Lightly stroking the lips or gently poking some skin regions can evoke mechanical itch in healthy human subjects. Sensitization of mechanical itch and persistent spontaneous itch are intractable symptoms in chronic itch patients. However, the underlying neural circuits are not well defined. We identified a subpopulation of excitatory interneurons expressing Urocortin 3::Cre (Ucn3 + ) in the dorsal spinal cord as a central node in the pathway that transmits acute mechanical itch and mechanical itch sensitization as well as persistent spontaneous itch under chronic itch conditions. This population receives peripheral inputs from Toll-like receptor 5-positive (TLR5 + ) Ab low-threshold mechanoreceptors and is directly innervated by inhibitory interneurons expressing neuropeptide Y::Cre (NPY + ) in the dorsal spinal cord. Reduced synaptic inhibition and increased intrinsic excitability of Ucn3 + neurons lead to chronic itch sensitization. Our study sheds new light on the neural basis of chronic itch and unveils novel avenues for developing mechanism-specific therapeutic advancements.

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The testing of candidate drugs to slow progression of Alzheimer’s disease (AD) requires clinical trials that are lengthy and expensive. Efforts to model the biochemical milieu of the AD brain may be greatly facilitated by combining two cutting edge technologies to generate three-dimensional (3D) human neuro-spheroid from induced pluripotent stem cells (iPSC) derived from AD subjects. We created iPSC from blood cells of five AD patients and differentiated them into 3D human neuronal culture. We characterized neuronal markers of our 3D neurons by immunocytochemical staining to validate the differentiation status. To block the generation of pathologic amyloid β peptides (Aβ), the 3D-differentiated AD neurons were treated with inhibitors targeting β-secretase (BACE1) and γ-secretases. As predicted, both BACE1 and γ-secretase inhibitors dramatically decreased Aβ generation in iPSC-derived neural cells derived from all five AD patients, under standard two-dimensional (2D) differentiation conditions. However, BACE1 and γ-secretase inhibitors showed less potency in decreasing Aβ levels in neural cells differentiated under 3D culture conditions. Interestingly, in a single subject AD1, we found that BACE1 inhibitor treatment was not able to significantly reduce Aβ42 levels. To investigate underlying molecular mechanisms, we performed proteomic analysis of 3D AD human neuronal cultures including AD1. Proteomic analysis revealed specific reduction of several proteins that might contribute to a poor inhibition of BACE1 in subject AD1. To our knowledge, this is the first iPSC-differentiated 3D neuro-spheroid model derived from AD patients’ blood. Our results demonstrate that our 3D human neuro-spheroid model can be a physiologically relevant and valid model for testing efficacy of AD drug.

For over two decades, fast-scan cyclic voltammetry (FSCV) has served as a reliable analytical method for monitoring dopamine release in near real-time in vivo. However, contemporary FSCV techniques have been limited to measure only rapid (on the order of seconds, i.e. phasic) changes in dopamine release evoked by either electrical stimulation or elicited by presentation of behaviorally salient stimuli, and not slower changes in the tonic extracellular levels of dopamine (i.e. basal concentrations). This is because FSCV is inherently a differential method that requires subtraction of prestimulation tonic levels of dopamine to measure phasic changes relative to a zeroed baseline. Here, we describe the development and application of a novel voltammetric technique, multiple cyclic square wave voltammetry (M-CSWV), for analytical quantification of tonic dopamine concentrations in vivo with relatively high temporal resolution (10 s). M-CSWV enriches the electrochemical information by generating two dimensional voltammograms which enable high sensitivity (limit of detection, 0.17 nM) and selectivity against ascorbic acid, and 3,4-dihydroxyphenylacetic acid (DOPAC), including changes in pH. Using M-CSWV, a tonic dopamine concentration of 120 ± 18 nM (n = 7 rats, ± SEM) was determined in the striatum of urethane anethetized rats. Pharmacological treatments to elevate dopamine by selectively inhibiting dopamine reuptake and to reduce DOPAC by inhibition of monoamine oxidase supported the selective detection of dopamine in vivo. Overall, M-CSWV offers a novel voltammetric technique to quantify levels and monitor changes in tonic dopamine concentrations in the brain to further our understanding of the role of dopamine in normal behavior and neuropsychiatric disorders.

Synaptic plasticity, the ability of synapses to change in strength, requires alterations in synaptic molecule compositions over time, and synapses undergo selective modifications on stimulation. Molecular motors operate in sorting/transport of neuronal proteins; however, the targeting mechanisms that guide and direct cargo delivery remain elusive. We addressed the impact of synaptic transmission on the regulation of intracellular microtubule (MT)-based transport. We show that increased neuronal activity, as induced through GlyR activity blockade, facilitates tubulin polyglutamylation, a posttranslational modification thought to represent a molecular traffic sign for transport. Also, GlyR activity blockade alters the binding of the MT-associated protein MAP2 to MTs. By using the kinesin (KIF5) and the postsynaptic protein gephyrin as models, we show that such changes of MT tracks are accompanied by reduced motor protein mobility and cargo delivery into neurites. Notably, the observed neurite targeting deficits are prevented on functional depletion or gene expression knockdown of neuronal polyglutamylase. Our data suggest a previously undescribed concept of synaptic transmission regulating MT-dependent cargo delivery.activity-dependent ͉ neuron ͉ polyglutamylation ͉ synapse ͉ strychnine

The significance of intracellular β-amyloid (Aβ 42 ) accumulation is increasingly recognized in Alzheimer's disease (AD) pathogenesis. Aβ removal mechanisms that have attracted attention include IDE/neprilysin degradation and antibody-mediated uptake by immune cells. However, the role of the ubiquitin-proteasome system (UPS) in the disposal of cellular Aβ has not been fully explored. The E3 ubiquitin ligase Parkin targets several proteins for UPS degradation, and Parkin mutations are the major cause of autosomal recessive Parkinson's disease. We tested whether Parkin has crossfunction to target misfolded proteins in AD for proteasome-dependent clearance in SH-SY5Y and primary neuronal cells. Wild-type Parkin greatly decreased steady-state levels of intracellular Aβ 42 , an action abrogated by proteasome inhibitors. Intracellular Aβ 42 accumulation decreased cell viability and proteasome activity. Accordingly, Parkin reversed both effects. Changes in mitochondrial ATP production from Aβ or Parkin did not account for their effects on the proteasome. Parkin knock-down led to accumulation of Aβ. In AD brain, Parkin was found to interact with Aβ and its levels were reduced. Thus, Parkin is cytoprotective, partially by increasing the removal of cellular Aβ through a proteasome-dependent pathway. Keywords amyloid; ubiquitin ligase; Parkin; Alzheimer's; proteasome Parkin is a 465-amino-acid protein containing an N-terminal ubiquitin-like (Ubl) domain linked to a C-terminal RING box (Shimura et al., 2000). Parkin functions as an E3 ubiquitin-protein ligase (Imai et al., 2000;Shimura et al., 2000; Zhang et al., 2000), facilitating the proteasomal degradation of misfolded proteins. Several Parkin gene mutations have been linked to autosomal-recessive Parkinsonism with juvenile onset (Kitada et al., 1998;Lucking et al., 2000).In cell culture systems, Parkin fusion proteins have been shown to interact with several proteins, including the α-synuclein-binding protein synphilin-1 (Chung et al., 2001) (Huynh et al., 2000), and α/β tubulin (Ren et al., 2003). Parkin has also been found to be upregulated during the integrated cellular response to misfolded protein-induced ER stress (Imai et al., 2001). Specific targets of Parkin activity having intrinsic toxic and aggregative properties include Pael-R, the Parkin-associated endothelin-like receptor (Imai et al., 2001), and possibly an O-glycosylated form of α-synuclein (Shimura et al., 2001). Thus, Parkin has been shown to suppress the toxicity of PAEL-R (Imai et al., 2001), mutated α-synuclein A30P (Petrucelli et al., 2002;Lo Bianco et al., 2004), and a poly(Q)-expanded mutant of ataxin-3 (Tsai et al., 2003). Deletions in the Parkin gene result in the accumulation of nonubiquitinated forms of α-synuclein and Pael-R in the brain (Imai et al., 2001;Shimura et al., 2001).The accumulation of neuronal β-amyloid (Aβ) is increasingly recognized as a critical factor in Alzheimer's disease (AD) and related pathologies (Hartmann, 1999;Wilson et al., 1999;Gouras et al., 2005). Solub...

Significance Synapses form and change in response to neuronal activity, and they dynamically exchange transmembrane proteins over time. Most synaptic proteins are synthesized in the cell body and undergo long-distance vesicular transport powered by molecular motors along microtubules. Here we show that two synaptic key proteins (GluA2 and N-cadherin) are simultaneously delivered within distinct transport vesicles through motor proteins. Our data suggest that multidomain cargo adaptors tether synaptic proteins destined for the same subcellular compartment. We propose that vesicular presorting is an alternative mechanism to efficiently supply synapses.

Energy loss due to ohmic heating is a major bottleneck limiting down-scaling and speed of nano-electronic devices, and harvesting ohmic heat for signal processing is a major challenge in modern electronics. Here, we demonstrate that thermal gradients arising from ohmic heating can be utilized for excitation of coherent auto-oscillations of magnetization and for generation of tunable microwave signals. The heat-driven dynamics is observed in Y3Fe5O12/Pt bilayer nanowires where ohmic heating of the Pt layer results in injection of pure spin current into the Y3Fe5O12 layer. This leads to excitation of auto-oscillations of the Y3Fe5O12 magnetization and generation of coherent microwave radiation. Our work paves the way towards spin caloritronic devices for microwave and magnonic applications.