Studies of NMDA receptor function and stoichiometry with truncated and tandem subunits

The subunits that compose eukaryotic glutamate ion channel receptors have three transmembrane domains (TMs) and terminate with intracellular tails that are important for controlling channel expression and localization. Truncation of NMDA receptor subunits before the final TM showed that this TM and intracellular tail region are necessary to form functional channels. However, it is shown here that these truncated subunits may be partially rescued by coexpressing the final TM and tail as a separate protein. The whole-cell currents so produced are somewhat lower than with full-length subunits, and they do not show the sag characteristic of currents from channels containing NR1 and NR2A subunits in the continued presence of an agonist. In addition, these truncated subunits were joined to full-length subunits to generate tandems. The functional expression of these tandems confirmed the tetrameric structure of NMDA receptors and also suggested that the subunits making up NMDA receptors are arranged as a dimer of dimers in the receptors with a 1-1-2-2 orientation of the subunits in the channel, and not in an alternating pattern of subunits around the pore. These results may redirect future studies into the mechanism of binding and gating in these receptors toward schemes including dimers, and may also be relevant to studies of glutamate receptor ion channels in general

Gene Therapy for Neurological Disease: State of the Art and Opportunities for Next-generation Approaches

Gene therapy for rare monogenetic neurological disorders is reaching clinics and offering hope to families affected by these diseases. There is also potential for gene therapy to offer new and effective treatments for common, non-genetic disorders. Treatments for Parkinson's Disease are in clinical trials, and treatments for refractory epilepsies are due to enter first-in-human clinical trials in 2022. Gene therapies for these disorders are based on delivering genes that address the mechanism of the disease, not repairing a mutated gene. Similar 'mechanistic' gene therapies could offer treatments to a wide range of neurological and neuropsychiatric diseases where there is a known mechanism that could be restored using gene therapy. However, the permanent nature of most gene therapies is a serious drawback for translation of gene therapies to a wide-range of diseases because it could present risk of irreversible adverse effects. Several lines of research are aimed at developing gene therapy approaches that allow for the treatment to be turned on and off, including: using proteins activated by exogenous ligands, and promoters turned on by activators. We review these approaches and propose an overall de-risking strategy for gene therapy for common neurological and psychiatric diseases. This approach is based on using a temporary mRNA-based treatment to initially assess efficacy and safety of the planned manipulation, and only following with permanent, virally-delivered treatment if the approach appears safe and effective

Conservation of alternative splicing in sodium channels reveals evolutionary focus on release from inactivation and structural insights into gating

Voltage-gated sodium channels are critical for neuronal activity, and highly intolerant to variation. Even mutations that cause subtle changes in the activity these channels are sufficient to cause devastating inherited neurological diseases, such as epilepsy and pain. However, these channels do vary in healthy tissue. Alternative splicing modifies sodium channels, but the functional relevance and adaptive significance of this splicing remain poorly understood. Here we use a conserved alternate exon encoding part of the first domain of sodium channels to compare how splicing modifies different channels, and to ask whether the functional consequences of this splicing have been preserved in different genes. Although the splicing event is highly conserved, one splice variant has been selectively removed from Nav1.1 in multiple mammalian species, suggesting that the functional variation in Nav1.1 is less well-tolerated. We show for three human channels (Nav1.1, Nav1.2 and Nav1.7) splicing modifies the return from inactivated to deactivated states, and the differences between splice variants are occluded by antiepileptic drugs that bind to and stabilize inactivated states. A model based on structural data can replicate these changes, and indicates that splicing may exploit a distinct role of the first domain to change channel availability, and that the first domain of all three sodium channels plays a role in determining the rate at which the inactivation domain dissociates. Taken together, our data suggest that the stability of inactivated states is under tight evolutionary control, but that in Nav1.1 faster recovery from inactivation is associated with negative selection in mammals

Tackling obstacles for gene therapy targeting neurons: disrupting perineural nets with hyaluronidase improves transduction.

Gene therapy has been proposed for many diseases in the nervous system. In most cases for successful treatment, therapeutic vectors must be able to transduce mature neurons. However, both in vivo, and in vitro, where preliminary characterisation of viral particles takes place, transduction of neurons is typically inefficient. One possible explanation is that the extracellular matrix (ECM), forming dense perineural nets (PNNs) around neurons, physically blocks access to the cell surface. We asked whether co-administration of lentiviral vectors with an enzyme that disrupts the ECM could improve transduction efficiency. Using hyaluronidase, an enzyme which degrades hyaluronic acid, a high molecular weight molecule of the ECM with mainly a scaffolding function, we show that in vitro in mixed primary cortical cultures, and also in vivo in rat cortex, hyaluronidase co-administration increased the percentage of transduced mature, NeuN-positive neurons. Moreover, hyaluronidase was effective at doses that showed no toxicity in vitro based on propidium iodide staining of treated cultures. Our data suggest that limited efficacy of neuronal transduction is partly due to PNNs surrounding neurons, and further that co-applying hyaluronidase may benefit applications where efficient transduction of neurons in vitro or in vivo is required

Defining the core proteome of the chloroplast envelope membranes

High-throughput protein localization studies require multiple strategies. Mass spectrometric analysis of defined cellular fractions is one of the complementary approaches to a diverse array of cell biological methods. In recent years, the protein content of different cellular (sub-)compartments was approached. Despite of all the efforts made, the analysis of membrane fractions remains difficult, in that the dissection of the proteomes of the envelope membranes of chloroplasts or mitochondria is often not reliable because sample purity is not always warranted. Moreover, proteomic studies are often restricted to single (model) species, and therefore limited in respect to differential individual evolution. In this study we analyzed the chloroplast envelope proteomes of different plant species, namely, the individual proteomes of inner and outer envelope (OE) membrane of Pisum sativum and the mixed envelope proteomes of Arabidopsis thaliana and Medicago sativa. The analysis of all three species yielded 341 identified proteins in total, 247 of them being unique. 39 proteins were genuine envelope proteins found in at least two species. Based on this and previous envelope studies we defined the core envelope proteome of chloroplasts. Comparing the general overlap of the available six independent studies (including ours) revealed only a number of 27 envelope proteins. Depending on the stringency of applied selection criteria we found 231 envelope proteins, while less stringent criteria increases this number to 649 putative envelope proteins. Based on the latter we provide a map of the outer and inner envelope core proteome, which includes many yet uncharacterized proteins predicted to be involved in transport, signaling, and response. Furthermore, a foundation for the functional characterization of yet unidentified functions of the inner and OE for further analyses is provided

CNS gene therapy: present developments and emerging trends accelerating industry-academia pathways

The recent success of first central nervous system gene therapies has reinvigorated the growing community of gene therapy researchers and strengthened the field's market position. We are witnessing an increase of clinical trials with long-term efficiency mainly for neurometabolic, neurodegenerative and neurodevelopmental diseases caused by loss-of-function mutations. The ever-expanding knowledge and accessibility to the most advanced tools allow enrichment of applications to more complex diseases. This gradually contributes towards sealing the gap between top diseases impacting current global health and those towards which gene therapy development is currently aimed. Here, we highlight innovative therapeutic approaches that have reached the clinics and outline the latest improvements of vector design and targeting. Finally, we address the pressing challenges faced by clinical trials and the direction they are heading

Dysfunction of the CaV2.1 calcium channel in cerebellar ataxias

Mutations in the CACNA1A gene are associated with episodic ataxia type 2 (EA2) and spinocerebellar ataxia type 6 (SCA6). CACNA1A encodes the α-subunit of the P/Q-type calcium channel or CaV2.1, which is highly enriched in the cerebellum. It is one of the main channels linked to synaptic transmission throughout the human central nervous system. Here, we compare recent advances in the understanding of the genetic changes that underlie EA2 and SCA6 and what these new findings suggest about the mechanism of the disease

Diagnosis of Ovarian Cancer Using Decision Tree Classification of Mass Spectral Data

Recent reports from our laboratory and others support the SELDI ProteinChip technology as a potential clinical diagnostic tool when combined with n-dimensional analyses algorithms. The objective of this study was to determine if the commercially available classification algorithm biomarker patterns software (BPS), which is based on a classification and regression tree (CART), would be effective in discriminating ovarian cancer from benign diseases and healthy controls. Serum protein mass spectrum profiles from 139 patients with either ovarian cancer, benign pelvic diseases, or healthy women were analyzed using the BPS software. A decision tree, using five protein peaks, resulted in an accuracy of 81.5% in the cross-validation analysis and 80% in a blinded set of samples in differentiating the ovarian cancer from the control groups. The potential, advantages, and drawbacks of the BPS system as a bioinformatic tool for the analysis of the SELDI high-dimensional proteomic data are discussed

A novel synaptopathy-defective synaptic vesicle protein trafficking in the mutant CHMP2B mouse model of frontotemporal dementia

Mutations in the ESCRT-III subunit CHMP2B cause frontotemporal dementia (FTD) and lead to impaired endolysosomal trafficking and lysosomal storage pathology in neurons. We investigated the effect of mutant CHMP2B on synaptic pathology, as ESCRT function was recently implicated in the degradation of synaptic vesicle (SV) proteins. We report here that expression of C-terminally truncated mutant CHMP2B results in a novel synaptopathy. This unique synaptic pathology is characterised by selective retention of presynaptic SV trafficking proteins in aged mutant CHMP2B transgenic mice, despite significant loss of postsynaptic proteins. Furthermore, ultrastructural analysis of primary cortical cultures from transgenic CHMP2B mice revealed a significant increase in the number of presynaptic endosomes, while neurons expressing mutant CHMP2B display defective SV recycling and alterations to functional SV pools. Therefore, we reveal how mutations in CHMP2B affect specific presynaptic proteins and SV recycling, identifying CHMP2B FTD as a novel synaptopathy. This novel synaptopathic mechanism of impaired SV physiology may be a key early event in multiple forms of FTD, since proteins that mediate the most common genetic forms of FTD all localise at the presynapse