The Stimulatory Effects of Intracellular α-Synuclein on Synaptic Transmission Are Attenuated by 2-Octahydroisoquinolin-2(1H)-ylethanamine

α-Synuclein (αSyn) species can be detected in synaptic boutons, where they play a crucial role in the pathogenesis of Parkinson’s Disease (PD). However, the effects of intracellular αSyn species on synaptic transmission have not been thoroughly studied. Here, using patch-clamp recordings in hippocampal neurons, we report that αSyn oligomers (αSynO), intracellularly delivered through the patch electrode, produced a fast and potent effect on synaptic transmission, causing a substantial increase in the frequency, amplitude and transferred charge of spontaneous synaptic currents. We also found an increase in the frequency of miniature synaptic currents, suggesting an effect located at the presynaptic site of the synapsis. Furthermore, our in silico approximation using docking analysis and molecular dynamics simulations showed an interaction between a previously described small anti-amyloid beta (Aβ) molecule, termed M30 (2-octahydroisoquinolin-2(1H)-ylethanamine), with a central hydrophobic region of αSyn. In line with this finding, our empirical data aimed to obtain oligomerization states with thioflavin T (ThT) and Western blot (WB) indicated that M30 interfered with αSyn aggregation and decreased the formation of higher-molecular-weight species. Furthermore, the effect of αSynO on synaptic physiology was also antagonized by M30, resulting in a decrease in the frequency, amplitude, and charge transferred of synaptic currents. Overall, the present results show an excitatory effect of intracellular αSyn low molecular-weight species, not previously described, that are able to affect synaptic transmission, and the potential of a small neuroactive molecule to interfere with the aggregation process and the synaptic effect of αSyn, suggesting that M30 could be a potential therapeutic strategy for synucleinopathies.

Interleukin-13 and its receptor are synaptic proteins involved in plasticity and neuroprotection

Immune system molecules are expressed by neurons, often for unknown functions. We have identified IL-13 and its receptor IL-13Ra1 as neuronal, synaptic proteins in mouse, rat, and human brains, whose engagement upregulates the phosphorylation of NMDAR and AMPAR subunits and, in turn, increases synaptic activity and CREB-mediated transcription. We demonstrate that increased IL-13 is a hallmark of traumatic brain injury (TBI) in mice as well as in two distinct cohorts of human patients. We also provide evidence that IL-13 upregulation protects neurons from excitotoxic death. We show IL-13 upregulation occurring in several cohorts of human brain samples and in CSF. Thus, IL-13 is a previously unrecognized physiological modulator of synaptic physiology of neuronal origin, with implications for the establishment of synaptic plasticity and the survival of neurons under injury conditions. Furthermore, we suggest that the neuroprotection afforded through the upregulation of IL-13 represents a new entry point for interventions in the pathophysiology of TBI.

The Cholinergic Lateral Line Efferent Synapse: Structural, Functional and Molecular Similarities With Those of the Cochlea

Vertebrate hair cell (HC) systems are innervated by efferent fibers that modulate their response to external stimuli. In mammals, the best studied efferent-HC synapse, the cholinergic medial olivocochlear (MOC) efferent system, makes direct synaptic contacts with HCs. The net effect of MOC activity is to hyperpolarize HCs through the activation of α9α10 nicotinic cholinergic receptors (nAChRs) and the subsequent activation of Ca2+-dependent SK2 potassium channels. A serious obstacle in research on many mammalian sensory systems in their native context is that their constituent neurons are difficult to access even in newborn animals, hampering circuit observation, mapping, or controlled manipulation. By contrast, fishes and amphibians have a superficial and accessible mechanosensory system, the lateral line (LL), which circumvents many of these problems. LL responsiveness is modulated by efferent neurons which aid to distinguish between external and self-generated stimuli. One component of the LL efferent system is cholinergic and its activation inhibits LL afferent activity, similar to what has been described for MOC efferents. The zebrafish (Danio rerio) has emerged as a powerful model system for studying human hearing and balance disorders, since LL HC are structurally and functionally analogous to cochlear HCs, but are optically and pharmacologically accessible within an intact specimen. Complementing mammalian studies, zebrafish have been used to gain significant insights into many facets of HC biology, including mechanotransduction and synaptic physiology as well as mechanisms of both hereditary and acquired HC dysfunction. With the rise of the zebrafish LL as a model in which to study auditory system function and disease, there has been an increased interest in studying its efferent system and evaluate the similarity between mammalian and piscine efferent synapses. Advances derived from studies in zebrafish include understanding the effect of the LL efferent system on HC and afferent activity, and revealing that an α9-containing nAChR, functionally coupled to SK channels, operates at the LL efferent synapse. In this review, we discuss the tools and findings of these recent investigations into zebrafish efferent-HC synapse, their commonalities with the mammalian counterpart and discuss several emerging areas for future studies.

HuD Regulates mRNA-circRNA-miRNA Networks in the Mouse Striatum Linked to Neuronal Development and Drug Addiction

The RNA-binding protein HuD (a.k.a., ELAVL4) is involved in neuronal development and synaptic plasticity mechanisms, including addiction-related processes such as cocaine conditioned-place preference (CPP) and food reward. The most studied function of this protein is mRNA stabilization; however, we have recently shown that HuD also regulates the levels of circular RNAs (circRNAs) in neurons. To examine the role of HuD in the control of coding and non-coding RNA networks associated with substance use, we identified sets of differentially expressed mRNAs, circRNAs and miRNAs in the striatum of HuD knockout (KO) mice. Our findings indicate that significantly downregulated mRNAs are enriched in biological pathways related to cell morphology and behavior. Furthermore, deletion of HuD altered the levels of 15 miRNAs associated with drug seeking. Using these sets of data, we predicted that a large number of upregulated miRNAs form competing endogenous RNA (ceRNA) networks with circRNAs and mRNAs associated with the neuronal development and synaptic plasticity proteins LSAMP and MARK3. Additionally, several downregulated miRNAs form ceRNA networks with mRNAs and circRNAs from MEF2D, PIK3R3, PTRPM and other neuronal proteins. Together, our results indicate that HuD regulates ceRNA networks controlling the levels of mRNAs associated with neuronal differentiation and synaptic physiology.

Hippocampal astroglial hypertrophy in mice subjected to early life maternal deprivation

Early-life stress (ELS), including chronic deprivation of maternal care, exerts persistent life-long effects on animal physiology and behavior, and is associated with several neurodevelopmental disorders. Long-lasting changes in neuronal plasticity and electrophysiology are documented extensively in the animal models of ELS. However, the role of astroglia in the lasting effects of ELS remains elusive. Astrocytes are intricately involved in the regulation of synaptic physiology and behavior. Moreover, astrocytes play a major role in the innate and adaptive immune responses in the central nervous system (CNS). The role of immune responses and neuroinflammation in the altered brain development and persistent adverse effects of ELS are beginning to be explored. Innate immune response in the CNS is characterized by a phenomenon called astrogliosis, a process in which astrocytes undergo hypertrophy, along with changes in gene expression and function. While the immune activation and neuroinflammatory changes concomitant with ELS, or in juveniles and young adults have been reported, it is unclear whether mice subjected to ELS exhibit astrogliosis-like alterations well into late-adulthood. Here, we subjected mice to maternal separation from postnatal day 2 to day 22 and performed comprehensive morphometric analysis of hippocampal astrocytes during late-adulthood. We found that the astrocytes in the stratum radiatum region of the CA1 hippocampal subfield from maternally separated mice exhibit significant hypertrophy as late as 8 months of age, revealing the crucial changes in astrocytes that manifest long after the cessation of ELS. This study highlights the persistence of neuroinflammatory changes in mice exposed to ELS.

Disruption of Drosophila larval muscle structure and function by UNC45 knockdown

Background Proper muscle function is heavily dependent on highly ordered protein complexes. UNC45 is a USC (named since this region is shared by three proteins UNC45/CRO1/She4P) chaperone that is necessary for myosin incorporation into the thick filaments. UNC45 is expressed throughout the entire Drosophila life cycle and it has been shown to be important during late embryogenesis when initial muscle development occurs. However, the effects of UNC45 manipulation at later developmental times, after muscle development, have not yet been studied. Main results UNC45 was knocked down with RNAi in a manner that permitted survival to the pupal stage, allowing for characterization of sarcomere organization in the well-studied third instar larvae. Second harmonic generation (SHG) microscopy revealed changes in the striated pattern of body wall muscles as well as a reduction of signal intensity. This observation was confirmed with immunofluorescence and electron microscopy imaging, showing diminished UNC45 signal and disorganization of myosin and z-disk proteins. Concomitant alterations in both synaptic physiology and locomotor function were also found. Both nerve-stimulated response and spontaneous vesicle release were negatively affected, while larval movement was impaired. Conclusions This study highlights the dependency of normal sarcomere structure on UNC45 expression. We confirm the known role of UNC45 for myosin localization and further show the I-Z-I complex is also disrupted. This suggests a broad need for UNC45 to maintain sarcomere integrity. Newly discovered changes in synaptic physiology reveal the likely presence of a homeostatic response to partially maintain synaptic strength and muscle function.

Recent Advances in Synaptosomal Proteomics in Alzheimer’s Disease

: The current meta-analysis of the cohort review was designed to elucidate the progress made in neuroproteomics of the synaptosome. The association of the comprehensive synaptic proteome and its link to physiological or pathological settings is rapidly mounting. Chemical synapses in the brain are focal hot spots for interneuronal signaling, signal transduction, and plasticity. Structurally, synapses comprise axon termini or the presynapse (vesicles filled with neurotransmitters that function as molecular signals), synaptic clefts (extracellular matrix and adhesion molecules), and post-synaptic density or PSD (with receptors for neurotransmitters that rely upon the chemical signaling). The pre- and post-synaptic clefts are responsible for mediating and regulating neurotransmitter release Their receptor binding and perception rely on chemical signals. Moreover, short- and long-term structural and functional alterations that are necessary for the optimal higher-order brain functions are also mainly dependent on the protein dynamics at the synapses. Not surprisingly, disruptions in synaptic physiology are considered as the major pathogenic mechanisms underlying the progression of several neurodegenerative disorders, including Alzheimer's disease. This review briefly discusses the subcellular fractionation protocols and the related biochemical approaches for the isolation of synaptic compartments. Besides, it discusses the progress made in understanding the pathological alterations in the synaptic proteome in neurodegenerative disorders, particularly focussing on Alzheimer's disease dementia.

NMDAR-dependent long-term depression is associated with increased short term plasticity through autophagy mediated loss of PSD-95

Long-term depression (LTD) of synaptic strength can take multiple forms and contribute to circuit remodeling, memory encoding or erasure. The generic term LTD encompasses various induction pathways, including activation of NMDA, mGlu or P2X receptors. However, the associated specific molecular mechanisms and effects on synaptic physiology are still unclear. We here compare how NMDAR- or P2XR-dependent LTD affect synaptic nanoscale organization and function in rodents. While both LTDs are associated with a loss and reorganization of synaptic AMPARs, only NMDAR-dependent LTD induction triggers a profound reorganization of PSD-95. This modification, which requires the autophagy machinery to remove the T19-phosphorylated form of PSD-95 from synapses, leads to an increase in AMPAR surface mobility. We demonstrate that these post-synaptic changes that occur specifically during NMDAR-dependent LTD result in an increased short-term plasticity improving neuronal responsiveness of depressed synapses. Our results establish that P2XR- and NMDAR-mediated LTD are associated to functionally distinct forms of LTD.

Chemical Stimulation of Rodent and Human Cortical Synaptosomes: Implications in Neurodegeneration

Synaptic plasticity events, including long-term potentiation (LTP), are often regarded as correlates of brain functions of memory and cognition. One of the central players in these plasticity-related phenomena is the α-amino-3-hydroxy-5-methylisoxazole-4-propionate receptor (AMPAR). Increased levels of AMPARs on postsynaptic membranes thus constitute a biochemical measure of LTP. Isolated synaptic terminals (synaptosomes) are an excellent ex vivo tool to monitor synaptic physiology in healthy and diseased brains, particularly in human research. We herein describe three protocols for chemically-induced LTP (cLTP) in synaptosomes from both rodent and human brain tissues. Two of these chemical stimulation protocols are described for the first time in synaptosomes. A pharmacological block of synaptosomal actin dynamics confirmed the efficiency of the cLTP protocols. Furthermore, the study prototypically evaluated the deficiency of cLTP in cortical synaptosomes obtained from human cases of early-onset Alzheimer’s disease (EOAD) and frontotemporal lobar degeneration (FLTD), as well as an animal model that mimics FLTD.

A novel mitragynine analog with low efficacy mu-opioid receptor agonism displays antinociception with attenuated adverse effects

Dried kratom leaves are anecdotally used for the treatment of pain, opioid dependence, and alcohol use disorder. We have previously shown that kratom and its natural products (mitragynine) and semi-synthetic analogs (7-hydroxy mitragynine (7OH) and mitragynine pseudoindoxyl) are mu opioid receptor (MOR) agonists that show minimal beta-arrestin2 recruitment. To further investigate the structure activity relationships of G-protein potency, efficacy, and beta-arrestin2 recruitment, we diversified the mitragynine/7OH templates at the C9, -10 and -12 positions of the aromatic ring of the indole moiety. Three lead C9 analogs, synthesized by swapping the 9-methoxy group with varied substituents, namely phenyl (SC11), methyl (SC12), 3-furanyl (SC13), were further characterized using a panel of in vitro and ex vivo electrophysiology assays. All three compounds were partial agonists with lower efficacy than both DAMGO and morphine in heterologous G-protein assays and synaptic physiology. SC11-13 also showed lower recruitment of both β-arrestin subtypes compared to DAMGO, and in assays with limited MOR receptor reserve, the G-protein efficacy of SC11, SC12 and SC13 was comparable to buprenorphine. In mouse models, at equianalgesic doses SC13 showed MOR-dependent analgesia with potency similar to morphine without respiratory depression, hyperlocomotion, constipation, or place conditioning. Taken together, these results suggest that MOR agonists with a G-protein efficacy profile similar to buprenorphine can be developed into opioids that are effective analgesics with greatly reduced liabilities.