Non-human primate model of amyotrophic lateral sclerosis with cytoplasmic mislocalization of TDP-43

Amyotrophic lateral sclerosis is a fatal neurodegenerative disease characterized by progressive motoneuron loss. Redistribution of transactive response deoxyribonucleic acid-binding protein 43 from the nucleus to the cytoplasm and the presence of cystatin C-positive Bunina bodies are considered pathological hallmarks of amyotrophic lateral sclerosis, but their significance has not been fully elucidated. Since all reported rodent transgenic models using wild-type transactive response deoxyribonucleic acid-binding protein 43 failed to recapitulate these features, we expected a species difference and aimed to make a non-human primate model of amyotrophic lateral sclerosis. We overexpressed wild-type human transactive response deoxyribonucleic acid-binding protein 43 in spinal cords of cynomolgus monkeys and rats by injecting adeno-associated virus vector into the cervical cord, and examined the phenotype using behavioural, electrophysiological, neuropathological and biochemical analyses. These monkeys developed progressive motor weakness and muscle atrophy with fasciculation in distal hand muscles first. They also showed regional cytoplasmic transactive response deoxyribonucleic acid-binding protein 43 mislocalization with loss of nuclear transactive response deoxyribonucleic acid-binding protein 43 staining in the lateral nuclear group of spinal cord innervating distal hand muscles and cystatin C-positive cytoplasmic aggregates, reminiscent of the spinal cord pathology of patients with amyotrophic lateral sclerosis. Transactive response deoxyribonucleic acid-binding protein 43 mislocalization was an early or presymptomatic event and was later associated with neuron loss. These findings suggest that the transactive response deoxyribonucleic acid-binding protein 43 mislocalization leads to α-motoneuron degeneration. Furthermore, truncation of transactive response deoxyribonucleic acid-binding protein 43 was not a prerequisite for motoneuronal degeneration, and phosphorylation of transactive response deoxyribonucleic acid-binding protein 43 occurred after degeneration had begun. In contrast, similarly prepared rat models expressed transactive response deoxyribonucleic acid-binding protein 43 only in the nucleus of motoneurons. There is thus a species difference in transactive response deoxyribonucleic acid-binding protein 43 pathology, and our monkey model recapitulates amyotrophic lateral sclerosis pathology to a greater extent than rodent models, providing a valuable tool for studying the pathogenesis of sporadic amyotrophic lateral sclerosis

The Autism-Related Protein PX-RICS Mediates GABAergic Synaptic Plasticity in Hippocampal Neurons and Emotional Learning in Mice

GABAergic dysfunction underlies many neurodevelopmental and psychiatric disorders. GABAergic synapses exhibit several forms of plasticity at both pre- and postsynaptic levels. NMDA receptor (NMDAR)–dependent inhibitory long-term potentiation (iLTP) at GABAergic postsynapses requires an increase in surface GABAARs through promoted exocytosis; however, the regulatory mechanisms and the neuropathological significance remain unclear. Here we report that the autism-related protein PX-RICS is involved in GABAAR transport driven during NMDAR–dependent GABAergic iLTP. Chemically induced iLTP elicited a rapid increase in surface GABAARs in wild-type mouse hippocampal neurons, but not in PX-RICS/RICS–deficient neurons. This increase in surface GABAARs required the PX-RICS/GABARAP/14–3-3 complex, as revealed by gene knockdown and rescue studies. iLTP induced CaMKII–dependent phosphorylation of PX-RICS to promote PX-RICS–14-3-3 assembly. Notably, PX-RICS/RICS–deficient mice showed impaired amygdala–dependent fear learning, which was ameliorated by potentiating GABAergic activity with clonazepam. Our results suggest that PX-RICS–mediated GABAAR trafficking is a key target for GABAergic plasticity and its dysfunction leads to atypical emotional processing underlying autism. Keywords: Autism, Inhibitory synaptic plasticity, Emotion, GABAA receptor, Trafficking, Amygdal

Development of Targeted Mass Spectrometry-Based Approaches for Quantitation of Proteins Enriched in the Postsynaptic Density (PSD)

The postsynaptic density (PSD) is a structural, electron-dense region of excitatory glutamatergic synapses, which is involved in a variety of cellular and signaling processes in neurons. The PSD is comprised of a large network of proteins, many of which have been implicated in a wide variety of neuropsychiatric disorders. Biochemical fractionation combined with mass spectrometry analyses have enabled an in-depth understanding of the protein composition of the PSD. However, the PSD composition may change rapidly in response to stimuli, and robust and reproducible methods to thoroughly quantify changes in protein abundance are warranted. Here, we report on the development of two types of targeted mass spectrometry-based assays for quantitation of PSD-enriched proteins. In total, we quantified 50 PSD proteins in a targeted, parallel reaction monitoring (PRM) assay using heavy-labeled, synthetic internal peptide standards and identified and quantified over 2100 proteins through a pre-determined spectral library using a data-independent acquisition (DIA) approach in PSD fractions isolated from mouse cortical brain tissue

PX-RICS mediates ER-to-Golgi transport of the N-cadherin/β-catenin complex

Cadherins mediate Ca2+-dependent cell–cell adhesion. Efficient export of cadherins from the endoplasmic reticulum (ER) is known to require complex formation with β-catenin. However, the molecular mechanisms underlying this requirement remain elusive. Here we show that PX-RICS, a β-catenin-interacting GTPase-activating protein (GAP) for Cdc42, mediates ER-to-Golgi transport of the N-cadherin/β-catenin complex. Knockdown of PX-RICS expression induced the accumulation of the N-cadherin/β-catenin complex in the ER and ER exit site, resulting in a decrease in cell–cell adhesion. PX-RICS was also required for ER-to-Golgi transport of the fibroblast growth factor-receptor 4 (FGFR4) associated with N-cadherin. PX-RICS-mediated ER-to-Golgi transport was dependent on its interaction with β-catenin, phosphatidylinositol-4-phosphate (PI4P), Cdc42, and its novel binding partner γ-aminobutyric acid type A receptor-associated protein (GABARAP). These results suggest that PX-RICS ensures the efficient entry of the N-cadherin/β-catenin complex into the secretory pathway, and thereby regulates the amount of N-cadherin available for cell adhesion and FGFR4-mediated signaling

The PX-RICS-14-3-3ζ/θ Complex Couples N-cadherin-β-Catenin with Dynein-Dynactin to Mediate Its Export from the Endoplasmic Reticulum*

We have recently shown that β-catenin-facilitated export of cadherins from the endoplasmic reticulum requires PX-RICS, a β-catenin-interacting GTPase-activating protein for Cdc42. Here we show that PX-RICS interacts with isoforms of 14-3-3 and couples the N-cadherin-β-catenin complex to the microtubule-based molecular motor dynein-dynactin. Similar to knockdown of PX-RICS, knockdown of either 14-3-3ζ or -θ resulted in the disappearance of N-cadherin and β-catenin from the cell-cell boundaries. Furthermore, we found that PX-RICS and 14-3-3ζ/θ are present in a large multiprotein complex that contains dynein-dynactin components as well as N-cadherin and β-catenin. Both RNAi- and dynamitin-mediated inhibition of dynein-dynactin function also led to the absence of N-cadherin and β-catenin at the cell-cell contact sites. Our results suggest that the PX-RICS-14-3-3ζ/θ complex links the N-cadherin-β-catenin cargo with the dynein-dynactin motor and thereby mediates its endoplasmic reticulum export