Rescue of synaptosomal glutamate release defects in tau transgenic mice by the tau aggregation inhibitor hydromethylthionine.

Glutamatergic neurotransmission, important for learning and memory, is disrupted in different ways in patients with Alzheimer's disease (AD) and frontotemporal dementia (FTD) tauopathies. We have previously reported that two tau transgenic mouse models, L1 and L66, produce different phenotypes resembling AD and FTD, respectively. The AD-like L1 model expresses the truncated core aggregation domain of the AD paired helical filament (PHF) form of tau (tau296-390) whereas the FTD-like L66 model expresses full-length tau carrying two mutations at P301S/G335D. We have used synaptosomes isolated from these mice to investigate K+-evoked glutamate release and, if abnormal, to determine responsiveness to hydromethylthionine, a tau aggregation inhibitor previously shown to reduce tau pathology in these models. We report that the transgenes in these two mouse lines cause opposite abnormalities in glutamate release. Over-expression of the core tau unit in L1 produces a significant reduction in glutamate release and a loss of Ca2+-dependency compared with wild-type control mice. Full-length mutant tau produces an increase in glutamate release that retains normal Ca2+-dependency. Chronic pre-treatment with hydromethylthionine normalises both reduced (L1) and excessive glutamate (L66) and restores normal Ca2+-dependency in L1 mice. This implies that both patterns of impairment are the result of tau aggregation, but that the direction and Ca2+-dependency of the abnormality is determined by expression of the disease-specific transgene. Our results lead to the conclusion that the tauopathies need not be considered a single entity in terms of the downstream effects of pathological aggregation of tau protein. In this case, directionally opposite abnormalities in glutamate release resulting from different types of tau aggregation in the two mouse models can be corrected by hydromethylthionine. This may help to explain the activity of hydromethylthionine on cognitive decline and brain atrophy in both AD and behavioural-variant FTD.

LETC inhibits α-Syn aggregation and ameliorates motor deficiencies in the L62 mouse model of synucleinopathy.

Alpha-Synuclein (α-Syn) aggregation is a pathological feature of synucleinopathies, neurodegenerative disorders that include Parkinson's disease (PD). Here, we explored the efficacy of N,N,N',N'-tetraethyl-10H-phenothiazine-3,7-diamine dihydrochloride (LETC), a protein aggregation inhibitor, on α-Syn aggregation. In both cellular models and transgenic mice, α-Syn aggregation was achieved by the overexpression of full-length human α-Syn fused with a signal sequence peptide. α-Syn accumulated in transfected DH60.21 neuroblastoma cells and α-Syn aggregation was inhibited by LETC with an EC50 of 0.066 ± 0.047 µM. Full-length human α-Syn overexpressing Line 62 (L62) mice accumulated neuronal α-Syn that was associated with a decreased motor performance in the open field and automated home cage. LETC, administered orally for 6 weeks at 10 mg/kg significantly decreased α-Syn-positive neurons in multiple brain regions and this resulted in a rescue of movement deficits in the open field in these mice. LETC however, did not improve activity deficits of L62 mice in the home cage environment. The results suggest that LETC may provide a potential disease modification therapy in synucleinopathies through the inhibition of α-Syn aggregation.

Assays for the Screening and Characterization of Tau Aggregation Inhibitors.

Aggregation of tau protein is a pathological hallmark of Alzheimer's disease and other neurodegenerative tauopathies. Inhibition of tau aggregation may provide a method for treatment of these disorders. Methods to identify tau aggregation inhibitors (TAIs) in vitro are useful and here we describe assays for TAIs using purified recombinant tau protein fragments in a cell-free immunoassay format and in a stably transfected cell model to create a more physiological environment.

High-SPL Air-Coupled Piezoelectric Micromachined Ultrasonic Transducers Based on 36% ScAlN Thin-Film.

In this paper, air-coupled piezoelectric micromachined ultrasonic transducers (PMUTs) using 36% scandium-doped aluminum nitride (ScAlN) thin-film are presented. ScAlN is known to exhibit higher piezoelectric properties compared to pure AlN leading to significant performance improvements in various piezoelectric micro-electromechanical systems (MEMS) applications including PMUTs. Here, the concentration of Sc in the actual sputtered 1- [Formula: see text]-thick ScAlN film was 36%, which is slightly below the maximum at the phase boundary. The ScAlN PMUTs were fabricated from an SOI wafer, where the dry etching of ScAlN film was optimized. The frequency response and displacement sensitivity of the PMUTs were characterized in the air using laser Doppler vibrometry confirming 2× higher transverse piezoelectric coefficient than AlN. The acoustic transmission and reception of the PMUTs were evaluated from a high-sensitivity microphone and pulse-echo measurements. The PMUTs were designed to operate below 100 kHz in order to mitigate the absorption loss, which resulted in a high transmit pressure of 105-dB sound pressure level (SPL) at 10 cm and only 30-dB attenuation at the 2-m range. Through implementing 36% ScAlN film, the presented PMUTs exhibited a large displacement and consequently, a high SPL compared to the state-of-the-art PMUTs and the bulk transducer considering the size and the excitation voltage.

Hybrid GMR Sensor Detecting 950 pT/sqrt(Hz) at 1 Hz and Room Temperature.

Advances in the magnetic sensing technology have been driven by the increasing demand for the capability of measuring ultrasensitive magnetic fields. Among other emerging applications, the detection of magnetic fields in the picotesla range is crucial for biomedical applications. In this work Picosense reports a millimeter-scale, low-power hybrid magnetoresistive-piezoelectric magnetometer with subnanotesla sensitivity at low frequency. Through an innovative noise-cancelation mechanism, the 1/f noise in the MR sensors is surpassed by the mechanical modulation of the external magnetic fields in the high frequency regime. A modulation efficiency of 13% was obtained enabling a final device's sensitivity of ~950 pT/Hz1/2 at 1 Hz. This hybrid device proved to be capable of measuring biomagnetic signals generated in the heart in an unshielded environment. This result paves the way for the development of a portable, contactless, low-cost and low-power magnetocardiography device.

Alpha-Synuclein transgenic mice, h-α-SynL62, display α-Syn aggregation and a dopaminergic phenotype reminiscent of Parkinson's disease.

Alpha-Synuclein (α-Syn) accumulation is considered a major risk factor for the development of synucleinopathies such as Parkinson's disease (PD) and dementia with Lewy bodies. We have generated mice overexpressing full-length human α-Syn fused to a membrane-targeting signal sequence under the control of the mouse Thy1-promotor. Three separate lines (L56, L58 and L62) with similar gene expression levels, but considerably heightened protein accumulation in L58 and L62, were established. In L62, there was widespread labelling of α-Syn immunoreactivity in brain including spinal cord, basal forebrain, cortex and striatum. Interestingly, there was no detectable α-Syn expression in dopaminergic neurones of the substantia nigra, but strong human α-Syn reactivity in glutamatergic synapses. The human α-Syn accumulated during aging and formed PK-resistant, thioflavin-binding aggregates. Mice displayed early onset bradykinesia and age progressive motor deficits. Functional alterations within the striatum were confirmed: L62 showed normal basal dopamine levels, but impaired dopamine release (upon amphetamine challenge) in the dorsal striatum measured by in vivo brain dialysis at 9 months of age. This impairment was coincident with a reduced response to amphetamine in the activity test. L62 further displayed greater sensitivity to low doses of the dopamine receptor 1 (D1) agonist SKF81297 but reacted normally to the D2 agonist quinpirole in the open field. Since accumulation of α-Syn aggregates in neurones and synapses and alterations in the dopaminergic tone are characteristics of PD, phenotypes reported for L62 present a good opportunity to further our understanding of motor dysfunction in PD and Lewy body dementia.

Ultrasonic Fingerprint Sensor With Transmit Beamforming Based on a PMUT Array Bonded to CMOS Circuitry.

In this paper, we present a single-chip 65 ×42 element ultrasonic pulse-echo fingerprint sensor with transmit (TX) beamforming based on piezoelectric micromachined ultrasonic transducers directly bonded to a CMOS readout application-specific integrated circuit (ASIC). The readout ASIC was realized in a standard 180-nm CMOS process with a 24-V high-voltage transistor option. Pulse-echo measurements are performed column-by-column in sequence using either one column or five columns to TX the ultrasonic pulse at 20 MHz. TX beamforming is used to focus the ultrasonic beam at the imaging plane where the finger is located, increasing the ultrasonic pressure and narrowing the 3-dB beamwidth to [Formula: see text], a factor of 6.4 narrower than nonbeamformed measurements. The surface of the sensor is coated with a poly-dimethylsiloxane (PDMS) layer to provide good acoustic impedance matching to skin. Scanning laser Doppler vibrometry of the PDMS surface was used to map the ultrasonic pressure field at the imaging surface, demonstrating the expected increase in pressure, and reduction in beamwidth. Imaging experiments were conducted using both PDMS phantoms and real fingerprints. The average image contrast is increased by a factor of 1.5 when beamforming is used.

A Protein Aggregation Inhibitor, Leuco-Methylthioninium Bis(Hydromethanesulfonate), Decreases α-Synuclein Inclusions in a Transgenic Mouse Model of Synucleinopathy.

α-Synuclein (α-Syn) aggregation is a pathological feature of synucleinopathies, neurodegenerative disorders that include Parkinson's disease (PD). We have tested whether N,N,N',N'-tetramethyl-10H-phenothiazine-3,7-diaminium bis(hydromethanesulfonate) (leuco-methylthioninium bis(hydromethanesulfonate); LMTM), a tau aggregation inhibitor, affects α-Syn aggregation in vitro and in vivo. Both cellular and transgenic models in which the expression of full-length human α-Syn (h-α-Syn) fused with a signal sequence peptide to promote α-Syn aggregation were used. Aggregated α-Syn was observed following differentiation of N1E-115 neuroblastoma cells transfected with h-α-Syn. The appearance of aggregated α-Syn was inhibited by LMTM, with an EC50 of 1.1 µM, with minimal effect on h-α-Syn mRNA levels being observed. Two independent lines of mice (L58 and L62) transgenic for the same fusion protein accumulated neuronal h-α-Syn that, with aging, developed into fibrillary inclusions characterized by both resistance to proteinase K (PK)-cleavage and their ability to bind thiazin red. There was a significant decrease in α-Syn-positive neurons in multiple brain regions following oral treatment of male and female mice with LMTM administered daily for 6 weeks at 5 and 15 mg MT/kg. The early aggregates of α-Syn and the late-stage fibrillar inclusions were both susceptible to inhibition by LMTM, a treatment that also resulted in the rescue of movement and anxiety-related traits in these mice. The results suggest that LMTM may provide a potential disease modification therapy in PD and other synucleinopathies through the inhibition of α-Syn aggregation.

Monolithic ultrasound fingerprint sensor.

This paper presents a 591×438-DPI ultrasonic fingerprint sensor. The sensor is based on a piezoelectric micromachined ultrasonic transducer (PMUT) array that is bonded at wafer-level to complementary metal oxide semiconductor (CMOS) signal processing electronics to produce a pulse-echo ultrasonic imager on a chip. To meet the 500-DPI standard for consumer fingerprint sensors, the PMUT pitch was reduced by approximately a factor of two relative to an earlier design. We conducted a systematic design study of the individual PMUT and array to achieve this scaling while maintaining a high fill-factor. The resulting 110×56-PMUT array, composed of 30×43-µm2 rectangular PMUTs, achieved a 51.7% fill-factor, three times greater than that of the previous design. Together with the custom CMOS ASIC, the sensor achieves 2 mV kPa-1 sensitivity, 15 kPa pressure output, 75 µm lateral resolution, and 150 µm axial resolution in a 4.6 mm×3.2 mm image. To the best of our knowledge, we have demonstrated the first MEMS ultrasonic fingerprint sensor capable of imaging epidermis and sub-surface layer fingerprints.

Assays for the Screening and Characterization of Tau Aggregation Inhibitors.

Aggregation of tau protein is a pathological hallmark of Alzheimer's disease and other neurodegenerative tauopathies. Inhibition of tau aggregation could provide a method for the treatment of these disorders. Methods to identify tau aggregation inhibitors (TAIs) in vitro are useful and here we describe assays for TAIs using purified recombinant tau protein fragments in a cell-free immunoassay format and a stably transfected cell model to create a more physiological environment.

Self-induced parametric amplification arising from nonlinear elastic coupling in a micromechanical resonating disk gyroscope.

Parametric amplification, resulting from intentionally varying a parameter in a resonator at twice its resonant frequency, has been successfully employed to increase the sensitivity of many micro- and nano-scale sensors. Here, we introduce the concept of self-induced parametric amplification, which arises naturally from nonlinear elastic coupling between the degenerate vibration modes in a micromechanical disk-resonator, and is not externally applied. The device functions as a gyroscope wherein angular rotation is detected from Coriolis coupling of elastic vibration energy from a driven vibration mode into a second degenerate sensing mode. While nonlinear elasticity in silicon resonators is extremely weak, in this high quality-factor device, ppm-level nonlinear elastic effects result in an order-of-magnitude increase in the observed sensitivity to Coriolis force relative to linear theory. Perfect degeneracy of the primary and secondary vibration modes is achieved through electrostatic frequency tuning, which also enables the phase and frequency of the parametric coupling to be varied, and we show that the resulting phase and frequency dependence of the amplification follow the theory of parametric resonance. We expect that this phenomenon will be useful for both fundamental studies of dynamic systems with low dissipation and for increasing signal-to-noise ratio in practical applications such as gyroscopes.

Cellular Models of Aggregation-dependent Template-directed Proteolysis to Characterize Tau Aggregation Inhibitors for Treatment of Alzheimer Disease.

Alzheimer disease (AD) is a degenerative tauopathy characterized by aggregation of Tau protein through the repeat domain to form intraneuronal paired helical filaments (PHFs). We report two cell models in which we control the inherent toxicity of the core Tau fragment. These models demonstrate the properties of prion-like recruitment of full-length Tau into an aggregation pathway in which template-directed, endogenous truncation propagates aggregation through the core Tau binding domain. We use these in combination with dissolution of native PHFs to quantify the activity of Tau aggregation inhibitors (TAIs). We report the synthesis of novel stable crystalline leucomethylthioninium salts (LMTX®), which overcome the pharmacokinetic limitations of methylthioninium chloride. LMTX®, as either a dihydromesylate or a dihydrobromide salt, retains TAI activity in vitro and disrupts PHFs isolated from AD brain tissues at 0.16 µM. The Ki value for intracellular TAI activity, which we have been able to determine for the first time, is 0.12 µM. These values are close to the steady state trough brain concentration of methylthioninium ion (0.18 µM) that is required to arrest progression of AD on clinical and imaging end points and the minimum brain concentration (0.13 µM) required to reverse behavioral deficits and pathology in Tau transgenic mice.

Effects of oxidized and reduced forms of methylthioninium in two transgenic mouse tauopathy models.

Given the repeated failure of amyloid-based approaches in Alzheimer's disease, there is increasing interest in tau-based therapeutics. Although methylthioninium (MT) treatment was found to be beneficial in tau transgenic models, the brain concentrations required to inhibit tau aggregation in vivo are unknown. The comparative efficacy of methylthioninium chloride (MTC) and leucomethylthioninium salts (LMTX; 5-75 mg/kg; oral administration for 3-8 weeks) was assessed in two novel transgenic tau mouse lines. Behavioural (spatial water maze, RotaRod motor performance) and histopathological (tau load per brain region) proxies were applied. Both MTC and LMTX dose-dependently rescued the learning impairment and restored behavioural flexibility in a spatial problem-solving water maze task in Line 1 (minimum effective dose: 35 mg MT/kg for MTC, 9 mg MT/kg for LMTX) and corrected motor learning in Line 66 (effective doses: 4 mg MT/kg). Simultaneously, both drugs reduced the number of tau-reactive neurons, particularly in the hippocampus and entorhinal cortex in Line 1 and in a more widespread manner in Line 66. MT levels in the brain followed a sigmoidal concentration-response relationship over a 10-fold range (0.13-1.38 µmol/l). These data establish that diaminophenothiazine compounds, like MT, can reverse both spatial and motor learning deficits and reduce the underlying tau pathology, and therefore offer the potential for treatment of tauopathies.

Complex disposition of methylthioninium redox forms determines efficacy in tau aggregation inhibitor therapy for Alzheimer's disease.

Methylthioninium (MT) is a tau aggregation inhibitor with therapeutic potential in Alzheimer's disease (AD). MT exists in equilibrium between reduced [leucomethylthioninium (LMT)] and oxidized (MT(+)) forms; as a chloride salt [methylthioninium chloride (MTC), "methylene blue"], it is stabilized in its MT(+) form. Although the results of a phase 2 study of MTC in 321 mild/moderate AD subjects identified a 138-mg MT/day dose as the minimum effective dose on cognitive and imaging end points, further clinical development of MT was delayed pending resolution of the unexpected lack of efficacy of the 228-mg MT/day dose. We hypothesized that the failure of dose response may depend on differences known at the time in dissolution in simulated gastric and intestinal fluids of the 100-mg MTC capsules used to deliver the 228-mg dose and reflect previously unsuspected differences in redox processing of MT at different levels in the gut. The synthesis of a novel chemical entity, LMTX (providing LMT in a stable anhydrous crystalline form), has enabled a systematic comparison of the pharmacokinetic properties of MTC and LMTX in preclinical and clinical studies. The quantity of MT released in water or gastric fluid within 60 minutes proved in retrospect to be an important determinant of clinical efficacy. A further factor was a dose-dependent limitation in the ability to absorb MT in the presence of food when delivered in the MT(+) form as MTC. A model is presented to account for the complexity of MT absorption, which may have relevance for other similar redox molecules.

A 32 × 32 optical phased array using polysilicon sub-wavelength high-contrast-grating mirrors.

We report on microelectromechanical systems (MEMS)-actuated 32 × 32 optical phased arrays (OPAs) with high fill-factors and microsecond response time. To reduce the mirror weight and temperature-dependent curvature, we use high-contrast-grating (HCG) mirrors comprising a single layer of sub-wavelength polysilicon gratings with 400 nm thickness, 1250 nm pitch, and 570 nm grating bar width. The mirror has a broad reflection band and a peak reflectivity of 99.9% at 1550 nm wavelength. With 20 × 20 µm2 pixels and 2 µm, the OPA has a total aperture of 702 × 702 µm2 and a fill factor of 85%. The OPA is electrostatically controlled by voltage and has a total field of view of ± 2°, an instantaneous field of view (beam width) of 0.14°, and a response time of 3.8 µs. The latter agrees well with the mechanical resonance frequency of the HCG mirror (0.42 MHz).

High speed optical phased array using high contrast grating all-pass filters.

We report a high speed 8x8 optical phased array using tunable 1550 nm all-pass filters with ultrathin high contrast gratings (HCGs) as the microelectromechanical-actuated top reflectors. The all-pass filter design enables a highly efficient phase tuning (1.7 π) with a small actuation voltage (10 V) and actuation displacement of the HCG (50 nm). The microelectromechanical HCG structure facilitates a high phase tuning speed >0.5 MHz. Beam steering is experimentally demonstrated with the optical phased array.

Faster-than-anticipated Na(+)/Cl(-) diffusion across lipid bilayers in vesicles.

Maintenance of electrochemical potential gradients across lipid membranes is critical for signal transduction and energy generation in biological systems. However, because ions with widely varying membrane permeabilities all contribute to the electrostatic potential, it can be difficult to measure the influence of diffusion of a single ion type across the bilayer. To understand the electrodiffusion of H(+) across lipid bilayers, we used a pH-sensitive fluorophore to monitor the lumenal pH in vesicles after a stepwise change in the bulk pH. In vesicles containing the ion channel gramicidin, the lumenal pH rapidly approached the external pH. In contrast, the lumen of intact vesicles showed a two stage pH response: an initial rapid change occurred over ~1min, followed by a much slower change over ~24h. We provide a quantitative interpretation of these results based on the Goldman-Hodgkin-Katz ion fluxes discharging the electrical capacitance of the bilayer membrane. This interpretation provides an estimate of the permeability of the membranes to Na(+) and Cl(-) ions of ~10(-8)cm/s, which is ~3 orders of magnitude faster than previous reports. We discuss possible mechanisms to account for this considerably higher permeability in vesicle membranes.

Optical phased array using high contrast gratings for two dimensional beamforming and beamsteering.

We have developed a microelectromechanical system (MEMS) optical phased array incorporating a high-index-contrast subwavelength grating (HCG) for beamforming and beamsteering in a range of ± 1.26° × 1.26°. Our approach needs only a thin single-layer HCG made of silicon, considerably improving its speed thanks to the low mass, and is suitable for high optical power applications. The measured resonant frequency of HCG is 0.32 MHz.

Optical beamsteering using an 8 × 8 MEMS phased array with closed-loop interferometric phase control.

We present a high-speed optical beamsteering system based on an 8x8 MEMS phased array. The system incorporates an in situ interferometer that provides a real-time, dynamic measure of the phase of each mirror in the array during beamsteering. A closed-loop phase-control algorithm results in <π/100 mirror phase accuracy and far field beam steering is shown. Stroboscopic measurement capabilities are demonstrated which allow us to show feedforward control to eliminate micromirror ringing.

Nanopore-spanning lipid bilayers on silicon nitride membranes that seal and selectively transport ions.

We report the formation of POPC lipid bilayers that span 130 nm pores in a freestanding silicon nitride film supported on a silicon substrate. These solvent-free lipid membranes self-assemble on organosilane-treated Si3N4 via the fusion of 200 nm unilamellar vesicles. Membrane fluidity is verified by fluorescence recovery after photobleaching (FRAP), and membrane resistance in excess of 1 GΩ is demonstrated using electrical impedance spectroscopy (EIS). An array of 40,000 membranes maintained high impedance over 72 h, followed by rupture of most of the membranes by 82 h. Membrane incorporation of gramicidin, a model ion channel, resulted in increased membrane conductance. This membrane conductance was diminished when the gramicidin channels were blocked with CaCl2, indicating that the change in membrane conductance results from gramicidin-mediated ion transport. These very stable, biologically functional pore-spanning membranes open many possibilities for silicon-based ion-channel devices for applications such as biosensors and high-throughput drug screening.