Dopamine Deficiency Reduces Striatal Cholinergic Interneuron Function in Models of Parkinson's Disease.

Motor and cognitive functions depend on the coordinated interactions between dopamine (DA) and acetylcholine (ACh) at striatal synapses. Increased ACh availability was assumed to accompany DA deficiency based on the outcome of pharmacological treatments and measurements in animals that were critically depleted of DA. Using Slc6a3DTR/+ diphtheria-toxin-sensitive mice, we demonstrate that a progressive and L-dopa-responsive DA deficiency reduces ACh availability and the transcription of hyperpolarization-activated cation (HCN) channels that encode the spike timing of ACh-releasing tonically active striatal interneurons (ChIs). Although the production and release of ACh and DA are reduced, the preponderance of ACh over DA contributes to the motor deficit. The increase in striatal ACh relative to DA is heightened via D1-type DA receptors that activate ChIs in response to DA release from residual axons. These results suggest that stabilizing the expression of HCN channels may improve ACh-DA reciprocity and motor function in Parkinson's disease (PD). VIDEO ABSTRACT.

Noonan Syndrome-Associated SHP2 Dephosphorylates GluN2B to Regulate NMDA Receptor Function.

Hyperactivating mutations in the non-receptor tyrosine phosphatase SHP2 cause Noonan syndrome (NS). NS is associated with cognitive deficits, but how hyperactivation of SHP2 in NS changes neuron function is not well understood. We find that mice bearing an NS-associated SHP2 allele (NS mice) have selectively impaired Schaffer collateral-CA1 NMDA (N-methyl-D-aspartate) receptor (NMDAR)-mediated neurotransmission and that residual NMDAR-mediated currents decay faster in NS mice because of reduced contribution of GluN1:GluN2B diheteromers. Consistent with altered GluN2B function, we identify GluN2B Y1252 as an NS-associated SHP2 substrate both in vitro and in vivo. Mutation of Y1252 does not alter recombinant GluN1:GluN2B receptor kinetics. Instead, phospho-Y1252 binds the actin-regulatory adaptor protein Nck2, and this interaction is required for proper NMDAR function. These results establish SHP2 and Nck2 as NMDAR regulatory proteins and strongly suggest that NMDAR dysfunction contributes to NS cognitive deficits.

A simple end-point assay for calcium channel activity.

Cellular calcium signaling events are transient. Hence they are observed in real time using fluorescence imaging or electrophysiological methods that require sophisticated instrumentation and specialized skills. For high throughput assays simple and inexpensive techniques are desirable. Many calcium channels that serve as drug targets have subtypes arising from diverse subunit combinations. These need to be targeted selectively for achieving efficacy and for avoiding side effects in therapies. This in turn increases the number of calcium channels that act as drug targets. We report a novel method for intracellular calcium sensing that utilizes the calcium dependent stable interaction between CaM kinase II (CaMKII) and its ligands such as the NMDA receptor subunit GluN2B. The CaMKII-GluN2B complex formed persists as a memory of the transient increase in calcium. In a cell-based assay system GFP-α-CaMKII expressed in the cytosol responds to calcium by translocating towards GluN2B sequence motif exogenously expressed on mitochondria or endoplasmic reticulum. The resulting punctate fluorescence pattern serves as the signal for intracellular calcium release. The pattern is stable, unaffected by sample processing and is observable without real time imaging. The activities of calcium channel proteins heterologously expressed in HEK-293 cells were detected with specificity using this technique. A calcium sensor vector and a calcium sensor cell line were developed as tools to perform this technique. This technique being simple and less expensive could significantly facilitate high throughput screening in calcium channel drug discovery.