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We developed a MEMS based sensor, Nanopin, for mechanical characterization of individual adherent cells. Nanopin consists of sensing tip that forms a contact with a cell, a displacement sensor, and an actuator. The feasibility of sensing was evaluated using various concentrations of agarose gel, and then the stiffness measurements of human carcinoma cells were conducted on different surfaces. After the measurements of cells, we confirmed that cells grow normally in an incubator.

Microtubule-associated protein (MAP) tau is a well-established hallmark of a large group of age related neurodegenerative diseases collectively called tauopathies. Under pathological conditions the equilibrium of tau binding to the MTs is perturbed, either by misregulation in the expression levels of specific tau isoforms or by MAPT gene mutations. Preclinical detection of such misregulated tau proteins in cerebrospinal fluid (CSF) is desirable for differential diagnosis and effective prognosis of neurodegeneration. Conventional tau protein detection methods utilize tau isoform-specific antibodies. Such immuno-based protocols, including enzyme-linked immunosorbent assay (ELISA) and Western blots have appropriate sensitivity and specificity, but often show high variability and are time consuming. Here, we established a non-immuno tau protein detection method utilizing microtubule (MT)-kinesin “gliding”assay. All the six tau isoforms expressed in the human brain (0N3R, 1N3R, 2N3R, 0N4R, 1N4R and 2N4R) and five MAPT gene mutants (V248L, G272V, P301L, V337M and R406W) were studied. The landing rate, binding density and gliding velocity of MTs with respect to each tau type were determined and are proposed as tau detection parameters. The detection parameters depicted the type of tau bound to the MTs. Furthermore, MT landing rate and density were found to be superior to gliding velocity in differentiating tau isoforms and mutants. The 3R vs. 4R isoforms, their admixtures, wild vs. mutant 2N4R and specific mutants were differentiated. Our data show that MT-kinesin gliding assay provides a convenient, lab-on-a-chip (LOC) compatible and antibody-free protocol for tau protein analysis.

Micro and nano systems (MNS) and Nano scaled devices, that are capable of handling fluids and to interact with DNA and proteins enable bio analysis at the “ultimate” molecular level and are prone to be coupled to IC Technology. This paper includes recent developments in this area, aimed at illustrating the diversity and potential of the MNS approach: (1) micromachined tweezers with sharp tips successfully captured a bundle of DNA molecules, allowing real-time observation of DNA degradation dynamics under therapeutic irradiations; (2) a simple method to fabricate FET silicon nanowires using only standard micro-electromechanical system (MEMS) processes is able to perform molecular level medical diagnosis; (3) a bio motor system, composed by microtubules and kinesin, reconstructed on a chip, can distinguish normal and abnormal tau-proteins related to Alzheimer’s disease; and (4) an advanced concept of molecular programing in which real molecular interactions emulate partial different equations and are able to solve complex systems such as a predator–prey ecosystem.

Previously, we reported the application of micromachined silicon nanotweezers (SNT) integrated with a comb-drive actuator and capacitive sensors for capturing and mechanical characterization of DNA bundles. Here, we demonstrate direct DNA amplification on such a MEMS structure with subsequent electrical and mechanical characterization of a single stranded DNA (ssDNA) bundle generated between the tips of SNT via isothermal rolling circle amplification (RCA) and dielectrophoresis (DEP). An in situ generated ssDNA bundle was visualized and evaluated via electrical conductivity (I-V) and mechanical frequency response measurements. Colloidal gold nanoparticles significantly enhanced (P < 0.01) the electrical properties of thin ssDNA bundles. The proposed technology allows direct in situ synthesis of DNA with a predefined sequence on the tips of a MEMS sensor device, such as SNT, followed by direct DNA electrical and mechanical characterization. In addition, our data provides a "proof-of-principle" for the feasibility of the on-chip label free DNA detection device that can be used for a variety of biomedical applications focused on sequence specific DNA detection.

This study combines the high-throughput capabilities of microfluidics with the sensitive measurements of microelectromechanical systems (MEMS) technology to perform biophysical characterization of circulating cells for diagnostic purposes. The proposed device includes a built-in microchannel that is probed by two opposing tips performing compression and sensing separately. Mechanical displacement of the compressing tip (up to a maximum of 14 &micro, m) and the sensing tip (with a quality factor of 8.9) are provided by two separate comb-drive actuators, and sensing is performed with a capacitive displacement sensor. The device is designed and developed for simultaneous electrical and mechanical measurements. As the device is capable of exchanging the liquid inside the channel, different solutions were tested consecutively. The performance of the device was evaluated by introducing varying concentrations of glucose (from 0.55 mM (0.1%) to 55.5 mM (10%)) and NaCl (from 0.1 mM to 10 mM) solutions in the microchannel and by monitoring changes in the mechanical and electrical properties. Moreover, we demonstrated biological sample handling by capturing single cancer cells. These results show three important capabilities of the proposed device: mechanical measurements, electrical measurements, and biological sample handling. Combined in one device, these features allow for high-throughput multi-parameter characterization of single cells.

Our earlier work showed that knockout of hematopoietic prostaglandin D synthase (HPGDS, an enzyme that produces prostaglandin D2) caused more adenomas in Apc(Min/+) mice. Conversely, highly expressed transgenic HPGDS allowed fewer tumors. Prostaglandin D2 (PGD2) binds to the prostaglandin D2 receptor known as PTGDR (or DP1). PGD2 metabolites bind to peroxisome proliferator-activated receptor γ (PPARG). We hypothesized that Ptgdr or Pparg knockouts may raise numbers of tumors, if these receptors take part in tumor suppression by PGD2. To assess, we produced Apc(Min/+) mice with and without Ptgdr knockouts (147 mice). In separate experiments, we produced Apc(Min/+) mice expressing transgenic lipocalin-type prostaglandin D synthase (PTGDS), with and without heterozygous Pparg knockouts (104 mice). Homozygous Ptgdr knockouts raised total numbers of tumors by 30-40% at 6 and 14 weeks. Colon tumors were not affected. Heterozygous Pparg knockouts alone did not affect tumor numbers in Apc(Min/+) mice. As mentioned above, our Pparg knockout assessment also included mice with highly expressed PTGDS transgenes. Apc(Min/+) mice with transgenic PTGDS had fewer large adenomas (63% of control) and lower levels of v-myc avian myelocytomatosis viral oncogene homolog (MYC) mRNA in the colon. Heterozygous Pparg knockouts appeared to blunt the tumor-suppressing effect of transgenic PTGDS. However, tumor suppression by PGD2 was more clearly mediated by receptor PTGDR in our experiments. The suppression mechanism did not appear to involve changes in microvessel density or slower proliferation of tumor cells. The data support a role for PGD2 signals acting through PTGDR in suppression of intestinal tumors.

We present a mesodissection platform that retains the advantages of laser-based dissection instrumentation with the speed and ease of manual dissection. Tissue dissection in clinical laboratories is often performed by manually scraping a physician-selected region from standard glass slide mounts. In this manner, costs associated with dissection remain low, but spatial resolution is compromised. In contrast, laser microdissection methods maintain spatial resolution that matches the requirements for analysis of important tissue heterogeneity but remains costly and labor intensive. We demonstrate a microfluidic tool for rapid extraction of histological regions of interest from formalin-fixed paraffin-embedded tissue, which uses a simple and automated method that is compatible with most downstream enzymatic reactions, including protocols used for next-generation DNA sequencing.

To progress in basic science and drug development, convenient methodology for detecting specific biological molecules and their interaction in living organism is in high demand. After more than 20 years of increasing research efforts, micro and nanotechnologies are now mature to propose a new class of miniature devices and principles enabling compartmentalized bioassays. Among them, this review proposes various examples that include array of electro-active microwells for highly parallel single cell analysis, cost-effective nanofluidic for DNA separation, parallel enzymatic reaction in 100 pL droplet and high-throughput platform for membrane proteins assays. The micro devices are presented with relevant experiments to foresee their future contribution to translational research and drug discovery.

Tau protein is a well-established biomarker for a group of neurodegenerative diseases collectively called tauopathies. So far, clinically relevant detection of tau species in cerebrospinal fluid (CSF) cannot be achieved without immunological methods. Recently, it was shown that different tau isoforms including the ones carrying various types of mutations affect microtubule (MT)–kinesin binding and velocity in an isoform specific manner. Here, based on these observations, we developed a microfluidic device to analyze tau mutations, isoforms and their ratios. The assay device consists of three regions: a MT reservoir which captures MTs from a solution to a kinesin-coated surface, a microchannel which guides gliding MTs, and an arrowhead-shaped collector which concentrates MTs. Tau-bound fluorescently labeled MTs (tau-MTs) were assayed, and the increase in fluorescence intensity (FI) corresponding to the total number of MTs accumulated was measured at the collector. We show that our device is capable of differentiating 3R and 4R tau isoform ratios and effects of point mutations within 5 minutes. Furthermore, radially oriented collector regions enable simultaneous FI measurements for six independent assays. Performing parallel assays in the proposed device with minimal image processing provides a cost-efficient, easy-to-use and fast tau detection platform.

There are currently no predictive methods to identify patients who suffered an initial brain injury and are at high risk of developing chronic epilepsy. Consequently, treatments aimed at epilepsy prevention that would target the underlying epileptogenic process are neither available nor being developed. After a brain injury or any other initial precipitating event (IPE) to the development of epilepsy, pathological changes may occur in forms of inflammation, damage in the blood brain barrier, neuron loss, gliosis, axon sprouting, etc., in multiple brain areas. Recent studies provide connections between various kinds of brain pathology and alterations in the peripheral blood transcriptome. In this review we discuss the possibility of using peripheral blood transcriptome biomarkers for the detection of epileptogenesis and consequently, subjects at high risk of developing epilepsy.

Single cell analysis (SCA) has become an essential part of basic and clinical research. A prerequisite to SCA studies is the isolation of individual cells from cell cultures. There are commercially available technologies including laser based systems and cell sorting instruments. However, they are typically complex and expensive making integration within standard lab/clinical sample processing workflows difficult. We have developed a universal platform for rapid acquisition of single cells from cultures and deposition into the single wells for further molecular analysis or clonal expansion. The instrument may also be used for tissue microdissection and measurements of individual cell adhesion. The main working principle is based on the capillary based vacuum pulse assisted technology (CTAS; UP8797644). Developed instrument collects individual cells from any adherent cultures grown in standard cell culture dishes in as small as 15 nl volume. It may be used with a wide range of inverted microscopes, so that the cells of interest can be identified based on morphology, location or fluorescent label. Automatic extraction and deposition algorithms were developed for acquisition of targeted cells and tissue regions of interest. Here, individual cells were collected from human neuroblastoma SH-SY5Y, CHO, and 3T3 cell lines. Collected cells were dispensed immediately into individual wells for clonal expansion. Clonal expansion and trypan blue assay revealed minimal effect on cellular viability (up to 99% when compared to controls). The benefits of the proposed technology include cost-efficiency, simple operation, complete workflow, and compatibility with a wide range of inverted microscopes and standard culture dishes.

Microelectromechanical systems (MEMS) have become an invaluable technology to advance the development of point-of-care (POC) devices for diagnostics and sample analyses. MEMS can transform sophisticated methods into compact and cost-effective microdevices that offer numerous advantages at many levels. Such devices include microchannels, microsensors, etc., that have been applied to various miniaturized POC products. Here we discuss some of the recent advances made in the use of MEMS devices for POC applications.

We report the successful demonstration of an on-chip tau detection system based on the difference in landing rate and binding density of microtubules (MTs) on a kinesin surface. Tau detection device comprises of a MT reservoir, channel and collector region with an overhang structure. We assayed MTs decorated with three tau types in the kinesin coated device. Since the increase in fluorescence intensity (FI) at the collector regions reflected the type of tau decorated on MTs, thus by measuring the FI we were able to distinguish wild 3R, 4R and P301L mutant tau.

Tau, a microtubule associated protein, aggregates into intracellular paired helical filaments (PHFs) by an unknown mechanism in Alzheimer's disease (AD) and other tauopathies. A contributing factor may be a failure to metabolize free cytosolic tau within the neuron. The buildup of tau may then drive the aggregation process through mass action. Therefore, proteases that normally degrade tau are of great interest. A recent genetic screen identified puromycin-sensitive aminopeptidase (PSA) as a potent modifier of tau-induced pathology and suggested PSA as a possible tau-degrading enzyme. Here we have extended these observations using human recombinant PSA purified from Escherichia coli. The enzymatic activity and characteristics of the purified PSA were verified using chromogenic substrates, metal ions, and several specific and nonspecific protease inhibitors, including puromycin. PSA was shown to digest recombinant human full-length tau in vitro, and this activity was hindered by puromycin. The mechanism of amino terminal degradation of tau was confirmed using a novel N-terminal cleavage-specific tau antibody (Tau-C6g, specific for cleavage between residues 13-14) and a C-terminal cleavage-specific tau antibody (Tau-C3). Additionally, PSA was able to digest soluble tau purified from normal human brain to a greater extent than either soluble or PHF tau purified from AD brain, indicating that post-translational modifications and/or polymerization of tau may affect its digestion by PSA. These results are consistent with observations that PSA modulates tau levels in vivo and suggest that this enzyme may be involved in tau degradation in human brain.

Summary Neurofibrillary tangles (NFT) containing tau are a hallmark of neurodegenerative diseases, including Alzheimer's disease (AD). NFT burden correlates with cognitive decline and neurodegeneration in AD. However, little is known about mechanisms that protect against tau-induced neurodegeneration. We used a cross species functional genomic approach to analyze gene expression in multiple brain regions in mouse, in parallel with validation in Drosophila , to identify tau modifiers, including the highly conserved protein puromycin-sensitive aminopeptidase (PSA/Npepps). PSA protected against tau-induced neurodegeneration in vivo, whereas PSA loss of function exacerbated neurodegeneration. We further show that human PSA directly proteolyzes tau in vitro. These data highlight the utility of using both evolutionarily distant species for genetic screening and functional assessment to identify modifiers of neurodegeneration. Further investigation is warranted in defining the role of PSA and other genes identified here as potential therapeutic targets in tauopathy.

The normal processes of learning and memory as well as the pathological progress of various neurological diseases may result in changes in gene expression in small, local populations of neurons in any given brain area, leading to the occurrence of specific patterns of electrical activity without easily detectable changes in the morphology of this brain area. One way of identifying these changes might be the comparison of gene expression of areas which generate and areas which do not generate specific patterns of electrical activity. A method for microbiopsy of limited (0.5-1.0 mm3) tissue samples from electrophysiologically identified areas of neurons generating epileptiform activity in the rat brain is described. Here we demonstrate that total RNA isolated from individual microbiopsy samples might be successfully used for microarray based gene expression analysis of any discretely localized neuronal group which can be identified electrophysiologically, including neurons in cortical columns, cell assemblies or other functional units.

Hoxd10 is expressed in the posterior spinal cord and hindlimbs of the mouse. Hoxd10, along with other Hox transcription factors, is thought to regulate the activity of genes involved in nervous system patterning and motor neuron development, but little is known about the downstream targets regulated by this gene. cDNA microarrays were used to investigate the transcriptional network regulated by Hoxd10 in homozygous knockout animals. Sixty-nine genes were identified with altered expression levels in mutant spinal cords. Among these were genes involved in such diverse cellular events as cellular communication, cell cycle control, development and differentiation, and neuronal survival. The expression of some of these genes was investigated using reverse transcriptase-polymerase chain reaction (RT-PCR) and in situ hybridization. Nine genes showed changes in expression of the same sign and similar magnitude using RT-PCR in Hoxd10 single mutant animals, with additional changes in expression seen in Hoxa10/Hoxd10 double mutant animals. In situ hybridization studies also demonstrated changes in expression consistent with microarray results. Analysis of putative promoter regions for Hox protein binding sites suggested that some genes may be direct Hoxd10 targets, whereas others likely are regulated through intermediate steps. Using cDNA microarrays to study a single gene knockout during critical developmental stages has identified a large number of genes regulated by Hoxd10, many of which would not have been approached as candidates for Hox gene regulation based on function or expression. © 2004 Wiley-Liss, Inc.

The genetic programs underlying neural stem cell (NSC) proliferation and pluripotentiality have only been partially elucidated. We compared the gene expression profile of proliferating neural stem cell cultures (NS) with cultures differentiated for 24 h (DC) to identify functionally coordinated alterations in gene expression associated with neural progenitor proliferation. The majority of differentially expressed genes (65%) were upregulated in NS relative to DC. Microarray analysis of this in vitro system was followed by high throughput screening in situ hybridization to identify genes enriched in the germinal neuroepithelium, so as to distinguish those expressed in neural progenitors from those expressed in more differentiated cells in vivo. NS cultures were characterized by the coordinate upregulation of genes involved in cell cycle progression, DNA synthesis, and metabolism, not simply related to general features of cell proliferation, since many of the genes identified were highly enriched in the CNS ventricular zones and not widely expressed in other proliferating tissues. Components of specific metabolic and signal transduction pathways, and several transcription factors, including Sox3, FoxM1, and PTTG1, were also enriched in neural progenitor cultures. We propose a putative network of gene expression linking cell cycle control to cell fate pathways, providing a framework for further investigations of neural stem cell proliferation and differentiation.

Tau protein is a biomarker for neurodegeneration. The microtubule (MT)-tau binding affinity varies according to the type of tau isoform and their degree of phosphorylation. We have utilized the difference in binding affinity of tau protein to MT to be evaluated by kinesin motor protein based MT gliding system, in order to detect and differentiate tau isoforms and their mutants. Evaluation parameters are landing rate and density of MTs on a kinesin-coated surface, and their strong correlation enables us to measure only landing rate to distinguish 2N3R and 2N4R (tau isoforms) and mutants. Secondly; we designed and fabricated a microstructure to detect tau-attached MTs, which is composed of a reservoir, parallel channels and collectors. Increase of fluorescent intensity by accumulation of MTs over time was successfully detected at the collector areas. Sensitive and rapid MT-kinesin based detection of tau isoforms (3R/4R) and mutated tau proteins on a microchip format will aid in differential diagnosis and early detection of neurodegenerative condition such as Alzheimer disease (AD).

We describe a novel type of complex genetic rearrangement in the iduronate-2-sulfatase (IDS) gene of a severely affected MPSII patient. Southern blot analysis indicated an intragenic deletion of exons 5 and 6. The deletion spans 5,581 bp. Sequencing of the deletion junctions revealed a complex rearrangement involving duplications and inversions. A remaining 20 bp fragment (c) from the intron 6 sequence and two duplicated IDS gene fragments of 314 bp (a) from intron 6/exon 7 boundary and 23 bp (b) from exon 7 were found between the deletion breakpoints. Fragments a and c were placed in an inverted orientation. We suggest that the described rearrangement is a result of a nonhomologous recombination event at sites with little homology. The proposed model explaining this recombinational event involves the formation of “tetra-loop” single-stranded DNA structure during replication. The complexity of the described rearrangement and the lack of large homologous sequences at the mutational breakpoints suggest that complex molecular intermediates are formed during illegitimate recombination. Hum Mutat 14:471–476, 1999. © 1999 Wiley-Liss, Inc.

We present a mutational analysis of the iduronate-2-sulfatase (IDS) gene of 36 Russian patients with Hunter syndrome. Among 29 mutant alleles, there were 19 missense mutations, 1 nonsense mutation, 6 mutations affecting splice sites, and 3 major structural alterations resulting in deletions. Of the 25 different mutations, 15 are novel and unique. Most of the missense mutations result in intermediate or severe phenotypes.

The interplay of genetics and the environment in the etiology of Alzheimer’s disease (AD) is not well understood. Now, Lazarov and coworkers (Lazarov et al., 2005 [this issue of Cell]) show that a simple paradigm of environmental enrichment alleviates amyloid burden and alters disease-associated gene expression changes in a double transgenic mouse model of AD.

The concept of a reconstructed microtubule kinesin-based transport system was originally introduced for studies of underlying biophysical mechanisms of intracellular transport and its potential applications in bioengineering at micro- and nanoscale levels. However, several technically challenging shortcomings prohibit its use in practical applications. One of them is the propensity of microtubules to bind various protein molecules creating “roadblocks” for kinesin molecule movement and subsequently preventing efficient delivery of the molecular cargo. The interruption in kinesin movement strictly depends on the specific type of “roadblock”, i.e. the microtubule associated protein (MAP). Therefore, we propose to use the “roadblock” effect as a molecular sensor that may be used for functional characterization of particular MAPs with respect to their role in MT-based transport and associated pathologies, such as neurodegeneration. Here, we applied a kinesin-based assay using a suspended MT design (sMT assay) to functionally characterize known MAP tau protein isoforms and common mutations found in familial frontotemporal dementia (FTD). The proposed sMT assay is compatible with an on-chip format and may be used for the routine characterization of MT associated molecules applicable to diagnostics and translational research.

A microfluidics device to detect microtubule-binding proteins was proposed using microtubule-kinesin based system. To provide “proof of principle”, attachment of microtubule associated tau protein was detected via the change in kinesin velocity. The proposed device is capable of multiplex parallel assay of several experimental samples by using the same kinesin-coated beads moving in the same channel with immobilized microtubules. The results showed that the device successfully detected the hindered kinesin motion due to tau attachment on microtubule (MT) surface.

Microtubule (MT) based intraneuronal transport deficiency is directly linked to neurodegeneration. Hence, the development of a reliable and sensitive in vitro approach permitting efficient analysis of MT-based transport is essential for our understanding of the underlying molecular mechanisms that may lead to novel therapeutic approaches for treating neurodegenerative diseases. Here, based on previously developed reconstructed MT–kinesin assay, we propose its “suspended” modification that shows higher sensitivity and lower experimental variability.

Here we describe the application of a recently developed high-resolution microcantilever biosensor resonating at the air–liquid interface for the continuous detection of antigen–antibody and enzyme–substrate interactions. The cantilever at the air–liquid interface demonstrated 50% higher quality factor and a 5.7-fold increase in signal-to-noise-ratio (SNR) compared with one immersed in the purified water. First, a label-free detection of a low molecular weight protein (insulin, 5.8 kDa) in physiological concentration was demonstrated. The liquid facing side of the cantilever was functionalized by coating its surface with insulin antibodies, while the opposite side was exposed to air. The meniscus membrane at the micro-slit around the cantilever sustained the liquid in the microchannel. After optimizing the process of surface functionalization, the resonance frequency shift was successfully measured for insulin solutions of 0.4, 2.0, and 6.3 ng ml^(−1). To demonstrate additional application of the device for monitoring enzymatic protein degradation, the liquid facing microcantilever surface was coated with human recombinant SOD1 (superoxide dismutase 1) and exposed to various concentrations of proteinase K solution, and the kinetics of the SOD1 digestion was continuously monitored. The results showed that it is a suitable tool for sensitive protein detection and analysis.

Author(s): Ren, Guijie; Ma, Zonghcai; Hui, Maria; Kudo, Lili C; Hui, Koon-Sea; Karsten, Stanislav L

A molecular detection system was realized using the change in the characteristics of the kinesin-coated bead motion along protein-attached microtubules. A hybrid bio/microfluidics device with a microtubule immobilized main channel and protein-injected sub-channels allowed us to compare different protein attachments and several control experiments with the same experimental conditions. As kinesin/microtubule system is an essential component of neurons, the proposed device is a promising tool for diagnostics of several neurodegenerative diseases.

Accumulation of neurotoxic hyperphosphorylated TAU protein is a major pathological hallmark of Alzheimer disease and other neurodegenerative dementias collectively called tauopathies. Puromycin-sensitive aminopeptidase (PSA/NPEPPS) is a novel modifier of TAU-induced neurodegeneration with neuroprotective effects via direct proteolysis of TAU protein. Here, to examine the effects of PSA/NPEPPS overexpression in vivo in the mammalian system, we generated and crossed BAC-PSA/NPEPPS transgenic mice with the TAU P301L mouse model of neurodegeneration. PSA/NPEPPS activity in the brain and peripheral tissues of human PSA/NPEPPS (hPSA) mice was elevated by ∼2–3-fold with no noticeable deleterious physiological effects. Double-transgenic animals for hPSA and TAU P301L transgenes demonstrated a distinct trend for delayed paralysis and showed significantly improved motor neuron counts, no gliosis and markedly reduced levels of total and hyperphosphorylated TAU in the spinal cord, brain stem, cortex, hippocampus and cerebellum of adult and aged animals when compared with TAU P301L mice. Furthermore, endogenous TAU protein abundance in human neuroblastoma SH-SY5Y cells was significantly reduced or augmented by overexpression or knockdown of PSA/NPEPPS, respectively. This study demonstrated that without showing neurotoxic effects, elevation of PSA/NPEPPS activity in vivo effectively blocks accumulation of soluble hyperphosphorylated TAU protein and slows down the disease progression in the mammalian system. Our data suggest that increasing PSA/NPEPPS activity may be a feasible therapeutic approach to eliminate accumulation of unwanted toxic substrates such as TAU.

Neither the molecular basis of the pathologic tendency of neuronal circuits to generate spontaneous seizures (epileptogenicity) nor anti-epileptogenic mechanisms that maintain a seizure-free state are well understood. Here, we performed transcriptomic analysis in the intrahippocampal kainate model of temporal lobe epilepsy in rats using both Agilent and Codelink microarray platforms to characterize the epileptic processes. The experimental design allowed subtraction of the confounding effects of the lesion, identification of expression changes associated with epileptogenicity, and genes upregulated by seizures with potential homeostatic anti-epileptogenic effects. Using differential expression analysis, we identified several hundred expression changes in chronic epilepsy, including candidate genes associated with epileptogenicity such as Bdnf and Kcnj13. To analyze these data from a systems perspective, we applied weighted gene co-expression network analysis (WGCNA) to identify groups of co-expressed genes (modules) and their central (hub) genes. One such module contained genes upregulated in the epileptogenic region, including multiple epileptogenicity candidate genes, and was found to be involved the protection of glial cells against oxidative stress, implicating glial oxidative stress in epileptogenicity. Another distinct module corresponded to the effects of chronic seizures and represented changes in neuronal synaptic vesicle trafficking. We found that the network structure and connectivity of one hub gene, Sv2a, showed significant changes between normal and epileptogenic tissue, becoming more highly connected in epileptic brain. Since Sv2a is a target of the antiepileptic levetiracetam, this module may be important in controlling seizure activity. Bioinformatic analysis of this module also revealed a potential mechanism for the observed transcriptional changes via generation of longer alternatively polyadenlyated transcripts through the upregulation of the RNA binding protein HuD. In summary, combining conventional statistical methods and network analysis allowed us to interpret the differentially regulated genes from a systems perspective, yielding new insight into several biological pathways underlying homeostatic anti-epileptogenic effects and epileptogenicity.

Results: We found that pseudophosphorylated tau aggregates in cells when Thr 212 is mutated to Glu (Ps-tau), suggesting that phosphorylation at this site facilitates tau self-assembly. The expression of tau pseudophosphorylated at Thr212, Thr231, and Ser262 disrupts the microtubules when co-transfected with fluorescent tubulin. EB-1 is a protein that binds to the growing end of the microtubules. Cells stably transfected with fluorescent EB-1 are used to visualize the dynamics of these filaments in the live cells. These cells were transfected with an inducible system to express tau upon withdraw of doxycycline (TET-off). The effect of Ps-tau on these cells was also studied. Conclusions: These findings suggest that tau phosphorylation at Thr 212 facilitates tau self-aggregation, and that the combination of phosphorylation at Thr212, 231 and Ser262 in the same tau molecule can trigger toxic reaction.

Advances in genomics and proteomics permit rapid identification of disease-relevant genes and proteins. Challenges include biological differences between animal models and human diseases, high discordance between DNA and protein expression data and a lack of experimental models to study human complex diseases. To overcome some of these limitations, we developed an integrative approach using animal models, postmortem human material and a combination of high-throughput microarray methods to identify novel molecular markers of amyotrophic lateral sclerosis (ALS). We used laser capture microdissection coupled with microarrays to identify early transcriptome changes occurring in spinal cord motor neurons or surrounding glial cells. Two models of familial motor neuron disease, SOD1(G93A) and TAU(P301L), transgenic mice were used at the presymptomatic stage. Identified gene expression changes were predominantly model-specific. However, several genes were regulated in both models. The relevance of identified genes as clinical biomarkers was tested in the peripheral blood transcriptome of presymptomatic SOD1(G93A) animals using custom-designed ALS microarray. To confirm the relevance of identified genes in human sporadic ALS (SALS), selected corresponding protein products were examined by high-throughput immunoassays using tissue microarrays constructed from human postmortem spinal cord tissues. Genes that were identified by these experiments and located within a linkage region associated with familial ALS/frontotemporal dementia were sequenced in several families. This large-scale gene and protein expression study pointing to distinct molecular mechanisms of TAU- and SOD1-induced motor neuron degeneration identified several new SALS-relevant proteins (CNGA3, CRB1, OTUB2, MMP14, SLK, DDX58, RSPO2) and putative blood biomarkers, including Nefh, Prph and Mgll.

Sanfilippo syndrome type B (mucopolysaccharidosis III B, MPS III B) is an autosomal recessive, neurodegenerative disease of children, characterized by profound mental retardation and dementia. The primary cause is mutation in the NAGLU gene, resulting in deficiency of α- N -acetylglucosaminidase and lysosomal accumulation of heparan sulfate. In the mouse model of MPS III B, neurons and microglia display the characteristic vacuolation of lysosomal storage of undegraded substrate, but neurons in the medial entorhinal cortex (MEC) display accumulation of several additional substances. We used whole genome microarray analysis to examine differential gene expression in MEC neurons isolated by laser capture microdissection from Naglu −/− and Naglu +/− mice. Neurons from the lateral entorhinal cortex (LEC) were used as tissue controls. The highest increase in gene expression (6- to 7-fold between mutant and control) in MEC and LEC neurons was that of Lyzs , which encodes lysozyme, but accumulation of lysozyme protein was seen in MEC neurons only. Because of a report that lysozyme induced the formation of hyperphosphorylated tau (P-tau) in cultured neurons, we searched for P-tau by immunohistochemistry. P-tau was found in MEC of Naglu −/− mice, in the same neurons as lysozyme. In older mutant mice, it was also seen in the dentate gyrus, an area important for memory. Electron microscopy of dentate gyrus neurons showed cytoplasmic inclusions of paired helical filaments, P-tau aggregates characteristic of tauopathies—a group of age-related dementias that include Alzheimer disease. Our findings indicate that the Sanfilippo syndrome type B should also be considered a tauopathy.

Neural stem cells (NSCs) can be isolated from different regions of the central nervous system. There has been controversy whether regional differences amongst stem and progenitor cells are cell intrinsic and whether these differences are maintained during expansion in culture. The identification of inherent regional differences has important implications for the use of these cells in neural repair. Here, we compared NSCs derived from the spinal cord and embryonic cortex. We found that while cultured cortical and spinal cord derived NSCs respond similarly to mitogens and are equally neuronogenic, they retain and maintain through multiple passages gene expression patterns indicative of the region from which they were isolated (e.g Emx2 and HoxD10). Further microarray analysis identified 229 genes that were differentially expressed between cortical and spinal cord derived neurospheres, including many Hox genes, Nuclear receptors, Irx3, Pace4, Lhx2, Emx2 and Ntrk2. NSCs in the cortex express LeX. However, in the embryonic spinal cord there are two lineally related populations of NSCs: one that expresses LeX and one that does not. The LeX negative population contains few markers of regional identity but is able to generate LeX expressing NSCs that express markers of regional identity. LeX positive cells do not give rise to LeX-negative NSCs. These results demonstrate that while both embryonic cortical and spinal cord NSCs have similar self-renewal properties and multipotency, they retain aspects of regional identity, even when passaged long-term in vitro. Furthermore, there is a population of a LeX negative NSC that is present in neurospheres derived from the embryonic spinal cord but not the cortex.

DNA microarrays pose specific challenges to those studying the central and peripheral nervous systems. Probably the most important involve difficulty in obtaining appropriate tissue for study, as well as the problems posed by cellular heterogeneity. This unit describes advances in the available technologies and provides protocols for cDNA microarray hybridization, including the use of PCR amplicons. Protocols are also provided for the two major methods for limiting cellular heterogeneity by study of RNA from single cell populations in high-throughput microarray studies, laser capture microdissection (LCM), and automated fluorescent cell sorting (FACS-array).

This unit focuses on cDNA microarrays, which are being increasingly applied to large-scale gene expression studies in the nervous system, concentrating on the steps of probe preparation and microarray hybridization. Four protocols for the attachment of fluorophores to cDNA (labeling) and cDNA hybridization onto microarrays are presented: direct labeling using either Klenow fragment or reverse transcriptase, and two signal amplification techniques represented by the tyramide signal amplification method (TSA), and detection and labeling of cDNA using PCR amplification. Basic issues in experimental design, sources of variability and potential pitfalls in microarray experiments are described. Anticipated results and follow up of microarray experiments, including confirmation of the results and troubleshooting are also discussed.

Intrinsic regulatory factors play critical roles in early cortical patterning, including the development of the anteroposterior (A-P) axis. To identify genes that are differentially expressed along the A-P axis of the developing cerebral cortex, we analyzed gene expression in presumptive frontal, parietal, and occipital cerebral walls of E12.5 mouse using complementary DNA microarrays. We identified 106 genes, including expressed sequence tags (ESTs), expressed in an A-P gradient in the embryonic brain and screened 88 by in situ hybridization for confirmation. Central nervous system (CNS) expression patterns of many of these genes were previously unknown. Others, such as Sfrp1, CoupTF1, and FABP7, were expressed in a manner consistent with previous studies, providing independent confirmation. Two related transcription factors, previously not implicated in CNS development, Fhl1 and Fhl2, were observed to be enriched in posterior and anterior telencephalon, respectively. We studied patterning gradients in Fhl1 knockout mice but observed no changes in gene expression related to A-P regionalization in the Fhl1 knockout mice. These data provide an important set of new candidates for studies of cortical patterning and maturation.

A major challenge in systems neuroscience is to perform precise molecular genetic analyses of a single neuronal population in the context of the complex mammalian brain. Existing technologies for profiling cell type‐specific gene expression are largely limited to immature or morphologically identifiable neurons. In this study, we developed a simple method using fluorescent activated cell sorting (FACS) to purify genetically labeled neurons from juvenile and adult mouse brains for gene expression profiling. We identify and verify a new set of differentially expressed genes in the striatonigral and striatopallidal neurons, two functionally and clinically important projection neuron subtypes in the basal ganglia. We further demonstrate that Ebf1 is a lineagespecific transcription factor essential to the differentiation of striatonigral neurons. Our study provides a general approach for profiling cell type‐specific gene expression in the mature mammalian brain and identifies a set of genes critical to the function and dysfunction of the striatal projection neuron circuit. The extraordinary complexity and heterogeneity of the mammalian nervous system significantly limits the power of the microarray for gene expression analyses. Because many distinct neuronal and non-neuronal cells are highly intermixed, microarray analyses of a given brain region either during development, behavioral or pharmacological manipulation or subsequent to a disease process only provide a composite view of gene expression. For example, if a gene is expressed at low or moderate levels in a single cell type, even a large change in its expression may not be detected because it is below the abundance level measurable by microarrays. Additionally, changes in gene expression in one cell type may be masked by apposing changes in another cell type. Thus, critical and significant gene expression alterations within a single neuronal population often elude detection in microarray analyses using complex brain tissues 1‐3 . Even when such expression changes