Mutations in the Aristaless-related homeobox (ARX) gene are found in a spectrum of epilepsy and X-linked intellectual disability disorders. During development Arx is expressed in pallial ventricular zone (VZ) progenitor cells where the excitatory projection neurons of the cortex are born. Arx−/Y mice were shown to have decreased proliferation in the cortical VZ resulting in smaller brains; however, the basis for this reduced proliferation was not established. To determine the role of ARX on cell cycle dynamics in cortical progenitor cells, we generated cerebral cortex-specific Arx mouse mutants (cKO). The loss of pallial Arx resulted in the reduction of cortical progenitor cells, particularly the proliferation of intermediate progenitor cells (IPCs) was affected. Later in development and postnatally cKO brains showed a reduction of upper layer but not deeper layer neurons consistent with the IPC defect. Transcriptional profile analysis of E14.5 Arx-ablated cortices compared with control revealed that CDKN1C, an inhibitor of cell cycle progression, is overexpressed in the cortical VZ and SVZ of Arx KOs throughout corticogenesis. We also identified ARX as a direct regulator of Cdkn1c transcription. Together these data support a model where ARX regulates the expansion of cortical progenitor cells through repression of Cdkn1c.
cell cycle; forebrain development; neurogenesis; neurodevelopmental disorders; TBR2
Mutations in the Aristaless related homeodomain transcription factor (ARX) are associated with a diverse set of X-linked mental retardation and epilepsy syndromes in humans. Although most studies have been focused on its function in the forebrain, ARX is also expressed in other regions of the developing nervous system including the floor plate (FP) of the spinal cord where its function is incompletely understood. To investigate the role of Arx in the FP, we performed gain-of-function studies in the chick using in ovo electroporation, and loss-of-function studies in Arx-deficient mice. We have found that Arx, in conjunction with FoxA2, directly induces Sonic hedgehog (Shh) expression through binding to a Shh floor plate enhancer (SFPE2). We also observed that FoxA2 induces Arx through its transcriptional activation domain whereas Nkx2.2, induced by Shh, abolishes this induction. Our data support a feedback loop model for Arx function; through interactions with FoxA2, Arx positively regulates Shh expression in the FP, and Shh signaling in turn activates Nkx2.2, which suppresses Arx expression. Furthermore, our data are evidence that Arx plays a role as a context dependent transcriptional activator, rather than a primary inducer of Shh expression, potentially explaining how mutations in ARX are associated with diverse, and often subtle, defects.
Spinal cord; neural tube; floor plate; development; Shh; Arx; FoxA2 and Nkx2.2
Rationale: Obesity and underweight are contraindications to lung
transplantation based on their associations with mortality in studies performed
before implementation of the lung allocation score (LAS)–based organ
allocation system in the United States
Objectives: To determine the associations of body mass index (BMI) and
plasma leptin levels with survival after lung transplantation.
Methods: We used multivariable-adjusted regression models to examine
associations between BMI and 1-year mortality in 9,073 adults who underwent lung
transplantation in the United States between May 2005 and June 2011, and plasma
leptin and mortality in 599 Lung Transplant Outcomes Group study participants. We
measured body fat and skeletal muscle mass using whole-body dual X-ray absorptiometry
in 142 adult lung transplant candidates.
Measurements and Main Results: Adjusted mortality rates were similar
among normal weight (BMI 18.5–24.9 kg/m2), overweight (BMI
25.0–29.9), and class I obese (BMI 30–34.9) transplant recipients.
Underweight (BMI < 18.5) was associated with a 35% increased rate of death (95%
confidence interval, 10–66%). Class II–III obesity (BMI ≥ 35
kg/m2) was associated with a nearly twofold increase in mortality
(hazard ratio, 1.9; 95% confidence interval, 1.3–2.8). Higher leptin levels
were associated with increased mortality after transplant surgery performed without
cardiopulmonary bypass (P for interaction = 0.03). A BMI
greater than or equal to 30 kg/m2 was 26% sensitive and 97% specific for
total body fat–defined obesity.
Conclusions: A BMI of 30.0–34.9 kg/m2 is not
associated with 1-year mortality after lung transplantation in the LAS era, perhaps
because of its low sensitivity for obesity. The association between leptin and
mortality suggests the need to validate alternative methods to measure obesity in
candidates for lung transplantation. A BMI greater than or equal to 30
kg/m2 may no longer contraindicate lung transplantation.
obesity; sarcopenia; adiposity; leptin; biomarker
Lung transplant (LT) aims to extend survival and improve patient-centered outcomes (PCOs) by reducing disability and improving health-related quality of life (HRQL). Few PCO instruments have been validated in LT populations. We aimed to develop and validate a shortened version of the Valued Life Activities (VLA) disability scale specific to LT.
We used data from 140 subjects participating in an ongoing cohort study of LT. Subjects completed a survey battery, including VLA items, and physical assessments before LT. To develop a shortened lung transplant specific VLA (LT-VLA), we iteratively deleted items from a longer 32-item VLA battery, retaining the instrument’s conceptual framework, scoring and performance characteristics. We evaluated LT-VLA validity by testing correlations with a HRQL measure (Short Form-12 Physical Function [SF-12 PF] subscale), FVC% predicted, and 6-minute walk distance (6MWD). Responsiveness was evaluated in 84 subjects who completed assessments before and after LT.
The 15-item LT-VLA scoring closely matched the longer VLA (correlations ≥0.96) and had excellent internal consistency (Cronbach’s alpha: 0.92). The LT-VLA required only 3 minutes or less to administer. The LT-VLA, measured as mean difficulty in performing each of the 15 activities queried, correlated with FVC% predicted (r= −0.30), 6MWD (r= −0.38) and SF-12 PF (r= −0.47) (all p<0.01). The LT-VLA mean difficulty was responsive to change from before to after LT: (63% improvement; effect size=1.60)
The LT-VLA is a short, easy to administer, valid, and responsive disease-specific PCO instrument that may be useful in clinical and research applications for lung transplantation.
lung transplantation; disability; patient centered outcomes; health-related quality of life
Mutations in the aristaless-related homeobox (ARX) gene result in a spectrum of structural and functional nervous system disorders including lissencephaly, movement disorders, intellectual disabilities, and epilepsy. Some patients also have symptoms indicative of hypothalamic dysfunction, however, little is known about the role of ARX in diencephalic development. To begin evaluating diencephalic defects we examined the expression of a panel of known genes and gene products that label specific diencephalic nuclei in two different Arx mutant mouse lines. Male mice engineered to have a polyalanine expansion mutation (Arx(GCG)7/Y) revealed no expression differences in any diencephalic nucleus when compared to wildtype littermates. In contrast, mice null for Arx (Arx−/Y) lost expression of specific markers of the thalamic reticular nucleus (TRN) and zona incerta (ZI), while retaining expression in other thalamic nuclei and in the hypothalamus. Tyrosine hydroxylase, a marker of the ZI’s dopaminergic A13 sub-nucleus, was among those lost, suggesting a requirement for Arx in normal TRN and ZI development, and for A13 dopaminergic fate, specifically. Since the ZI and A13 regions make connections to several hypothalamic nuclei, such mis-specification may contribute to the “hypothalamic dysfunction” observed in some patients.
Arx; zona incerta; thalamic reticular nucleus; A13; dopaminergic fate; diencephalon
Normal cerebral cortical function requires a highly ordered balance between projection neurons and interneurons. During development these two neuronal populations migrate from distinct progenitor zones to form the cerebral cortex, with interneurons originating in the more distant ganglionic eminences. Moreover, deficits in interneurons have been linked to a variety of neurodevelopmental disorders underscoring the importance of understanding interneuron development and function. We, and others, have identified SDF1 signaling as one important modulator of interneuron migration speed and leading process branching behavior in mice, although how SDF1 signaling impacts these behaviors remains unknown. We previously found SDF1 inhibited leading process branching while increasing the rate of migration. We have now mechanistically linked SDF1 modulation of leading process branching behavior to a dual regulation of both actin and microtubule organization. We find SDF1 consolidates actin at the leading process tip by de-repressing calpain protease and increasing proteolysis of branched-actin-supporting cortactin. Additionally, SDF1 stabilizes the microtubule array in the leading process through activation of the microtubule-associated protein doublecortin (DCX). DCX stabilizes the microtubule array by bundling microtubules within the leading process, reducing branching. These data provide mechanistic insight into the regulation of interneuron leading process dynamics during neuronal migration in mice and provides insight into how cortactin and DCX, a known human neuronal migration disorder gene, participate in this process.
cortactin; cytoskeleton; DCX; interneuron; migration; SDF1
Leber’s congenital amaurosis (LCA) is a group of severe inherited retinal degenerations that are symptomatic in infancy and lead to total blindness in adulthood. Recent clinical trials using recombinant adeno-associated virus serotype 2 (rAAV2) successfully reversed blindness in patients with LCA caused by RPE65 mutations after one subretinal injection. However, it was unclear whether treatment of the second eye in the same manner would be safe and efficacious, given the potential for a complicating immune response after the first injection. Here, we evaluated the immunological and functional consequences of readministration of rAAV2-hRPE65v2 to the contralateral eye using large animal models. Neither RPE65-mutant (affected; RPE65−/−) nor unaffected animals developed antibodies against the transgene product, but all developed neutralizing antibodies against the AAV2 capsid in sera and intraocular fluid after subretinal injection. Cell-mediated immune responses were benign, with only 1 of 10 animals in the study developing a persistent T cell immune response to AAV2, a response that was mediated by CD4+ T cells. Sequential bilateral injection caused minimal inflammation and improved visual function in affected animals. Thus, subretinal readministration of rAAV2 in animals is safe and effective, even in the setting of preexisting immunity to the vector, a parameter that has been used to exclude patients from gene therapy trials.
Mutations in human LIS1 cause abnormal neuronal migration and a smooth brain phenotype known as lissencephaly. Lis1+/− (Pafah1b1) mice show defective lamination in the cerebral cortex and hippocampal formation, whereas homozygous mutations result in embryonic lethality. Given that Lis1 is highly expressed in embryonic neurons, we hypothesized that sympathetic and parasympathetic preganglionic neurons (SPNs and PPNs) would exhibit migratory defects in Lis1+/− mice. The initial radial migration of SPNs and PPNs that occurs together with somatic motor neurons appeared unaffected in Lis1+/− mice. The subsequent dorsally directed tangential migration, however, was aberrant in a subset of these neurons. At all embryonic ages analyzed, the distribution of SPNs and PPNs in Lis1+/− mice was elongated dorsoventrally compared with Lis1+/+ mice. Individual cell bodies of ectopic preganglionic neurons were found in the ventral spinal cord with their leading processes oriented along their dorsal migratory trajectory. By birth, Lis1+/− SPNs and PPNs were separated into distinct groups, those that were correctly, and those incorrectly positioned in the intermediate horn. As mispositioned SPNs and PPNs still were detected in P30 Lis1+/− mice, we conclude that these neurons ceased migration prematurely. Additionally, we found that a dorsally located group of somatic motor neurons in the lumbar spinal cord, the retrodorsolateral nucleus, showed delayed migration in Lis1+/− mice. These results suggest that Lis1 is required for the dorsally directed tangential migration of many sympathetic and parasympathetic preganglionic neurons and a subset of somatic motor neurons.
lissencephaly; pafah1b1; reeler; sympathetic preganglionic neurons; parasympathetic preganglionic neurons; somatic motor neurons
Belatacept is a novel immunosuppressant that blocks a T-cell costimulation pathway and is approved for use in adult kidney transplant recipients. Its safety and efficacy have not been established after lung transplantation. We present a case of a lung transplant recipient treated with belatacept. A 56-year-old man underwent bilateral lung retransplantation for bronchiolitis obliterans syndrome (BOS). In the third year posttransplant, he developed hemolytic uremic syndrome (HUS) attributed to tacrolimus. Tacrolimus was changed to sirolimus. One month later, he presented with worsening renal function and HUS attributed to sirolimus. Plasmapheresis and steroid pulse were initiated with clinical improvement, and sirolimus was switched to belatacept. He experienced no episodes of cellular rejection but developed recurrent BOS. Complications during treatment included anemia and recurrent pneumonias. The safety and efficacy of belatacept in lung transplantation remains unclear; further studies are needed.
Belatacept; lung transplant; novel immunosuppression; outcomes
Lung disease (LD) is the leading cause of death in systemic sclerosis (SSc). The diagnosis of SSc-related LD (SSc-LD) is often a contraindication to lung transplantation (LT) due to concerns that extra-pulmonary involvement will yield worse outcomes. We sought to evaluate post-transplant outcomes in persons with SSc-LD with esophageal involvement compared to persons with non-connective tissue disease related interstitial lung disease (nCTD-ILD).
From 1998-2012, persons undergoing LT for SSc-LD were age and gender matched in a 2:1 fashion to controls undergoing LT for nCTD-ILD. Esophageal function was assessed by pH testing and manometry. We defined esophageal dysfunction as the presence of a DeMeester score >14 or dysmotility more severe than “mild non-specific disorder”. The primary outcome was post-transplant survival. Secondary outcomes included freedom from bronchiolitis obliterans syndrome (fBOS) and rates of acute rejection. Survival and fBOS were estimated with Kaplan-Meier methods. Acute rejection was compared with Students t-test.
Survival was similar in 23 persons with SSc-LD and 46 controls who underwent LT (p=0.47). For the SSc-LD group, 1- and 5-year survival was 83% and 76% compared to 91% and 64% in the nCTD-ILD group. There were no differences in fBOS (p=0.83). Rates of acute rejection were less in SSc-ILD (p=0.05). Esophageal dysfunction was not associated with worse outcomes (p>0.55).
Persons with SSc-LD appear to have similar survival and fBOS as persons transplanted for nCTD-ILD. The risk of acute rejection after transplant may be reduced in persons with SSc-LD. Esophageal involvement does not appear to impact outcomes.
Lung Transplantation; Systemic Sclerosis; Interstitial Lung Disease; survival; esophageal dysmotility; bronchiolitis obliterans syndrome
Rationale: Lung transplantation offers great promise for otherwise terminal lung diseases, but the development of bronchiolitis obliterans syndrome (BOS) continues to limit survival. Although acute rejection and lymphocytic bronchiolitis have been identified as risk factors for the development of BOS, it is unclear whether large-airway lymphocytic inflammation conveys the same risk.
Objectives: We evaluated lymphocytic bronchitis on endobronchial biopsies as a risk factor for BOS and mortality.
Methods: Endobronchial biopsies were collected and graded during surveillance after lung transplantation. We assessed samples with negative cultures collected in the first 90 days from 298 subjects and compared large-airway lymphocytic bronchitis assessed by a 0–2 “E-score” and with standard A and BR pathology scores for acute rejection and small-airway lymphocytic bronchiolitis, respectively.
Measurements and Main Results: We found surprisingly little association between large- and small-airway lymphocytic inflammation scores from a given bronchoscopy. Endobronchial lymphocytic bronchitis was more prevalent in subjects in BOS stage 0p and BOS stages 1–3 at the time of biopsy. Within 90 days after transplantation, increasing maximum E-score was associated with greater risk of BOS (adjusted hazard ratio, 1.76; 95% confidence interval, 1.11–2.78; P = 0.02) and in this analysis 90-day maximum E-scores were the only score type predictive of BOS (P < 0.01).
Conclusions: These results support a multicenter study to evaluate endoscopic biopsies for the identification of patients at increased risk for BOS. The association of endobronchial lymphocytic inflammation and BOS may have mechanistic implications.
bronchiolitis obliterans; graft rejection; bronchoscopy; lung transplantation
Community acquired respiratory virus (CARV) infections in lung transplant recipients (LTR) have been associated with adverse outcomes, including acute rejection (AR) and decline in allograft function, in some but not all studies.
Spirometry and transbronchial biopsy results of LTR diagnosed with CARV infection over a 2-year period were extracted from clinical records. Primary outcomes, studied at 1-2.5 months post-infection, were: (1) incidence of biopsy-proven AR (grade>A0) and (2) allograft function, defined by forced expiratory volume in 1 second (FEV1). A reference group of biopsies (n=526) collected during the study period established the baseline incidence of AR. Rhinovirus (RV) and non-rhinovirus (non-RV) infections were analyzed as subgroups.
87 cases of CARV infection were identified in 59 subjects. Incidences of AR were similar in the post-CARV and reference groups, and did not differ significantly after RV vs non-RV infection. Allograft function declined significantly after non-RV infection, but not after RV infection.
In LTR, CARV infections other than RV are associated with allograft dysfunction at 1-2.5 months after infection. However, CARVs do not appear associated with AR at this time point. The impact of specific CARVs on lung allografts, including the development of chronic allograft rejection, merits further study.
Lung transplantation; graft rejection; adenoviridae infections; influenza; paramyxoviridae infections; picornaviridae infections
Mutations affecting proteolipid protein 1 (PLP1), the major protein in central nervous system myelin, cause the X-linked leukodystrophy Pelizaeus–Merzbacher disease (PMD). We describe the neuropathologic findings in a series of eight male PMD subjects with confirmed PLP1 mutations, including duplications, complete gene deletion, missense and exon-skipping. While PLP1 mutations have effects on oligodendrocytes that result in mutation-specific degrees of dysmyelination, our findings indicate that there are also unexpected effects in the central nervous system resulting in neuronal loss. Although length-dependent axonal degeneration has been described in PLP1 null mutations, there have been no reports on neuronal degeneration in PMD patients. We now demonstrate widespread neuronal loss in PMD. The patterns of neuronal loss appear to be dependent on the mutation type, suggesting selective vulnerability of neuronal populations that depends on the nature of the PLP1 disturbance. Nigral neurons, which were not affected in patients with either null or severe misfolding mutations, and thalamic neurons appear particularly vulnerable in PLP1 duplication and deletion patients, while hippocampal neuronal loss was prominent in a patient with complete PLP1 gene deletion. All subjects showed cerebellar neuronal loss. The patterns of neuronal involvement may explain some clinical findings, such as ataxia, being more prominent in PMD than in other leukodystrophies. While the precise pathogenetic mechanisms are not known, these observations suggest that defective glial functions contribute to neuronal pathology.
Cutaneous squamous cell carcinoma (cSCC) is the most common malignancy after solid organ transplantation, with an increased risk of recurrence and metastasis over the general population. The newly updated 7th edition American Joint Committee on Cancer (AJCC) staging system for cSCC is based on consensus expert opinion and requires validation in large cohort studies and in specific patient subpopulations.
Our objective was to evaluate the risk of cSCC recurrence in a high-risk population of heart and lung transplant recipients, based on the 7th edition AJCC staging system.
We performed a 10-year retrospective cohort study of all primary cSCC diagnosed in heart and lung transplant recipients at a tertiary care academic dermatology center.
The cumulative incidence of local recurrence was 4% for cSCC in situ and 19% for Stage I cSCC at 5 years, and 54% for Stage II cSCC at 3 years. Stage II tumors had a 10-fold greater risk of recurrence than stage I, and a 43-fold greater risk of recurrence than in situ tumors.
This study is limited to a specific patient subgroup at a tertiary care center, and may not be generalizable to all populations.
Heart and lung transplant recipients are at high risk for local recurrence of cSCC. These data substantiate the prognostic accuracy of the newly updated 7th edition AJCC staging system for stage 0, I and II cSCC in this population and demonstrate the aggressive behavior of this cancer in immunosuppressed patients.
Squamous Cell Carcinoma; Lung Transplantation; Heart Transplantation; Staging
Lung transplant recipients (LTR) have an increased risk of cutaneous squamous cell carcinoma (SCC) due to immunosuppressive therapy. Voriconazole, which is associated with phototoxic side effects in some patients, may be an additional risk factor for SCC in this population.
To test whether voriconazole is a risk factor for developing SCC in LTR, we evaluated cumulative exposure to voriconazole in 327 adults who underwent lung transplantation at a single center between 1991 and 2010. Voriconazole exposure was assessed as a time-varying covariate. We analyzed risk of developing SCC over time using survival analysis methods.
Exposure to voriconazole was associated with a 2.6-fold increased risk for SCC. This phenomenon was dose-dependent; the risk for SCC increased by 5.6% with each 60-day exposure at a standard dose of 200mg twice daily. At five years posttransplant, voriconazole conferred an absolute risk increase for SCC of 28%.
These results suggest that caution should be taken when using voriconazole in LTR, as this drug increases the already high risk for SCC in this population.
Voriconazole; Squamous Cell Carcinoma; Lung Transplantation; Skin Cancer; Risk Factors
The relationship of mast cells to the pathogenesis of lung fibrosis remains undefined despite recognition of their presence in the lungs of patients with pulmonary fibrosis. This study was performed to characterize the relationship of mast cells to fibrotic lung diseases.
Methods and results
Lung tissues from patients with idiopathic pulmonary fibrosis (IPF), chronic hypersensitivity pneumonitis (HP), systemic sclerosis (SSc)-related interstitial lung disease (ILD) and normal individuals were subjected to chymase immunostaining and the mast cell density quantified. Eosinophils were quantified by immunostaining for eosinophil peroxidase. Changes in lung function were correlated with mast cell density. Lung tissue obtained from IPF patients had a higher density of chymase-immunoreactive mast cells than that from patients with HP, SSc-related ILD or normal lungs. IPF lung tissue had a higher density of eosinophils than normal lung. There was no correlation between mast cell density and eosinophil density in IPF lung. IPF patients with high mast cell density had a slower rate of decline in forced vital capacity (FVC) than IPF patients with low mast cell density.
Mast cell density in IPF lungs is higher than in other fibrotic lung diseases and normal lungs. Increased mast cell density in IPF may predict slower disease progression.
chloroacetate esterase; eosinophil; hypersensitivity pneumonitis; idiopathic interstitial pneumonia; systemic sclerosis
Intrauterine growth retardation (IUGR) is associated with neurological deficits including cerebral palsy and cognitive and behavioral disabilities. The pathogenesis involves oxidative stress that leads to periventricular white matter injury with a paucity of mature oligodendrocytes and hypomyelination. The molecular mechanisms underlying this damage remain poorly understood. We employed a rat model of IUGR created by bilateral ligation of the uterine artery at embryonic day 19 that results in fetal growth retardation and oxidative stress in the developing brain. The IUGR rat pups showed significant delays in oligodendrocyte differentiation and myelination that resolved by 8 weeks. Bone morphogenetic protein 4 (BMP4), which inhibits oligodendrocyte maturation, was elevated in IUGR brains at postnatal time points and returned to near normal by adulthood. Despite the apparent recovery, behavioral deficiencies were found in 8-week-old female animals, suggesting that the early transient myelination defects have permanent effects. In support of these in vivo data, oligodendrocyte precursor cells cultured from postnatal IUGR rats retained increased BMP4 expression and impaired differentiation that was reversed with the BMP inhibitor noggin. Oxidants in oligodendrocyte cultures increased BMP expression, which decreased differentiation; however, abrogating BMP signaling with noggin in vitro and in BMP-deficient mice prevented these effects. Together, these findings suggest that IUGR results in delayed myelination through the generation of oxidative stress that leads to BMP4 upregulation.
Bone morphogenetic protein (BMP); Intrauterine growth retardation; Myelin; Oligodendrocytes; Oxidative stress; Periventricular white matter injury
Microcephaly is a neurodevelopmental disorder causing significantly reduced cerebral cortex size. Many known microcephaly gene products localize to centrosomes, regulating cell fate and proliferation. Here, we identify and characterize a nuclear zinc finger protein, ZNF335/NIF-1, as a causative gene for severe microcephaly, small somatic size, and neonatal death. Znf335-null mice are embryonically lethal and conditional knockout leads to severely reduced cortical size. RNA-interference and postmortem human studies show that Znf335 is essential for neural progenitor self-renewal, neurogenesis, and neuronal differentiation. ZNF335 is a component of a vertebrate-specific, trithorax H3K4-methylation complex, directly regulating REST/NRSF, a master regulator of neural gene expression and cell fate, as well as other essential neural-specific genes. Our results reveal ZNF335 as an essential link between H3K4 complexes and REST/NRSF, and provide the first direct genetic evidence that this pathway regulates human neurogenesis and neuronal differentiation.
Progressive multisystem disease should invoke consideration of potential mitochondrial etiologies. Mitochondrial disease can affect any organ system at any time, particularly involving neurologic, cardiac, muscular, gastroenterologic, and/or ophthalmologic manifestations. We report here a 19-year-old Caucasian man who was followed since birth in multiple pediatric subspecialty clinics for myelomeningocele complications. However, he progressively developed a host of additional problems that were not readily attributable to his neural tube defect but involved developmental, ophthalmologic, cardiac, muscular, endocrine, and intermediary metabolic manifestations. Clinical diagnostic testing limited to analysis for common point mutations and deletions in his blood mitochondrial DNA (mtDNA) was not revealing. Skeletal muscle biopsy revealed abnormal mitochondrial morphology and immunostaining, mitochondrial proliferation, and mildly reduced respiratory chain complex I–III activity. Whole mitochondrial genome sequencing analysis in muscle identified an apparently homoplasmic, novel, 12264C>T transition in the tRNA serine (AGY) gene. The pathogenicity of this mutation was supported by identification of it being present at low heteroplasmy load in his blood (34 percent) as well as in blood from his maternal grandmother (1 percent). Interestingly, the proband developed severe nuclear cataracts that proved to be homoplasmic for the pathogenic mtDNA 12264C>T mutation. This case highlights the value of pursuing whole mitochondrial genome sequencing in symptomatic tissues in the diagnostic evaluation of suspected mitochondrial disease. Furthermore, it is the first report to directly implicate an mtDNA mutation in the pathogenesis of ocular cataracts and clearly illustrates the important contribution of normal metabolic activity to the function of the ocular lens.
Polyalanine (poly-A) tracts exist in 494 annotated proteins; to date, expansions in these tracts have been associated with nine human diseases. The pathogenetic mechanism by which a poly-A tract results in these various human disorders remains uncertain. To understand the role of this mutation type, we investigated the change in functional properties of the transcription factor Arx when it has an expanded poly-A tract (ArxE), a mutation associated with infantile spasms and intellectual disabilities in humans. We found that although ArxE functions normally in the dorsal brain, its function in subpallial-derived populations of neurons is compromised. These contrasting functions are associated with the misregulation of Arx targets through the loss of the ability of ArxE to interact with the Arx cofactor Tle1. Our data demonstrate a novel mechanism for poly-A expansion diseases: the misregulation of a subset of target genes normally regulated by a transcription factor.
Mutations in the Aristaless-related homeobox gene (ARX) are associated with a wide variety of neurologic disorders including lissencephaly, hydrocephaly, West syndrome, Partington syndrome, and X-linked intellectual disability with or without epilepsy. A genotype-phenotype correlation exists for ARX mutations, however the molecular basis for this association has not been investigated. To begin understanding the molecular basis for ARX mutations, we tested the DNA binding sequence preference and transcriptional repression activity for Arx, deletion mutants and mutants associated with various neurologic disorders. We found DNA binding preferences of Arx are influenced by the amino acid sequences adjacent to the homeodomain. Mutations in the homeodomain show a loss DNA binding activity, while the T333N and P353R homeodomain mutants still possess DNA binding activities, although less than wild type. Transcription repression activity, the primary function of ARX, is reduced in all mutants except the L343Q, which has no DNA binding activity and does not functionally repress Arx targets. These data indicate that mutations in the homeodomain result in not only a loss of DNA binding activity but also loss of transcriptional repression activity. Our results provide novel insights into the pathogenesis of ARX related disorders and possible directions to pursue potential therapeutic interventions.
ARX; lissencephaly; X-linked intellectual disability and Homeodomain
Malformations of cortical development are frequently identified in surgical resections for intractable epilepsy. Among the more frequently identified are cortical dysplasia, pachygyria and polymicrogyria. The pathogenesis of these common developmental anomalies remains uncertain. Polymicrogyria is particularly vexing because there are multiple described forms (2, 4 and 6 layer) that have been attributed to multiple etiologies (e.g. ischemic, genetic, infectious, and toxic). We reviewed the pathology in 19 cases and performed cortical laminar analysis in 10 of these cases. Our data indicate that a defining feature of polymicrogyria is fusion of the molecular layer and that most often there is a well-defined grey matter-white matter junction. Unexpectedly, the cortical lamina were normally positioned but there were reduced neuronal populations within these lamina, particularly in the subgranular layers. Based on these data, we propose that the categorization of polymicrogyria according to the number of lamina is artificial and should be abandoned and polymicrogyria should be defined according to the presence or absence of coexisting neuropathological features. Furthermore, our data indicate that polymicrogyria is not a cell migration disorder and rather that it should be considered a post-migration malformation of cortical development.
Cell migration; Cerebral cortex; Cortical lamina; Malformation of cortical development; Polymicrogyria; Seizures
Sizn1 (Zcchc12) is a transcriptional co-activator that positively modulates BMP (Bone Morphogenic Protein) signaling through its interaction with Smad family members and CBP. We have demonstrated a role for Sizn1 in basal forebrain cholinergic neuron specific gene expression. Furthermore, mutations in SIZN1 have been associated with X-linked mental retardation. Given the defined role of SIZN1 in mental retardation, knowing its complete forebrain expression pattern is essential to further elucidating its role in cognition. To better define the dynamic expression pattern of Sizn1 during forebrain development, we investigated its expression in mouse brain development from embryonic day 8.0 (E8.0) to adult. We found that Sizn1 is primarily restricted to the ventral forebrain including the medial ganglionic eminence, the septum, amygdala, and striatum. In addition, Sizn1 expression is detected in the cortical hem and Pallial-subpallial boundary (PSB; anti-hem); both sources of Cajal-Retzius cells. Sizn1 expression in the dorsal forebrain is restricted to a subset of cells in the marginal zone that also express Reln, indicative of Cajal-Retzius cells. These data provide novel information on brain regions and cell types that express Sizn1, facilitating further investigations into the function of Sizn1 in both development and the pathogenesis of mental retardation.
Cell migration is required for normal embryonic development, yet how cells navigate complex paths while integrating multiple guidance cues remains poorly understood. During brain development, interneurons migrate from the ventral ganglionic eminence to the cerebral cortex within several migratory streams. They must exit these streams to invade the cortical plate. While SDF1-signaling is necessary for normal interneuron stream migration, how they switch from tangential stream migration to invade the cortical plate is unknown. Here we demonstrate that SDF1-signaling reduces interneuron branching frequency by reducing cAMP levels via a Gi-signaling pathway using an in vitro mouse explant system, resulting in the maintenance of stream migration. Blocking SDF1-signaling, or increasing branching frequency, results in stream exit and cortical plate invasion in mouse brain slices. These data support a novel model to understand how migrating interneurons switch from tangential migration to invade the cortical plate in which reducing SDF1-signaling increases leading process branching and slows the migration rate, permitting migrating interneurons to sense cortically directed guidance cues.
Interneuron; migration; branching; SDF1; guidance
Mammalian forebrain development requires extensive migration, yet the mechanisms through which migrating neurons sense and respond to guidance cues are not well understood. Similar to the axon growth cone, the leading process and branches of neurons may guide migration, but the cytoskeletal events that regulate branching are unknown. We have previously shown that loss of microtubule-associated protein Lis1 reduces branching during migration compared with wild-type neurons. Using time-lapse imaging of Lis1+/− and Lis1+/+ cells migrating from medial ganglionic eminence explant cultures, we show that the branching defect is not due to a failure to initiate branches but a defect in the stabilization of new branches. The leading processes of Lis1+/− neurons have reduced expression of stabilized, acetylated microtubules compared with Lis1+/+ neurons. To determine whether Lis1 modulates branch stability through its role as the noncatalytic β regulatory subunit of platelet-activating factor (PAF) acetylhydrolase 1b, exogenous PAF was applied to wild-type cells. Excess PAF added to wild-type neurons phenocopies the branch instability observed in Lis1+/− neurons, and a PAF antagonist rescues leading process branching in Lis1+/− neurons. These data highlight a role for Lis1, acting through the PAF pathway, in leading process branching and microtubule stabilization.
branching; leading process; microtubules; migration; PAFAH1b