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1.  Neuronal Tsc1/2 complex controls autophagy through AMPK-dependent regulation of ULK1 
Human Molecular Genetics  2014;23(14):3865-3874.
Tuberous sclerosis complex (TSC) is a disorder arising from mutation in the TSC1 or TSC2 gene, characterized by the development of hamartomas in various organs and neurological manifestations including epilepsy, intellectual disability and autism. TSC1/2 protein complex negatively regulates the mammalian target of rapamycin complex 1 (mTORC1) a master regulator of protein synthesis, cell growth and autophagy. Autophagy is a cellular quality-control process that sequesters cytosolic material in double membrane vesicles called autophagosomes and degrades it in autolysosomes. Previous studies in dividing cells have shown that mTORC1 blocks autophagy through inhibition of Unc-51-like-kinase1/2 (ULK1/2). Despite the fact that autophagy plays critical roles in neuronal homeostasis, little is known on the regulation of autophagy in neurons. Here we show that unlike in non-neuronal cells, Tsc2-deficient neurons have increased autolysosome accumulation and autophagic flux despite mTORC1-dependent inhibition of ULK1. Our data demonstrate that loss of Tsc2 results in autophagic activity via AMPK-dependent activation of ULK1. Thus, in Tsc2-knockdown neurons AMPK activation is the dominant regulator of autophagy. Notably, increased AMPK activity and autophagy activation are also found in the brains of Tsc1-conditional mouse models and in cortical tubers resected from TSC patients. Together, our findings indicate that neuronal Tsc1/2 complex activity is required for the coordinated regulation of autophagy by AMPK. By uncovering the autophagy dysfunction associated with Tsc2 loss in neurons, our work sheds light on a previously uncharacterized cellular mechanism that contributes to altered neuronal homeostasis in TSC disease.
PMCID: PMC4065158  PMID: 24599401
2.  Cerebellar associative sensory learning defects in five mouse autism models 
eLife  null;4:e06085.
Sensory integration difficulties have been reported in autism, but their underlying brain-circuit mechanisms are underexplored. Using five autism-related mouse models, Shank3+/ΔC, Mecp2R308/Y, Cntnap2−/−, L7-Tsc1 (L7/Pcp2Cre::Tsc1flox/+), and patDp(15q11-13)/+, we report specific perturbations in delay eyeblink conditioning, a form of associative sensory learning requiring cerebellar plasticity. By distinguishing perturbations in the probability and characteristics of learned responses, we found that probability was reduced in Cntnap2−/−, patDp(15q11-13)/+, and L7/Pcp2Cre::Tsc1flox/+, which are associated with Purkinje-cell/deep-nuclear gene expression, along with Shank3+/ΔC. Amplitudes were smaller in L7/Pcp2Cre::Tsc1flox/+ as well as Shank3+/ΔC and Mecp2R308/Y, which are associated with granule cell pathway expression. Shank3+/ΔC and Mecp2R308/Y also showed aberrant response timing and reduced Purkinje-cell dendritic spine density. Overall, our observations are potentially accounted for by defects in instructed learning in the olivocerebellar loop and response representation in the granule cell pathway. Our findings indicate that defects in associative temporal binding of sensory events are widespread in autism mouse models.
eLife digest
On a windy day, hearing the sound of wind makes many individuals squint in anticipation in order to protect their eyes. Linking two sensations that arrive within a split second of one another, such as sound and the feeling of wind, is a type of learning that requires the cerebellum, a region found at the base of the brain. When done in a laboratory setting, this particular form of learning has been dubbed eyeblink conditioning.
Individuals with autism tend to have difficulties with appropriate matching of different senses. For example, they have trouble identifying a video that goes with a spoken soundtrack. They also do not learn eyeblink conditioning the same way that other individuals do. However, it is not known which circuits in the brain are responsible for their difficulty. Kloth et al. now investigate this issue by asking whether versions of genes that increase the risk of autism in humans also disrupt eyeblink conditioning in mice. They tested five types of mouse model, each with a different genetic mutation that has previously been linked to autism. All five of these mutations cause defects in different cell types of the cerebellum, and all mice have abnormal social and habitual behaviors, similar to autistic people.
The tests involved shining a bright light at the mice, which was followed, a split second later, by a puff of air that always causes the mice to blink. After this had occurred dozens of times, the mice started to blink earlier, as soon as the light appeared, in anticipation of the puff of air. To test whether the mice had successfully learned to respond to just the bright light, the light was also occasionally flashed without a puff of air.
Kloth et al. found that the mice generally performed poorly in eyeblink conditioning, although in different ways depending on which cell types of the cerebellum were affected by the genetic mutations. Some mice blinked too soon or too late after the light appeared; others blinked weakly or less frequently; and some did not blink at all. This suggests that autism can affect the processing of sensory information in the cerebellum in different ways.
This work is important because it demonstrates that a form of split-second multisensory learning is generally disrupted by autism genes. If defects in cerebellar learning are present early in life, they could keep autistic children from learning about the world around them, and drive their developing brains off track. Hundreds of autism genes have been found. Linking these genes to a single brain region identifies the cerebellum as an important anatomical target for future diagnosis and intervention.
PMCID: PMC4512177  PMID: 26158416
autism spectrum disorder; associative learning; cerebellum; mouse
3.  Early developmental trajectories associated with ASD in infants with tuberous sclerosis complex 
Neurology  2014;83(2):160-168.
We performed a longitudinal cohort study of infants with tuberous sclerosis complex (TSC), with the overarching goal of defining early clinical, behavioral, and biological markers of autism spectrum disorder (ASD) in this high-risk population.
Infants with TSC and typically developing controls were recruited as early as 3 months of age and followed longitudinally until 36 months of age. Data gathered at each time point included detailed seizure history, developmental testing using the Mullen Scales of Early Learning, and social-communication assessments using the Autism Observation Scale for Infants. At 18 to 36 months, a diagnostic evaluation for ASD was performed using the Autism Diagnostic Observation Schedule.
Infants with TSC demonstrated delays confined to nonverbal abilities, particularly in the visual domain, which then generalized to more global delays by age 9 months. Twenty-two of 40 infants with TSC were diagnosed with ASD. Both 12-month cognitive ability and developmental trajectories over the second and third years of life differentiated the groups. By 12 months of age, the ASD group demonstrated significantly greater cognitive delays and a significant decline in nonverbal IQ from 12 to 36 months.
This prospective study characterizes early developmental markers of ASD in infants with TSC. The early delay in visual reception and fine motor ability in the TSC group as a whole, coupled with the decline in nonverbal ability in infants diagnosed with ASD, suggests a domain-specific pathway to ASD that can inform more targeted interventions for these high-risk infants.
PMCID: PMC4117170  PMID: 24920850
4.  Copy number variation plays an important role in clinical epilepsy 
Annals of neurology  2014;75(6):943-958.
To evaluate the role of copy number abnormalities detectable by chromosomal microarray (CMA) testing in patients with epilepsy at a tertiary care center.
We identified patients with ICD-9 codes for epilepsy or seizures and clinical CMA testing performed between October 2006 and February 2011 at Boston Children’s Hospital. We reviewed medical records and included patients meeting criteria for epilepsy. We phenotypically characterized patients with epilepsy-associated abnormalities on CMA.
Of 973 patients who had CMA and ICD-9 codes for epilepsy or seizures, 805 patients satisfied criteria for epilepsy. We observed 437 copy number variants (CNVs) in 323 patients (1–4 per patient), including 185 (42%) deletions and 252 (58%) duplications. Forty (9%) were confirmed de novo, 186 (43%) were inherited, and parental data were unavailable for 211 (48%). Excluding full chromosome trisomies, CNV size ranged from 18 kb to 142 Mb, and 34% were over 500 kb. In at least 40 cases (5%), the epilepsy phenotype was explained by a CNV, including 29 patients with epilepsy-associated syndromes and 11 with likely disease-associated CNVs involving epilepsy genes or “hotspots.” We observed numerous recurrent CNVs including 10 involving loss or gain of Xp22.31, a region described in patients with and without epilepsy.
Copy number abnormalities play an important role in patients with epilepsy. Given that the diagnostic yield of CMA for epilepsy patients is similar to the yield in autism spectrum disorders and in prenatal diagnosis, for which published guidelines recommend testing with CMA, we recommend the implementation of CMA in the evaluation of unexplained epilepsy.
PMCID: PMC4487364  PMID: 24811917
Epilepsy; array comparative genomic hybridization (aCGH); chromosomal microarray; copy number variants; deletions; duplications; chromosomal abnormalities
5.  Tuberous Sclerosis Associated Neuropsychiatric Disorders (TAND) and the TAND Checklist 
Pediatric neurology  2014;52(1):25-35.
Tuberous sclerosis complex is a multisystem genetic disorder with a range of physical manifestations that require evaluation, surveillance, and management. Individuals with tuberous sclerosis complex also have a range of behavioral, psychiatric, intellectual, academic, neuropsychologic, and psychosocial difficulties. These may represent the greatest burden of the disease. Around 90% of individuals with tuberous sclerosis complex will have some of these difficulties during their lifetime, yet only about 20% ever receive evaluation and treatment. The Neuropsychiatry Panel at the 2012 Tuberous Sclerosis Complex International Consensus Conference expressed concern about the significant “treatment gap” and about confusion regarding terminology relating to the biopsychosocial difficulties associated with tuberous sclerosis complex.
The Tuberous Sclerosis Complex Neuropsychiatry Panel coined the term TAND—tuberous sclerosis complex-associated neuropsychiatric disorders—to bring together these multidimensional manifestations of the disorder, and recommended annual screening for TAND. In addition, the Panel agreed to develop a TAND Checklist as a guide for screening.
Here, we present an outline of the conceptualization of TAND, rationale for the structure of the TAND Checklist, and include the full US English version of the TAND Checklist.
We hope that the unified term TAND and the TAND Checklist will raise awareness of the importance of tuberous sclerosis complex-associated neuropsychiatric disorders and of the major burden of disease associated with it, provide a shared language and a simple tool to describe and evaluate the different levels of TAND, alert clinical teams and families or individuals of the importance of screening, assessment, and treatment of TAND, and provide a shared framework for future studies of tuberous sclerosis complex-associated neuropsychiatric disorders.
PMCID: PMC4427347  PMID: 25532776
behavior; psychiatric disorders; autism; mental health; neurocognition; learning disorders; neuropsychological; psycho-social
6.  Atypical Face Processing in Children With Tuberous Sclerosis Complex 
Journal of child neurology  2012;28(12):1569-1576.
There is a high incidence of autism in tuberous sclerosis complex. Given the evidence of impaired face processing in autism, the authors sought to investigate electrophysiological markers of face processing in children with tuberous sclerosis complex. The authors studied 19 children with tuberous sclerosis complex under age 4, and 20 age-matched controls, using a familiar–unfamiliar faces paradigm. Of the children, 6 with tuberous sclerosis complex (32%) had autism. Children with tuberous sclerosis complex showed a longer N290 latency than controls (276 ms vs 259 ms, P = .05) and also failed to show the expected hemispheric differences in face processing. The longest N290 latency was seen in (1) children with autism and tuberous sclerosis complex and (2) children with temporal lobe tubers. This study is the first to quantify atypical face processing in children with tuberous sclerosis complex. This functional impairment may provide insight into a mechanism underlying a pathway to autism in tuberous sclerosis complex.
PMCID: PMC4422391  PMID: 23143725
tuberous sclerosis complex; autism; evoked potentials; cortical tubers
7.  The role of n-butyl-2-cyanoacrylate in the repair of traumatic diaphragmatic injuries 
Diaphragmatic injuries either by blunt or penetrating trauma require prompt surgical intervention and are often exigent to repair. N-butyl-2-cyanoacrylate (n-butyl-2-CA) is a tissue adhesive which has gained wide application in many areas of surgery including emergency. To repair the extensive injuries of the diaphragm it may be necessary the use of synthetic mesh by fixing it with sutures or staples. The use of tissue adhesives may circumvent the potential problems associated with mesh fixation. This study aimed to evaluate the efficacy and safety of tissue adhesives usage for mesh fixation in diaphragmatic injury repair. Twenty-four rats were divided into 3 groups each of them containing 8 rats. A 1- cm diaphragmatic defect was created in all rats. The defect was repaired by polypropylene suture in Group I, by mesh fixed with sutures in group II and by mesh fixed with n-butyl-2-CA in group III. The rats were sacrificed after 1 month. The episode of hernia and the adhesions were assessed by adhesion density score. Also, the abscess and inflammation in the repaired tissue were evaluated microscopically. The Kruskal-Wallis test was performed for the histopathological analysis. No diaphragmatic hernia was detected in any group. While Group III had higher adhesion density scores than group I (P: 0.027), there were no differences between group III and II (P: 0.317) and group II and I (P = 0.095) regarding adhesion density scores. The inflammation grade was higher in group III than group I and II (P < 0.001) and was higher in group II than group I (P < 0.05). There was no differences between each groups, concerning microabcsess formation (P > 0.05). Repair of traumatic diaphragmatic injury in penetrating wound, with polypropylene mesh fixed by n-butyl-2-CA in rats appears to be as efficacious and safe as conventional methods in early period. However, further experimental and clinical study are needed to compare the long-term results of adhesive mesh repair with those of the traditional sutured techniques.
PMCID: PMC4483802  PMID: 26131179
Diaphragmatic injury; repair; synthetic graft; cyanoacrylate
8.  Mechanism-based Treatment in Tuberous Sclerosis Complex 
Pediatric neurology  2013;50(4):290-296.
Tuberous Sclerosis Complex is a genetic multi-system disorder that affects the brain in almost every patient. It is caused by a mutation in the TSC1 or TSC2 genes, which regulate mTOR, a key player in control of cellular growth and protein synthesis. The most frequent neurologic symptoms are seizures, which occur in up to 90% of patients and are often intractable, followed by autism spectrum disorders, intellectual disability, ADHD and sleep problems. Conventional treatment has frequently proven insufficient for neurologic and behavioral symptoms, particularly seizure control. This review focuses on the role of TSC/mTOR in neuronal development and network formation, and recent mechanism-based treatment approaches.
We performed a literature review to identify ongoing therapeutical challenges and novel strategies.
To achieve a better quality of life for many patients, current therapy approaches are directed at restoring dysregulated mTOR signaling. Animal studies have provided insight into aberrant neuronal network formation caused by constitutive activation of the mTOR pathway, and initial studies in TSC patients using MR diffusion tensor imaging and EEG support a model of impaired neuronal connectivity in TSC. Rapamycin, an mTOR inhibitor, has been used successfully in Tsc-deficient mice to prevent and treat seizures and behavioral abnormalities. There is recent evidence in humans of improved seizure control with mTOR inhibitors.
Current research provides insight into aberrant neuronal connectivity in TSC and the role of mTOR inhibitors as a promising therapeutical approach.
PMCID: PMC3959246  PMID: 24486221
TSC; rapamycin; neuronal connectivity; mTOR
9.  Minimally invasive management of anastomotic leak after bariatric Roux-en-Y gastric bypass 
Journal of Minimal Access Surgery  2015;11(2):160-162.
The aim of this retrospective study was to examine the anastomotic erosion due to drain and success of fibrin sealant in its management. Between 2013 and 2014, 102 patients underwent LRYGB and gastrojejunal anastomotic leak occurred due to drain erosion in 2 of them. The diagnosis was established with saliva drainage and was confirmed by upper gastrointestinal series. The absence of hemodynamic instability was directed us to conservative treatment. During the endoscopy, dehiscence was assessed and fibrin sealant was applied. The leaks healed progressively in a few days, and the drains removed within 6 days. Seven and 9 days later, the patients were discharged without any problem. Anastomotic leaks after bariatric surgery can cause severe morbidity, cost, and effects quality of life. Hemodynamically stable and drained patients are candidates for conservative methods. Endoscopic injection of fibrin sealant has been successful in closing gastric leaks.
PMCID: PMC4392494  PMID: 25883461
Drain erosion; endoscopy; fibrin sealant; gastrojejunal leak; morbid obesity
10.  Somatic Mutations in Cerebral Cortical Malformations 
The New England journal of medicine  2014;371(8):733-743.
Although there is increasing recognition of the role of somatic mutations in genetic disorders, the prevalence of somatic mutations in neurodevelopmental disease and the optimal techniques to detect somatic mosaicism have not been systematically evaluated.
Using a customized panel of known and candidate genes associated with brain malformations, we applied targeted high-coverage sequencing (depth, ≥200×) to leukocyte-derived DNA samples from 158 persons with brain malformations, including the double-cortex syndrome (subcortical band heterotopia, 30 persons), polymicrogyria with megalencephaly (20), periventricular nodular heterotopia (61), and pachygyria (47). We validated candidate mutations with the use of Sanger sequencing and, for variants present at unequal read depths, subcloning followed by colony sequencing.
Validated, causal mutations were found in 27 persons (17%; range, 10 to 30% for each phenotype). Mutations were somatic in 8 of the 27 (30%), predominantly in persons with the double-cortex syndrome (in whom we found mutations in DCX and LIS1), persons with periventricular nodular heterotopia (FLNA), and persons with pachygyria (TUBB2B). Of the somatic mutations we detected, 5 (63%) were undetectable with the use of traditional Sanger sequencing but were validated through subcloning and subsequent sequencing of the subcloned DNA. We found potentially causal mutations in the candidate genes DYNC1H1, KIF5C, and other kinesin genes in persons with pachygyria.
Targeted sequencing was found to be useful for detecting somatic mutations in patients with brain malformations. High-coverage sequencing panels provide an important complement to whole-exome and whole-genome sequencing in the evaluation of somatic mutations in neuropsychiatric disease. (Funded by the National Institute of Neurological Disorders and Stroke and others.)
PMCID: PMC4274952  PMID: 25140959
11.  Isolated Vitamin D Deficiency Is Not Associated with Nonthyroidal Illness Syndrome, but with Thyroid Autoimmunity 
The Scientific World Journal  2015;2015:239815.
Aim. This study aimed to compare thyroid functions, thyroid autoantibodies, and the existence of nonthyroidal illness syndrome (NTIS) according to vitamin D level. Materials and Methods. The study included age- and BMI-matched healthy volunteers with and without vitamin D deficiency. In addition, the nonthyroidal illness syndrome status was evaluated. Results. Anti-TPO positivity was significantly more common in those with severe and moderate vitamin D deficiency, as compared to those with a normal 25(OH)D level. Furthermore, TSH levels were significantly lower in those with severe and moderate vitamin D deficiency than in those with a normal 25(OH)D level. In addition, there was a significant weak inverse correlation between anti-TPO positivity and the 25(OH)D level and a positive correlation between the TSH level and 25(OH)D level. Only 1 thyroid function test result was compatible with NTIS among the participants with moderate vitamin D deficiency; therefore the difference was not significant. Conclusions. The prevalence of thyroid autoantibody positivity was higher in those with severe and moderate vitamin D deficiency than in those with a normal 25(OH)D level. Additional large-scale studies must be conducted to determine if vitamin D deficiency plays a causal role in the pathogenesis of Hashimoto's thyroiditis and NTIS.
PMCID: PMC4306373  PMID: 25654127
12.  Same-day colonoscopy preparation with Senna alkaloids and bisacodyl tablets: A pilot study 
World Journal of Gastroenterology : WJG  2014;20(41):15382-15386.
AIM: To evaluate the efficacy of same-day bowel preparation with Senna alkaloids combined with bisacodyl tablets in routine colonoscopy procedures.
METHODS: Between March and June 2013, a same-day bowel preparation was implemented in our endoscopy unit. The preparation consisted of a semi-liquid, fiber-free diet one day prior to the procedure, with two bisacodyl tablets after lunch and dinner, and 250 mL of Senna alkaloid with 1.5 L of drinking water at 6 am the day of the procedure. The quality control parameters of colonoscopy were evaluated and implemented according to the guidelines of the American Society for Gastrointestinal Endoscopy. The pre-procedure, during-procedure and post-procedure patient data were collected and analyzed: (1) pre-procedure (age, gender, comorbid diseases, colonoscopy indications, complete lack of compliance with the bowel preparation protocol); (2) during-procedure (sedation dose, duration of colonoscopy, withdrawal time, cecal intubation rate, polyp detection rate, Boston Bowel Preparation Scores and presence of foam and clear liquid); and (3) post-procedure (visual analogue scale score, pain during the procedure, patient satisfaction and premature withdrawal due to the insufficient bowel preparation).
RESULTS: A total of 75 patients were included in this study with a mean age of 54.64 ± 13.29 years; 53.3% (40/75) were female and 46.7% (35/75) were male. A complete lack of compliance with the bowel preparation protocol was seen in 6.7% of patients (5/75). The mean total duration of colonoscopy was 16.12 ± 6.51 min, and the mean withdrawal time was 8.89 ± 4.07 min. The cecal intubation rate was 93.8% (61/64) and the polyp detection rate was 40% (30/75). The mean Boston Bowel Preparation Score was 7.38 ± 1.81, with the following distribution: right colon, 2.34 ± 0.89; transverse colon, 2.52 ± 0.67; left colon, 2.52 ± 0.63. The mean visual analogue scale score was 4.59 ± 1.57. Due to insufficient bowel preparation, seven patients (7/75; 9.3%) were asked to repeat the procedure. Of these, five patients had poor or modest compliance with the protocol, and two patients reported constipation. Premature withdrawal due to insufficient bowel preparation was 2.7% (2/75). The overall satisfaction with the protocol was 86.7% (65/75), with patients reporting they would prefer the same protocol in a repeat procedure.
CONCLUSION: The same-day administration of Senna alkaloids appears to be a safe and effective bowel cleansing protocol for colonoscopy procedures.
PMCID: PMC4223273  PMID: 25386088
Bisacodyl; Bowel preparation; Colonoscopy; Same-day preparation; Senna alkaloid
13.  A Clinical Prediction Formula for Apnea-Hypopnea Index 
Objectives. There are many studies regarding unnecessary polysomnography (PSG) when obstructive sleep apnea syndrome (OSAS) is suspected. In order to reduce unnecessary PSG, this study aims to predict the apnea-hypopnea index (AHI) via simple clinical data for patients who complain of OSAS symptoms. Method. Demographic, anthropometric, physical examination and laboratory data of a total of 390 patients (290 men, average age 50 ± 11) who were subject to diagnostic PSG were obtained and evaluated retrospectively. The relationship between these data and the PSG results was analyzed. A multivariate linear regression analysis was performed step by step to identify independent AHI predictors. Results. Useful parameters were found in this analysis in terms of body mass index (BMI), waist circumference (WC), neck circumference (NC), oxygen saturation measured by pulse oximetry (SpO2), and tonsil size (TS) to predict the AHI. The formula derived from these parameters was the predicted AHI = (0.797 × BMI) + (2.286 × NC) − (1.272 × SpO2) + (5.114 × TS) + (0.314 × WC). Conclusion. This study showed a strong correlation between AHI score and indicators of obesity. This formula, in terms of predicting the AHI for patients who complain about snoring, witnessed apneas, and excessive daytime sleepiness, may be used to predict OSAS prior to PSG and prevent unnecessary PSG.
PMCID: PMC4199210  PMID: 25349613
14.  Both Maternal and Pup Genotype Influence Ultrasonic Vocalizations and Early Developmental Milestones in Tsc2+/− Mice 
Tuberous sclerosis complex (TSC) is an autosomal dominant disorder characterized by tumor growth and neuropsychological symptoms such as autistic behavior, developmental delay, and epilepsy. While research has shed light on the biochemical and genetic etiology of TSC, the pathogenesis of the neurologic and behavioral manifestations remains poorly understood. TSC patients have a greatly increased risk of developmental delay and autism spectrum disorder, rendering the relationship between the two sets of symptoms an extremely pertinent issue for clinicians. We have expanded on previous observations of aberrant vocalizations in Tsc2+/− mice by testing vocalization output and developmental milestones systematically during the early postnatal period. In this study, we have demonstrated that Tsc2 haploinsufficiency in either dams or their pups results in a pattern of developmental delay in sensorimotor milestones and ultrasonic vocalizations.
PMCID: PMC4137560  PMID: 25165580
15.  Impaired Language Pathways in Tuberous Sclerosis Complex Patients with Autism Spectrum Disorders 
Cerebral Cortex (New York, NY)  2012;23(7):1526-1532.
The purpose of this study was to examine the relationship between language pathways and autism spectrum disorders (ASDs) in patients with tuberous sclerosis complex (TSC). An advanced diffusion-weighted magnetic resonance imaging (MRI) was performed on 42 patients with TSC and 42 age-matched controls. Using a validated automatic method, white matter language pathways were identified and microstructural characteristics were extracted, including fractional anisotropy (FA) and mean diffusivity (MD). Among 42 patients with TSC, 12 had ASD (29%). After controlling for age, TSC patients without ASD had a lower FA than controls in the arcuate fasciculus (AF); TSC patients with ASD had even a smaller FA, lower than the FA for those without ASD. Similarly, TSC patients without ASD had a greater MD than controls in the AF; TSC patients with ASD had even a higher MD, greater than the MD in those without ASD. It remains unclear why some patients with TSC develop ASD, while others have better language and socio-behavioral outcomes. Our results suggest that language pathway microstructure may serve as a marker of the risk of ASD in TSC patients. Impaired microstructure in language pathways of TSC patients may indicate the development of ASD, although prospective studies of language pathway development and ASD diagnosis in TSC remain essential.
PMCID: PMC3673171  PMID: 22661408
arcuate fasciculus; diffusion tensor imaging; neuroanatomy; tractography; white matter
16.  Diffusion tensor imaging and related techniques in tuberous sclerosis complex: review and future directions 
Future neurology  2013;8(5):583-597.
In this article, the authors aim to introduce the nonradiologist to diffusion tensor imaging (DTI) and its applications to both clinical and research aspects of tuberous sclerosis complex. Tuberous sclerosis complex is a genetic neurocutaneous syndrome with variable and unpredictable neurological comorbidity that includes refractory epilepsy, intellectual disability, behavioral abnormalities and autism spectrum disorder. DTI is a method for modeling water diffusion in tissue and can noninvasively characterize microstructural properties of the brain. In tuberous sclerosis complex, DTI measures reflect well-known pathological changes. Clinically, DTI can assist with detecting the epileptogenic tuber. For research, DTI has a putative role in identifying potential disease biomarkers, as DTI abnormalities of the white matter are associated with neurocognitive morbidity including autism. If indeed DTI changes parallel phenotypical changes related to the investigational treatment of epilepsy, cognition and behavior with mTOR inhibitors, it will facilitate future clinical trials.
PMCID: PMC3904372  PMID: 24489482
autism spectrum disorders; behavior; cognition; diffusion tensor imaging; epilepsy; MRI; mTOR serine–threonine kinases; tuberous sclerosis complex
17.  Characterizing the DIstribution of Anisotropic MicrO-structural eNvironments with Diffusion-weighted imaging (DIAMOND) 
Diffusion-weighted imaging (DWI) enables investigation of the brain microstructure by probing natural barriers to diffusion in tissues. In this work, we propose a novel generative model of the DW signal based on considerations of the tissue microstructure that gives rise to the diffusion attenuation. We consider that the DW signal can be described as the sum of a large number of individual homogeneous spin packets, each of them undergoing local 3-D Gaussian diffusion represented by a diffusion tensor. We consider that each voxel contains a number of large scale microstructural environments and describe each of them via a matrix-variate Gamma distribution of spin packets. Our novel model of DIstribution of Anisotropic MicrOstructural eNvironments in DWI (DIAMOND) is derived from first principles. It enables characterization of the extra-cellular space, of each individual white matter fascicle in each voxel and provides a novel measure of the microstructure heterogeneity. We determine the number of fascicles at each voxel with a novel model selection framework based upon the minimization of the generalization error. We evaluate our approach with numerous in-vivo experiments, with cross-testing and with pathological DW-MRI. We show that DIAMOND may provide novel biomarkers that captures the tissue integrity.
PMCID: PMC4029840  PMID: 24505801
18.  RASA1 functions in EPHB4 signaling pathway to suppress endothelial mTORC1 activity 
The Journal of Clinical Investigation  2014;124(6):2774-2784.
Vascular malformations are linked to mutations in RAS p21 protein activator 1 (RASA1, also known as p120RasGAP); however, due to the global expression of this gene, it is unclear how these mutations specifically affect the vasculature. Here, we tested the hypothesis that RASA1 performs a critical effector function downstream of the endothelial receptor EPHB4. In zebrafish models, we found that either RASA1 or EPHB4 deficiency induced strikingly similar abnormalities in blood vessel formation and function. Expression of WT EPHB4 receptor or engineered receptors with altered RASA1 binding revealed that the ability of EPHB4 to recruit RASA1 is required to restore blood flow in EPHB4-deficient animals. Analysis of EPHB4-deficient zebrafish tissue lysates revealed that mTORC1 is robustly overactivated, and pharmacological inhibition of mTORC1 in these animals rescued both vessel structure and function. Furthermore, overexpression of mTORC1 in endothelial cells exacerbated vascular phenotypes in animals with reduced EPHB4 or RASA1, suggesting a functional EPHB4/RASA1/mTORC1 signaling axis in endothelial cells. Tissue samples from patients with arteriovenous malformations displayed strong endothelial phospho-S6 staining, indicating increased mTORC1 activity. These results indicate that deregulation of EPHB4/RASA1/mTORC1 signaling in endothelial cells promotes vascular malformation and suggest that mTORC1 inhibitors, many of which are approved for the treatment of certain cancers, should be further explored as a potential strategy to treat patients with vascular malformations.
PMCID: PMC4089464  PMID: 24837431
19.  The use of fibrin glue without surgery in the treatment of pilonidal sinus disease 
Pilonidal sinus disease is a common disabling condition affecting the natal clefts of the buttocks. We analyze the role of fibrin glue in the treatment of selected patients with pilonidal sinus disease. Forty patients diagnosed with pilonidal sinus disease at Vakif Gureba Training and Research Hospital were treated between December 2007 and December 2011. Recurrence was noted in four patients (10%). Ninety percent of patients had no recurrence one year later (95% confidence interval: 0.85-0.95). This procedure is suggested as a first line of treatment for patients with no prior history of infection and who have only one sinus orifice.
PMCID: PMC4057859  PMID: 24955180
Fibrin glue; pilonidal sinus
20.  A Mathematical Framework for the Registration and Analysis of Multi-Fascicle Models for Population Studies of the Brain Microstructure 
Diffusion tensor imaging (DTI) is unable to represent the diffusion signal arising from multiple crossing fascicles and freely diffusing water molecules. Generative models of the diffusion signal, such as multi-fascicle models, overcome this limitation by providing a parametric representation for the signal contribution of each population of water molecules. These models are of great interest in population studies to characterize and compare the brain microstructural properties. Central to population studies is the construction of an atlas and the registration of all subjects to it. However, the appropriate definition of registration and atlasing methods for multi-fascicle models have proven challenging. This paper proposes a mathematical framework to register and analyze multi-fascicle models. Specifically, we define novel operators to achieve interpolation, smoothing and averaging of multi-fascicle models. We also define a novel similarity metric to spatially align multi-fascicle models. Our framework enables simultaneous comparisons of different microstructural properties that are confounded in conventional DTI. The framework is validated on multi-fascicle models from 24 healthy subjects and 38 patients with tuberous sclerosis complex, 10 of whom have autism. We demonstrate the use of the multi-fascicle models registration and analysis framework in a population study of autism spectrum disorder.
PMCID: PMC3984609  PMID: 24235301
Diffusion magnetic resonance imaging (MRI); interpolation; microstructure; multi-fascicle; multi-tensor; population studies; registration
21.  A TSC signaling node at the peroxisome regulates mTORC1 and autophagy in response to ROS 
Nature cell biology  2013;15(10):1186-1196.
Subcellular localization is emerging as an important mechanism for mTORC1 regulation. We report that the tuberous sclerosis complex (TSC) signaling node, TSC1, TSC2 and Rheb, localizes to peroxisomes, where it regulates mTORC1 in response to reactive oxygen species (ROS). TSC1 and TSC2 were bound by PEX19 and PEX5, respectively, and peroxisome-localized TSC functioned as a Rheb GAP to suppress mTORC1 and induce autophagy. Naturally occurring pathogenic mutations in TSC2 decreased PEX5 binding, abrogated peroxisome localization, Rheb GAP activity, and suppression of mTORC1 by ROS. Cells lacking peroxisomes were deficient in mTORC1 repression by ROS and peroxisome-localization deficient TSC2 mutants caused polarity defects and formation of multiple axons in neurons. These data identify a role for TSC in responding to ROS at the peroxisome, and identify the peroxisome as a signaling organelle involved in regulation of mTORC1.
PMCID: PMC3789865  PMID: 23955302
22.  Divergent dysregulation of gene expression in murine models of fragile X syndrome and tuberous sclerosis 
Molecular Autism  2014;5:16.
Fragile X syndrome and tuberous sclerosis are genetic syndromes that both have a high rate of comorbidity with autism spectrum disorder (ASD). Several lines of evidence suggest that these two monogenic disorders may converge at a molecular level through the dysfunction of activity-dependent synaptic plasticity.
To explore the characteristics of transcriptomic changes in these monogenic disorders, we profiled genome-wide gene expression levels in cerebellum and blood from murine models of fragile X syndrome and tuberous sclerosis.
Differentially expressed genes and enriched pathways were distinct for the two murine models examined, with the exception of immune response-related pathways. In the cerebellum of the Fmr1 knockout (Fmr1-KO) model, the neuroactive ligand receptor interaction pathway and gene sets associated with synaptic plasticity such as long-term potentiation, gap junction, and axon guidance were the most significantly perturbed pathways. The phosphatidylinositol signaling pathway was significantly dysregulated in both cerebellum and blood of Fmr1-KO mice. In Tsc2 heterozygous (+/−) mice, immune system-related pathways, genes encoding ribosomal proteins, and glycolipid metabolism pathways were significantly changed in both tissues.
Our data suggest that distinct molecular pathways may be involved in ASD with known but different genetic causes and that blood gene expression profiles of Fmr1-KO and Tsc2+/− mice mirror some, but not all, of the perturbed molecular pathways in the brain.
PMCID: PMC3940253  PMID: 24564913
Fragile X syndrome; Tuberous sclerosis; Autism; Cerebellum; Blood; Gene expression; Murine model
23.  Effect of orally administered simvastatin on prevention of postoperative adhesion in rats 
Aim: Formation of adhesions in the abdominal region appearing after abdominal pelvic surgery lead to infertility, chronic pelvic pain, intestinal obstructions, difficulty and morbidity at the following operations, and increased morbidity. The aim of our study is to examine the effectiveness of orally administered simvastatin on preventing the postoperative adhesion. Materials and methods: 20 male Wistar Albino rats weighing 230-250 gr were used. The rats were housed for 12 hours day and 12 hours night cycles in cages and were divided into two groups, namely study and control group. Microscopic evaluation of adhesion was assessed under 5 main topics which are the signs of inflammatory response; inflammation, activation, fibroblast activity, vascularity, presence of giant cell. Activation was scored as follows: (0) no activation, (1) while activation was accepted as present the score for other parameters was evaluated between 0 to 3 according to the increased severity. After evaluating all topics separately, the average of all scores has been assessed in both groups. Results: As a result of the macroscopic evaluation of postoperative intra-abdominal adhesions, the percentage of adhesion in simvastatin applied group was found to be 0.8 ± 0.17. This value was calculated as 0.6 ± 0.2 in the control group. Regarding the severity of adhesion, while in the simvastatin applied group the value was found to be 9.1 ± 4, in the control group it was 6.8 ± 3. The general adhesion score was found to be 7.7 ± 4.2 in simvastatin applied group and 5.1 ± 3.7 in control group. Conclusion: In this experimental study it was showed that orally administered simvastatin has no significant effect on preventing formation of postoperative adhesions.
PMCID: PMC3931595  PMID: 24600496
Surgery; adhesion; simvastatin
24.  An MRI Study of Cerebellar Volume in Tuberous Sclerosis Complex 
Pediatric neurology  2013;48(2):105-110.
The cerebellum plays an important role in motor learning and cognition, and structural cerebellar abnormalities have been associated with cognitive impairment. In tuberous sclerosis complex, neurological outcome is highly variable, and no consistent imaging or pathological determinant of cognition has been firmly established. The cerebellum calls for specific attention as mouse models of tuberous sclerosis complex have demonstrated a loss of cerebellar Purkinje cells and cases of human histological data have demonstrated a similar loss in patients. We hypothesized that there might be a common cerebellar finding in tuberous sclerosis complex that could be measured as morphometric changes with magnetic resonance imaging. Using a robust, automated image analysis procedure, we studied 36 patients with tuberous sclerosis complex and age-matched controls and observed significant volume loss among patients in the cerebellar cortices and vermis. Furthermore, this effect was strongest in a subgroup of 19 patients with a known, pathogenic mutation of the tuberous sclerosis 2 gene and impacted all cerebellar structures. We conclude that patients with tuberous sclerosis complex exhibit volume loss in the cerebellum, and this loss is larger and more widespread in patients with a tuberous sclerosis 2 mutation.
PMCID: PMC3763730  PMID: 23337002
25.  Prenatal rapamycin results in early and late behavioral abnormalities in wildtype C57Bl/6 mice 
Behavior genetics  2012;43(1):51-59.
Mammalian target of rapamycin (mTOR) signaling has been shown to be deregulated in a number of genetic, neurodevelopmental disorders including Tuberous Sclerosis Complex, Neurofibromatosis, Fragile X, and Rett syndromes. As a result, mTOR inhibitors, such as rapamycin and its analogs, offer potential therapeutic avenues for these disorders. Some of these disorders – such as Tuberous Sclerosis Complex – can be diagnosed prenatally. Thus, prenatal administration of these inhibitors could potentially prevent the development of the devastating symptoms associated with these disorders. To assess the possible detrimental effects of prenatal rapamycin treatment, we evaluated both early and late behavioral effects of a single rapamycin treatment at embryonic day 16.5 in wildtype C57Bl/6 mice. This treatment adversely impacted early developmental milestones as well as motor function in adult animals. Rapamycin also resulted in anxiety-like behaviors during both early development and adulthood but did not affect adult social behaviors. Together, these results indicate that a single, prenatal rapamycin treatment not only adversely affects early postnatal development but also results in long lasting negative effects, persisting into adulthood. These findings are of importance in considering prenatal administration of rapamycin and related drugs in the treatment of patients with neurogenetic, neurodevelopmental disorders.
PMCID: PMC3554236  PMID: 23229624
mTOR; tuberous sclerosis; embryonic; mouse

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