Tau depletion in human neurons mitigates Aß-driven toxicity.

Alzheimer's disease (AD) is an age-related neurodegenerative condition and the most common type of dementia, characterised by pathological accumulation of extracellular plaques and intracellular neurofibrillary tangles that mainly consist of amyloid-ß (Aß) and hyperphosphorylated tau aggregates, respectively. Previous studies in mouse models with a targeted knock-out of the microtubule-associated protein tau (Mapt) gene demonstrated that Aß-driven toxicity is tau-dependent. However, human cellular models with chronic tau lowering remain unexplored. In this study, we generated stable tau-depleted human induced pluripotent stem cell (iPSC) isogenic panels from two healthy individuals using CRISPR-Cas9 technology. We then differentiated these iPSCs into cortical neurons in vitro in co-culture with primary rat cortical astrocytes before conducting electrophysiological and imaging experiments for a wide range of disease-relevant phenotypes. Both AD brain derived and recombinant Aß were used in this study to elicit toxic responses from the iPSC-derived cortical neurons. We showed that tau depletion in human iPSC-derived cortical neurons caused considerable reductions in neuronal activity without affecting synaptic density. We also observed neurite outgrowth impairments in two of the tau-depleted lines used. Finally, tau depletion protected neurons from adverse effects by mitigating the impact of exogenous Aß-induced hyperactivity, deficits in retrograde axonal transport of mitochondria, and neurodegeneration. Our study established stable human iPSC isogenic panels with chronic tau depletion from two healthy individuals. Cortical neurons derived from these iPSC lines showed that tau is essential in Aß-driven hyperactivity, axonal transport deficits, and neurodegeneration, consistent with studies conducted in Mapt-/- mouse models. These findings highlight the protective effects of chronic tau lowering strategies in AD pathogenesis and reinforce the potential in clinical settings. The tau-depleted human iPSC models can now be applied at scale to investigate the involvement of tau in disease-relevant pathways and cell types.

A Regional and Projection-Specific Role of RGSz1 in the Ventrolateral Periaqueductal Grey in the Modulation of Morphine Reward.

Opioid analgesics exert their therapeutic and adverse effects by activating µ opioid receptors (MOPR); however, functional responses to MOPR activation are modulated by distinct signal transduction complexes within the brain. The ventrolateral periaqueductal gray (vlPAG) plays a critical role in modulation of nociception and analgesia, but the exact intracellular pathways associated with opioid responses in this region are not fully understood. We previously showed that knockout of the signal transduction modulator Regulator of G protein Signaling z1 (RGSz1) enhanced analgesic responses to opioids, whereas it decreased the rewarding efficacy of morphine. Here, we applied viral mediated gene transfer methodology and delivered adeno-associated virus (AAV) expressing Cre recombinase to the vlPAG of RGSz1fl\fl mice to demonstrate that downregulation of RGSz1 in this region decreases sensitivity to morphine in the place preference paradigm, under pain-free as well as neuropathic pain states. We also used retrograde viral vectors along with flippase-dependent Cre vectors to conditionally downregulate RGSz1 in vlPAG projections to the ventral tegmental area (VTA) and show that downregulation of RGSz1 prevents the development of place conditioning to low morphine doses. Consistent with the role for RGSz1 as a negative modulator of MOPR activity, RGSz1KO enhances opioid-induced cAMP inhibition in periaqueductal gray (PAG) membranes. Furthermore, using a new generation of bioluminescence resonance energy transfer (BRET) sensors, we demonstrate that RGSz1 modulates Gαz but not other Gαi family subunits and selectively impedes MOPR-mediated Gαz signaling events invoked by morphine and other opioids. Our work highlights a regional and circuit-specific role of the G protein-signaling modulator RGSz1 in morphine reward, providing insights on midbrain intracellular pathways that control addiction-related behaviors. SIGNIFICANCE STATEMENT: This study used advanced genetic mouse models to highlight the role of the signal transduction modulator named RGSz1 in responses to clinically used opioid analgesics. We show that RGSz1 controls the rewarding efficacy of opioids by actions in ventrolateral periaqueductal gray projections to the ventral tegmental area, a key component of the midbrain dopamine pathway. These studies highlight novel mechanisms by which pain-modulating structures control the rewarding efficacy of opioids.

FOXR1 regulates stress response pathways and is necessary for proper brain development.

The forkhead box (Fox) family of transcription factors are highly conserved and play essential roles in a wide range of cellular and developmental processes. We report an individual with severe neurological symptoms including postnatal microcephaly, progressive brain atrophy and global developmental delay associated with a de novo missense variant (M280L) in the FOXR1 gene. At the protein level, M280L impaired FOXR1 expression and induced a nuclear aggregate phenotype due to protein misfolding and proteolysis. RNAseq and pathway analysis showed that FOXR1 acts as a transcriptional activator and repressor with central roles in heat shock response, chaperone cofactor-dependent protein refolding and cellular response to stress pathways. Indeed, FOXR1 expression is increased in response to cellular stress, a process in which it directly controls HSPA6, HSPA1A and DHRS2 transcripts. The M280L mutant compromises FOXR1's ability to respond to stress, in part due to impaired regulation of downstream target genes that are involved in the stress response pathway. Quantitative PCR of mouse embryo tissues show Foxr1 expression in the embryonic brain. Using CRISPR/Cas9 gene editing, we found that deletion of mouse Foxr1 leads to a severe survival deficit while surviving newborn Foxr1 knockout mice have reduced body weight. Further examination of newborn Foxr1 knockout brains revealed a decrease in cortical thickness and enlarged ventricles compared to littermate wild-type mice, suggesting that loss of Foxr1 leads to atypical brain development. Combined, these results suggest FOXR1 plays a role in cellular stress response pathways and is necessary for normal brain development.

A promising chemical series of positive allosteric modulators of the µ-opioid receptor that enhance the antinociceptive efficacy of opioids but not their adverse effects.

Positive allosteric modulators (PAMs) of the µ-opioid receptor (MOR) have been proposed to exhibit therapeutic potential by maximizing the analgesic properties of clinically used opioid drugs while limiting their adverse effects or risk of overdose as a result of using lower drug doses. We herein report in vitro and in vivo characterization of two small molecules from a chemical series of MOR PAMs that exhibit: (i) MOR PAM activity and receptor subtype selectivity in vitro, (ii) a differential potentiation of the antinociceptive effect of oxycodone, morphine, and methadone in mouse models of pain that roughly correlates with in vitro activity, and (iii) a lack of potentiation of adverse effects associated with opioid administration, such as somatic withdrawal, respiratory depression, and analgesic tolerance. This series of MOR PAMs holds promise for the development of adjuncts to opioid therapy to mitigate against overdose and opioid use disorders.

Regulators of G Protein Signaling in Analgesia and Addiction.

Regulator of G protein signaling (RGS) proteins are multifunctional proteins expressed in peripheral and neuronal cells, playing critical roles in development, physiologic processes, and pharmacological responses. RGS proteins primarily act as GTPase accelerators for activated Gα subunits of G-protein coupled receptors, but they may also modulate signal transduction by several other mechanisms. Over the last two decades, preclinical work identified members of the RGS family with unique and critical roles in intracellular responses to drugs of abuse. New information has emerged on the mechanisms by which RGS proteins modulate the efficacy of opioid analgesics in a brain region- and agonist-selective fashion. There has also been progress in the understanding of the protein complexes and signal transduction pathways regulated by RGS proteins in addiction and analgesia circuits. In this review, we summarize findings on the mechanisms by which RGS proteins modulate functional responses to opioids in models of analgesia and addiction. We also discuss reports on the regulation and function of RGS proteins in models of psychostimulant addiction. Using information from preclinical studies performed over the last 20 years, we highlight the diverse mechanisms by which RGS protein complexes control plasticity in response to opioid and psychostimulant drug exposure; we further discuss how the understanding of these pathways may lead to new opportunities for therapeutic interventions in G protein pathways. SIGNIFICANCE STATEMENT: Regulator of G protein signaling (RGS) proteins are signal transduction modulators, expressed widely in various tissues, including brain regions mediating addiction and analgesia. Evidence from preclinical work suggests that members of the RGS family act by unique mechanisms in specific brain regions to control drug-induced plasticity. This review highlights interesting findings on the regulation and function of RGS proteins in models of analgesia and addiction.