StARD9 is a novel lysosomal kinesin required for membrane tubulation, cholesterol transport and Purkinje cell survival.

The pathological accumulation of cholesterol is a signature feature of Niemann-Pick type C (NPC) disease, in which excessive lipid levels induce Purkinje cell death in the cerebellum. NPC1 encodes a lysosomal cholesterol-binding protein, and mutations in NPC1 drive cholesterol accumulation in late endosomes and lysosomes (LE/Ls). However, the fundamental role of NPC proteins in LE/L cholesterol transport remains unclear. Here, we demonstrate that NPC1 mutations impair the projection of cholesterol-containing membrane tubules from the surface of LE/Ls. A proteomic survey of purified LE/Ls identified StARD9 as a novel lysosomal kinesin responsible for LE/L tubulation. StARD9 contains an N-terminal kinesin domain, a C-terminal StART domain, and a dileucine signal shared with other lysosome-associated membrane proteins. Depletion of StARD9 disrupts LE/L tubulation, paralyzes bidirectional LE/L motility and induces accumulation of cholesterol in LE/Ls. Finally, a novel StARD9 knock-out mouse recapitulates the progressive loss of Purkinje cells in the cerebellum. Together, these studies identify StARD9 as a microtubule motor protein responsible for LE/L tubulation and provide support for a novel model of LE/L cholesterol transport that becomes impaired in NPC disease.

Quantitative susceptibility mapping of basal ganglia iron is associated with cognitive and motor functions that distinguish spinocerebellar ataxia type 6 and type 3.

Background: In spinocerebellar ataxia type 3 (SCA3), volume loss has been reported in the basal ganglia, an iron-rich brain region, but iron content has not been examined. Recent studies have reported that patients with SCA6 have markedly decreased iron content in the cerebellar dentate, coupled with severe volume loss. Changing brain iron levels can disrupt cognitive and motor functions, yet this has not been examined in the SCAs, a disease in which iron-rich regions are affected. Methods: In the present study, we used quantitative susceptibility mapping (QSM) to measure tissue magnetic susceptibility (indicating iron concentration), structural volume, and normalized susceptibility mass (indicating iron content) in the cerebellar dentate and basal ganglia in people with SCA3 (n = 10) and SCA6 (n = 6) and healthy controls (n = 9). Data were acquired using a 7T Philips MRI scanner. Supplemental measures assessed motor, cognitive, and mood domains. Results: Putamen volume was lower in both SCA groups relative to controls, replicating prior findings. Dentate susceptibility mass and volume in SCA6 was lower than in SCA3 or controls, also replicating prior findings. The novel finding was that higher basal ganglia susceptibility mass in SCA6 correlated with lower cognitive performance and greater motor impairment, an association that was not observed in SCA3. Cerebellar dentate susceptibility mass, however, had the opposite relationship with cognition and motor function in SCA6, suggesting that, as dentate iron is depleted, it relocated to the basal ganglia, which contributed to cognitive and motor decline. By contrast, basal ganglia volume loss, rather than iron content, appeared to drive changes in motor function in SCA3. Conclusion: The associations of higher basal ganglia iron with lower motor and cognitive function in SCA6 but not in SCA3 suggest the potential for using brain iron deposition profiles beyond the cerebellar dentate to assess disease states within the cerebellar ataxias. Moreover, the role of the basal ganglia deserves greater attention as a contributor to pathologic and phenotypic changes associated with SCA.

The association between educational attainment and SCA 3 age of onset and disease course.

BACKGROUND: The number of trinucleotide CAG repeats is inversely correlated with the age at onset (AAO) of motor symptoms in individuals with Spinocerebellar Ataxia type 3 (SCA 3) and may be responsible for 50%-60% of the variability in AAO. Drawing from a social determinants of health model, we sought to determine if educational attainment further contributes to the AAO and motor symptom progression of SCA 3. METHODS: We performed a retrospective chart review in which twenty individuals met criteria for inclusion and had been seen by an ataxia specialist at our hospital between January 2005 and July 2019. AAO of motor symptoms and Scale for Assessment and Rating of Ataxia (SARA) scores were used as primary outcome measures. RESULTS: Using a linear regression, we found that having greater CAG repeat length and greater than 16 years of education results in an earlier AAO. The importance of the CAG repeat length on AAO, however, is greater amongst individuals with lower education. Using a linear mixed model evaluating SARA score over time with AAO, we found that less than 16 years of education is associated with faster progression of the disease. CONCLUSION: In our group of SCA 3 patients, level of education correlated with both the AAO and SARA scores. Though our findings need to be confirmed with a larger cohort, our study suggests that level of education can have a strong influence on health outcomes in SCA 3 and possibly other groups of patients with ataxia.

Characterization of basal ganglia volume changes in the context of HIV and polysubstance use.

HIV and psychoactive substances can impact the integrity of the basal ganglia (BG), a neural substrate of cognition, motor control, and reward-seeking behaviors. This study assessed BG gray matter (GM) volume as a function of polysubstance (stimulant and opioid) use and HIV status. We hypothesized that comorbid polysubstance use and HIV seropositivity would alter BG GM volume differently than would polysubstance use or HIV status alone. We collected structural MRI scans, substance use history, and HIV diagnoses. Participants who had HIV (HIV +), a history of polysubstance dependence (POLY +), both, or neither completed assessments for cognition, motor function, and risk-taking behaviors (N = 93). All three clinical groups showed a left-lateralized pattern of GM reduction in the BG relative to controls. However, in the HIV + /POLY + group, stimulant use was associated with increased GM volume within the globus pallidus and putamen. This surpassed the effects from opioid use, as indicated by decreased GM volume throughout the BG in the HIV-/POLY + group. Motor learning was impaired in all three clinical groups, and in the HIV + /POLY + group, motor learning was associated with increased caudate and putamen GM volume. We also observed associations between BG GM volume and risk-taking behaviors in the HIV + /POLY- and HIV-/POLY + groups. The effects of substance use on the BG differed as a function of substance type used, HIV seropositivity, and BG subregion. Although BG volume decreased in association with HIV and opioid use, stimulants can, inversely, lead to BG volume increases within the context of HIV.

Quality of Life Changes Following the Onset of Cerebellar Ataxia: Symptoms and Concerns Self-reported by Ataxia Patients and Informants.

Semi-structured interviews of patient accounts and caregiver, or informant, perspectives are a beneficial resource for patients suffering from diseases with complex symptomatology, such as cerebellar ataxia. The aim of this study was to identify, quantify, and compare the ways in which cerebellar ataxia patients' and informants' quality of life had changed as a result of living with ataxia. Using a semi-structured interview, responses were collected from patients and informants regarding motor, cognitive, and psychosocial variables. Responses were also collected from patients and informants to open-ended questions that were subsequently categorized into 15 quality of life themes that best represented changes experienced by the patients and informants. Ataxia patients and informants agreed as to the severity of posture/gait, daily activities/fine motor tasks, speech/feeding/swallowing, and oculomotor/vision impairment. It was also demonstrated that severity ratings for specific motor-related functions strongly correlated with corresponding functions within the International Cooperative Ataxia Rating Scale (ICARS), and that this interview identified frequency associations between motor impairments and specific psychosocial difficulties, which could be useful for prognostic purposes. Overall, the information obtained from this study characterized the symptoms and challenges to ataxia patients and their caregivers, which could serve as a useful educational resource for those affected by ataxia, clinicians, and researchers.

Neurotoxicity in Gulf War Illness and the potential role of glutamate.

Shortly after the Gulf War in 1990-1991, service men and women began reporting multiple symptoms ranging from persistent headaches, widespread pain, chronic fatigue, cognitive dysfunction, mood dysregulation, gastrointestinal issues, skin abnormalities, and respiratory problems. This prompted the Centers for Disease Control and Prevention (CDC) to initially classify the disorder as chronic multi-symptom illness (CMI), where it later became known as Gulf War Illness (GWI). Researchers and healthcare professionals since the early 1990s have been working extensively on alleviating the symptoms expressed in GWI as well as attempting to understand the mechanisms behind this illness. Scientific literature as well as reports from GWI veterans indicate that the toxic exposures during deployment may be responsible for the symptoms. These toxic exposures potentially include nerve agents, pyridostigmine bromide pills, pesticides, munitions with depleted uranium, and burning oil well fires. GWI currently affects 25-32 % of the 697,000 American troops who were stationed overseas during the short conflict. The purpose of this paper is to review the literature on neurotoxic exposures in Gulf War Illness, to explain how these exposures may lead to glutamate excitotoxicity, which has been implicated in the majority of the symptoms characterizing the illness, and to propose a novel treatment option for GWI.

Incorporating yeast biosensors into paper-based analytical tools for pharmaceutical analysis.

Paper-based devices serve to address many analytical questions both inside and outside of the laboratory setting. For the first time, yeast is used to construct a whole-cell, paper-based biosensor device. This biologically based paper analytical device (BioPAD) is sensitive to antibiotics in the tetracycline family, and it could potentially address questions of pharmaceutical quality as well as antibiotic contamination in liquids. Our BioPAD can qualitatively discriminate the presence/absence of doxycycline over a range of 30-10,000 µg/mL. In an analysis of a doxycycline dosage form (tablet) commonly used for malaria prophylaxis, BioPADs identified the presence of antibiotic with 92 and 95 % sensitivity, evaluated by eye and computer-assisted image analysis, respectively, with no false positives by either method. BioPADs were found to remain viable for at least 415 days when stored at 4 °C. This research demonstrates the utility of whole yeast cells in paper-based pharmaceutical testing, and it highlights the potential for the development of yeast-based BioPADs to address a range of qualitative analytical questions, especially in low resource settings.

LmaPA2G4, a homolog of human Ebp1, is an essential gene and inhibits cell proliferation in L. major.

We have identified LmaPA2G4, a homolog of the human proliferation-associated 2G4 protein (also termed Ebp1), in a phosphoproteomic screening. Multiple sequence alignment and cluster analysis revealed that LmaPA2G4 is a non-peptidase member of the M24 family of metallopeptidases. This pseudoenzyme is structurally related to methionine aminopeptidases. A null mutant system based on negative selection allowed us to demonstrate that LmaPA2G4 is an essential gene in Leishmania major. Over-expression of LmaPA2G4 did not alter cell morphology or the ability to differentiate into metacyclic and amastigote stages. Interestingly, the over-expression affected cell proliferation and virulence in mouse footpad analysis. LmaPA2G4 binds a synthetic double-stranded RNA polyriboinosinic polyribocytidylic acid [poly(I∶C)] as shown in an electrophoretic mobility shift assay (EMSA). Quantitative proteomics revealed that the over-expression of LmaPA2G4 led to accumulation of factors involved in translation initiation and elongation. Significantly, we found a strong reduction of de novo protein biosynthesis in transgenic parasites using a non-radioactive metabolic labeling assay. In conclusion, LmaPA2G4 is an essential gene and is potentially implicated in fundamental biological mechanisms, such as translation, making it an attractive target for therapeutic intervention.

Kinetics and mechanism of (•)OH mediated degradation of dimethyl phthalate in aqueous solution: experimental and theoretical studies.

The hydroxyl radical ((•)OH) is one of the main oxidative species in aqueous phase advanced oxidation processes, and its initial reactions with organic pollutants are important to understand the transformation and fate of organics in water environments. Insights into the kinetics and mechanism of (•)OH mediated degradation of the model environmental endocrine disruptor, dimethyl phthalate (DMP), have been obtained using radiolysis experiments and computational methods. The bimolecular rate constant for the (•)OH reaction with DMP was determined to be (3.2 ± 0.1) × 10(9) M(-1)s(-1). The possible reaction mechanisms of radical adduct formation (RAF), hydrogen atom transfer (HAT), and single electron transfer (SET) were considered. By comparing the experimental absorption spectra with the computational results, it was concluded that the RAF and HAT were the dominant reaction pathways, and OH-adducts ((•)DMPOH1, (•)DMPOH2) and methyl type radicals (•)DMP(-H)α were identified as dominated intermediates. Computational results confirmed the identification of transient species with maximum absorption around 260 nm as (•)DMPOH1 and (•)DMP(-H)α, and these radical intermediates then converted to monohydroxylated dimethyl phthalates and monomethyl phthalates. Experimental and computational analyses which elucidated the mechanism of (•)OH-mediated degradation of DMP are discussed in detail.

Rare immediate hypersensitivity to rifampicin in a patient with tuberculosis requiring drug discontinuation.

A 47-year-old Turkish female patient was diagnosed with tuberculosis of the sacro-iliac joints and terminal ileum. She developed a severe adverse drug reaction while taking first-line tuberculosis therapy consisting of isoniazid, pyrazinamide and rifampicin as Rifater and ethambutol. Within 5 min of ingestion she developed pruritic rash, angioedema and breathing difficulties, resulting in an A&E admission. The tuberculosis therapy was discontinued. Intradermal and oral challenge tests for rifampicin were conducted but abandoned early on due to reactions which included audible wheeze, vomiting, throat pain and violent rigours. Clinical manifestations were swiftly treated with appropriate medications. This resulted in a change to the tuberculosis treatment regime, where streptomycin, isoniazid, ethambutol and pyrazinamide were given for 2 months and isoniazid and ethambutol for 12 months. Allergic reactions to rifampicin are rare and should be distinguished from flushing due to pyrazinamide. Prompt diagnosis and treatment by clinicians can be life saving.

Cutting edge: Evidence for a dynamically driven T cell signaling mechanism.

T cells use the αß TCR to bind peptides presented by MHC proteins (pMHC) on APCs. Formation of a TCR-pMHC complex initiates T cell signaling via a poorly understood process, potentially involving changes in oligomeric state, altered interactions with CD3 subunits, and mechanical stress. These mechanisms could be facilitated by binding-induced changes in the TCR, but the nature and extent of any such alterations are unclear. Using hydrogen/deuterium exchange, we demonstrate that ligation globally rigidifies the TCR, which via entropic and packing effects will promote associations with neighboring proteins and enhance the stability of existing complexes. TCR regions implicated in lateral associations and signaling are particularly affected. Computational modeling demonstrated a high degree of dynamic coupling between the TCR constant and variable domains that is dampened upon ligation. These results raise the possibility that TCR triggering could involve a dynamically driven, allosteric mechanism.

Zwint-1 is a novel Aurora B substrate required for the assembly of a dynein-binding platform on kinetochores.

Aurora B (AurB) is a mitotic kinase responsible for multiple aspects of mitotic progression, including assembly of the outer kinetochore. Cytoplasmic dynein is an abundant kinetochore protein whose recruitment to kinetochores requires phosphorylation. To assess whether AurB regulates recruitment of dynein to kinetochores, we inhibited AurB using ZM447439 or a kinase-dead AurB construct. Inhibition of AurB reduced accumulation of dynein at kinetochores substantially; however, this reflected a loss of dynein-associated proteins rather than a defect in dynein phosphorylation. We determined that AurB inhibition affected recruitment of the ROD, ZW10, zwilch (RZZ) complex to kinetochores but not zwint-1 or more-proximal kinetochore proteins. AurB phosphorylated zwint-1 but not ZW10 in vitro, and three novel phosphorylation sites were identified by tandem mass spectrometry analysis. Expression of a triple-Ala zwint-1 mutant blocked kinetochore assembly of RZZ-dependent proteins and induced defects in chromosome movement during prometaphase. Expression of a triple-Glu zwint-1 mutant rendered cells resistant to AurB inhibition during prometaphase. However, cells expressing the triple-Glu mutant failed to satisfy the spindle assembly checkpoint (SAC) at metaphase because poleward streaming of dynein/dynactin/RZZ was inhibited. These studies identify zwint-1 as a novel AurB substrate required for kinetochore assembly and for proper SAC silencing at metaphase.

Probing interactions between CLIP-170, EB1, and microtubules.

Cytoplasmic linker protein 170 (CLIP-170) is a microtubule (MT) plus-end tracking protein (+TIP) that dynamically localizes to the MT plus end and regulates MT dynamics. The mechanisms of these activities remain unclear because the CLIP-170-MT interaction is poorly understood, and even less is known about how CLIP-170 and other +TIPs act together as a network. CLIP-170 binds to the acidic C-terminal tail of alpha-tubulin. However, the observation that CLIP-170 has two CAP-Gly (cytoskeleton-associated protein glycine-rich) motifs and multiple serine-rich regions suggests that a single CLIP-170 molecule has multiple tubulin binding sites, and that these sites might bind to multiple parts of the tubulin dimer. Using a combination of chemical cross-linking and mass spectrometry, we find that CLIP-170 binds to both alpha-tubulin and beta-tubulin, and that binding is not limited to the acidic C-terminal tails. We provide evidence that these additional binding sites include the H12 helices of both alpha-tubulin and beta-tubulin and are significant for CLIP-170 activity. Previous work has shown that CLIP-170 binds to end-binding protein 1 (EB1) via the EB1 C-terminus, which mimics the acidic C-terminal tail of tubulin. We find that CLIP-170 can utilize its multiple tubulin binding sites to bind to EB1 and MT simultaneously. These observations help to explain how CLIP-170 can nucleate MTs and alter MT dynamics, and they contribute to understanding the significance and properties of the +TIP network.

The role of Drosophila ninaG oxidoreductase in visual pigment chromophore biogenesis.

We previously reported (Sarfare, S., Ahmad, S. T., Joyce, M. V., Boggess, B., and O'Tousa, J. E. (2005) J. Biol. Chem. 280, 11895-11901) that the Drosophila ninaG gene encodes an oxidoreductase involved in the biosynthesis of the (3S)-3-hydroxyretinal serving as chromophore for Rh1 rhodopsin and that ninaG mutant flies expressing Rh4 as the major opsin accumulate large amounts of a different retinoid. Here, we show that this unknown retinoid is 11-cis-3-hydroxyretinol. Reversed phase high performance liquid chromatography coupled with a photodiode array UV-visible absorbance detector and mass spectrometer revealed a major product eluting at a retention time, t(r), of 3.5 min with a lambda(max) of approximately 324 nm and with a base peak in the mass spectrum at m/z 285. These observations are identical with those of the 3-hydroxyretinol standard. The base peak in the electrospray ionization mass spectrum arises from the loss of a water molecule from the protonated molecule at m/z 303 because of fragmentation in the ion source. These results suggest that 11-cis-3-hydroxyretinol is an intermediate required for chromophore biogenesis in Drosophila. We further show that ninaG mutants fed on retinal as the sole source of vitamin A are able to synthesize 3-hydroxyretinoids. Thus, the NinaG oxidoreductase is not responsible for the initial hydroxylation of the retinal ring but rather acts in a subsequent step in chromophore production. These data are used to review chromophore biosynthesis and propose that NinaG acts in the conversion of (3R)-3-hydroxyretinol to the 3S enantiomer.

Treatment of recalcitrant idiopathic muscular torticollis in infants with botulinum toxin type a.

Congenital muscular torticollis (CMT) is the most common form of torticollis in children, significantly outnumbering orthopedic, neurologic, and ocular causes. CMT may present as a palpable sternomastoid tumor (SMT) or a simple tightness of the sternocleidomastoid muscle (SCM), designated as idiopathic muscular torticollis (IMT). Muscular torticollis has been associated with positional plagiocephaly in neonates who slept in the supine position. We have had difficulty in treating some of these combined cases by traditional methods such as physiotherapy, stretching exercises, and molding helmets. In November 2000, we began injecting botulinum toxin type A in cases in which there was persistent IMT, despite significant physical therapy input. The 15 patients included in this retrospective study all presented with IMT and positional plagiocephaly; all had responded poorly to conservative treatment, including physiotherapy, stretching exercises, or use of a helmet. In the attempt to avoid progression to surgical release, these patients were treated with botulinum toxin injected into the affected SCM and subsequent additional physiotherapy. All appeared to respond well, and a retrospective analysis of this treatment strategy was undertaken. Information gathered included a questionnaire, skull-shape tracings, and photographs. Independent outcome assessment data were then obtained from the regional child development teams and community physiotherapists. These results show that 14 of 15 children with recalcitrant IMT and positional plagiocephaly treated with botulinum toxin obtained sufficient improvement in neck range of motion and head position as to make surgical release of the muscle unnecessary. Our conclusion is that the use of botulinum toxin is a safe and effective adjunct to physical therapy in treating recalcitrant IMT; in selected cases, it may obviate the need for surgical release of a tight but nonfibrotic SCM.

The Drosophila ninaG oxidoreductase acts in visual pigment chromophore production.

The Drosophila ninaG mutant is characterized by low levels of Rh1 rhodopsin, because of the inability to transport this rhodopsin from the endoplasmic reticulum to the rhabdomere. ninaG mutants do not affect the biogenesis of the minor opsins Rh4 and Rh6. A genetic analysis placed the ninaG gene within the 86E4-86E6 chromosomal region. A sequence analysis of the 15 open reading frames within this region from the ninaG(P330) mutant allele identified a stop codon in the CG6728 gene. Germ-line transformation of the CG6728 genomic region rescued the ninaG mutant phenotypes, confirming that CG6728 corresponds to the ninaG gene. The NinaG protein belongs to the glucose-methanol-choline oxidoreductase family of flavin adenine dinucleotide-binding enzymes catalyzing hydroxylation and oxidation of a variety of small organic molecules. High performance liquid chromatography analysis of retinoids was used to gain insight into the in vivo role of the NinaG oxidoreductase. The results show that when Rh1 is expressed as the major rhodopsin, ninaG flies fail to accumulate 3-hydroxyretinal. Further, in transgenic flies expressing Rh4 as the major rhodopsin, 3-hydroxyretinal is the major retinoid in ninaG+, but a different retinoid profile is observed in ninaG(P330). These results indicate that the ninaG oxidoreductase acts in the biochemical pathway responsible for conversion of retinal to the rhodopsin chromophore, 3-hydroxyretinal.

Detection of G-quartet structure in a DNA aptamer stationary phase using a fluorescent dye.

The fluorescent porphyrin dye N-methylmesoporphyrin IX (NMM) was used to provide direct evidence of intramolecular G-quartet formation by an oligonucleotide immobilized at the inner surface of a fused silica capillary. The oligonucleotide is the thrombin-binding DNA aptamer, which has been used in several analytical applications, including a stationary phase for open tubular capillary electrochromatography. Spectroscopic studies of the dye in batch solutions of the aptamer and of an oligonucleotide with the same base composition, but in a different, "scrambled" sequence that does not form an intramolecular G-quartet, provided evidence of selective fluorescence enhancement of NMM by the aptamer in the intramolecular G-quartet structure. On-column experiments compared results for injections of NMM onto an aptamer-coated capillary, a capillary coated with the scrambled sequence oligonucleotide, and a bare fused silica capillary. Results show that while NMM adsorbs to both coated capillaries, the selective fluorescence enhancement provides evidence of the intramolecular G-quartet structure on the aptamer-coated capillary.