Assorted dysfunctions of endosomal alkali cation/proton exchanger SLC9A6 variants linked to Christianson syndrome.

Genetic screening has identified numerous variants of the endosomal solute carrier family 9 member A6 (SLC9A6)/(Na+,K+)/H+ exchanger 6 (NHE6) gene that cause Christianson syndrome, a debilitating X-linked developmental disorder associated with a range of neurological, somatic, and behavioral symptoms. Many of these variants cause complete loss of NHE6 expression, but how subtler missense substitutions or nonsense mutations that partially truncate its C-terminal cytoplasmic regulatory domain impair NHE6 activity and endosomal function are poorly understood. Here, we describe the molecular and cellular consequences of six unique mutations located in the N-terminal cytoplasmic segment (A9S), the membrane ion translocation domain (L188P and G383D), and the C-terminal regulatory domain (E547*, R568Q, and W570*) of human NHE6 that purportedly cause disease. Using a heterologous NHE6-deficient cell expression system, we show that the biochemical, catalytic, and cellular properties of the A9S and R568Q variants were largely indistinguishable from those of the WT transporter, which obscured their disease significance. By contrast, the L188P, G383D, E547*, and W570* mutants exhibited variable deficiencies in biosynthetic post-translational maturation, membrane sorting, pH homeostasis in recycling endosomes, and cargo trafficking, and they also triggered apoptosis. These findings broaden our understanding of the molecular dysfunctions of distinct NHE6 variants associated with Christianson syndrome.

Roles of Collagen XXV and Its Putative Receptors PTPσ/δ in Intramuscular Motor Innervation and Congenital Cranial Dysinnervation Disorder.

Intramuscular motor innervation is an essential process in neuromuscular development. Recently, mutations in COL25A1, encoding CLAC-P/collagen XXV, have been linked to the development of a congenital cranial dysinnervation disorder (CCDD). Yet the molecular mechanisms of intramuscular innervation and the etiology of CCDD related to COL25A1 have remained elusive. Here, we report that muscle-derived collagen XXV is indispensable for intramuscular innervation. In developing skeletal muscles, Col25a1 expression is tightly regulated by muscle excitation. In vitro and cell-based assays reveal a direct interaction between collagen XXV and receptor protein tyrosine phosphatases (PTPs) σ and δ. Motor explant assays show that expression of collagen XXV in target cells attracts motor axons, but this is inhibited by exogenous PTPσ/δ. CCDD mutations attenuate motor axon attraction by reducing collagen XXV-PTPσ/δ interaction. Overall, our study identifies PTPσ/δ as putative receptors for collagen XXV, implicating collagen XXV and PTPσ/δ in intramuscular innervation and a developmental ocular motor disorder.

Amyloid clearance defect in ApoE4 astrocytes is reversed by epigenetic correction of endosomal pH.

Endosomes have emerged as a central hub and pathogenic driver of Alzheimer's disease (AD). The earliest brain cytopathology in neurodegeneration, occurring decades before amyloid plaques and cognitive decline, is an expansion in the size and number of endosomal compartments. The strongest genetic risk factor for sporadic AD is the ε4 allele of Apolipoprotein E (ApoE4). Previous studies have shown that ApoE4 potentiates presymptomatic endosomal dysfunction and defective endocytic clearance of amyloid beta (Aß), although how these two pathways are linked at a cellular and mechanistic level has been unclear. Here, we show that aberrant endosomal acidification in ApoE4 astrocytes traps the low-density lipoprotein receptor-related protein (LRP1) within intracellular compartments, leading to loss of surface expression and Aß clearance. Pathological endosome acidification is caused by ε4 risk allele-selective down-regulation of the Na+/H+ exchanger isoform NHE6, which functions as a critical leak pathway for endosomal protons. In vivo, the NHE6 knockout (NHE6KO) mouse model showed elevated Aß in the brain, consistent with a causal effect. Increased nuclear translocation of histone deacetylase 4 (HDAC4) in ApoE4 astrocytes, compared with the nonpathogenic ApoE3 allele, suggested a mechanistic basis for transcriptional down-regulation of NHE6. HDAC inhibitors that restored NHE6 expression normalized ApoE4-specific defects in endosomal pH, LRP1 trafficking, and amyloid clearance. Thus, NHE6 is a downstream effector of ApoE4 and emerges as a promising therapeutic target in AD. These observations have prognostic implications for patients who have Christianson syndrome with loss of function mutations in NHE6 and exhibit prominent glial pathology and progressive hallmarks of neurodegeneration.

Expression of MyoD, insulin like growth factor binding protein, thioredoxin and p27 in secondarily overacting inferior oblique muscles with superior oblique palsy.

BACKGOUND: To identify and compare specific protein levels between overacting inferior oblique (IO) muscles in superior oblique (SO) palsy patients and normal IO muscles. METHODS: We obtained 20 IO muscle samples from SO palsy patients with IO overaction ≥ + 3 who underwent IO myectomies (IOOA group), and 20 IO samples from brain death donors whose IO had functioned normally, according to their ophthalmological chart review (control group). We used MyoD for identifying satellite cell activation, insulin-like growth factor binding protein 5 (IGFBP5) for IGF effects, thioredoxin for oxidative stress, and p27 for satellite cell activation or oxidative stress in both groups. Using immunohistochemistry and Western blot, we compared expression levels of the four proteins (MyoD, IGFBP5, thioredoxin, and p27). RESULTS: Levels of thioredoxin and p27 were decreased significantly in the IOOA group. MyoD and IGFBP5 levels showed no significant difference between the groups. CONCLUSIONS: Based on these findings, the overacting IOs of patients with SO palsy had been under oxidative stress status versus normal IOs. Pathologically overacting extraocular muscles may have an increased risk of oxidative stress compared with normal extraocular muscles.

Tendinous muscle insertions (scleromuscular junctions of the recti muscles) in patients with ocular alignment problems.

BACKGROUND: The purpose of this study was to prove the hypothesis whether the scleromuscular junction of extraocular recti muscle is tendinous. PATIENTS AND METHODS: Muscle samples of the 41 extraocular recti muscles of 33 patients and 4 muscle-/eye-matched samples from 2 postmortem eyes, were processed for light/electron microscopy and immunohistochemistry with antibodies against desmin, smooth-muscle actin and muscle regulating proteins like myf3 and myf4 (myogenin), tenascin C and for 8 samples against collagens I to IV. RESULTS: Histological examination of the muscle samples confirmed a thick collagen-structured tissue, specific for muscle tendon; without appearance of muscle tissue. This was confirmed by immunohistochemistry with antibodies against desmin, smooth-muscle actin, myf3 and myf4 (myogenin) and for eight samples with collagens I to IV. Anti-tenascin C marker was only strongly positive in the connective tissue of the blood vessel walls. Electron microscopy demonstrated collagen bundles composed of parallel oriented fibrils with a moderate amount of ground substance. CONCLUSIONS: The absence of contractile fibers at the sclerotendinous junction is an entirely normal finding in humans and cannot be related to ocular alignment pathogenesis.

Horizontal Gaze Palsy With Progressive Scoliosis and Severe Keratoconus With a Compound Heterozygous Mutation in ROBO3.

The authors report the case of a child with horizontal gaze palsy with progressive scoliosis and keratoconus. ROBO3 analysis identified compound heterozygous mutations. Keratoconus surgical approach resulted in visual acuity improvement in both eyes. The previously unreported occurrence of keratoconus with horizontal gaze palsy with progressive scoliosis suggests that ophthalmologic assessment should search for signs of this ectasia in these patients.

CFEOM1-associated kinesin KIF21A is a cortical microtubule growth inhibitor.

Mechanisms controlling microtubule dynamics at the cell cortex play a crucial role in cell morphogenesis and neuronal development. Here, we identified kinesin-4 KIF21A as an inhibitor of microtubule growth at the cell cortex. In vitro, KIF21A suppresses microtubule growth and inhibits catastrophes. In cells, KIF21A restricts microtubule growth and participates in organizing microtubule arrays at the cell edge. KIF21A is recruited to the cortex by KANK1, which coclusters with liprin-α1/ß1 and the components of the LL5ß-containing cortical microtubule attachment complexes. Mutations in KIF21A have been linked to congenital fibrosis of the extraocular muscles type 1 (CFEOM1), a dominant disorder associated with neurodevelopmental defects. CFEOM1-associated mutations relieve autoinhibition of the KIF21A motor, and this results in enhanced KIF21A accumulation in axonal growth cones, aberrant axon morphology, and reduced responsiveness to inhibitory cues. Our study provides mechanistic insight into cortical microtubule regulation and suggests that altered microtubule dynamics contribute to CFEOM1 pathogenesis.

Different molecular expression in thymoma with ocular or generalized myasthenia gravis.

BACKGROUND: Increasing evidence supports a link between expressions of CD4, CD25, Foxp3, and CXCL13 in thymic hyperplasia with myasthenia gravis (MG) patients. Herein, we investigated the expressions of these molecules in thymoma patients with ocular MG (OMG) or generalized MG (GMG). METHODS: A total of 58 thymoma patients with MG (23 GMG and 35 OMG) and 73 thymoma patients without MG were analyzed using immunohistochemistry for CD4, CD25, Foxp3 and CXCL13. RESULTS: OMG was more frequent than GMG in late-onset thymoma males (P=0.037), but no difference was observed in females (P=0.128). There was no significant difference of Foxp3 expression among all types of thymoma from patients with OMG and Non-MG. Compared to patients with OMG, a decreased Foxp3 expression was seen in types AB, B1 and B2 thymoma with GMG, with the decrease in the former two types reaching significance (P=0.001, 0.043, respectively). However, a significantly increased expression of CXCL13 was observed in types B1 and B2 thymoma patients with GMG (P=0.027, 0.048, respectively, compared to those with OMG). Furthermore, the CXCL13 expression in type AB thymoma patients with GMG was higher than those with Non-MG (P=0.003).There were no differences among expressions of CD4, CD25, Foxp3, and CXCL13 in type A and B3 thymoma patients, regardless of with OMG, GMG or Non-MG. CONCLUSION: Differential expressions of Foxp3 and CXCL13 in various types of thymoma patients with OMG or GMG might suggest the differential immunological processes underlying the two subtype of MG.

Human extraocular muscles in ALS.

PURPOSE. To investigate the general morphology, fiber type content, and myosin heavy chain (MyHC) composition of extraocular muscles (EOMs) from postmortem donors with amyotrophic lateral sclerosis (ALS) and to evaluate whether EOMs are affected or truly spared in this disease. METHODS. EOM and limb muscle samples obtained at autopsy from ALS donors and EOM samples from four control donors were processed for immunohistochemistry with monoclonal antibodies against distinct MyHC isoforms and analyzed by SDS-PAGE. In addition, hematoxylin and eosin staining and nicotinamide tetrazolium reductase (NADH-TR) activity were studied. RESULTS. Wide heterogeneity was observed in the appearance of the different EOMs from each single donor and between donors, irrespective of ALS type or onset. Pathologic morphologic findings in ALS EOMs included presence of atrophic and hypertrophic fibers, either clustered in groups or scattered; increased amounts of connective tissue; and areas of fatty replacement. The population of fibers stained with anti-MyHCslow tonic was smaller than that of MyHCIpositive fibers and was mostly located in the orbital layer in most of the ALS EOM samples, whereas an identical staining pattern for both fiber populations was observed in the control specimens. MyHCembryonic was notably absent from the ALS EOMs. CONCLUSIONS. The EOMs showed signs of involvement with altered fiber type composition, contractile protein content, and cellular architecture. However, when compared to the limb muscles, the EOMs were remarkably preserved. EOMs are a useful model for the study of the pathophysiology of ALS.

Unidirectional startle responses and disrupted left-right co-ordination of motor behaviors in robo3 mutant zebrafish.

The Roundabout (Robo) family of receptors and their Slit ligands play well-established roles in axonal guidance, including in humans where horizontal gaze palsy with progressive scoliosis (HGPPS) is caused by mutations in the robo3 gene. Although significant progress has been made toward understanding the mechanism by which Robo receptors establish commissural projections in the central nervous system, less is known about how these projections contribute to neural circuits mediating behavior. In this study, we report cloning of the zebrafish behavioral mutant twitch twice and show that twitch twice encodes robo3. We show that in mutant hindbrains the axons of an identified pair of neurons, the Mauthner cells, fail to cross the midline. The Mauthner neurons are essential for the startle response, and in twitch twice/robo3 mutants misguidance of the Mauthner axons results in a unidirectional startle response. Moreover, we show that twitch twice mutants exhibit normal visual acuity but display defects in horizontal eye movements, suggesting a specific and critical role for twitch twice/robo3 in sensory-guided behavior.

Dual involvement of G-substrate in motor learning revealed by gene deletion.

In this study, we generated mice lacking the gene for G-substrate, a specific substrate for cGMP-dependent protein kinase uniquely located in cerebellar Purkinje cells, and explored their specific functional deficits. G-substrate-deficient Purkinje cells in slices obtained at postnatal weeks (PWs) 10-15 maintained electrophysiological properties essentially similar to those from WT littermates. Conjunction of parallel fiber stimulation and depolarizing pulses induced long-term depression (LTD) normally. At younger ages, however, LTD attenuated temporarily at PW6 and recovered thereafter. In parallel with LTD, short-term (1 h) adaptation of optokinetic eye movement response (OKR) temporarily diminished at PW6. Young adult G-substrate knockout mice tested at PW12 exhibited no significant differences from their WT littermates in terms of brain structure, general behavior, locomotor behavior on a rotor rod or treadmill, eyeblink conditioning, dynamic characteristics of OKR, or short-term OKR adaptation. One unique change detected was a modest but significant attenuation in the long-term (5 days) adaptation of OKR. The present results support the concept that LTD is causal to short-term adaptation and reveal the dual functional involvement of G-substrate in neuronal mechanisms of the cerebellum for both short-term and long-term adaptation.

Amyotrophic lateral sclerosis-plus syndrome with TAR DNA-binding protein-43 pathology.

BACKGROUND: Amyotrophic lateral sclerosis (ALS)-Plus syndromes meet clinical criteria for ALS but also include 1 or more additional features such as dementia, geographic clustering, extrapyramidal signs, objective sensory loss, autonomic dysfunction, cerebellar degeneration, or ocular motility disturbance. METHODS: We performed a whole-brain and spinal cord pathologic analysis in a patient with an ALS-Plus syndrome that included repetitive behaviors along with extrapyramidal and supranuclear ocular motility disturbances resembling the clinical phenotype of progressive supranuclear palsy. RESULTS: There was motoneuron cell loss and degeneration of the corticospinal tracts. Bunina bodies were present. TAR DNA-binding protein-43 pathology was diffuse. Significant tau pathology was absent. CONCLUSIONS: TAR DNA-binding protein-43 disorders can produce a clinical spectrum of neurodegeneration that includes ALS, frontotemporal lobar degeneration, and ALS with frontotemporal lobar degeneration. The present case illustrates that isolated TAR DNA-binding protein-43 disorders can produce an ALS-Plus syndrome with extrapyramidal features and supranuclear gaze palsy resembling progressive supranuclear palsy.

A preliminary validation study of diffusion tensor imaging as a measure of functional brain injury.

BACKGROUND: Diffusion tensor imaging (DTI) characterizes multiple sclerosis (MS) tissue injury, although it has remained unproven whether DTI changes in disease have functional consequences. The medial longitudinal fasciculus (MLF) is a key brainstem pathway for ocular adduction and is commonly injured in patients with MS, typically resulting in internuclear ophthalmoparesis. OBJECTIVE: To validate DTI as a physiologically relevant measure of brain tissue integrity. DESIGN: A correlation study of ocular dysmotility and DTI conducted between January 2004 and September 2004. SETTING: Multiple Sclerosis Center, University of Texas Southwestern Medical Center, Dallas. Patients Six patients with chronic, unilateral, or bilateral internuclear ophthalmoparesis and 10 healthy control subjects. Main Outcome Measure We used infrared oculography to correlate the velocity versional dysconjugacy index, defined as the ratio of the velocity of the abducting to adducting eye movements during horizontal saccades, and DTI measures within the MLF as measured through an anatomical overlay. Overall diffusion was measured by mean diffusivity, and anisotropy was measured by the lattice index. RESULTS: Within the pontine MLF, the mean diffusivity was increased compared with healthy controls (P < .005), whereas the pontine lattice index was decreased (P < .03). Correlations were observed between the velocity versional dysconjugacy index and the mean diffusivity (left: r = 0.65, P < .01; right: r = 0.46, P = .07). Similar correlations were found between the versional dysconjugacy index and the lattice index (left: r = -0.43, P = .09; right: r = -0.65, P <.01). CONCLUSIONS: We identified DTI evidence of pathologic disruption of a small brainstem fiber pathway, which is crucial for accurate horizontal eye movements. In this small study, we observed correlations between the DTI changes and oculomotor dysfunction. Our preliminary observations provide criterion validity of DTI as a surrogate marker of brain tissue integrity.

A novel mutation of aprataxin associated with ataxia ocular apraxia type 1: phenotypical and genotypical characterization.

Ataxia oculomotor apraxia type 1 (AOA1) is the most common form of autosomal recessive ataxia in Japan, and the second in Portugal after Friedreich ataxia. AOA1 is typically characterized by early-onset cerebellar ataxia, oculomotor apraxia, hypoalbuminemia, hypercholesterolemia and late axonal sensori-motor neuropathy. AOA1 is associated with the aprataxin gene (APTX) encoding a protein involved in DNA repair. We characterized a novel homozygous missense mutation of APTX in a 34 year-old female patient born from consanguineous parents. The mutation, a Val230Gly caused by a c.689 T>G substitution, involved the histidine-triad (HIT) domain of the protein, affected a phylogenetically conserved amino acid and was absent in the control population. We described the clinical and neurophysiological features, the findings at structural and functional brain imaging, and the pathological picture of the sural nerve biopsy. The report emphasized the genetical and phenotypical heterogeneity of AOA1 by demonstrating atypical features such as absence of oculomotor apraxia and signs of pyramidal involvement. Expression studies by Western blotting on fibroblasts demonstrated that the homozygous Val230Gly mutation was associated with decreased levels of APTX indicating a loss-of-function mechanism.

Calcium uptake and release through sarcoplasmic reticulum in the inferior oblique muscles of patients with inferior oblique overaction.

We characterized and compared the characteristics of Ca2+ movements through the sarcoplasmic reticulum of inferior oblique muscles in the various conditions including primary inferior oblique overaction (IOOA), secondary IOOA, and controls, so as to further understand the pathogenesis of primary IOOA. Of 15 specimens obtained through inferior oblique myectomy, six were from primary IOOA, 6 from secondary IOOA, and the remaining 3 were controls from enucleated eyes. Ryanodine binding assays were performed, and Ca2+ uptake rates, calsequestrins and SERCA levels were determined. Ryanodine bindings and sarcoplasmic reticulum Ca2+ uptake rates were significantly decreased in primary IOOA (p < 0.05). Western blot analysis conducted to quantify calsequestrins and SERCA, found no significant difference between primary IOOA, secondary IOOA, and the controls. Increased intracellular Ca2+ concentration due to reduced sarcoplasmic reticulum Ca2+ uptake may play a role in primary IOOA.

Calcium Uptake and Release through Sarcoplasmic Reticulum in the Inferior Oblique Muscles of Patients with Inferior Oblique Overaction

We characterized and compared the characteristics of Ca2+ movements through the sarcoplasmic reticulum of inferior oblique muscles in the various conditions including primary inferior oblique overaction (IOOA), secondary IOOA, and controls, so as to further understand the pathogenesis of primary IOOA. Of 15 specimens obtained through inferior oblique myectomy, six were from primary IOOA, 6 from secondary IOOA, and the remaining 3 were controls from enucleated eyes. Ryanodine binding assays were performed, and Ca2+ uptake rates, calsequestrins and SERCA levels were determined. Ryanodine bindings and sarcoplasmic reticulum Ca2+ uptake rates were significantly decreased in primary IOOA (p < 0.05). Western blot analysis conducted to quantify calsequestrins and SERCA, found no significant difference between primary IOOA, secondary IOOA, and the controls. Increased intracellular Ca2+ concentration due to reduced sarcoplasmic reticulum Ca2+ uptake may play a role in primary IOOA.