Distinctive transcriptome alterations of prefrontal pyramidal neurons in schizophrenia and schizoaffective disorder.

Schizophrenia is associated with alterations in working memory that reflect dysfunction of dorsolateral prefrontal cortex (DLPFC) circuitry. Working memory depends on the activity of excitatory pyramidal cells in DLPFC layer 3 and, to a lesser extent, in layer 5. Although many studies have profiled gene expression in DLPFC gray matter in schizophrenia, little is known about cell-type-specific transcript expression in these two populations of pyramidal cells. We hypothesized that interrogating gene expression, specifically in DLPFC layer 3 or 5 pyramidal cells, would reveal new and/or more robust schizophrenia-associated differences that would provide new insights into the nature of pyramidal cell dysfunction in the illness. We also sought to determine the impact of other variables, such as a diagnosis of schizoaffective disorder or medication use at the time of death, on the patterns of gene expression in pyramidal neurons. Individual pyramidal cells in DLPFC layers 3 or 5 were captured by laser microdissection from 36 subjects with schizophrenia or schizoaffective disorder and matched normal comparison subjects. The mRNA from cell collections was subjected to transcriptome profiling by microarray followed by quantitative PCR validation. Expression of genes involved in mitochondrial (MT) or ubiquitin-proteasome system (UPS) functions were markedly downregulated in the patient group (P-values for MT-related and UPS-related pathways were <10(-7) and <10(-5), respectively). MT-related gene alterations were more prominent in layer 3 pyramidal cells, whereas UPS-related gene alterations were more prominent in layer 5 pyramidal cells. Many of these alterations were not present, or found to a lesser degree, in samples of DLPFC gray matter from the same subjects, suggesting that they are pyramidal cell specific. Furthermore, these findings principally reflected alterations in the schizophrenia subjects were not present or present to a lesser degree in the schizoaffective disorder subjects (diagnosis of schizoaffective disorder was the most significant covariate, P<10(-6)) and were not attributable to factors frequently comorbid with schizophrenia. In summary, our findings reveal expression deficits in MT- and UPS-related genes specific to layer 3 and/or layer 5 pyramidal cells in the DLPFC of schizophrenia subjects. These cell type-specific transcriptome signatures are not characteristic of schizoaffective disorder, providing a potential molecular-cellular basis of differences in clinical phenotypes.

Hypoxia-induced immortalization of primary cells depends on Tfcp2L1 expression.

Cellular senescence is a stress response mechanism that induces proliferative arrest. Hypoxia can bypass senescence and extend the lifespan of primary cells, mainly by decreasing oxidative damage. However, how hypoxia promotes these effects prior to malignant transformation is unknown. Here we observed that the lifespan of mouse embryonic fibroblasts (MEFs) is increased when they are cultured in hypoxia by reducing the expression of p16INK4a, p15INK4b and p21Cip1. We found that proliferating MEFs in hypoxia overexpress Tfcp2l1, which is a main regulator of pluripotency and self-renewal in embryonic stem cells, as well as stemness genes including Oct3/4, Sox2 and Nanog. Tfcp2l1 expression is lost during culture in normoxia, and its expression in hypoxia is regulated by Hif1α. Consistently, its overexpression in hypoxic levels increases the lifespan of MEFs and promotes the overexpression of stemness genes. ATAC-seq and Chip-seq experiments showed that Tfcp2l1 regulates genes that control proliferation and stemness such as Sox2, Sox9, Jarid2 and Ezh2. Additionally, Tfcp2l1 can replicate the hypoxic effect of increasing cellular reprogramming. Altogether, our data suggest that the activation of Tfcp2l1 by hypoxia contributes to immortalization prior to malignant transformation, facilitating tumorigenesis and dedifferentiation by regulating Sox2, Sox9, and Jarid2.

Consensus for tinnitus patient assessment and treatment outcome measurement: Tinnitus Research Initiative meeting, Regensburg, July 2006.

There is widespread recognition that consistency between research centres in the ways that patients with tinnitus are assessed and outcomes following interventions are measured would facilitate more effective co-operation and more meaningful evaluations and comparisons of outcomes. At the first Tinnitus Research Initiative meeting held in Regensburg in July 2006 an attempt was made through workshops to gain a consensus both for patient assessments and for outcome measurements. It is hoped that this will contribute towards better cooperation between research centres in finding and evaluating treatments for tinnitus by allowing better comparability between studies.

Identification of B-KSR1, a novel brain-specific isoform of KSR1 that functions in neuronal signaling.

Kinase suppressor of Ras (KSR) is an evolutionarily conserved component of Ras-dependent signaling pathways. Here, we report the identification of B-KSR1, a novel splice variant of murine KSR1 that is highly expressed in brain-derived tissues. B-KSR1 protein is detectable in mouse brain throughout embryogenesis, is most abundant in adult forebrain neurons, and is complexed with activated mitogen-activated protein kinase (MAPK) and MEK in brain tissues. Expression of B-KSR1 in PC12 cells resulted in accelerated nerve growth factor (NGF)-induced neuronal differentiation and detectable epidermal growth factor (EGF)-induced neurite outgrowth. Sustained MAPK activity was observed in cells stimulated with either NGF or EGF, and all effects on neurite outgrowth could be blocked by the MEK inhibitor PD98059. In B-KSR1-expressing cells, the MAPK-B-KSR1 interaction was inducible and correlated with MAPK activation, while the MEK-B-KSR1 interaction was constitutive. Further examination of the MEK-B-KSR1 interaction revealed that all genetically identified loss-of-function mutations in the catalytic domain severely diminished MEK binding. Moreover, B-KSR1 mutants defective in MEK binding were unable to augment neurite outgrowth. Together, these findings demonstrate the functional importance of MEK binding and indicate that B-KSR1 may function to transduce Ras-dependent signals that are required for neuronal differentiation or that are involved in the normal functioning of the mature central nervous system.

Identification of constitutive and ras-inducible phosphorylation sites of KSR: implications for 14-3-3 binding, mitogen-activated protein kinase binding, and KSR overexpression.

Genetic and biochemical studies have identified kinase suppressor of Ras (KSR) to be a conserved component of Ras-dependent signaling pathways. To better understand the role of KSR in signal transduction, we have initiated studies investigating the effect of phosphorylation and protein interactions on KSR function. Here, we report the identification of five in vivo phosphorylation sites of KSR. In serum-starved cells, KSR contains two constitutive sites of phosphorylation (Ser297 and Ser392), which mediate the binding of KSR to the 14-3-3 family of proteins. In the presence of activated Ras, KSR contains three additional sites of phosphorylation (Thr260, Thr274, and Ser443), all of which match the consensus motif (Px[S/T]P) for phosphorylation by mitogen-activated protein kinase (MAPK). Further, we find that treatment of cells with the MEK inhibitor PD98059 blocks phosphorylation of the Ras-inducible sites and that activated MAPK associates with KSR in a Ras-dependent manner. Together, these findings indicate that KSR is an in vivo substrate of MAPK. Mutation of the identified phosphorylation sites did not alter the ability of KSR to facilitate Ras signaling in Xenopus oocytes, suggesting that phosphorylation at these sites may serve other functional roles, such as regulating catalytic activity. Interestingly, during the course of this study, we found that the biological effect of KSR varied dramatically with the level of KSR protein expressed. In Xenopus oocytes, KSR functioned as a positive regulator of Ras signaling when expressed at low levels, whereas at high levels of expression, KSR blocked Ras-dependent signal transduction. Likewise, overexpression of Drosophila KSR blocked R7 photoreceptor formation in the Drosophila eye. Therefore, the biological function of KSR as a positive effector of Ras-dependent signaling appears to be dependent on maintaining KSR protein expression at low or near-physiological levels.

The epsilon isoform of protein kinase C is an oncogene when overexpressed in rat fibroblasts.

We have overproduced the Ca(2+)-independent protein kinase C isoform, nPKC epsilon, in Rat 6 embryo fibroblasts, and examined the effects of this novel isoform on cell growth and transformation. As compared to vector control cell lines expressing only the hygromycin resistance gene, the nPKC epsilon overproducing cell lines exhibited a 7-13-fold increase in Ca(2+)-independent enzyme activity. Detailed analysis of seven individual nPKC epsilon overproducing clones indicated that those clones that expressed very high activity displayed a number of disorders in growth control, including: formation of dense foci in monolayer culture, decreased doubling time, increased saturation density, decreased serum requirement, growth in soft agar, and tumor formation in nude mice. These findings are in contrast to previous studies from our laboratory indicating that stable expression of high levels of cPKC beta 1 produced only a partially transformed phenotype (Housey et al., 1988). Taken together, these results provide the first direct evidence that distinct isoforms of PKC can exert different effects on growth control and malignant transformation in the same cell type.

PKC epsilon functions as an oncogene by enhancing activation of the Raf kinase.

Previous studies have indicated that PKCepsilon behaves as an oncogene when overproduced in rodent fibroblasts (Cacace et al., 1993; Mishak et al., 1993). In the present study, Western blot analysis revealed that the hyperphosphorylated form of Raf kinase was present at a high level in PKCepsilon overproducing R6 rat fibroblasts but not in R6 fibroblasts overproducing PKCalpha or beta1. Extracts from the PKCepsilon overproducing cells also exhibited a marked increase in Raf-1 kinase and MAP-kinase activity. To investigate the significance of these findings, dominant negative mutants of ras (N17) or raf (301-1) were stably expressed in early passage control and PKCepsilon-transformed R6 fibroblasts, by transduction using retrovirus-derived constructs. Dominant negative raf expressing clones exhibited a flat morphology, a decreased saturation density, and decreased growth in soft agar. In addition, these reverted clones exhibited decreased Raf kinase activity. In contrast, dominant negative ras expressing clones remained highly transformed. In addition, PKCepsilon was detected in Raf-1 immunoprecipitates indicating that PKCepsilon forms a complex with Raf-1 in vivo. Taken together, these results suggest that PKCepsilon functions as an oncogene in R6 cells by enhancing activation of the Raf-1 kinase.

Overexpression of cyclin D1 in rat fibroblasts causes abnormalities in growth control, cell cycle progression and gene expression.

Cyclin D1, a putative G1 cyclin, has been implicated in cell cycle control. The human cyclin D1 gene is located on chromosome 11q13 where DNA rearrangement and amplification have been detected in several types of human cancer. Previous studies demonstrated that the cyclin D1 gene is not only rearranged or amplified but also overexpressed in some of these human tumors and tumor-derived cell lines. To further address the roles of cyclin D1 in cell cycle control and tumorigenesis, we have stably overexpressed the human cyclin D1 cDNA in Rat6 embryo fibroblasts by using retrovirus mediated transduction. The cyclin D1 protein was overproduced about 10-fold and was localized predominately in the nucleus. Cyclin D1 overexpressing cells displayed a decrease in the duration of the G1 phase, decreased cell size, and induced tumors when injected into athymic (nude) mice. In addition, overexpression of cyclin D1 in Rat6 cells perturbed the expression of several cellular growth-related genes including c-myc, c-jun, and cyclin A, but not cyclin D3. Taken together, these results indicate that deregulated expression of the cyclin D1 gene can cause disturbances in cell cycle control and gene expression and also enhance tumorigenesis.

Stable overexpression of cyclin D1 in a human mammary epithelial cell line prolongs the S-phase and inhibits growth.

Amplification and/or increased expression of cyclin D1 occurs in an appreciable fraction of primary human breast carcinomas and several other types of human cancer. In addition, overexpression of cyclin D1 in rodent fibroblasts enhances growth and malignant transformation. The present study demonstrates that the extent of amplification and expression of cyclin D1 varies widely amongst a series of cell lines established from normal human mammary epithelium or human breast carcinomas. The HBL-100 mammary epithelial cell line did not display amplification or increased expression of cyclin D1. We used retrovirus-mediated transduction to obtain derivatives of this cell line that stably expressed relatively high levels of an exogenous cyclin D1 cDNA. These derivatives displayed an increased doubling time, decreased saturation density, decreased cloning efficiency, decreased anchorage-independent growth, an increased fraction of cells in the S-phase, and decreased tumorigenicity. Thus, increased expression of cyclin D1 in this cell line markedly inhibits rather than enhances growth, which may be due to the prolongation of S-phase.

Effects of suramin-related and other clinically therapeutic polyanions on protein kinase C activity.

The mechanism of the antineoplastic effects of suramin may involve interference with signal transduction, but in general is not well understood. We examined several polyanions to determine their effects on the kinase activity of the protein kinase C (PKC) beta1 and other PKC isoforms. Similar to suramin, a phosphorothioate oligodeoxynucleotide 28-mer homopolymer of cytidine (SdC28) inhibited the phosphatidylserine and Ca2+-dependent phosphorylation of an epidermal growth factor receptor octapeptide substrate. The inhibition by suramin was mixed competitive/noncompetitive with respect to ATP, but uncompetitive with respect to substrate. In contrast, the inhibition by SdC28 was competitive with respect to substrate (Ki = 5.4 microM) and not competitive with respect to ATP. The PKC alpha and beta1 isoforms were inhibited to the same extent with SdC28, while PKC epsilon was not inhibited. SdC28, in the absence of lipid cofactor, stimulated substrate phosphorylation, and in the absence of substrate induced PKC beta1 autophosphorylation. Similar behavior was seen with another polyanion, the polysulfated carbohydrate pentosan polysulfate (polyxylyl hydrogen sulfate). H4, a bis-naphthalene disulfonate tetraanion structurally related to suramin, also inhibited kinase activity but was not competitive with respect to ATP. Dianions closely related to H4 failed to inhibit PKC beta1, suggesting that multiple (>2) negative charges are required. The interactions of polyanions with PKC are complex, and are dependent on the molecular structure of the polyanion, the presence of cofactors, and the PKC isoform.

Auditory dysfunction in Friedreich ataxia: result of spiral ganglion degeneration.

We performed behavioral audiometric tests and brainstem auditory evoked potentials in four patients with Friedreich ataxia. None of the patients had symptomatic hearing difficulties. Results of the audiometric tests pointed to a disorder of the eighth nerve. In none of the patients could we elicit normal-appearing waves of the brainstem auditory evoked potentials. These abnormalities could be attributed to degeneration of spiral ganglion neurons. Our patients had useful and functional hearing despite very abnormal brainstem auditory evoked potentials.

Brainstem auditory evoked potentials: a comparison of two high-frequency filter settings.

Filter bandpass characteristics are important elements in all modalities of evoked potential recordings. We studied the brainstem auditory evoked potentials by changing the bandpass of the recording system between 150 to 1500 Hz and 150 to 3000 Hz. The 150- to 1500-Hz bandpass prolonged the absolute latencies of the various waves, whereas the interpeak latencies (I to III, IIII to V, III to IV/V, I to V, or I to IV/V) were not significantly altered. The 150- to 1500-Hz filter reduced notched or multipeaked wave forms by decreasing the raw high-frequency input activity.

Assessing short-term recognition memory with forced-choice psychophysical methods.

The rationale and methodology for using computer-controlled forced-choice psychophysical methods to assess short-term recognition memory in human subjects are presented. Here, we use non-verbal computer-synthesized auditory and visual stimuli with an adaptive psychophysical procedure. Sequence-length thresholds (SLTs, span lengths) for randomly generated binary auditory and visual-sequential patterns and simultaneous visual-spatial patterns are determined to assess short-term memory capacity. The SLTs can also be used to equate for initial retention level for delayed matching-to-sample (DMS) or delayed matching-to-non-sample (DMNS) tasks which assess memory decay. The DMS/DMNS tasks have also been modified for use with the forced-choice paradigm. In contrast to many verbal paradigms requiring immediate ordered recall, non-verbal stimuli in a forced-choice paradigm provide a more direct measure of sensory memory because long-term memory, complex encoding/decoding processes, and motor-sequencing factors are minimized or avoided. Furthermore, the forced-choice recognition memory tasks are applicable over a broad age range, are less sensitive to socio-economic factors and educational level, and avoid complex instructions. Taken together, these factors enhance the applicability of these tasks in children and adults with CNS lesions, particularly where cognitive status may be compromised.

Some poststimulatory effects on the whole nerve action potential.

Short-term poststimulatory effects on the N1 component of the auditory-nerve compound action potential (CAP) were investigated in Mongolian gerbils. Some effects of conditioner level, conditioner frequency and probe level were assessed. In most cases, poststimulatory decrements occurred. The decrements were independent of probe intensity when the conditioner was close in frequency and of equal or less intensity than the probe. At some other conditioner frequencies, decrements decreased as probe intensity increased. Departures from these relationships occurred when a more intense conditioner was applied. In some instances, increases in CAP amplitude were also observed. The roles of single unit responses including spread of cochlear excitation, and possible mechanisms for poststimulatory facilitation are discussed.

T complex hemispheric asymmetries: effects of stimulus intensity.

The T complex component of the human auditory evoked potential (AEP) is thought to be produced in auditory cortex, on the posterior lateral surface of the temporal lobe. Recorded over temporal scalp, it consists of an 80-90 ms positive peak, Ta, and a 120-140 negative peak, Tb. As part of an effort to develop the clinical usefulness of the T complex in assessing auditory cortical function, we studied the effects of change in monaural stimulus intensity (20-80 dB SL) on T complex latency, amplitude, and hemispheric differences in normal adults. Ta and Tb peak latencies decreased as stimulus intensity increased. These latency changes were not dependent on ear or hemisphere. Right hemisphere Ta latency was shorter with contralateral than with ipsilateral stimulation; while left hemisphere Ta latency was not dependent on the ear stimulated. Tb latency was shorter over the left hemisphere, and over the contralateral hemisphere. Ta-b amplitude increased as stimulus intensity increased. This amplitude change was not dependent on ear or hemisphere. Ta-b amplitudes were larger over the right hemisphere and over the contralateral hemisphere. Hemispheric asymmetries were not significantly affected by stimulus intensity.

Anomalous cross-modal plasticity following posterior fossa surgery: some speculations on gaze-evoked tinnitus.

A unique and intriguing form of subjective tinnitus evoked by eye gaze is reviewed. A new perspective is presented because this condition is sufficiently different from other forms of subjective tinnitus and its manifestation cannot be adequately explained by existing models or conceptual frameworks. Our examination of this topic considers pathophysiologic changes in the central nervous system in the context of deafferentation-induced plasticity. Potential neuroanatomical areas contributing to this effect include a number of distributed and functionally diverse areas in the brainstem and neocortex involved in the auditory control of eye movements. We also consider contemporary psychophysical methods to evaluate the perceptual correlates of this phenomenon and tools for the development of objective tinnitus measurements. Although theoretical and speculative in nature, this article is intended to stimulate interest in, advance knowledge of, and provide a better understanding about this condition.

Clinical applications of otoacoustic emissions in sudden hearing loss.

Sudden hearing loss (SHL) is a controversial topic for which no definitive practical guidelines exist. Studies employing vasodilators, plasma expanders, anticoagulants, and carbogen inhalations have shown no improvement over the rate of spontaneous recovery. At present, there is insufficient evidence to support medical treatment for SHL, except steroid therapy in selected patients. Distortion product otoacoustic emissions (DPOAEs) are sensitive to cochlear disorders and are absent in ischemic injury to the cochlea, but can persist in cochlear neuritis. In a prospective study of 10 patients who presented to Albany Medical Center from 1995 to 1996, three patients with intact DPOAEs at presentation had an average improvement of 33 dB in the pure-tone average (PTA) of 0.5, 1.0, and 2.0 kHz with steroid therapy, whereas five of seven patients with absent DPOAEs had no improvement in hearing despite steroid therapy in six patients. The presence of DPOAEs may be a useful prognostic factor that positively correlates with recovery from SHL.

Advances in the development of the interferometric otoscope.

Present measurement techniques for middle ear function have inherent limitations because they are either spatially insensitive (acoustic immittance) or descriptive and qualitative in nature (otoscopy). By integrating advances in electrooptic technology (fiber optics, miniature diode lasers, solid-state detector arrays) and digital processing, further advances are possible. On the basis of measurements taken with electronic speckle-pattern interferometry on human temporal bones and models, we demonstrate quantitative static and dynamic vibration/displacement characteristics of the tympanic membrane with high spatial resolution. Our presentation emphasizes advantages of optically based methods and demonstrates computerized signal processing capable of fringe localization, enhancement, and counting. Miniaturization and real-time digital image processing in the clinical setting is the goal of this research.

Estimation of formant frequencies in infant cry.

The formant frequencies (F1, F2, F3) of normal infant crying were measured using three different estimation techniques: sound spectrography, linear predictive coding (LPC), and power spectrum analysis. Results found all three techniques to be highly similar for estimation of F1. However, the techniques differed significantly in the estimation of F2 and F3. Power spectrum analysis tended to yield the highest F2 and F3 values, while LPC consistently provided the lowest F2 and F3 values. Based on the results of the study, serious questions arise whether formant estimates of cry are accurate or appropriate for use as a metric of infant vocal tract resonance.

Swallowing disorders in a population of children with cerebral palsy.

One of the disabilities in patients with cerebral palsy (CP) is dysphagia. To establish the prevalence of dysphagia in a population of children with CP, and to determine if any factors are related to dysphagia, we studied 56 CP patients, 5-21 years, enrolled in a primary school for the disabled. Fifteen patients (27%) had either radiographic or clinical evidence of dysphagia. These 15 patients were compared to the remaining 41 patients without dysphagia. Using data obtained from chart review and interviews with speech pathologists, several factors that contributed to dysphagia were found. These included: bite reflexes, slowness of oral intake, poor trunk control, inability to feed independently, anticonvulsant medication, coughing with meals, choking, and pneumonia. We also noted trends in the following factors: presence of tongue thrusting, presence of drooling, severity of CP, poor head control, severity of mental retardation, seizures, and speech disorders. Factors not related to the presence of dysphagia include: subject age, cause of CP, and type of CP. Early, aggressive work-up and identification in CP patients with the risk factors outlined above can reduce the associated pulmonary complications.