Cultured Mouse Neurons Research Articles

CNTnC and fYTnC2, Genetically Encoded Green Calcium Indicators Based on Troponin C from Fast Animals

NTnC-like green fluorescent genetically encoded calcium indicators (GECIs) with two calcium ion binding sites were constructed using the insertion of truncated troponin C (TnC) from Opsanus tau into green fluorescent proteins (GFPs). These GECIs are small proteins containing the N- and C-termini of GFP; they exert a limited effect on the cellular free calcium ion concentration; and in contrast to calmodulin-based calcium indicators they lack undesired interactions with intracellular proteins in neurons. The available TnC-based NTnC or YTnC GECIs had either an inverted response and high brightness but a limited dynamic range or a positive response and fast kinetics in neurons but lower brightness and an enhanced but still limited dF/F dynamic range. Here, we solved the crystal structure of NTnC at 2.5 Å resolution. Based on this structure, we developed positive NTnC2 and inverted iNTnC2 GECIs with a large dF/F dynamic range in vitro but very slow rise and decay kinetics in neurons. To overcome their slow responsiveness, we swapped TnC from O. tau in NTnC2 with truncated troponin C proteins from the muscles of fast animals, namely, the falcon, hummingbird, cheetah, bat, rattlesnake, and ant, and then optimized the resulting constructs using directed molecular evolution. Characterization of the engineered variants using purified proteins, mammalian cells, and neuronal cultures revealed cNTnC GECI with truncated TnC from Calypte anna (hummingbird) to have the largest dF/F fluorescence response and fast dissociation kinetics in neuronal cultures. In addition, based on the insertion of truncated TnCs from fast animals into YTnC2, we developed fYTnC2 GECI with TnC from Falco peregrinus (falcon). The purified proteins cNTnC and fYTnC2 had 8- and 6-fold higher molecular brightness and 7- and 6-fold larger dF/F responses to the increase in Ca2+ ion concentration than YTnC, respectively. cNTnC GECI was also 4-fold more photostable than YTnC and fYTnC2 GECIs. Finally, we assessed the developed GECIs in primary mouse neuronal cultures stimulated with an external electric field; in these conditions, cNTnC had a 2.4-fold higher dF/F fluorescence response than YTnC and fYTnC2 and was the same or slightly slower (1.4-fold) than fYTnC2 and YTnC in the rise and decay half-times, respectively.

Huntingtin exon 1 deletion does not alter the subcellular distribution of huntingtin and gene transcription in mice.

Huntington disease (HD) is caused by the expansion of CAG triplet repeats in exon 1 of the huntingtin (HTT) gene, which also encodes the first 17 amino acids (N-17) that can modulate the toxicity of the expanded polyQ repeat. N-17 are conserved in a wide range of species and are found to influence the subcellular distribution of mutant Htt. Moreover, N-17 is subject to many posttranslational modifications that may regulate the function, stability, and distribution of HTT. However, the function of Htt exon 1 and its influence on the normal Htt remains to be fully investigated. By investigating a knock-in mouse model that lacks Htt exon1, we found that deletion of Htt exon1 does not affect the survival of mice and differentiation of cultured mouse neurons. Furthermore, the lack of Htt exon 1 does not alter the subcellular distribution of Htt, autophagy protein expression, and global gene transcription in the mouse brain. These results suggest that removing the entire exon 1 of Htt could be a therapeutic approach to eliminate expanded polyQ toxicity.

Thebromine Targets Adenosine Receptors to Control Hippocampal Neuronal Function and Damage.

Theobromine is a caffeine metabolite most abundant in dark chocolate, of which consumption is linked with a lower risk of cognitive decline. However, the mechanisms through which theobromine affects neuronal function remain ill-defined. Using electrophysiological recordings in mouse hippocampal synapses, we now characterized the impact of a realistic concentration of theobromine on synaptic transmission and plasticity. Theobromine (30 μM) facilitated synaptic transmission while decreasing the magnitude of long-term potentiation (LTP), with both effects being blunted by adenosine deaminase (2 U/mL). The pharmacological blockade of A1R with DPCPX (100 nM) eliminated the theobromine-dependent facilitation of synaptic transmission, whereas the A2AR antagonist SCH58261 (50 nM), as well as the genetic deletion of A2AR, abrogated the theobromine-induced impairment of LTP. Furthermore, theobromine prevented LTP deficits and neuronal loss, respectively, in mouse hippocampal slices and neuronal cultures exposed to Aβ1–42 peptides, considered a culprit of Alzheimer’s disease. Overall, these results indicate that theobromine affects information flow via the antagonism of adenosine receptors, normalizing synaptic plasticity and affording neuroprotection in dementia-related conditions in a manner similar to caffeine.

TFG mutation induces haploinsufficiency and drives axonal Charcot-Marie-Tooth disease by causing neurite degeneration.

TFG-related axonal Charcot-Marie-Tooth (CMT) disease is a late-onset, autosomal dominant, hereditary motor, and sensory neuropathy characterized by slowly progressive weakness and atrophy of the distal muscles. The objective of this study was to determine the common pathogenic mechanism of TFG-related CMT type 2 (CMT2) caused by different mutations and establish a direct association between TFG haploinsufficiency and neurodegeneration. Three individuals carrying the TFG p.G269V mutation but with varying disease durations were studied. The effect of the p.G269V mutation was confirmed by analyzing protein samples extracted from the blood of two individuals. The functional consequences of both CMT2 mutant gene products were evaluated in vitro. The effect of TFG deficiency in the nervous system was examined using zebrafish models and cultured mouse neurons. Overexpression of p.G269V TFG failed to enhance soluble TFG levels by generating insoluble TFG aggregates. TFG deficiency disrupted neurite outgrowth and induced neuronal apoptosis both in vivo and in vitro and further impaired locomotor capacity in zebrafish, which was consistent with the phenotype in patients. Wnt signaling was activated as a protective factor in response to TFG deficiency. CMT2-related TFG mutation induces TFG haploinsufficiency within cells and drives disease by causing progressive neurite degeneration.

Sphingosine 1-Phoshpate Receptors are Located in Synapses and Control Spontaneous Activity of Mouse Neurons in Culture

Sphingosine-1-phosphate (S1P) is best known for its roles as vascular and immune regulator. Besides, it is also present in the central nervous system (CNS) where it can act as neuromodulator via five S1P receptors (S1PRs), and thus control neurotransmitter release. The distribution of S1PRs in the active zone and postsynaptic density of CNS synapses remains unknown. In the current study, we investigated the localization of S1PR1-5 in synapses of the mouse cortex. Cortical nerve terminals purified in a sucrose gradient were endowed with all five S1PRs. Further subcellular fractionation of cortical nerve terminals revealed S1PR2 and S1PR4 immunoreactivity in the active zone of presynaptic nerve terminals. Interestingly, only S1PR2 and S1PR3 immunoreactivity was found in the postsynaptic density. All receptors were present outside the active zone of nerve terminals. Neurons in the mouse cortex and primary neurons in culture showed immunoreactivity against all five S1PRs, and Ca2+ imaging revealed that S1P inhibits spontaneous neuronal activity in a dose-dependent fashion. When testing selective agonists for each of the receptors, we found that only S1PR1, S1PR2 and S1PR4 control spontaneous neuronal activity. We conclude that S1PR2 and S1PR4 are located in the active zone of nerve terminals and inhibit neuronal activity. Future studies need to test whether these receptors modulate stimulation-induced neurotransmitter release.

Improvement of Cognitive Function in Ovariectomized Rats by Human Neural Stem Cells Overexpressing Choline Acetyltransferase via Secretion of NGF and BDNF.

Menopause is associated with memory deficits attributed to reduced serum estrogen levels. We evaluated whether an increase in brain-derived neurotrophic factor (BDNF) and nerve-growth factor (NGF) levels, through transplantation of choline acetyltransferase (ChAT)-overexpressing neural stem cells (F3.ChAT), improved learning and memory in ovariectomized rats. PD13 mouse neuronal primary culture cells were treated with estradiol or co-cultured with F3.ChAT cells; choline transporter1 (CHT1), ChAT, and vesicular acetylcholine transporter (VAChT) expression was evaluated using real-time PCR. The relationship between estrogen receptors (ERs) and neurotrophin family members was analyzed using immunohistochemistry. After the transplantation of F3.ChAT cells into OVx rats, we evaluated the memory, ACh level, and the expression of ER, neurotrophin family proteins, and cholinergic system. Estradiol upregulated CHT1, ChAT, and VAChT expression in ER; they were co-localized with BDNF, NGF, and TrkB. Co-culture with F3.ChAT upregulated CHT1, ChAT, and VAChT by activating the neurotrophin signalling pathway. Transplantation of F3.ChAT cells in OVX animals increased the ACh level in the CSF and improved memory deficit. In addition, it increased the expression of ERs, neurotrophin signaling, and the cholinergic system in the brains of OVX animals. Therefore, the estradiol deficiency induced memory loss by the down-regulation of the neurotrophin family and F3.ChAT could ameliorate the cognitive impairment owing to the loss or reduction of estradiol.

ToxCast chemical library Wnt screen identifies diethanolamine as an activator of neural progenitor proliferation.

Numerous autism spectrum disorder (ASD) risk genes are associated with Wnt signaling, suggesting that brain development may be especially sensitive to genetic perturbation of this pathway. Additionally, valproic acid, which modulates Wnt signaling, increases risk for ASD when taken during pregnancy. We previously found that an autism‐linked gain‐of‐function UBE3A T485A mutant construct hyperactivated canonical Wnt signaling, providing a genetic means to elevate Wnt signaling above baseline levels. To identify environmental use chemicals that enhance or suppress Wnt signaling, we screened the ToxCast Phase I and II libraries in cells expressing this autism‐linked UBE3A T485A gain‐of‐function mutant construct. Using structural comparisons, we identify classes of chemicals that stimulated Wnt signaling, including ethanolamines, as well as chemicals that inhibited Wnt signaling, such as agricultural pesticides, and synthetic hormone analogs. To prioritize chemicals for follow‐up, we leveraged predicted human exposure data, and identified diethanolamine (DEA) as a chemical that stimulates Wnt signaling in UBE3A T485A –transfected cells, and has a high potential for prenatal exposure in humans. DEA enhanced proliferation in primary human neural progenitor cell lines (phNPC), but did not affect expression of canonical Wnt target genes in NPCs or primary mouse neuron cultures. Instead, we found DEA increased expression of the H3K9 methylation sensitive gene CALB1, consistent with competitive inhibition of the methyl donor enzymatic pathways.

SARS-CoV-2 Infects Primary Neurons from Human ACE2 Expressing Mice and Upregulates Genes Involved in the Inflammatory and Necroptotic Pathways.

Transgenic mice expressing human angiotensin-converting enzyme 2 under the cytokeratin 18 promoter (K18-hACE2) have been extensively used to investigate the pathogenesis and tissue tropism of severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) infection. Neuroinvasion and the replication of SARS-CoV-2 within the central nervous system (CNS) of K18-hACE2 mice is associated with increased mortality; although, the mechanisms by which this occurs remain unclear. In this study, we generated primary neuronal cultures from K18-hACE2 mice to investigate the effects of a SARS-CoV-2 infection. We also evaluated the immunological response to SARS-CoV-2 infection in the CNS of K18-hACE2 mice and mouse neuronal cultures. Our data show that neuronal cultures obtained from K18-hACE2 mice are permissive to SARS-CoV-2 infection and support productive virus replication. Furthermore, SARS-CoV-2 infection upregulated the expression of genes involved in innate immunity and inflammation, including IFN-α, ISG-15, CXCL10, CCL2, IL-6 and TNF-α, in the neurons and mouse brains. In addition, we found that SARS-CoV-2 infection of neurons and mouse brains activates the ZBP1/pMLKL-regulated necroptosis pathway. Together, our data provide insights into the neuropathogenesis of SARS-CoV-2 infection in K18-hACE2 mice.

Simulated traumatic brain injury in in-vitro mouse neuronal and brain endothelial cell culture models

Traumatic brain injury can lead to fatal outcomes such as disability and death. Every year, it affects many patients all over the world. Not only the primary ischemic event, but also the subsequent reperfusion can cause severe brain injury. This so-called ischemia/reperfusion injury combined with mechanical forces lead to cellular disruption. Hence, this paper describes a special in-vitro model, mimicking traumatic brain injury by combining both hypoxia/reoxygenation and compression to simulate ischemia/reperfusion injury as well as the mechanical effects that occur concurrently when suffering traumatic brain injury. Through this approach, stroke, concussion, and traumatic brain injury can be studied on different cell lines in a simplified way. We used two primary mouse brain cell cultures, namely neurons and endothelial cells. Our results show that for the different cell types, different timelines of hypoxia and compression need to be explored to achieve the optimal amount of cellular damage in order to effectively mimic traumatic brain injury. Thus, this model will be useful to test potential treatments of brain injury in future in-vitro studies.

In Vitro and In Silico Analysis of the Residence Time of Serotonin 5-HT7 Receptor Ligands with Arylpiperazine Structure: A Structure-Kinetics Relationship Study.

During the last decade, the kinetics of drug-target interaction has received increasing attention as an important pharmacological parameter in the drug development process. Several studies have suggested that the lipophilicity of a molecule can play an important role. To date, this aspect has been studied for several G protein-coupled receptors (GPCRs) ligands but not for the 5-HT7 receptor (5-HT7R), a GPCR proposed as a valid therapeutic target in neurodevelopmental and neuropsychiatric disorders associated with abnormal neuronal connectivity. In this study, we report on structure-kinetics relationships of a set of arylpiperazine-based 5-HT7R ligands. We found that it is not the overall lipophilicity of the molecule that influences drug-target interaction kinetics but rather the position of polar groups within the molecule. Next, we performed a combination of molecular docking studies and molecular dynamics simulations to gain insights into structure-kinetics relationships. These studies did not suggest specific contact patterns between the ligands and the receptor-binding site as determinants for compounds kinetics. Finally, we compared the abilities of two 5-HT7R agonists with similar receptor-binding affinities and different residence times to stimulate the 5-HT7R-mediated neurite outgrowth in mouse neuronal primary cultures and found that the compounds induced the effect with different timing. This study provides the first insights into the binding kinetics of arylpiperazine-based 5-HT7R ligands that can be helpful to design new 5-HT7R ligands with fine-tuning of the kinetic profile.

N-Acetylcysteine Induces Apoptotic, Oxidative and Excitotoxic Neuronal Death in Mouse Cortical Cultures.

N-acetylcysteine (NAC) has been used as an antioxidant to prevent oxidative cell death. However, we found NAC itself to induce neuronal death in mouse cortical cultures. Therefore, the current study was performed to investigate the mechanism of neuronal death caused by NAC. Cell death was assessed by measuring lactate dehydrogenase efflux to bathing media after 24-48 h exposure to NAC. NAC (0.1-10 mM) induced neuronal death in a concentration- and exposure time-dependent manner. However, NAC did not injure astrocytes even at a concentration of 10 mM. Also, 10 mM NAC markedly attenuated oxidative astrocyte death induced by 0.5 mM diethyl maleate or 0.25 mM H2O2. The NMDA receptor antagonist MK-801 (10 µM) markedly attenuated the neuronal death caused by 10 mM NAC, while NBQX did not affect the neuronal death. Cycloheximide (a protein synthesis inhibitor, 0.1 µg/mL) and z-VAD-FMK (a caspase inhibitor, 100 µM) also significantly attenuated neuronal death. Apoptotic features such as chromatin condensation, nuclear fragmentation, and caspase 3 activation were observed 1 h after the NAC treatment. The neuronal death induced by 1 or 10 mM NAC was significantly attenuated by the treatment with 100 µM Trolox or 1 mM ascorbic acid. NAC induced the generation of intracellular reactive oxygen species (ROS), as measured by the fluorescent dye 2′,7′-dichlorofluorescein diacetate. The ROS generation was almost completely abolished by treatment with Trolox or ascorbic acid. These findings demonstrate that NAC can cause oxidative, apoptotic, and excitotoxic neuronal death in mouse neuronal cultures.

The neuronal retromer can regulate both neuronal and microglial phenotypes of Alzheimer's disease.

SUMMARYDisruption of retromer-dependent endosomal trafficking is considered pathogenic in late-onset Alzheimer’s disease (AD). Here, to investigate this disruption in the intact brain, we turn to a genetic mouse model where the retromer core protein VPS35 is depleted in hippocampal neurons, and then we replete VPS35 using an optimized viral vector protocol. The VPS35 depletion-repletion studies strengthen the causal link between the neuronal retromer and AD-associated neuronal phenotypes, including the acceleration of amyloid precursor protein cleavage and the loss of synaptic glutamate receptors. Moreover, the studies show that the neuronal retromer can regulate a distinct, dystrophic, microglia morphology, phenotypic of hippocampal microglia in AD. Finally, the neuronal and, in part, the microglia responses to VPS35 depletion were found to occur independent of tau. Showing that the neuronal retromer can regulate AD-associated pathologies in two of AD’s principal cell types strengthens the link, and clarifies the mechanism, between endosomal trafficking and late-onset sporadic AD.

VLDLR and ApoER2 are receptors for multiple alphaviruses

Alphaviruses, like many other arthropod-borne viruses, infect vertebrate species and insect vectors separated by hundreds of millions of years of evolutionary history. Entry into evolutionarily divergent host cells can be accomplished by recognition of different cellular receptors in different species, or by binding to receptors that are highly conserved across species. Although multiple alphavirus receptors have been described1–3, most are not shared among vertebrate and invertebrate hosts. Here we identify the very low-density lipoprotein receptor (VLDLR) as a receptor for the prototypic alphavirus Semliki forest virus. We show that the E2 and E1 glycoproteins (E2–E1) of Semliki forest virus, eastern equine encephalitis virus and Sindbis virus interact with the ligand-binding domains (LBDs) of VLDLR and apolipoprotein E receptor 2 (ApoER2), two closely related receptors. Ectopic expression of either protein facilitates cellular attachment, and internalization of virus-like particles, a VLDLR LBD–Fc fusion protein or a ligand-binding antagonist block Semliki forest virus E2–E1-mediated infection of human and mouse neurons in culture. The administration of a VLDLR LBD–Fc fusion protein has protective activity against rapidly fatal Semliki forest virus infection in mouse neonates. We further show that invertebrate receptor orthologues from mosquitoes and worms can serve as functional alphavirus receptors. We propose that the ability of some alphaviruses to infect a wide range of hosts is a result of their engagement of evolutionarily conserved lipoprotein receptors and contributes to their pathogenesis.

A new look at Alzheimer’s genetics: APP dampens neuronal excitability by regulating the size and cellular location of the axon initial segment

Early-onset forms of familial Alzheimer's disease (FAD) are usually caused by mutations in three known disease genes: the amyloid precursor protein (APP), and two presenilin genes (PSEN1 and PSEN2). APP is a single-transmembrane protein whose normal biological function is largely unknown. The congruence of FAD genetics and the presence of amyloid plaque pathology has focused attention on the APP proteolytic fragment Aβ, even while the normal function of APP remains largely unexplored. To fill this gap in our understanding, have studied the axon initial segment (AIS), a specialized region of a neuron that initiates an action potential.Immunocytochemistry of the two AIS scaffold proteins, Ankyrin G and βIV-spectrin, was performed on sections from the R1.40 mouse model of AD (B6.129-Tg(APPSw)40Btla/Mmjax) and from human frontal cortex samples (Braak Stage V-VI). We measured the length of the AIS and the intensity of their immunostaining. APP-overexpressing cultures will be recorded on the multielectrode-array (MEA) wells for electrophysiology studies RESULT: In frontal cortex of human AD cases and R1.40 mice, the AIS was shorter than in controls. In vitro, the AIS of cultured mouse neurons transfected with wild type or Swedish APP (APPSwe ) was shorter and also shifted distally along the axon. Both changes were greater with APPSwe than with wild type APP. Significantly, the addition of either Aβ oligomers or fibrils elicited no response. By transfecting neurons cultured in MEA wells, we showed that the observed shifts in the AIS altered neuronal firing patterns. We observed an overall decrease in mean spike number but not in spike amplitude.Our data suggest that the APP protein, but not the Aβ peptide, regulates neuronal activity through its effects on the position and length AIS. As APP is a neuronal stress response protein, our hypothesis is that age, trauma or genetic mutation leads to APP overexpression. While short term overexpression may protect against excitotoxicity, chronically elevated APP would permanently retard neuronal activity and serve as a substrate for dementia. This study argues for a reinterpretation of APP mutations as support for the role of the Aβ peptide in AD.

Targeting microRNA‐485‐3p blocks Alzheimer's disease progression

AbstractBackgroundAlzheimer’s disease (AD) is a form of dementia characterized by progressive memory decline and cognitive dysfunction, which affects more than 44 million people worldwide. Currently, there is no effective therapy for AD despite its increasing global incidence; thus, effective treatment strategies for AD are urgently needed. While several drugs that decrease amyloid beta (Aβ) production or increase Aβ clearance in the brain have been identified, treatment with these drugs is poorly correlated with improvements in AD severity and cognitive dysfunction.MethodExpression of miR‐485‐3p was analysed by real‐time PCR in the human frontal cortex (8 healthy controls (HC), 7 AD patients), precentral gyrus (6 HC, 8 AD), cerebrospinal fluid (CSF) (6 HC, 7 AD), plasma exosomes (10 HC, 17 mild cognitive impairment (MCI), 12 AD). Aβ 1–42 plaque immunofluorescence and tau pathology were imaged in primary cultured mouse neurons after lentivirus‐derived miR485‐3p transduction. MiR‐485‐3p antisense oligonucleotide (ASO, 1.5 μg) or control oligonucleotide formulated with the in vivo jetPEI reagent was injected into 8‐month‐old 5XFAD mice by ICV injection once weekly for two weeks. Behavioral tests were performed at 8 months and their brain pathology was examined after 8‐week‐washout at 10 months.ResultWe found that the miR‐485‐3p is overexpressed in brain tissues and CSF of AD patients, and its ASO reduces Aβ plaques, tau pathology, neuroinflammation, and cognitive decline in a transgenic mouse model of AD. Mechanistically, miR‐485‐3p ASO enhanced Aβ clearance via CD36‐mediated phagocytosis of Aβ in vitro and in vivo. We found that miR‐485‐3p ASO reduces apoptosis, which effectively decreases truncated tau levels. Further, miR‐485‐3p ASO reduced secretion of pro‐inflammatory cytokines, including IL‐1β and TNF‐α, and eventually relieved cognitive impairment.ConclusionCollectively, our findings suggest that miR‐485‐3p is a useful biomarker as well as a causative factor of the inflammatory pathophysiology of AD; furthermore, miR‐485‐3p ASO represents a candidate therapy for AD pathology and cognitive decline, establishing a new paradigm in the AD field.

A novel specific Aβ42 oligomer antibody ameliorates the neurotoxicity of patient‐derived brain extracts

AbstractBackgroundIn Alzheimer’s disease (AD), the neurotoxicity of amyloid‐β 1‐42 (Aβ42) stems mostly from soluble oligomeric aggregates of this peptide. However, the study of these aggregates has been hampered by their unstable and elusive nature, thus questioning the very definition of them as distinct targets for drug development. Here, we assess the toxicity of physiologically‐derived Aβ using extracts from deceased patients and controls in primary neuron cultures. By using a monoclonal antibody with a unique binding profile, ALZ‐201, we demonstrate that all of the Aβ toxicity observed in human‐derived brain extracts stem from soluble Aβ42 aggregates.MethodBrain tissue samples from 10 individuals were confirmed as AD (n=7) and non‐AD (n=3) cases using IHC staining for Aβ and p‐Tau aggregates. Extracts were prepared as described in Hong et al. 2018 (doi:10.1007/s00401‐018‐1846‐7) and immunodepleted with either the antibody 4G8 (removes all Aβ), ALZ‐201 (100% specific for non‐fibrillar soluble Aβ42 aggregates) or an isotype control to ALZ‐201 (does not bind any Aβ). Fractions were biochemically characterized, and toxicity assays were performed in primary mouse neuronal cultures using an automated cell imaging platform.ResultThe total Aβ content was higher in AD cases, as expected. The toxicity of the patient extracts exceeded those obtained from non‐AD cases with respect to neuronal loss and morphological determinants such as synapse density and measures of neurite complexity. Immunodepletion using 4G8 dramatically reduced Aβ in the AD patient samples and the control samples. This is in contrast to immunodepletion using ALZ‐201 or the isotype control, for which there was no detectable difference. Despite this remarkable difference in Aβ content, the therapeutic effect of depletion with 4G8 and ALZ‐201 was comparable, whereas there was no effect after depletion using the isotype control.ConclusionThe results demonstrate that very small amounts of soluble aggregated Aβ42 likely account for a large part of the toxicity in AD patients. The unique antibody, ALZ‐201, is capable of depleting these without targeting other forms of Aβ. Since this natural toxic form of Aβ is extremely low in abundance, this feature may be a critical attribute for achieving a true therapeutic effect in actual patients.

PARP-1 activation leads to cytosolic accumulation of TDP-43 in neurons

Oxidative stress in neurodegenerative disease leads to poly(ADP-ribose) polymerase 1 (PARP-1) overactivation and subsequent cell death via excessive generation of Poly(ADP-ribose) polymer (PAR). PAR binds to neurodegenerative disease linked protein TAR DNA binding protein of 43 kDa (TDP-43). However, the consequence of this interaction is not yet fully understood. TDP-43 translocates from the nucleus to the cytoplasm in response to oxidative stress, but the mechanism of stress-induced translocation remains unknown. We used N-methyl-N-nitroso-N′-nitroguanidine (MNNG) and oxygen-glucose deprivation (OGD) in mouse neuronal cultures to activate PARP-1 and observed that pharmacological inhibition of PARP-1 blocked the cytosolic translocation of TDP-43. PARP-1 inhibition is also neuroprotective against both MNNG and OGD, suggesting that PARP inhibitors could play a role in the neuroprotective role in neurodegenerative diseases involving TDP-43. Together, these data present the novel finding that TDP-43 translocation depends on PARP-1 activation and set a ground for future research of how PARP-1 activation or PAR binding to TDP-43 may facilitate its cytosolic accumulation.

Glucuronidation of Efavirenz and its Cytochrome P450‐dependent Metabolites in Brain Microsomes and Neurons

Efavirenz (EFV) is a non-nucleoside reverse transcriptase inhibitor (NNRTI) anti-HIV medication that is glucuronidated by UDP-glucuronosyl transferases (UGTs). Brain penetrant anti-HIV drugs, such as EFV, are essential to combatting HIV in the brain. Persons with HIV often suffer from neurocognitive deficits, known as HIV-associated neurocognitive disorder (HAND). In addition to HAND, EFV itself has been shown to lead to severe neurological adverse events that can result the discontinuation of EFV-based therapies. Despite the importance of this drug and the observed adverse side effects, its metabolism in the brain is virtually unknown. Furthermore, UGT expression and activity in the brain remains poorly understood. We tested whether EFV and/or its cytochrome P450-dependent metabolites –8-hydroxyefavirenz (8-OHEFV) and 8,14-dihydroxyefavirenz (8,14-diOHEFV) undergo glucuronidation in the brain. To investigate this, brain microsomes and primary mouse neurons in culture were incubated with EFV, 8-hydroxyefavirenz (8-OHEFV), or 8,14-dihydroxy efavirenz (8,14-diOHEFV). Glucuronidated metabolites were detected using ultra-high performance liquid chromatography tandem mass spectrometry (UHPLC MS/MS). Incubating pooled brain microsomes from mice, cynomolgus macaques, and humans with 10µM EFV or one of its cytochrome P450-dependent metabolites for 30 min, we observed the formation of glucuronidated metabolites of 8-OHEFV and 8,14-diOHEFV for all three species. Interestingly, the cynomolgus macaque microsomes were the only microsomes to yield the EFV N-glucuronide metabolite, indicating interspecies differences in UGT activity toward EFV. In primary C57BL6/J neurons treated with a drug concentration of 50µM for 3-4 hours, we observed glucuronidation of 8-OHEFV, but not EFV. This was commensurate with our findings using mouse brain microsomes. These data lend insight into EFV disposition in brain across humans, mice, and cynomolgus macaques, and can be leveraged to inform understanding of EFV-mediated neurotoxicity.

MicroRNA-338 inhibition protects against focal cerebral ischemia and preserves mitochondrial function in vitro in astrocytes and neurons via COX4I1

Brain-enriched microRNA-338 (miR-338) is known to play a central role in brain mitochondrial function, however the role of miR-338 in stroke injury remains unknown. This study investigated the role of miR-338 in injury from transient focal cerebral ischemia in mice, and in cell survival and mitochondrial function after in vitro ischemia in astrocyte and neuronal cultures. Pre-treatment of mice with intracerebroventricular injection of miR-338 antagomir 24 h prior to 1 h of middle cerebral artery occlusion (MCAO) significantly reduced infarct size and improved neurological score at both 24 h and 7d after injury. Levels of the miR-338 target cytochrome-c oxidase subunit 4I1 (COX4I1), which plays an essential role in maintaining brain mitochondrial ATP production, were increased in miR-338 antagomir-treated mice. Mouse primary astrocyte cell cultures subjected to glucose deprivation exhibited increased cell survival when pre-treated with miR-338 inhibitor, and greater cell death with miR-338 mimic. Decreased miR-338 levels were associated with increased ATP production, augmented cytochrome c oxidative (CcO) activity and preservation of COX4I1. In vitro protection with miR-338 inhibitor was blocked by concurrent knockdown of COX4I1 with small interfering RNA. Parallel studies in mouse neuronal N2a cultures resulted in preserved ATP content and CcO activity with miR-338 inhibition, indicating a shared miR-338-dependent response to ischemic stress between brain cell types. These results suggest that miR-338 inhibition and/or COX4I1-targeted therapies may be novel clinical strategies to protect against stroke injury via preservation of mitochondrial function in multiple cell types.

Semaphorin3F Drives Dendritic Spine Pruning Through Rho-GTPase Signaling.

Dendritic spines of cortical pyramidal neurons are initially overproduced then remodeled substantially in the adolescent brain to achieve appropriate excitatory balance in mature circuits. Here we investigated the molecular mechanism of developmental spine pruning by Semaphorin 3F (Sema3F) and its holoreceptor complex, which consists of immunoglobulin-class adhesion molecule NrCAM, Neuropilin-2 (Npn2), and PlexinA3 (PlexA3) signaling subunits. Structure-function studies of the NrCAM-Npn2 interface showed that NrCAM stabilizes binding between Npn2 and PlexA3 necessary for Sema3F-induced spine pruning. Using a mouse neuronal culture system, we identified a dual signaling pathway for Sema3F-induced pruning, which involves activation of Tiam1-Rac1-PAK1-3 -LIMK1/2-Cofilin1 and RhoA-ROCK1/2-Myosin II in dendritic spines. Inhibitors of actin remodeling impaired spine collapse in the cortical neurons. Elucidation of these pathways expands our understanding of critical events that sculpt neuronal networks and may provide insight into how interruptions to these pathways could lead to spine dysgenesis in diseases such as autism, bipolar disorder, and schizophrenia.