The world’s human population ages rapidly thanks to the great advance in modern medicine. While more and more body system diseases become treatable and curable, age-related neurodegenerative diseases remain poorly understood mechanistically, and are desperately in need of preventive and therapeutic interventions. Biomarker development consists of a key part of concerted effort in combating neurodegenerative diseases. In many chronic neurodegenerative conditions, neuronal damage/death occurs long before the onset of disease symptoms, and abnormal proteolysis may either play an active role or be a companying event of neuronal injury. Increased spectrin cleavage yielding elevated spectrin breakdown products (SBDPs) by calcium-sensitive proteases such as calpain and caspases has been established in conditions associated with acute neuronal damage such as traumatic brain injury (TBI). Here we review literature regarding spectrin expression and metabolism in the brain, and propose a potential use of SBDPs as biomarkers for neurodegenerative diseases such as Alzheimer’s diseases.
Aging; Alzheimer’s disease; Apoptosis; Autophagy; Calcium; Calpain; Caspases; Membrane skeleton; Neuroplasticity; Neurodegeneration; Proteolysis
High brain and acute leukemia, cytoplasmic (BAALC) expression defines an important risk factor in cytogenetically normal acute myeloid leukemia (CN-AML). The prognostic value of BAALC expression in relation to other molecular prognosticators was analyzed in 326 CN-AML patients (<65 years). At diagnosis, high BAALC expression was associated with prognostically adverse mutations: FLT3 internal tandem duplication (FLT3-ITD) with an FLT3-ITD/FLT3 wild-type (wt) ratio of ⩾0.5 (P=0.001), partial tandem duplications within the MLL gene (MLL-PTD) (P=0.002), RUNX1 mutations (mut) (P<0.001) and WT1mut (P=0.001), while it was negatively associated with NPM1mut (P<0.001). However, high BAALC expression was also associated with prognostically favorable biallelic CEBPA (P=0.001). Survival analysis revealed an independent adverse prognostic impact of high BAALC expression on overall survival (OS) and event-free survival (EFS), and also on OS when eliminating the effect of allogeneic stem cell transplantation (SCT) (OSTXcens). Furthermore, we analyzed BAALC expression in 416 diagnostic and follow-up samples of 66 patients. During follow-up, BAALC expression correlated with mutational load or expression levels, respectively, of other minimal residual disease markers: FLT3-ITD (r=0.650, P<0.001), MLL-PTD (r=0.728, P<0.001), NPM1mut (r=0.599, P<0.001) and RUNX1mut (r=0.889, P<0.001). Moreover, a reduction in BAALC expression after the second cycle of induction chemotherapy was associated with improved EFS. Thus, our data underline the utility of BAALC expression as a marker for prognostic risk stratification and detection of residual disease in CN-AML.
BAALC expression; CN-AML; prognosis; MRD
Therapeutic products that depend on the use of an in vitro diagnostic biomarker test to confirm their effectiveness are increasingly being developed. Use of biomarkers is particularly meaningful in the context of selecting the patient population where the therapeutic treatment is believed to be efficacious (patient enrichment). Currently available 'research-use-only' assays for Alzheimer's disease diagnosis all suffer from non-analyte and analyte-specific interferences. The impact of these interferences on the outcome of the assays is not well understood. The confounding factors are hampering correct value determination in biological samples and are intrinsic to the assay concept, the assay design, the presence in the sample of heterophilic antibodies and auto-antibodies, or might be the result of the therapeutic approach. This review focuses on the importance of assay interferences and considers how these might be minimized with the final aim of making the assays more acceptable as in vitro diagnostic biomarker tests for theranostic use.
Neurobiochemical marker levels in blood after traumatic brain injury (TBI) may reflect structural changes detected by neuroimaging. This study evaluates whether correlations between neuronal (ubiquitin carboxy-terminal hydrolase-L1 [UCH-L1]) and glial (glial fibrillary acidic protein [GFAP]) biomarkers may be used as an indicator for differing intracranial pathologies after brain trauma. In 59 patients with severe TBI (Glasgow Coma Scale [GCS] score≤8) serum samples were obtained at the time of hospital admission and analyzed for UCH-L1 and GFAP. Glial neuronal ratio (GNR) was evaluated as the ratio between GFAP and UCH-L1 concentrations. A logistic regression analysis was used to identify variables associated with type of injury. GNR had a median of 0.85 and was positively correlated with age (R=0.45, p=0.003). Twenty-nine patients presented with diffuse injury and 30 with focal mass lesions as assessed by CT scan at admission and classified according to the Marshall Classification. GNR was significantly higher in the focal mass lesion group compared with the diffuse injury group (1.77 versus 0.48, respectively; p=0.003). Receiver operating characteristic curve analysis showed that GNR discriminated between types of injury (area under the curve [AUC]=0.72; p=0.003). GNR was more accurate earlier (≤12 h after injury) than later (AUC=0.80; p=0.002). Increased GNR was independently associated with type of injury, but not age, gender, GCS score, or mechanism of injury. GNR was significantly higher in patients who died, but was not an independent predictor of death. The data from the present study indicate that GNR provides valuable information about different injury pathways, which may be of diagnostic significance. In addition, GNR may help to identify different pathophysiological mechanisms following different types of brain trauma, with implications for therapeutic interventions.
biomarkers; computed tomography; diagnostic; glial neuronal ratio; traumatic brain injury
The somatodendritic A-current, ISA, in hippocampal CA1 pyramidal neurons regulates the processing of synaptic inputs and the amplitude of back propagating action potentials into the dendritic tree, as well as the action potential firing properties at the soma. In this study, we have used RNA interference and over-expression to show that expression of the Kv4.2 gene specifically regulates the ISA component of A-current in these neurons. In dissociated hippocampal pyramidal neuron cultures, or organotypic cultured CA1 pyramidal neurons, the expression level of Kv4.2 is such that the ISA channels are maintained in the population at a peak conductance of approximately 950 pS/pF. Suppression of Kv4.2 transcripts in hippocampal pyramidal neurons using an RNA interference vector suppresses ISA current by 60% in 2 days, similar to the effect of expressing dominant-negative Kv4 channel constructs. Increasing the expression of Kv4.2 in these neurons increases the level of ISA to 170% of the normal set point without altering the biophysical properties. Our results establish a specific role for native Kv4.2 transcripts in forming and maintaining ISA current at characteristic levels in hippocampal pyramidal neurons.
A-current; dominant–negative; hippocampal pyramidal neurons; Kv4 potassium channels; RNA interference; viral vectors
In Poland, because breast cancer detection is delayed, patients usually undergo amputation or breast reconstruction. Surgeons believe that delayed reconstruction yields better aesthetic results compared with immediate reconstruction after mastectomy. Reconstruction is achieved by using either the patient’s own tissues or a tissue expander, which is later exchanged for a prosthesis/expandable implant. The two-stage reconstruction method (expander and prosthesis) is considered to be optimal because the implant position can be corrected. This study evaluated the aesthetic results of 54 patients who underwent the two-stage breast reconstruction method.
Presently, breast cancer detection is delayed in Poland and, thus, the only other option for patients is amputation and breast reconstruction (immediate or delayed). Reconstructive methods are based on using the patient’s own tissue (pedicled or free myocutaneous flaps) or implants (a tissue expander, which is later exchanged for a prosthesis or an expandable implant).
To evaluate the aesthetic results of a delayed two-stage breast reconstruction with the use of implants (expander and prosthesis) in patients who have previously undergone cancer-related mastectomy.
From 2006 to 2009, 54 patients (34 to 65 years of age) underwent reconstruction at least one year after their mastectomy and adjuvant chemotherapy; three women also received x-ray therapy. All women underwent a two-stage treatment with a tissue expander, which was later exchanged for a prosthesis.
Outcomes of the surgery (evaluated by the physician and the patient at least six months after all stages of reconstruction) were found to be very good in 42 patients and good in 12 patients. After amputation and x-ray therapy in two cases, a fistula developed, which necessitated implant removal.
After amputation, breast reconstruction with implants (expander and prosthesis) provides good aesthetic results. The method is mildly burdening to the patient and does not cause severe scarring. Symmetrization of the second breast is often recommended; however, the cost is not covered by the national health system. In principle, earlier x-ray therapy disqualifies the application of implants. Dividing reconstruction into two stages (expander and prosthesis) allows for possible correction of prosthesis placement.
Breast implant; Breast reconstruction; Delayed; Prosthesis; Tissue expander; Two stage
Neurons of the intercalated cell clusters (ITCs) represent an important relay site for information flow within amygdala nuclei. These neurons receive mainly glutamatergic inputs from the basolateral amygdala at their dendritic domains and provide feed-forward inhibition to the central nucleus. Voltage-gated potassium channels type-4.2 (Kv4.2) are main players in dendritic signal processing and integration providing a key component of the A currents. In this study, the subcellular localization and distribution of the Kv4.2 was studied in ITC neurons by means of light- and electron microscopy, and compared to other types of central principal neurons. Several ultrastructural immunolocalization techniques were applied including pre-embedding techniques and, most importantly, SDS-digested freeze-fracture replica labeling. We found Kv4.2 densely expressed in somato-dendritic domains of ITC neurons where they show a differential distribution pattern as revealed by nearest neighbor analysis. Comparing ITC neurons with hippocampal pyramidal and cerebellar granule cells, a cell type- and domain-dependent organization in Kv4.2 distribution was observed. Kv4.2 subunits were localized to extrasynaptic sites where they were found to influence intrasynaptic NMDA receptor subunit expression. In samples of Kv4.2 knockout mice, the frequency of NR1-positive synapses containing the NR2B subunit was significantly increased. This indicates a strong, yet indirect effect of Kv4.2 on the synaptic content of NMDA receptor subtypes, and a likely role in synaptic plasticity at ITC neurons.
Voltage-gated potassium channel; Immuno-electron microscopy; Freeze-fracture replica; Nearest neighbor analysis; Glutamatergic synapse
Clinical and experimental studies have demonstrated that seizures can cause molecular and cellular responses resulting in neuronal damage. At present, there are no valid tests for assessing organic damage to the brain associated with seizure. The aim of this study was to investigate cerebrospinal fluid (CSF) and plasma concentrations of Ubiquitin carboxy-terminal hydrolase L1 (UCH-L1), a sensitive indicator of acute injury to brain neurons, in patients with tonic–clonic or partial secondarily generalized seizures due to various etiologies.
CSF and plasma concentrations of UCH-L1 were assessed in 52 patients within 48 hours after epileptic seizure and in 19 controls using ELISA assays.
CSF obtained within 48 hours after seizure or status epilepticus (SE) presented significantly higher levels of UCH-L1 compared to controls (p = 0.008). Plasma UCH-L1 concentrations were negatively correlated with time to sample withdrawal. An analysis conducted using only the first 12 hours post-seizure revealed significant differences between concentrations of UCH-L1 in plasma and controls (p = 0.025). CSF and plasma concentrations were strongly correlated with age in patients with seizure, but not in control patients. Plasma UCH-L1 levels were also significantly higher in patients after recurrent seizures (n = 4) than in those after one or two seizures (p = 0.013 and p = 0.024, respectively).
Our results suggest that determining levels of neuronal proteins may provide valuable information on the assessment of brain damage following seizure. These data might allow clinicians to make more accurate therapeutic decisions, to identify patients at risk of progression and, ultimately, to provide new opportunities for monitoring therapy and targeted therapeutic interventions.
Biomarkers; UCH-L1; Epileptic seizures; Neuronal damage
Spectrins line the intracellular surface of plasmalemma and play a critical role in supporting cytoskeletal stability and flexibility. Spectrins can be proteolytically degraded by calpains and caspases, yielding breakdown products (SBDPs) of various molecular sizes, with SBDP120 being largely derived from caspase-3 cleavage. SBDPs are putative biomarkers for traumatic brain injury. The levels of SBDPs also elevate in the brain during aging and perhaps in Alzheimer’s disease (AD), although the cellular basis for this change is currently unclear. Here we examined age-related SBDP120 alteration in forebrain neurons in rats and in the triple transgenic model of AD (3×Tg-AD) relative to non-transgenic controls. SBDP120 immunoreactivity (IR) was found in cortical neuronal somata in aged rats, and was prominent in the proximal dendrites of the olfactory bulb mitral cells. Western blot and densitometric analyses in wild-type mice revealed an age-related elevation of intraneuronal SBDP120 in the forebrain which was more robust in their 3×Tg-AD counterparts. The intraneuronal SBDP120 occurrence was not spatiotemporally correlated with transgenic amyloid precursor protein (APP) expression, β-amyloid plaque development, or phosphorylated tau expression over various forebrain regions or lamina. No microscopically detectable in situ activated caspase-3 was found in the nuclei of SBDP120-containing neurons. The present study demonstrates the age-dependent intraneuronal presence of an αII-spectrin cleavage fragment in mammalian forebrain which is exacerbated in a transgenic model of AD. This novel neuronal alteration indicates that impairments in membrane protein metabolism, possibly due to neuronal calcium mishandling and/or enhancement of calcium sensitive proteolysis, occur during aging and in transgenic AD mice.
Compressive sensing can provide a promising framework for accelerating fMRI image acquisition by allowing reconstructions from a limited number of frequency-domain samples. Unfortunately, the majority of compressive sensing studies are based on stochastic sampling geometries that cannot guarantee fast acquisitions that are needed for fMRI. The purpose of this study is to provide a comprehensive optimization framework that can be used to determine the optimal 2D stochastic or deterministic sampling geometry, as well as to provide optimal reconstruction parameter values for guaranteeing image quality in the reconstructed images.
We investigate the use of frequency-space (k-space) sampling based on: (i) 2D deterministic geometries of dyadic phase encoding (DPE) and spiral low pass (SLP) geometries, and (ii) 2D stochastic geometries based on random phase encoding (RPE) and random samples on a PDF (RSP). Overall, we consider over 36 frequency-sampling geometries at different sampling rates. For each geometry, we compute optimal reconstructions of single BOLD fMRI ON & OFF images, as well as BOLD fMRI activity maps based on the difference between the ON and OFF images. We also provide an optimization framework for determining the optimal parameters and sampling geometry prior to scanning.
For each geometry, we show that reconstruction parameter optimization converged after just a few iterations. Parameter optimization led to significant image quality improvements. For activity detection, retaining only 20.3% of the samples using SLP gave a mean PSNR value of 57.58 dB. We also validated this result with the use of the Structural Similarity Index Matrix (SSIM) image quality metric. SSIM gave an excellent mean value of 0.9747 (max = 1). This indicates that excellent reconstruction results can be achieved. Median parameter values also gave excellent reconstruction results for the ON/OFF images using the SLP sampling geometry (mean SSIM > =0.93). Here, median parameter values were obtained using mean-SSIM optimization. This approach was also validated using leave-one-out.
We have found that compressive sensing parameter optimization can dramatically improve fMRI image reconstruction quality. Furthermore, 2D MRI scanning based on the SLP geometries consistently gave the best image reconstruction results. The implication of this result is that less complex sampling geometries will suffice over random sampling. We have also found that we can obtain stable parameter regions that can be used to achieve specific levels of image reconstruction quality when combined with specific k-space sampling geometries. Furthermore, median parameter values can be used to obtain excellent reconstruction results.
Compressive sensing; MRI; fMRI; Numerical optimization
Traumatic brain injury is a major health and socioeconomic problem that affects all societies. However, traditional approaches to the classification of clinical severity are the subject of debate and are being supplemented with structural and functional neuroimaging, as the need for biomarkers that reflect elements of the pathogenetic process is widely recognized. Basic science research and developments in the field of proteomics have greatly advanced our knowledge of the mechanisms involved in damage and have led to the discovery and rapid detection of new biomarkers that were not available previously. However, translating this research for patients' benefits remains a challenge. In this article, we summarize new developments, current knowledge and controversies, focusing on the potential role of these biomarkers as diagnostic, prognostic and monitoring tools of brain-injured patients.
biomarkers; clinical management; diagnostics; ELISA; prognostics; proteomics; traumatic brain injury
The German Multicenter EPO Stroke Trial, which investigated safety and efficacy of erythropoietin (EPO) treatment in ischemic stroke, was formally declared a negative study. Exploratory subgroup analysis, however, revealed that patients not receiving thrombolysis most likely benefited from EPO during clinical recovery, a result demonstrated in the findings of the Göttingen EPO Stroke Study. The present work investigated whether the positive signal on clinical outcome in this patient subgroup was mirrored by respective poststroke biomarker profiles. All patients of the German Multicenter EPO Stroke Trial nonqualifying for thrombolysis were included if they (a) were treated per protocol and (b) had at least two of the five follow-up blood samples for circulating damage markers drawn (n = 163). The glial markers S100B and glial fibrillary acid protein (GFAP) and the neuronal marker ubiquitin C-terminal hydrolase (UCH-L1) were measured by enzyme-linked immunosorbent assay in serum on d 1, 2, 3, 4 and 7 poststroke. All biomarkers increased poststroke. Overall, EPO-treated patients had significantly lower concentrations (area under the curve) over 7 d of observation, as reflected by the composite score of all three markers (Cronbach α = 0.811) and by UCH-L1. S100B and GFAP showed a similar tendency. To conclude, serum biomarker profiles, as an outcome measure of brain damage, corroborate an advantageous effect of EPO in ischemic stroke. In particular, reduction in the neuronal damage marker UCH-L1 may reflect neuroprotection by EPO.
A prominent characteristic of the inherited intellectual impairment disease fragile X syndrome (FXS) is neuronal hyperexcitability, resulting in a variety of symptoms, such as hyperactivity, increased sensitivity to sensory stimuli, and a high incidence of epileptic seizures. These symptoms account for a significant part of the disease pattern, but the underlying molecular mechanisms of neuronal hyperexcitability in FXS remain poorly understood. FXS is caused by loss of expression of fragile X mental retardation protein (FMRP), which regulates synaptic protein synthesis and is a key player to limit signaling pathways downstream of metabotropic glutamate receptors 1/5 (mGlu1/5). Recent findings suggest that FMRP might also directly regulate voltage-gated potassium channels. Here, we show that total and plasma membrane protein levels of Kv4.2, the major potassium channel regulating hippocampal neuronal excitability, are reduced in the brain of an FXS mouse model. Antagonizing mGlu1/5 activity with MPEP partially rescues reduced surface Kv4.2 levels in Fmr1 KO, suggesting that excess mGlu1/5 signal activity contributes to Kv4.2 dysregulation. As an additional mechanism, we show that FMRP is a positive regulator of Kv4.2 mRNA translation and protein expression and associates with Kv4.2 mRNA in vivo and in vitro. Our results suggest that absence of FMRP-mediated positive control of Kv4.2 mRNA translation, protein expression and plasma membrane levels might contribute to excess neuronal excitability in Fmr1 KO mice, and thus imply a potential mechanism underlying FXS-associated epilepsy.
Fragile X Syndrome; FMRP; Kv4.2; potassium channels; mRNA translation
To determine the prevalence of Internet use by patients considering rhinoplasty and to identify the impact of such information on their decisions.
Prospective analysis of the impact of information received via the Internet by patients considering rhinoplasty on their decision-making prior to surgery. Eighty six patients, scheduled for post-traumatic or aesthetic rhinoplasty, received a questionnaire, consisting of 19 questions, which sought to evaluate their perception of the importance of the Internet information to them and also collected relevant demographic and sociological data.
Respondents searched online for descriptions of medical procedures, information about how to contact other patients, pre- and post-operative pictures and making contact with a doctor. Patients considering aesthetic rhinoplasty received medical information from a third party or via the Internet. Individuals requiring post-traumatic surgical treatment were usually referred by family doctor.
Patients planning nasal aesthetic surgery form their opinions after consulting friends and searching the Internet, which can act as an important medical assistance.
Internet; Information resources; Rhinoplasty
Short-term synaptic plasticity is a defining feature of neuronal activity, but the underlying molecular mechanisms are poorly understood. Depression of synaptic activity might be due to limited vesicle availability, whereas facilitation is thought to result from elevated calcium levels. However, it is unclear whether the strength and direction (facilitation versus depression) of plasticity at a given synapse result from preexisting synaptic strength or whether they are regulated by separate mechanisms. Here we show, in rat hippocampal cell cultures, that increases in the calcium binding protein neuronal calcium sensor-1 (NCS-1) can switch paired-pulse depression to facilitation without altering basal synaptic transmission or initial neurotransmitter release probability. Facilitation persisted during high-frequency trains of stimulation, indicating that NCS-1 can recruit ‘dormant’ vesicles. Our results suggest that NCS-1 acts as a calcium sensor for short-term plasticity by facilitating neurotransmitter output independent of initial release. We conclude that separate mechanisms are responsible for determining basal synaptic strength and short-term plasticity.
Tumoural angiogenesis is essential for the growth and spread of breast cancer cells. Therefore the aim of this study was to assess the diagnostic performance of angiogenesis markers in tumours and there reflecting levels in serum of breast cancer patients. Angiogenin, Ang2, fibroblast growth factor basic, intercellular adhesion molecule (ICAM)-1, keratinocyte growth factor (KGF), platelet-derived growth factor-BB, and VEGF-A were measured using a FASTQuant angiogenic growth factor multiplex protein assay. We observed that breast cancer tumours exhibited high levels of PDGF-BB, bFGF and VEGF, and extremely high levels of TIMP-1 and Ang-2, whereas in serum we found significantly higher levels of Ang-2, PDGF-BB, bFGF, ICAM-1 and VEGF in patients with breast cancer compared to the benign breast diseases patients. Moreover, some of these angiogenesis markers evaluated in tumour and serum of breast cancer patients exhibited association with standard clinical parameters, ER status as well as MVD of tumours. Angiogenesis markers play important roles in tumour growth, invasion and metastasis. Our results suggest that analysis of angiogenesis markers in tumour and serum of breast cancer patients using multiplex protein assay can improve diagnosis and prognosis in this diseases.
Breast cancer; Angiogenesis; Cancer progression; Multiplex protein assay
We report the first successful insertion of an engineered, high-affinity α-bungarotoxin (Bgtx) binding site into a voltage-gated ion channel, Kv4.2, using a short, intra-protein embedded sequence (GGWRYYESSLEPYPDGG), derived from a previously described mimotope peptide, HAP. A major benefit to this approach is the ability to live-image the distribution and fate of functional channels on the plasma membrane surface. The Bgtx binding sequence was introduced into the putative extracellular loop between the S1 and S2 transmembrane domains of Kv4.2. Following co-expression with KChIP3 in tsA201 cells, S1–S2 HAP-tagged channels express at levels comparable to wild-type Kv4.2, and their activation and inactivation kinetics are minimally altered under most conditions. Binding assays, as well as live staining of surface-expressed Kv4.2 channels with fluorescent-Bgtx, readily demonstrate specific binding of Bgtx to HAP-tagged Kv4.2 expressed on the surface of tsA201 cells. Similar live-imaging results were obtained with HAP-tagged Kv4.2 transfected into hippocampal neurons in primary culture suggesting applicability for future in vivo studies. Furthermore, the activation kinetics of S1–S2-tagged Kv4.2 channels are minimally affected by the binding of Bgtx, suggesting a limited role if any for the S1–S2 loop in voltage sensing or gating associated conformational changes. Successful functional insertion of the HAP sequence into the S1–S2 linker of Kv4.2 suggests that other related channels may similarly be amenable to this tagging strategy.
Kv4.2; S1-S2 loop; alpha-bungarotoxin; trafficking; ion channel; mutagenesis
The aim of this study was to compare the aesthetic results of post traumatic rhinoplasty among Europeans with populations of healthy Caucasians described in earlier reports, and to collate correct parameters of nasal shape in healthy representatives of both sexes and various races for improvement in aesthetic results of surgery. 3-D scanning of the face was performed in 54 patients after posttraumatic septorhinoplasty. Analysis of 3D model was based on two indices of the nasal proportions and four angles of the region from 18 anthropological points. Parameters of the nasal shape in addition to gender were compared to average values of healthy Caucasian population, described before. Normal characteristics of the nose among individuals of three races from previous studies were also compared to one another. In females, mean height and width of the nose as well as length of both nostrils was smaller. Neither were there any significant differences in width of the nostrils and length of the nasal pyramid nor nasal prominence. Nasal proportions were similar in both sexes. Some of the nasal angles differed in addition to gender. Posttraumatic rhinoplasty resulted in correct shape of the nose similar to the healthy Caucasian population. The aesthetic nose differ among healthy individuals of the three races analysed. While preoperative planning is important, knowledge of the normal values of parameters characterising shape in both genders is equally important as the individual differences in relation to the whole face.
Posttraumatic; Distortion of nose; Rhinoplasty; Sexual dimorphism; Caucasian race; Preoperative planning; Aesthetic nose
Schizophrenia is the major psychiatry disorder, which the exact cause remains unknown. However, it is well known that dopamine-mediated neurotransmission imbalance is associated with this pathology and the main target of antipsychotics is the dopamine receptor D2. Recently, it was described alteration in levels of two dopamine signaling related proteins in schizophrenic prefrontal cortex (PFC): Neuronal Calcium Sensor-1 (NCS-1) and DARPP-32. NCS-1, which is upregulated in PFC of schizophrenics, inhibits D2 internalization. DARPP-32, which is decreased in PFC of schizophrenics, is a key downstream effector in transducing dopamine signaling. We previously demonstrated that antipsychotics do not change levels of both proteins in rat's brain. However, since NCS-1 and DARPP-32 levels are not altered in wild type rats, we treated wild type PC12 cells (PC12 WT) and PC12 cells stably overexpressing NCS-1 (PC12 Clone) with antipsychotics to investigate if NCS-1 upregulation modulates DARPP-32 expression in response to antipsychotics treatment.
We chronically treated both PC12 WT and PC12 Clone cells with typical (Haloperidol) or atypical (Clozapine and Risperidone) antipsychotics for 14 days. Using western blot technique we observed that there is no change in NCS-1 and DARPP-32 protein levels in both PC12 WT and PC12 Clone cells after typical and atypical antipsychotic treatments.
Because we observed no alteration in NCS-1 and DARPP-32 levels in both PC12 WT and Clone cells treated with typical or atypical antipsychotics, we suggest that the alteration in levels of both proteins in schizophrenic's PFC is related to psychopathology but not with antipsychotic treatment.
Changes in neuronal excitability, synaptic efficacy and generally in cell signaling often result from insertion of key molecules into plasma membrane (PM). Many of the techniques used for monitoring PM insertion lack either spatial or temporal resolution.
We improved the imaging method based on time-lapse total internal reflection fluorescence (TIRF) microscopy and pHluorin tagging by supplementing it with a repetitive extracellular acidification protocol. We illustrate the applicability of this method by showing that brief activation of NMDA receptors ("chemical LTP") in cultured hippocampal neurons induced a persistent PM insertion of glutamate receptors containing the pHluorin-tagged GluR-A(flip) subunits.
The repetitive acidification technique provides a more accurate way of monitoring the PM-inserted fraction of fluorescently tagged molecules and offers a good temporal and spatial resolution.
Transgenic mice with mosaic, Golgi-staining-like expression of enhanced green fluorescent protein (EGFP) have been very useful in studying the dynamics of neuronal structure and function. In order to further investigate the molecular events regulating structural plasticity, it would be useful to express multiple proteins in the same sparse neurons, allowing co-expression of functional proteins or co-labeling of subcellular compartments with other fluorescent proteins. However, it has been difficult to obtain reproducible expression in the same subset of neurons for direct comparison of neurons expressing different functional proteins.
Here we describe a Cre-transgenic line that allows reproducible expression of transgenic proteins of choice in a small number of neurons of the adult cortex, hippocampus, striatum, olfactory bulb, subiculum, hypothalamus, superior colliculus and amygdala. We show that using these Cre-transgenic mice, multiple Cre-dependent transgenes can be expressed together in the same isolated neurons. We also describe a Cre-dependent transgenic line expressing a membrane associated EGFP (EGFP-F). Crossed with the Cre-transgenic line, EGFP-F expression starts in the adolescent forebrain, is present in dendrites, dendritic protrusions, axons and boutons and is strong enough for acute or chronic in vivo imaging.
This triple transgenic approach will aid the morphological and functional characterization of neurons in various Cre-dependent transgenic mice.
Neurons in the avian cochlear nucleus, nucleus magnocellularis (NM), are highly sensitive to manipulations of afferent input, and removal of afferent activity through cochlear ablation results in the death of approximately 20-40% of ipsilateral NM neurons. The intracellular cascades that determine whether an individual NM neuron will die or survive are not fully understood. One early event observed in NM following deafferentation is a rapid rise in intracellular calcium concentration. In most cellular systems, the activity of calcium-binding proteins is believed to accommodate calcium influx. The calcium-binding protein, neuronal calcium sensor-1 (NCS-1), is an intracellular neuronal calcium sensor belonging to the EF-hand superfamily. NCS-1 has been implicated in calcium-dependent regulation of signaling cascades. To evaluate NCS-1 action in NM neurons, the localization of NCS-1 protein was examined. Double-label immunofluorescence experiments revealed that NCS-1 expression is evident in both the presynaptic nerve terminal and postsynaptic NM neuron. The postsynaptic expression of NCS-1 typically appears to be closely associated with the cell membrane. This close proximity of NCS-1 to the postsynaptic membrane could allow NCS-1 to function as a modulator of postsynaptic signaling events. Following deafferentation, NM neurons were more likely to show diffuse cytoplasmic NCS-1 labeling. This increase in the number of cells showing diffuse cytoplasmic labeling was observed 12 and 24 h following cochlea ablation, but was not observed 4 days following surgery. This activity-dependent regulation of NCS-1 subcellular localization suggests it may be associated with, or influenced by, processes important for the survival of NM neurons.
deafferentation; nucleus magnocellularis; calcium binding proteins; frequenin; auditory system; cell death
Neuronal development requires highly coordinated regulation of the cytoskeleton within the developing axon. This dynamic regulation manifests itself in axonal branching, turning, and pathfinding, presynaptic differentiation, and growth cone collapse and extension. Semaphorin 3A (Sema3A), a secreted guidance cue that primarily acts to repel axons from inappropriate targets, induces cytoskeletal rearrangements that results in growth cone collapse 1. These effects require intra-axonal mRNA translation. Here we show that transcripts for RhoA, a small GTPase that regulates the actin cytoskeleton, are localized to developing axons and growth cones, and this localization is mediated by an axonal targeting element located in the RhoA 3’UTR. Sema3A induces intra-axonal translation of RhoA mRNA and this local translation of RhoA is necessary and sufficient for Sema3A-mediated growth cone collapse. These studies indicate that local RhoA translation regulates the neuronal cytoskeleton and identify a novel mechanism for the regulation of RhoA signaling.
We demonstrate that recycling through the endocytic recycling compartment (ERC) is an essential step in FcεRI-induced activation of extracellular signal-regulated kinase (ERK)1/2. We show that ERK1/2 acquires perinuclear localization and colocalizes with Rab 11 and internalized transferrin in FcεRI-activated cells. Moreover, a close correlation exists between the amount of ERC-localized ERK1/2 and the amount of phospho-ERK1/2 that resides in the nucleus. We further show that by activating phosphatidylinositol 4-kinase β (PI4Kβ) and increasing the cellular level of phosphatidylinositol(4) phosphate, neuronal calcium sensor-1 (NCS-1), a calmodulin-related protein, stimulates recycling and thereby enhances FcεRI-triggered activation and nuclear translocation of ERK1/2. Conversely, NCS-1 short hairpin RNA, a kinase dead (KD) mutant of PI4Kβ (KD-PI4Kβ), the pleckstrin homology (PH) domain of FAPP1 as well as RNA interference of synaptotagmin IX or monensin, which inhibit export from the ERC, abrogate FcεRI-induced activation of ERK1/2. Consistently, NCS-1 also enhances, whereas both KD-PI4Kβ and FAPP1-PH domain inhibit, FcεRI-induced release of arachidonic acid/metabolites, a downstream target of ERK1/2 in mast cells. Together, our results demonstrate a novel role for NCS-1 and PI4Kβ in regulating ERK1/2 signaling and inflammatory reactions in mast cells. Our results further identify the ERC as a crucial determinant in controlling ERK1/2 signaling.