Quantification of microtubule stutters: dynamic instability behaviors that are strongly associated with catastrophe.

Microtubules (MTs) are cytoskeletal fibers that undergo dynamic instability (DI), a remarkable process involving phases of growth and shortening separated by stochastic transitions called catastrophe and rescue. Dissecting DI mechanism(s) requires first characterizing and quantifying these dynamics, a subjective process that often ignores complexity in MT behavior. We present a Statistical Tool for Automated Dynamic Instability Analysis (STADIA) that identifies and quantifies not only growth and shortening, but also a category of intermediate behaviors that we term "stutters." During stutters, the rate of MT length change tends to be smaller in magnitude than during typical growth or shortening phases. Quantifying stutters and other behaviors with STADIA demonstrates that stutters precede most catastrophes in our in vitro experiments and dimer-scale MT simulations, suggesting that stutters are mechanistically involved in catastrophes. Related to this idea, we show that the anticatastrophe factor CLASP2γ works by promoting the return of stuttering MTs to growth. STADIA enables more comprehensive and data-driven analysis of MT dynamics compared with previous methods. The treatment of stutters as distinct and quantifiable DI behaviors provides new opportunities for analyzing mechanisms of MT dynamics and their regulation by binding proteins.

Elevated iron concentration in putamen and cortical speech motor network in developmental stuttering.

Theoretical accounts of developmental stuttering implicate dysfunctional cortico-striatal-thalamo-cortical motor loops through the putamen. However, the analysis of conventional MRI brain scans in individuals who stutter has failed to yield strong support for this theory in terms of reliable differences in the structure or function of the basal ganglia. Here, we performed quantitative mapping of brain tissue, which can be used to measure iron content alongside markers sensitive to myelin and thereby offers particular sensitivity to the measurement of iron-rich structures such as the basal ganglia. Analysis of these quantitative maps in 41 men and women who stutter and 32 individuals who are typically fluent revealed significant group differences in maps of R2*, indicative of higher iron content in individuals who stutter in the left putamen and in left hemisphere cortical regions important for speech motor control. Higher iron levels in brain tissue in individuals who stutter could reflect elevated dopamine levels or lysosomal dysfunction, both of which are implicated in stuttering. This study represents the first use of these quantitative measures in developmental stuttering and provides new evidence of microstructural differences in the basal ganglia and connected frontal cortical regions.

Abrogating ALIX Interactions Results in Stuttering of the ESCRT Machinery.

Endosomal sorting complexes required for transport (ESCRT) proteins assemble on budding cellular membranes and catalyze their fission. Using live imaging of HIV virions budding from cells, we followed recruitment of ESCRT proteins ALIX, CHMP4B and VPS4. We report that the ESCRT proteins transiently co-localize with virions after completion of virion assembly for durations of 45 ± 30 s. We show that mutagenizing the YP domain of Gag which is the primary ALIX binding site or depleting ALIX from cells results in multiple recruitments of the full ESCRT machinery on the same virion (referred to as stuttering where the number of recruitments to the same virion >3). The stuttering recruitments are approximately 4 ± 3 min apart and have the same stoichiometry of ESCRTs and same residence time (45 ± 30 s) as the single recruitments in wild type interactions. Our observations suggest a role for ALIX during fission and question the linear model of ESCRT recruitment, suggesting instead a more complex co-assembly model.

Amino acids profiles of children who stutter compared to their fluent sibling.

Purpose: This study scrutinises the abundances of 9 neurological-related amino acids of the scalp hair of 35 (5 females, 30 males) children who stutter and 30 normally fluent children (age and sex matched).Methods: Samples of hair from children who stutter aged (5-9 years) were collected from Speech Clinic at King Abdullah Hospital. The control subjects were selected from the same families of children who stutter to exclude the effect of nutritional, environmental, and biological factors. Amino Acid Analyser was used to measure the concentrations of amino acids in acid hydrolysed hair samples.Results: results indicated that the concentrations of threonine, tyrosine, and isoleucine (p = 0.001, 0.001, 0.02 respectively) are lower in hair samples of people who stutter compared with samples of normal fluent speakers.Conclusion: The findings of this study could introduce a new treatment protocol including the supplementation of reduced nutritional elements.KEY POINTSConcentrations of amino acids (threonine, isoleucine, and tyrosine) in the hair samples of people who stutter were significantly lower than the concentrations in the hair samples of control group.Concentrations of amino acids (histidine and glutamate) in hair samples of people who stutter were lower than control group with low significant values.The concentrations of amino acids (phenylalanine, serine, glycine, aspartate, glutamate) were not significantly different between hair samples of stuttering group and control group.The findings of the current study will be helpful in designing a new treatment method based on the supplementation of deficient amino acids.

Neurofilament-lysosomal genetic intersections in the cortical network of stuttering.

The neurobiological underpinnings of stuttering, a speech disorder characterized by disrupted speech fluency, remain unclear. While recent developments in the field have afforded researchers the ability to pinpoint several genetic profiles associated with stuttering, how these specific genetic backgrounds impact neuronal circuits and how they generate or facilitate the emergence of stuttered speech remains unknown. In this study, we identified the large-scale cortical network that characterizes stuttering using functional connectivity MRI and graph theory. We performed a spatial similarity analysis that examines whether the topology of the stuttering cortical network intersects with genetic expression levels of previously reported genes for stuttering from the protein-coding transcriptome data of the Allen Human Brain Atlas. We found that GNPTG - a gene involved in the mannose-6-phosphate lysosomal targeting pathways - was significantly co-localized with the stuttering cortical network. An enrichment analysis demonstrated that the genes identified with the stuttering cortical network shared a significantly overrepresented biological functionality of Neurofilament Cytoskeleton Organization (NEFH, NEFL and INA). The relationship between lysosomal pathways, cytoskeleton organization, and stuttering, was investigated by comparing the genetic interactome between GNPTG and the neurofilament genes implicated in the current study. We found that genes of the interactome network, including CDK5, SNCA, and ACTB, act as functional links between lysosomal and neurofilament genes. These findings support the notion that stuttering is due to a lysosomal dysfunction, which has deleterious effects on the neurofilament organization of the speech neuronal circuits. They help to elucidate the intriguing, unsolved link between lysosomal mutations and the presence of stuttering.

A fNIRS Investigation of Speech Planning and Execution in Adults Who Stutter.

Our study aimed to determine the neural correlates of speech planning and execution in adults who stutter (AWS). Fifteen AWS and 15 controls (CON) completed two tasks that either manipulated speech planning or execution processing loads. Functional near-infrared spectroscopy (fNIRS) was used to measure changes in blood flow concentrations during each task, thus providing an indirect measure of neural activity. An image-based reconstruction technique was used to analyze the results and facilitate their interpretation in the context of previous functional neuroimaging studies of AWS that used positron emission tomography (PET) or functional magnetic resonance imaging (fMRI). For planning, we compared neural activity associated with high versus low planning load in AWS and CON. For execution, we compared the neural activity associated with overt versus covert naming in AWS and CON. Broadly, group level effects corroborate previous PET/fMRI findings including under-activation in left-hemisphere perisylvian speech-language networks and over-activation in right-hemisphere homologs. Increased planning load revealed atypical left-hemisphere activation in AWS, whereas increased execution load yielded atypical right fronto-temporo-parietal and bilateral motor activation in AWS. Our results add to the limited literature differentiating speech planning versus execution processes in AWS.

The concentrations of bioelements in the hair samples of Jordanian children who stutter.

OBJECTIVES: This study investigates the levels of 15 bioelements (calcium, copper, chromium, sodium, iron, magnesium, manganese, zinc, cobalt, selenium, molybdenum, vanadium, potassium, boron, and lithium) in the hair species of Jordanian stutterer. METHODS: The subjects of the study included 25 cases of stuttering, and 25 normal children (age and sex matched). The severity of stuttering (low, moderate, and severe) were assessed using Stuttering Severity Instrument, Fourth edition (SSI-4). Hair samples of subjects were cut, washed, dried, physically degraded, hydrolyzed, and analyzed by Inductively Coupled Plasma Mass Spectrometry (ICP-MS). RESULTS: Results indicated that the levels of bioelements (Calcium, Copper, Chromium, Magnesium, Manganese, Cobalt, Selenium, Molybdenum, Vanadium, Boron, and Lithium) were significantly lesser in the hair samples of stuttering group than the control group. CONCLUSION: The findings of the current study could support the use of biochemical analyses as diagnostic biomarker for stuttering.

Reduced fractional anisotropy in the anterior corpus callosum is associated with reduced speech fluency in persistent developmental stuttering.

Developmental stuttering is a speech disorder that severely limits one's ability to communicate. White matter anomalies were reported in stuttering, but their functional significance is unclear. We analyzed the relation between white matter properties and speech fluency in adults who stutter (AWS). We used diffusion tensor imaging with tract-based spatial statistics, and examined group differences as well as correlations with behavioral fluency measures. We detected a region in the anterior corpus callosum with significantly lower fractional anisotropy in AWS relative to controls. Within the AWS group, reduced anisotropy in that region is associated with reduced fluency. A statistically significant interaction was found between group and age in two additional regions: the left Rolandic operculum and the left posterior corpus callosum. Our findings suggest that anterior callosal anomaly in stuttering may represent a maladaptive reduction in interhemispheric inhibition, possibly leading to a disadvantageous recruitment of right frontal cortex in speech production.

A study of the role of the FOXP2 and CNTNAP2 genes in persistent developmental stuttering.

A number of speech disorders including stuttering have been shown to have important genetic contributions, as indicated by high heritability estimates from twin and other studies. We studied the potential contribution to stuttering from variants in the FOXP2 gene, which have previously been associated with developmental verbal dyspraxia, and from variants in the CNTNAP2 gene, which have been associated with specific language impairment (SLI). DNA sequence analysis of these two genes in a group of 602 unrelated cases, all with familial persistent developmental stuttering, revealed no excess of potentially deleterious coding sequence variants in the cases compared to a matched group of 487 well characterized neurologically normal controls. This was compared to the distribution of variants in the GNPTAB, GNPTG, and NAGPA genes which have previously been associated with persistent stuttering. Using an expanded subject data set, we again found that NAGPA showed significantly different mutation frequencies in North Americans of European descent (p=0.0091) and a significant difference existed in the mutation frequency of GNPTAB in Brazilians (p=0.00050). No significant differences in mutation frequency in the FOXP2 and CNTNAP2 genes were observed between cases and controls. To examine the pattern of expression of these five genes in the human brain, real time quantitative reverse transcription PCR was performed on RNA purified from 27 different human brain regions. The expression patterns of FOXP2 and CNTNAP2 were generally different from those of GNPTAB, GNPTG and NAPGA in terms of relatively lower expression in the cerebellum. This study provides an improved estimate of the contribution of mutations in GNPTAB, GNPTG and NAGPA to persistent stuttering, and suggests that variants in FOXP2 and CNTNAP2 are not involved in the genesis of familial persistent stuttering. This, together with the different brain expression patterns of GNPTAB, GNPTG, and NAGPA compared to that of FOXP2 and CNTNAP2, suggests that the genetic neuropathological origins of stuttering differ from those of verbal dyspraxia and SLI.

The role of the DRD2 C957T polymorphism in neuroticism in persons who stutter and healthy controls.

The present study investigates for the first time the influence of the DRD2 C957T polymorphism on personality in persons who stutter. In a recent study, the CC genotype of this single nucleotide polymorphism has been associated with stuttering, which could not be replicated in a follow-up study. Here, we demonstrate, in N=105 persons who stutter, that carriers of the CC and the CT genotype significantly have the highest neuroticism scores. This shows that the inclusion of personality measures in the investigation of the biological underpinnings of stuttering represents an important new avenue. In healthy control persons, a sex by C+/- allele interaction effect could be demonstrated. Female but not male carriers of the C+ variant report the highest neuroticism scores. Because neuroticism has been reported to be associated with stuttering before, the present data support the idea that this personality trait acts as an endophenotype for stuttering, contributing towards bridging the gap from gene variation to the complex pathology. This idea is supported by an additional path model showing that the polymorphism DRD2 C957T influences the self-reported severity of stuttering mainly by its influence on neuroticism (independent of the variable sex).

Developing physiologic stress profiles for school-age children who stutter.

PURPOSE: Physiologic reactivity profiles were generated for 9 school-age children with a history of stuttering. Utilizing salivary sampling, stress biomarkers cortisol and alpha-amylase were measured in response to normal daily stressors. Children with a history of stuttering were characterized as high or low autonomic reactors when compared to age-normed references established for cortisol and alpha-amylase levels. METHOD: Salivary samples were collected four times daily over three consecutive days from children (6 boys and 3 girls) aged 6-11 years. Samples were measured for two important stress biomarkers, cortisol and alpha-amylase. Following laboratory training on appropriate sampling technique using the Salivette, families collected all samples in their homes. RESULTS: Compared to published references established for normal children, children with a history of stuttering exhibited mean cortisol and alpha-amylase levels that were significantly lower, though within normal limits. CONCLUSIONS: Results reflect an initial investigation into the use of salivary sampling to measure reactivity in children who stutter. As children who stutter have historically been excluded from physiologic stress studies, salivary sampling appears to provide an innovative and minimally invasive option for investigators. Though interpreted with caution secondary to the small sample size, initial findings suggest that in response to normal daily stressors, school-age children with a history of stuttering do not exhibit significantly elevated stress biomarkers. EDUCATIONAL OBJECTIVES: The reader will be able to: (1) identify two biomarkers that reflect activation of the body's stress response; (2) discuss possible implications the generation of individualized stress profiles may have on the treatment of stuttering; and (3) discuss and evaluate the research needs associated with the inclusion of children in physiological studies of fluency.

Copper in developmental stuttering.

It has previously been reported that men with developmental stuttering showed reduced concentration of copper in the blood, and a negative correlation between the copper level and the severity of stuttering. Disorders of copper metabolism may result in dysfunction of the basal ganglia system and dystonia, a motor disorder sharing some traits of stuttering. It has been shown that copper ions affect the dopamine and the GABA systems. With this background we investigated the plasma level of copper, the copper binding protein ceruloplasmin, and the estimated level of free copper in stuttering adults. Sixteen men with developmental stuttering were compared with 16 men without speech problems. The samples were assayed in one batch in a pseudorandom and counterbalanced order. No significant differences were found between stuttering men and the control group in any of the biological variables, and no negative correlation between copper and the general severity of stuttering was shown. On the contrary, an explorative analysis resulted in a positive correlation between high plasma copper and superfluous muscular activity during stuttering (r=0.51, p=0.04). This result indicates that there is no relation between developmental stuttering and low plasma copper in the main population of stuttering adults.

Stuttering and the basal ganglia circuits: a critical review of possible relations.

UNLABELLED: The possible relation between stuttering and the basal ganglia is discussed. Important clues to the pathophysiology of stuttering are given by conditions known to alleviate dysfluency, like the rhythm effect, chorus speech, and singing. Information regarding pharmacologic trials, lesion studies, brain imaging, genetics, and developmental changes of the nervous system is reviewed. The symptoms of stuttering are compared with basal ganglia motor disorders like Parkinson's disease and dystonia. It is proposed that the basal ganglia-thalamocortical motor circuits through the putamen are likely to play a key role in stuttering. The core dysfunction in stuttering is suggested to be impaired ability of the basal ganglia to produce timing cues for the initiation of the next motor segment in speech. Similarities between stuttering and dystonia are indicated, and possible relations to the dopamine system are discussed, as well as the interaction between the cerebral cortex and the basal ganglia. Behavioral and pharmacologic information suggests the existence of subtypes of stuttering. LEARNING OUTCOMES: As a result of this activity, the reader will (1) become familiar with the research regarding the basal ganglia system relating to speech motor control; (2) become familiar with the research on stuttering with indications of basal ganglia involvement; and (3) be able to discuss basal ganglia mechanisms with relevance for theory of stuttering.

Slower and incomplete retrieval of speech motor plans is the proximal source of stuttering: stutters occur when syllable motor plans stored in memory are concatenated to produce the utterance motor plan.

This paper describes a hypothesis about stuttering that distinguishes between two methods of assembling the speech motor plan for an utterance - speech construction, which involves real-time preparation of the speech motor plan and speech concatenation, which involves retrieval and chaining of syllable motor plans stored in memory. It is hypothesized that stutters are significantly reduced when people who stutter (PWS) speak in novel speaking patterns such as whispering and prolonged speech because these unaccustomed speaking tasks require speech construction. In contrast, when PWS speak in their habitual speech pattern, concatenating syllable motor plans stored in memory, they stutter. Reaction time studies and the effect of word frequency on stutter rate show that PWS are slower in retrieving stored motor plans during speech concatenation. On the other hand, conditions that positively prime the retrieval of motor plans - phonological priming, reading, production of serial speech, speech shadowing, and stuttering adaptation tasks - speed up the retrieval of motor plans and, therefore, reduce the amount of stutters. The slower retrieval of syllable motor plans may lead to an incomplete specification of the utterance motor plan. Stutters result when the articulatory system attempts to execute an underspecified utterance motor plan.

Increased dopamine activity associated with stuttering.

Position emission tomography using 6-FDOPA as a marker of presynaptic dopaminergic activity was used to investigate the role of the dopamine system in stuttering. Three patients with moderate to severe developmental stuttering were compared with six normal controls. Stuttering subjects showed significantly higher 6-FDOPA uptake than normal controls in medial prefrontal cortex, deep orbital cortex, insular cortex, extended amygdala, auditory cortex and caudate tail. Elevated 6-FDOPA uptake in ventral limbic cortical and subcortical regions is compatible with the hypothesis that stuttering is associated with an overactive presynaptic dopamine system in brain regions that modulate verbalization.

The acute effect of haloperidol and apomorphine on the severity of stuttering.

In studies of the acute effects of haloperidol on the severity of stuttering in 12 subjects not in treatment at the time of drug evaluation, a single 0.5 mg haloperidol injection was found to increase fluency in 9 to 12 subjects, as compared with saline placebo. The average improvement in those subjects who improved was 25% on reading and 40% on spontaneous speech. Side effects from this dose of haloperidol were minimal. The effects of apomorphine on speech were not statistically significant, but increased fluency was seen in a number of subjects on the reading test. The results of this study suggest that acute drug evaluation studies may be valuable in determining the effects of various psychotropic agents on the severity of stuttering. The increased fluency after haloperidol, an agent which is felt to turn off the dopaminergic system via postsynaptic blockade, and after low-dose apomorphine, which appears to inhibit the dopamine system via presynaptic effects is consistent with a role for central dopaminergic systems in the pathogenesis of stuttering. Further studies are needed to confirm this hypothesis.