Pisgah Astronomical Research Institute

For the promise of human induced pluripotent stem cells (iPSCs) to be realized, it is necessary to ask if and how efficiently they may be differentiated to functional cells of various lineages. Here, we have directly compared the neural-differentiation capacity of human iPSCs and embryonic stem cells (ESCs). We have shown that human iPSCs use the same transcriptional network to generate neuroepithelia and functionally appropriate neuronal types over the same developmental time course as hESCs in response to the same set of morphogens; however, they do it with significantly reduced efficiency and increased variability. These results were consistent across iPSC lines and independent of the set of reprogramming transgenes used to derive iPSCs as well as the presence or absence of reprogramming transgenes in iPSCs. These findings, which show a need for improving differentiation potency of iPSCs, suggest the possibility of employing human iPSCs in pathological studies, therapeutic screening, and autologous cell transplantation.

Astrocytes have a higher antioxidant potential in comparison to neurons. Pathways associated with this selective advantage include the transcriptional regulation of antioxidant enzymes via the action of the Cap'n'Collar transcription factor Nrf2 at the antioxidant response element (ARE). Here we show that Nrf2 overexpression can reengineer neurons to express this glial pathway and enhance antioxidant gene expression. However, Nrf2-mediated protection from oxidative stress is conferred primarily by glia in mixed cultures. The antioxidant properties of Nrf2-overexpressing glia are more pronounced than those of neurons, and a relatively small number of these glia (< 1% of total cell number added) could protect fully cocultured naive neurons from oxidative glutamate toxicity associated with glutathione (GSH) depletion. Microarray and biochemical analyses indicate a coordinated upregulation of enzymes involved in GSH biosynthesis (xCT cystine antiporter, gamma-glutamylcysteine synthetase, and GSH synthase), use (glutathione S-transferase and glutathione reductase), and export (multidrug resistance protein 1) with Nrf2 overexpression, leading to an increase in both media and intracellular GSH. Selective inhibition of glial GSH synthesis and the supplementation of media GSH indicated that an Nrf2-dependent increase in glial GSH synthesis was both necessary and sufficient for the protection of neurons, respectively. Neuroprotection was not limited to overexpression of Nrf2, because activation of endogenous glial Nrf2 by the small molecule ARE inducer, tert-butylhydroquinone, also protected against oxidative glutamate toxicity.

The antioxidant responsive element (ARE) is a cis-acting regulatory element of genes encoding phase II detoxification enzymes and antioxidant proteins, such as NAD(P)H: quinone oxidoreductase 1, glutathione S-transferases, and glutamate-cysteine ligase. Interestingly, it has been reported that Nrf2 (NF-E2-related factor 2) regulates a wide array of ARE-driven genes in various cell types. Nrf2 is a basic leucine zipper transcription factor, which was originally identified as a binding protein of locus control region of beta-globin gene. The DNA binding sequence of Nrf2 and ARE sequence are very similar, and many studies demonstrated that Nrf2 binds to the ARE sites leading to up-regulation of downstream genes. The function of Nrf2 and its downstream target genes suggests that the Nrf2-ARE pathway is important in the cellular antioxidant defense system. In support of this, many studies showed a critical role of Nrf2 in cellular protection and anti-carcinogenicity, implying that the Nrf2-ARE pathway may serve as a therapeutic target for neurodegenerative diseases and cancers, in which oxidative stress is closely implicated.

Oxidative stress has been implicated in the etiology of Parkinson's disease (PD) and in the 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) animal model of PD. It is known that under conditions of oxidative stress, the transcription factor NF-E2-related factor (Nrf2) binds to antioxidant response element (ARE) to induce antioxidant and phase II detoxification enzymes. To investigate the role of Nrf2 in the process of MPTP-induced toxicity, mice expressing the human placental alkaline phosphatase (hPAP) gene driven by a promoter containing a core ARE sequence (ARE-hPAP) were used. ARE-hPAP mice were injected (30 mg/kg) once per day for 5 days and killed 7 days after the last MPTP injection. In response to this design, ARE-dependent gene expression was decreased in striatum whereas it was increased in substantia nigra. The same MPTP protocol was applied in Nrf2(+/+) and Nrf2(-/-) mice; Nrf2 deficiency increases MPTP sensitivity. Furthermore, we evaluated the potential for astrocytic Nrf2 overexpression to protect from MPTP toxicity. Transgenic mice with Nrf2 under control of the astrocyte-specific promoter for the glial fribillary acidic protein (GFAP-Nrf2) on both a Nrf2(+/+) and Nrf2(-/-) background were administered MPTP. In the latter case, only the astrocytes expressed Nrf2. Independent of background, MPTP-mediated toxicity was abolished in GFAP-Nrf2 mice. These striking results indicate that Nrf2 expression restricted to astrocytes is sufficient to protect against MPTP and astrocytic modulation of the Nrf2-ARE pathway is a promising target for therapeutics aimed at reducing or preventing neuronal death in PD.

Binding of the transcription factor nuclear factor E2-related factor 2 (Nrf2) to the antioxidant response element (ARE) in neural cells results in the induction of a battery of genes that can coordinate a protective response against a variety of oxidative stressors. In this study, tert-butylhydroquinone (tBHQ) and sulforaphane were used as activators of this pathway. Consistent with previous studies, treatment of primary cortical cultures from ARE reporter mice revealed selective promoter activity in astrocytes. This activation protected neurons from hydrogen peroxide and nonexcitotoxic glutamate toxicity. tBHQ treatment of cultures from Nrf2 knock-out animals resulted in neither ARE activation nor neuroprotection. By reintroducing Nrf2 via infection with a replication-deficient adenovirus (ad), both the genetic response and neuroprotection were rescued. Conversely, infection with adenovirus encoding dominant-negative (DN) Nrf2 (ad-DN-Nrf2) or pretreatment with the selective phosphatidylinositol-3 kinase inhibitor LY294002 inhibited the tBHQ-mediated promoter response and corresponding neuroprotection. Interestingly, the adenoviral infection showed a high selectivity for astrocytes over neurons. In an attempt to reveal some of the cell type-specific changes resulting from ARE activation, cultures were infected with adenovirus encoding green fluorescent protein (GFP) (ad-GFP) or ad-DN-Nrf2 (containing GFP) before tBHQ treatment. A glia-enriched population of GFP-infected cells was then isolated from a population of uninfected neurons using cell-sorting technology. Microarray analysis was used to evaluate potential glial versus neuron-specific contributions to the neuroprotective effects of ARE activation and Nrf2 dependence. Strikingly, the change in neuronal gene expression after tBHQ treatment was dependent on Nrf2 activity in the astrocytes. This suggests that Nrf2-dependent genetic changes alter neuron-glia interactions resulting in neuroprotection.

Activation of the transcription factor Nrf2 in astrocytes coordinates the upregulation of antioxidant defenses and confers protection to neighboring neurons. Dominant mutations in Cu/Zn-superoxide dismutase (SOD1) cause familial forms of amyotrophic lateral sclerosis (ALS), a fatal disorder characterized by the progressive loss of motor neurons. Non-neuronal cells, including astrocytes, shape motor neuron survival in ALS and are a potential target to prevent motor neuron degeneration. The protective effect of Nrf2 activation in astrocytes has never been examined in a chronic model of neurodegeneration. We generated transgenic mice over-expressing Nrf2 selectively in astrocytes using the glial fibrillary acidic protein (GFAP) promoter. The toxicity of astrocytes expressing ALS-linked mutant hSOD1 to cocultured motor neurons was reversed by Nrf2 over-expression. Motor neuron protection depended on increased glutathione secretion from astrocytes. This protective effect was also observed by crossing the GFAP-Nrf2 mice with two ALS-mouse models. Over-expression of Nrf2 in astrocytes significantly delayed onset and extended survival. These findings demonstrate that Nrf2 activation in astrocytes is a viable therapeutic target to prevent chronic neurodegeneration.

OBJECTIVE: The goal was to estimate the prevalence of cerebral palsy and cerebral palsy subtypes among children in 3 areas of the United States by using a population-based surveillance system. METHODS: Using methods developed by the Centers for Disease Control and Prevention Metropolitan Atlanta Developmental Disabilities Surveillance Program, investigators from the Autism and Developmental Disabilities Monitoring Network conducted surveillance of cerebral palsy among 8-year-old children living in northern Alabama, metropolitan Atlanta, and southeastern Wisconsin in 2002 (N = 114,897). Cross-sectional data were collected through retrospective record review from multiple sources. Cases were linked to birth certificate and census files to obtain additional information. Period prevalence estimates were calculated per 1000 children 8 years of age. RESULTS: The average prevalence of cerebral palsy across the 3 sites was 3.6 cases per 1000, with notably similar site-specific prevalence estimates (3.3 cases per 1000 in Wisconsin, 3.7 cases per 1000 in Alabama, and 3.8 cases per 1000 in Georgia). At all sites, prevalence was higher in boys than girls (overall boy/girl ratio: 1.4:1). Also, at all sites, the prevalence of cerebral palsy was highest in black non-Hispanic children and lowest in Hispanic children. At all sites, the prevalence among children living in low- and middle-income neighborhoods was higher than that among children living in high-income neighborhoods. Spastic cerebral palsy was the most common subtype (77% of all cases), with bilateral spastic cerebral palsy dominating the spastic group (70%). CONCLUSION: These findings contribute new knowledge to the epidemiology of cerebral palsy in the United States. The similarities in prevalence rates and patterns of cerebral palsy reported for 8-year-old children at 3 geographically distinct sites provide evidence of the reliability of the surveillance methods used by the Autism and Developmental Disabilities Monitoring Network.

How a naive human neuroepithelial cell becomes an electrophysiologically active neuron remains unknown. Here, we describe the early physiological development of neurons differentiating from naive human embryonic stem (hES) cells. We found that differentiating neuronal cells progressively decrease their resting membrane potential, gain characteristic Na+ and K+ currents, and fire mature action potentials by 7 weeks of differentiation. This is similar to the maturation pattern observed in animals, albeit on a greatly expanded time scale. An additional 3 weeks of differentiation resulted in neurons that could fire repetitive trains of action potentials in response to depolarizing current pulses. The onset of spontaneous synaptic activity also occurred after 7 weeks of differentiation, in association with the differentiation of astrocytes within the culture. Cocultures of hES cell-derived neuroepithelial cells with exogenous astrocytes significantly accelerated the onset of synaptic currents but did not alter action potential generation. These findings suggest that the development of membrane characteristics and action potentials depend on the intrinsic maturation of Na+ and K+ currents, whereas synaptic transmission is enhanced by astrocytes, which may be achieved independently of the maturation of action potentials. Furthermore, we found that although astrocyte-conditioned medium accelerated synaptic protein localization, it did not increase synaptic activity, suggesting a contact-dependent mechanism by which astrocytes augment synaptic activity. These results lay the foundation for future studies examining the functional development of human neurons and provide support for the potential application of human cells in restorative neuronal therapies.

The antioxidant-responsive element (ARE) plays an important role in the induction of phase II detoxifying enzymes including NADPH:quinone oxidoreductase (NQO1). We report herein that activation of the human NQO1-ARE (hNQO1-ARE) by tert-butylhydroquinone (tBHQ) is mediated by phosphatidylinositol 3-kinase (PI3-kinase), not extracellular signal-regulated kinase (Erk1/2), in IMR-32 human neuroblastoma cells. Treatment with tBHQ significantly increased NQO1 protein without activation of Erk1/2. In addition, PD 98059 (a selective mitogen-activated kinase/Erk kinase inhibitor) did not inhibit hNQO1-ARE-luciferase expression or NQO1 protein induction by tBHQ. Pretreatment with LY 294002 (a selective PI3-kinase inhibitor), however, inhibited both hNQO1-ARE-luciferase expression and endogenous NQO1 protein induction. In support of a role for PI3-kinase in ARE activation we show that: 1) transfection of IMR-32 cells with constitutively active PI3-kinase selectively activated the ARE in a dose-dependent manner that was completely inhibited by treatment with LY 294002; 2) pretreatment of cells with the PI3-kinase inhibitors, LY 294002 and wortmannin, significantly decreased NF-E2-related factor 2 (Nrf2) nuclear translocation induced by tBHQ; and 3) ARE activation by constitutively active PI3-kinase was blocked completely by dominant negative Nrf2. Taken together, these data clearly show that ARE activation by tBHQ depends on PI3-kinase, which lies upstream of Nrf2.

Down syndrome (trisomy 21) is the most common genetic cause of intellectual disability, but the precise molecular mechanisms underlying impaired cognition remain unclear. Elucidation of these mechanisms has been hindered by the lack of a model system that contains full trisomy of chromosome 21 (Ts21) in a human genome that enables normal gene regulation. To overcome this limitation, we created Ts21-induced pluripotent stem cells (iPSCs) from two sets of Ts21 human fibroblasts. One of the fibroblast lines had low level mosaicism for Ts21 and yielded Ts21 iPSCs and an isogenic control that is disomic for human chromosome 21 (HSA21). Differentiation of all Ts21 iPSCs yielded similar numbers of neurons expressing markers characteristic of dorsal forebrain neurons that were functionally similar to controls. Expression profiling of Ts21 iPSCs and their neuronal derivatives revealed changes in HSA21 genes consistent with the presence of 50% more genetic material as well as changes in non-HSA21 genes that suggested compensatory responses to oxidative stress. Ts21 neurons displayed reduced synaptic activity, affecting excitatory and inhibitory synapses equally. Thus, Ts21 iPSCs and neurons display unique developmental defects that are consistent with cognitive deficits in individuals with Down syndrome and may enable discovery of the underlying causes of and treatments for this disorder.

Best disease (BD) is an inherited degenerative disease of the human macula that results in progressive and irreversible central vision loss. It is caused by mutations in the retinal pigment epithelium (RPE) gene BESTROPHIN1 (BEST1), which, through mechanism(s) that remain unclear, lead to the accumulation of subretinal fluid and autofluorescent waste products from shed photoreceptor outer segments (POSs). We employed human iPS cell (hiPSC) technology to generate RPE from BD patients and unaffected siblings in order to examine the cellular and molecular processes underlying this disease. Consistent with the clinical phenotype of BD, RPE from mutant hiPSCs displayed disrupted fluid flux and increased accrual of autofluorescent material after long-term POS feeding when compared with hiPSC-RPE from unaffected siblings. On a molecular level, RHODOPSIN degradation after POS feeding was delayed in BD hiPSC-RPE relative to unaffected sibling hiPSC-RPE, directly implicating impaired POS handling in the pathophysiology of the disease. In addition, stimulated calcium responses differed between BD and normal sibling hiPSC-RPE, as did oxidative stress levels after chronic POS feeding. Subcellular localization, fractionation and co-immunoprecipitation experiments in hiPSC-RPE and human prenatal RPE further linked BEST1 to the regulation and release of endoplasmic reticulum calcium stores. Since calcium signaling and oxidative stress are critical regulators of fluid flow and protein degradation, these findings likely contribute to the clinical picture of BD. In a larger context, this report demonstrates the potential to use patient-specific hiPSCs to model and study maculopathies, an important class of blinding disorders in humans.

The disease mechanism of Rett syndrome (RTT) is not well understood. Studies in RTT mouse models have suggested a non-cell-autonomous role for astrocytes in RTT pathogenesis. However, it is not clear whether this is also true for human RTT astrocytes. To establish an in vitro human RTT model, we previously generated isogenic induced pluripotent stem cell (iPSC) lines from several RTT patients carrying different disease-causing mutations. Here, we show that these RTT iPSC lines can be efficiently differentiated into astroglial progenitors and glial fibrillary acidic protein-expressing (GFAP(+)) astrocytes that maintain isogenic status, that mutant RTT astrocytes carrying three different RTT mutations and their conditioned media have adverse effects on the morphology and function of wild-type neurons and that the glial effect on neuronal morphology is independent of the intrinsic neuronal deficit in mutant neurons. Moreover, we show that both insulin-like growth factor 1 (IGF-1) and GPE (a peptide containing the first 3 amino acids of IGF-1) are able to partially rescue the neuronal deficits caused by mutant RTT astrocytes. Our findings confirm the critical glial contribution to RTT pathology, reveal potential cellular targets of IGF-1 therapy and further validate patient-specific iPSCs and their derivatives as valuable tools to study RTT disease mechanism.

Alpha synuclein (SYN) is a central player in the pathogenesis of sporadic and familial Parkinson's disease (PD). SYN aggregation and oxidative stress are associated and enhance each other's toxicity. It is unknown whether the redox-sensitive transcription factor nuclear factor erythroid 2-related factor 2 (Nrf2) plays a role against the toxicity of SYN. To examine this, mice selectively overexpressing Nrf2 in astrocytes (GFAP-Nrf2) were crossed with mice selectively expressing human mutant SYN (hSYN(A53T)) in neurons. Increased astrocytic Nrf2 delayed the onset and extended the life span of the hSYN(A53T) mice. This correlated with increased motor neuron survival, reduced oxidative stress, and attenuated gliosis in the spinal cord, as well as a dramatic decrease in total hSYN(A53T) and phosphorylated (Ser129) hSYN(A53T) in Triton-insoluble aggregates. Furthermore, Nrf2 in astrocytes delayed chaperone-mediated autophagy and macroautophagy dysfunction observed in the hSYN(A53T) mice. Our data suggest that Nrf2 in astrocytes provides neuroprotection against hSYN(A53T)-mediated toxicity by promoting the degradation of hSYN(A53T) through the autophagy-lysosome pathway in vivo. Thus, activation of the Nrf2 pathway in astrocytes is a potential target to develop therapeutic strategies for treating pathologic synucleinopathies including PD.

PURPOSE: This study compared movement characteristics of markers attached to the jaw, lower lip, tongue blade, and dorsum during production of selected English vowels by normal speakers and speakers with dysarthria due to amyotrophic lateral sclerosis (ALS) or Parkinson disease (PD). The study asked the following questions: (a) Are movement measures different for healthy controls and speakers with ALS or PD, and (b) Are articulatory profiles comparable for speakers with ALS and speakers with PD? METHOD: Nineteen healthy controls and 15 speakers with dysarthria participated in this study. The severity of dysarthria varied across individuals and between the 2 disorder groups. The stimuli were 10 words (i.e., seed, feed, big, dish, too, shoo, bad, cat, box, and dog) embedded into sentences read at a comfortable reading rate. Movement data were collected using the X-ray microbeam. Movement measures included distances, durations, and average speeds of vowel-related movement strokes. RESULTS: Differences were found (a) between speakers with ALS and healthy controls and (b) between speakers with ALS and PD, particularly in movement speed. Tongue movements in PD and ALS were more consistently different from healthy controls than jaw and lower lip movements. This study showed that the effects of neurologic disease on vowel production are often articulator-, vowel-, and context-specific. CONCLUSIONS: Differences in severity between the speakers with PD and ALS may have accounted for some of the differences in movement characteristics between the groups. These factors need to be carefully considered when describing the nature of speech disorder and developing empirically based evaluation and treatment strategies for dysarthria.

Physical stimuli can act in either a synergistic or antagonistic manner to regulate cell fate decisions, but it is less clear whether insoluble signals alone can direct human pluripotent stem (hPS) cell differentiation into specialized cell types. We previously reported that stiff materials promote nuclear localization of the Yes-associated protein (YAP) transcriptional coactivator and support long-term self-renewal of hPS cells. Here, we show that even in the presence of soluble pluripotency factors, compliant substrata inhibit the nuclear localization of YAP and promote highly efficient differentiation of hPS cells into postmitotic neurons. In the absence of neurogenic factors, the effective substrata produce neurons rapidly (2 wk) and more efficiently (>75%) than conventional differentiation methods. The neurons derived from substrate induction express mature markers and possess action potentials. The hPS differentiation observed on compliant surfaces could be recapitulated on stiff surfaces by adding small-molecule inhibitors of F-actin polymerization or by depleting YAP. These studies reveal that the matrix alone can mediate differentiation of hPS cells into a mature cell type, independent of soluble inductive factors. That mechanical cues can override soluble signals suggests that their contributions to early tissue development and lineage commitment are profound.

Mutations in the gene for the astrocyte specific intermediate filament, glial fibrillary acidic protein (GFAP), cause the rare leukodystrophy Alexander disease (AxD). To study the pathology of this primary astrocyte defect, we have generated knock-in mice with missense mutations homologous to those found in humans. In this report, we show that mice with GFAP-R76H and -R236H mutations develop Rosenthal fibers, the hallmark protein aggregates observed in astrocytes in AxD, in the hippocampus, corpus callosum, olfactory bulbs, subpial, and periventricular regions. Astrocytes in these areas appear reactive and total GFAP expression is elevated. Although general white matter architecture and myelination appear normal, when crossed with an antioxidant response element reporter line, the mutant mice show a distinct pattern of reporter-gene induction that is especially prominent in the corpus callosum, and histochemical staining reveals accumulation of iron in the same region. The mutant mice have a normal lifespan and show no overt behavioral defects, but are more susceptible to kainate-induced seizures. Although these mice demonstrate increased GFAP expression by themselves, further elevation of GFAP via crosses to GFAP transgenic animals leads to a shift in GFAP solubility, an increased stress response, and ultimately death. The mice do not display the full spectrum of pathology observed in human infantile AxD, but may more closely resemble the adult form of the disease. These studies provide formal proof linking GFAP mutations with Rosenthal fibers and oxidative stress, and correlate gliosis and GFAP protein levels to the severity of the disease.

In Brief Objectives: To measure sound source localization in children who have sequential bilateral cochlear implants (BICIs); to determine whether localization accuracy correlates with performance on a right-left discrimination task (i.e., spatial acuity); to determine whether there is a measurable bilateral benefit on a sound source identification task (i.e., localization accuracy) by comparing performance under bilateral and unilateral listening conditions; and to determine whether sound source localization continues to improve with longer durations of bilateral experience. Design: Two groups of children participated in this study: a group of 21 children who received BICIs in sequential procedures (5 to 14 years) and a group of 7 typically developing children with normal acoustic hearing (5 years). Testing was conducted in a large sound-treated booth with loudspeakers positioned on a horizontal arc with a radius of 1.2 m. Children participated in two experiments that assessed spatial hearing skills. Spatial hearing acuity was assessed with a discrimination task in which listeners determined whether a sound source was presented on the right or left side of center; the smallest angle at which performance on this task was reliably above chance is the minimum audible angle. Sound localization accuracy was assessed with a sound source identification task in which children identified the perceived position of the sound source from a multiloudspeaker array (7 or 15); errors are quantified using the root mean square (RMS) error. Results: Sound localization accuracy was highly variable among the children with BICIs, with RMS errors ranging from 19 to 56°. Performance of the normal hearing group, with RMS errors ranging from 9 to 29° was significantly better. Within the BICI group, in 11 of 21 children, RMS errors were smaller in the bilateral versus unilateral listening condition, indicating bilateral benefit. There was a significant correlation between spatial acuity and sound localization accuracy (R2 = 0.68, p < 0.01), suggesting that children who achieve small RMS errors tend to have the smallest minimum audible angles. Although there was large intersubject variability, testing of 11 children in the BICI group at two sequential visits revealed a subset of children who show improvement in spatial hearing skills over time. Conclusions: A subset of children who use sequential BICIs can acquire sound localization abilities, even after long intervals between activation of hearing in the first- and second-implanted ears. This suggests that children with activation of the second implant later in life may be capable of developing spatial hearing abilities. The large variability in performance among the children with BICIs suggests that maturation of sound localization abilities in children with BICIs may be dependent on various individual subject factors such as age of implantation and chronological age. This study aimed to document spatial hearing skills in 21 children (5-14 years old) who had 3-28 months of experience with sequential bilateral cochlear implants (BICIs). Children were centered in front of a horizontal arc, with speakers located from ±70° at 10° intervals, and asked to identify the location of an auditory stimulus on two spatial hearing tasks: right-left discrimination (quantified by minimal audible angle) and sound source identification (quantified by the root-mean-square error). Although there was large individual variability in performance, results showed emerging spatial hearing skills in a subset of children who use sequential BICIs.

Epidemiological studies of children and adolescents with intellectual disability have found 30 to 50% exhibiting clinically significant behavior problems. Few studies, however, have assessed young children, included a cognitively typical comparison group, assessed for specific disorders, and/or studied family correlates of diagnosis. We assessed 236 5-year-old children--95 with developmental delay (DD) and 141 with typical development--for clinical diagnoses using a structured interview. Every disorder assessed was more prevalent in the DD group. The percent of children meeting criteria for Attention Deficit Hyperactivity Disorder (ADHD) most highly differentiated the two groups (ratio = 3.21:1). There was high stability from externalizing behavior problems at age 3 to ADHD diagnoses at age 5 in both groups. In regression analyses, parenting stress at child age 3 related to later ADHD diagnosis in both groups and maternal scaffolding (sensitive teaching) also predicted ADHD in the DD group.

Down syndrome (DS), caused by trisomy of chromosome 21, occurs in 1 of every 800 live births. Early defects in cortical development likely account for the cognitive impairments in DS, although the underlying molecular mechanism remains elusive. Here, we performed histological assays and unbiased single-cell RNA-Seq (scRNA-Seq) analysis on cerebral organoids derived from 4 euploid cell lines and from induced pluripotent stem cells (iPSCs) from 3 individuals with trisomy 21 to explore cell-type-specific abnormalities associated with DS during early brain development. We found that neurogenesis was significantly affected, given the diminished proliferation and decreased expression of layer II and IV markers in cortical neurons in the subcortical regions; this may have been responsible for the reduced size of the organoids. Furthermore, suppression of the DSCAM/PAK1 pathway, which showed enhanced activity in DS, using CRISPR/Cas9, CRISPR interference (CRISPRi), or small-molecule inhibitor treatment reversed abnormal neurogenesis, thereby increasing the size of organoids derived from DS iPSCs. Our study demonstrates that 3D cortical organoids developed in vitro are a valuable model of DS and provide a direct link between dysregulation of the DSCAM/PAK1 pathway and developmental brain defects in DS.

PURPOSE: Darley, Aronson, and Brown (1969a, 1969b) detailed methods and results of auditory-perceptual assessment for speakers with dysarthrias of varying etiology. They reported adequate listener reliability for use of the rating system as a tool for differential diagnosis, but several more recent studies have raised concerns about listener reliability using this approach. METHOD: In the present study, the authors examined intrarater and interrater agreement for perceptual ratings of 47 speakers with various dysarthria types by 2 listener groups (inexperienced and experienced). The entire set of perceptual features proposed by Darley et al. was rated based on a 40-s conversational speech sample. RESULTS: No differences in levels of agreement were found between the listener groups. Agreement was within 1 scale value or better for 67% of the pairwise comparisons. Levels of agreement were lower when the average rating fell in the mid-range of the scale compared with samples that had an average rating near either of the scale endpoints; agreement was above chance level. No significant differences in agreement were found between the perceptual features. DISCUSSION: The levels of listener agreement that were found indicate that auditory-perceptual ratings show promise during clinical assessment for identifying salient features of dysarthria for speakers with various etiologies.