was 326 per FSHRO subject (range 178-430). Bone mineral density and whole-body composition were analyzed with DXA. Psychological and sexual well-being was assessed with Beck Depression Inventory (BDI21), Generalized Anxiety Disorder 7 (GAD-7) and Female Sexual Function Index (FSFI) questionnaires. RESULTS: HT was initiated late (median 18 years of age) compared with normal puberty and the median time of use was shorter (20-22 years) than the normal fertile period. Osteopenia was detected in 9/14 of the FSHRO women despite HT. No major risk factors for CVD or diabetes were found. CONCLUSIONS: HT of 20 years seems to be associated with a similar cardiovascular and metabolic risk factor profile as in the population control group. However, optimal bone health may require an early-onset and longer period of HT, which would better correspond to the natural fertile period.

Novel interactions of CLN3 protein link Batten disease to dysregulation of fodrin-Na+, K+ ATPase complex.

Juvenile neuronal ceroid lipofuscinosis (JNCL, Batten disease) is the most common progressive neurodegenerative disorder of childhood. CLN3, the transmembrane protein underlying JNCL, is proposed to participate in multiple cellular events including membrane trafficking and cytoskeletal functions. We demonstrate here that CLN3 interacts with the plasma membrane-associated cytoskeletal and endocytic fodrin and the associated Na(+), K(+) ATPase. The ion pumping activity of Na(+), K(+) ATPase was unchanged in Cln3(-/-) mouse primary neurons. However, the immunostaining pattern of fodrin appeared abnormal in JNCL fibroblasts and Cln3(-/-) mouse brains suggesting disturbances in the fodrin cytoskeleton. Furthermore, the basal subcellular distribution as well as ouabain-induced endocytosis of neuron-specific Na(+), K(+) ATPase were remarkably affected in Cln3(-/-) mouse primary neurons. These data suggest that CLN3 is involved in the regulation of plasma membrane fodrin cytoskeleton and consequently, the plasma membrane association of Na(+), K(+) ATPase. Most of the processes regulated by multifunctional fodrin and Na(+), K(+) ATPase are also affected in JNCL and Cln3-deficiency implicating that dysregulation of fodrin cytoskeleton and non-pumping functions of Na(+), K(+) ATPase may play a role in the neuronal degeneration in JNCL.

Deficiency of the INCL protein Ppt1 results in changes in ectopic F1-ATP synthase and altered cholesterol metabolism.

Infantile neuronal ceroid lipofuscinosis (INCL) is a severe neurodegenerative disease caused by deficiency of palmitoyl protein thioesterase 1 (PPT1). INCL results in dramatic loss of thalamocortical neurons, but the disease mechanism has remained elusive. In the present work we describe the first interaction partner of PPT1, the F(1)-complex of the mitochondrial ATP synthase, by co-purification and in vitro-binding assays. In addition to mitochondria, subunits of F(1)-complex have been reported to localize in the plasma membrane, and to be capable of acting as receptors for various ligands such as apolipoprotein A-1. We verified here the plasma membrane localization of F(1)-subunits on mouse primary neurons and fibroblasts by cell surface biotinylation and TIRF-microscopy. To gain further insight into the Ppt1-mediated properties of the F(1)-complex, we utilized the Ppt1-deficient Ppt1(Delta ex4) mice. While no changes in the mitochondrial function could be detected in the brain of the Ppt1(Delta ex4) mice, the levels of F(1)-subunits alpha and beta on the plasma membrane were specifically increased in the Ppt1(Delta ex4) neurons. Significant changes were also detected in the apolipoprotein A-I uptake by the Ppt1(Delta ex4) neurons and the serum lipid composition in the Ppt1(Delta ex4) mice. These data indicate neuron-specific changes for F(1)-complex in the Ppt1-deficient cells and give clues for a possible link between lipid metabolism and neurodegeneration in INCL.

Palmitoyl protein thioesterase 1 (Ppt1)-deficient mouse neurons show alterations in cholesterol metabolism and calcium homeostasis prior to synaptic dysfunction.

Infantile neuronal ceroid lipofuscinosis (INCL) is a severe neurodegenerative disorder of children, characterized by selective death of neocortical neurons. To understand early disease mechanisms in INCL, we have studied Ppt1(Deltaex4) knock-out mouse neurons in culture and acute brain slices. Global transcript profiling showed deregulation of key neuronal functions in knock-out mice including cholesterol metabolism, neuronal maturation, and calcium homeostasis. Cholesterol metabolism showed major changes; sterol biosynthesis was enhanced and steady-state amounts of sterols were altered at the cellular level. Changes were also present in early maturation of Ppt1(Deltaex4) neurons indicated by increased proliferative capacity of neuronal stem cells. Knock-out neurons presented unaltered electrophysiological properties suggesting uncompromised synaptic function in young animals. However, knock-out neurons exhibited more efficient recovery from glutamate-induced calcium transients, possibly indicating neuroprotective activation. This study established that the neuronal deregulation in INCL is linked to neuronal maturation, lipid metabolism and calcium homeostasis.

Batten disease (JNCL) is linked to disturbances in mitochondrial, cytoskeletal, and synaptic compartments.

Intracellular pathways leading to neuronal degeneration are poorly understood in the juvenile neuronal ceroid lipofuscinosis (JNCL, Batten disease), caused by mutations in the CLN3 gene. To elucidate the early pathology, we carried out comparative global transcript profiling of the embryonic, primary cultures of the Cln3-/- mouse neurons. Statistical and functional analyses delineated three major cellular pathways or compartments affected: mitochondrial glucose metabolism, cytoskeleton, and synaptosome. Further functional studies showed a slight mitochondrial dysfunction and abnormalities in the microtubule cytoskeleton plus-end components. Synaptic dysfunction was also indicated by the pathway analysis, and by the gross upregulation of the G protein beta 1 subunit, known to regulate synaptic transmission via the voltage-gated calcium channels. Intracellular calcium imaging showed a delay in the recovery from depolarization in the Cln3-/- neurons, when the N-type Ca2+ channels had been blocked. The data suggests a link between the mitochondrial dysfunction and cytoskeleton-mediated presynaptic inhibition, thus providing a foundation for further investigation of the disease mechanism underlying JNCL disease.

Palmitoyl protein thioesterase 1 (PPT1) deficiency causes endocytic defects connected to abnormal saposin processing.

Infantile neuronal ceroid lipofuscinosis (INCL) is a severe neurodegenerative disorder of the childhood caused by mutations in the gene encoding palmitoyl protein thioesterase 1 (PPT1). PPT1 localizes to late endosomes/lysosomes of non-neuronal cells and in neurons also to presynaptic areas. PPT1-deficiency causes massive death of cortical neurons and most tissues show an accumulation of saposins A and D. We have here studied endocytic pathways, saposin localization and processing in PPT1-deficient fibroblasts to elucidate the cellular defects resulting in accumulation of specific saposins. We show that PPT1-deficiency causes a defect in fluid-phase and receptor-mediated endocytosis, whereas marker uptake and recycling endocytosis remain intact. Furthermore, we show that saposins A and D are more abundant and relocalized in PPT-deficient fibroblasts and mouse primary neurons. Metabolic labeling and immunoprecipitation analyses revealed hypersecretion and abnormal processing of prosaposin, implying that the accumulation of saposins may result from endocytic defects. We show for the first time a connection between saposin storage and a defect in the endocytic pathway of INCL cells. These data provide new insights into the metabolism of PPT1-deficient cells and offer a basis for further studies on cellular processes causing neuronal death in INCL and other neurodegenerative diseases.

Interconnections of CLN3, Hook1 and Rab proteins link Batten disease to defects in the endocytic pathway.

The endosomal/lysosomal transmembrane protein CLN3 is mutated in the Batten disease (juvenile neuronal ceroid lipofuscinosis, JNCL). However, the molecular mechanism of JNCL pathogenesis and the exact function of the CLN3 protein have remained unclear. Previous studies have shown that deletion of BTN1, the yeast orthologue of CLN3, leads to increased expression of BTN2. BTN2 encodes Btn2p, a proposed homologue to a novel microtubule-binding protein Hook1, which regulates endocytosis in Drosophila. We analysed here the putative interconnection between CLN3 and Hook1 in the mammalian cells and discovered that overexpression of human CLN3 induces aggregation of Hook1 protein, potentially by mediating its dissociation from the microtubules. Using in vitro binding assay we were able to demonstrate a weak interaction between Hook1 and the cytoplasmic segments of CLN3. We also found receptor-mediated endocytosis to be defective in CLN3-deficient JNCL fibroblasts, connecting CLN3, Hook1 and endocytosis in the mammalian system. Moreover, co-immunoprecipitation experiments showed that Hook1 physically interacts with endocytic Rab7, Rab9 and Rab11, hence delineating a manifold role for mammalian Hook1 in membrane trafficking events. These novel interactions between the microtubule-binding Hook1 and the large family of Rab GTPases also suggest a link between CLN3 function, microtubule cytoskeleton and endocytic membrane trafficking.