Copper induces neuron-sparing, ferredoxin 1-independent astrocyte toxicity mediated by oxidative stress.

Copper is an essential enzyme cofactor in oxidative metabolism, anti-oxidant defenses, and neurotransmitter synthesis. However, intracellular copper, when improperly buffered, can also lead to cell death. Given the growing interest in the use of copper in the presence of the ionophore elesclomol (CuES) for the treatment of gliomas, we investigated the effect of this compound on the surround parenchyma-namely neurons and astrocytes in vitro. Here, we show that astrocytes were highly sensitive to CuES toxicity while neurons were surprisingly resistant, a vulnerability profile that is opposite of what has been described for zinc and other toxins. Bolstering these findings, a human astrocytic cell line was similarly sensitive to CuES. Modifications of cellular metabolic pathways implicated in cuproptosis, a form of copper-regulated cell death, such as inhibition of mitochondrial respiration or knock-down of ferredoxin 1 (FDX1), did not block CuES toxicity to astrocytes. CuES toxicity was also unaffected by inhibitors of apoptosis, necrosis or ferroptosis. However, we did detect the presence of lipid peroxidation products in CuES-treated astrocytes, indicating that oxidative stress is a mediator of CuES-induced glial toxicity. Indeed, treatment with anti-oxidants mitigated CuES-induced cell death in astrocytes indicating that oxidative stress is a mediator of CuES-induced glial toxicity. Lastly, prior induction of metallothioneins 1 and 2 in astrocytes with zinc plus pyrithione was strikingly protective against CuES toxicity. As neurons express high levels of metallothioneins basally, these results may partially account for their resistance to CuES toxicity. These results demonstrate a unique toxic response to copper in glial cells which contrasts with the cell selectivity profile of zinc, another biologically relevant metal.

Microglial reprogramming by Hv1 antagonism protects neurons from inflammatory and glutamate toxicity.

Although the precise mechanisms determining the neurotoxic or neuroprotective activation phenotypes in microglia remain poorly characterized, metabolic changes in these cells appear critical for these processes. As cellular metabolism can be tightly regulated by changes in intracellular pH, we tested whether pharmacological targeting of the microglial voltage-gated proton channel 1 (Hv1), an important regulator of intracellular pH, is critical for activated microglial reprogramming. Using a mouse microglial cell line and mouse primary microglia cultures, either alone, or co-cultured with rat cerebrocortical neurons, we characterized in detail the microglial activation profile in the absence and presence of Hv1 inhibition. We observed that activated microglia neurotoxicity was mainly attributable to the release of tumor necrosis factor alpha, reactive oxygen species, and zinc. Strikingly, pharmacological inhibition of Hv1 largely abrogated inflammatory neurotoxicity not only by reducing the production of cytotoxic mediators but also by promoting neurotrophic molecule production and restraining excessive phagocytic activity. Importantly, the Hv1-sensitive change from a pro-inflammatory to a neuroprotective phenotype was associated with metabolic reprogramming, particularly via a boost in NADH availability and a reduction in lactate. Most critically, Hv1 antagonism not only reduced inflammatory neurotoxicity but also promoted microglia-dependent neuroprotection against a separate excitotoxic injury. Our results strongly suggest that Hv1 blockers may provide an important therapeutic tool against a wide range of inflammatory neurodegenerative disorders.

Intracellular zinc signaling influences NMDA receptor function by enhancing the interaction of ZnT1 with GluN2A.

Zinc, loaded into glutamate-containing presynaptic vesicles and released into the synapse in an activity-dependent manner, modulates neurotransmission through its actions on postsynaptic targets, prominently via high-affinity inhibition of GluN2A-containing NMDA receptors. Recently, we identified a postsynaptic transport mechanism that regulates endogenous zinc inhibition of NMDARs. In this new model of zinc regulation, the postsynaptic transporter ZnT1 mediates zinc inhibition of NMDARs by binding to GluN2A. Through this interaction, ZnT1, a transporter that moves zinc from the cytoplasm to the extracellular domain, generates a zinc microdomain that modulates NMDAR-mediated neurotransmission. As ZnT1 expression is transcriptionally driven by the metal-responsive transcription factor 1 (MTF-1), we found that intracellular zinc strongly drives MTF-1 in cortical neurons in vitro and increases the number of GluN2A-ZnT1 interactions, thereby enhancing tonic zinc inhibition of NMDAR-mediated currents. Importantly, this effect is absent when the interaction between GluN2A and ZnT1 is disrupted by a cell-permeable peptide. These results suggest that zinc-regulated gene expression can dynamically regulate NMDAR-mediated synaptic processes.

NUP62 localizes to ALS/FTLD pathological assemblies and contributes to TDP-43 insolubility.

A G4C2 hexanucleotide repeat expansion in the C9orf72 gene is the most common genetic cause of ALS and FTLD (C9-ALS/FTLD) with cytoplasmic TDP-43 inclusions observed in regions of neurodegeneration. The accumulation of repetitive RNAs and dipeptide repeat protein (DPR) are two proposed mechanisms of toxicity in C9-ALS/FTLD and linked to impaired nucleocytoplasmic transport. Nucleocytoplasmic transport is regulated by the phenylalanine-glycine nucleoporins (FG nups) that comprise the nuclear pore complex (NPC) permeability barrier. However, the relationship between FG nups and TDP-43 pathology remains elusive. Our studies show that nuclear depletion and cytoplasmic mislocalization of one FG nup, NUP62, is linked to TDP-43 mislocalization in C9-ALS/FTLD iPSC neurons. Poly-glycine arginine (GR) DPR accumulation initiates the formation of cytoplasmic RNA granules that recruit NUP62 and TDP-43. Cytoplasmic NUP62 and TDP-43 interactions promotes their insolubility and NUP62:TDP-43 inclusions are frequently found in C9orf72 ALS/FTLD as well as sporadic ALS/FTLD postmortem CNS tissue. Our findings indicate NUP62 cytoplasmic mislocalization contributes to TDP-43 proteinopathy in ALS/FTLD.

Local translation in primary afferents and its contribution to pain.

ABSTRACT: Chronic pain remains a significant problem due to its prevalence, impact, and limited therapeutic options. Progress in addressing chronic pain is dependent on a better understanding of underlying mechanisms. Although the available evidence suggests that changes within the central nervous system contribute to the initiation and maintenance of chronic pain, it also suggests that the primary afferent plays a critical role in all phases of the manifestation of chronic pain in most of those who suffer. Most notable among the changes in primary afferents is an increase in excitability or sensitization. A number of mechanisms have been identified that contribute to primary afferent sensitization with evidence for both increases in pronociceptive signaling molecules, such as voltage-gated sodium channels, and decreases in antinociceptive signaling molecules, such as voltage-dependent or calcium-dependent potassium channels. Furthermore, these changes in signaling molecules seem to reflect changes in gene expression as well as posttranslational processing. A mechanism of sensitization that has received far less attention, however, is the local or axonal translation of these signaling molecules. A growing body of evidence indicates that this process not only is dynamically regulated but also contributes to the initiation and maintenance of chronic pain. Here, we review the biology of local translation in primary afferents and its relevance to pain pathobiology.

Imprecision in Precision Medicine: Differential Response of a Disease-Linked GluN2A Mutant to NMDA Channel Blockers.

Mutations in N-methyl-d-aspartate receptors (NMDAR) subunits have been implicated in a growing number of human neurodevelopmental disorders. Previously, a de novo mutation in GRIN2A, encoding the GluN2A subunit, was identified in a patient with severe epilepsy and developmental delay. This missense mutation, which leads to GluN2A-P552R, produces significant dendrotoxicity in transfected rodent cortical neurons, as evidenced by pronounced dendritic blebbing. This injurious process can be prevented by treatment with the NMDA antagonist memantine. Given the increasing use of FDA approved NMDA antagonists to treat patients with GRIN mutations, who may have seizures refractory to traditional anti-epileptic drugs, we investigated whether additional NMDA antagonists were effective in attenuating neurotoxicity associated with GluN2A-P552R expression. Intriguingly, we found that while treatment with memantine can effectively block GluN2A-P552R-mediated dendrotoxicity, treatment with ketamine does not, despite the fact that both drugs work as open NMDAR channel blockers. Interestingly, we found that neurons expressing GluN2A-P552R were more vulnerable to an excitotoxic insult-an effect that, in this case, could be equally rescued by both memantine and ketamine. These findings suggest that GluN2A-P552R induced dendrotoxicity and increased vulnerability to excitotoxic stress are mediated through two distinct mechanisms. The differences between memantine and ketamine in halting GluN2A-P552R dendrotoxicity could not be explained by NMDA antagonist induced changes in MAP or Src kinase activation, previously shown to participate in NMDA-induced excitotoxicity. Our findings strongly suggest that not all NMDA antagonists may be of equal clinical utility in treating GRIN2A-mediated neurological disorders, despite a shared mechanism of action.

The Multifaceted Roles of Zinc in Neuronal Mitochondrial Dysfunction.

Zinc is a highly abundant cation in the brain, essential for cellular functions, including transcription, enzymatic activity, and cell signaling. However, zinc can also trigger injurious cascades in neurons, contributing to the pathology of neurodegenerative diseases. Mitochondria, critical for meeting the high energy demands of the central nervous system (CNS), are a principal target of the deleterious actions of zinc. An increasing body of work suggests that intracellular zinc can, under certain circumstances, contribute to neuronal damage by inhibiting mitochondrial energy processes, including dissipation of the mitochondrial membrane potential (MMP), leading to ATP depletion. Additional consequences of zinc-mediated mitochondrial damage include reactive oxygen species (ROS) generation, mitochondrial permeability transition, and excitotoxic calcium deregulation. Zinc can also induce mitochondrial fission, resulting in mitochondrial fragmentation, as well as inhibition of mitochondrial motility. Here, we review the known mechanisms responsible for the deleterious actions of zinc on the organelle, within the context of neuronal injury associated with neurodegenerative processes. Elucidating the critical contributions of zinc-induced mitochondrial defects to neurotoxicity and neurodegeneration may provide insight into novel therapeutic targets in the clinical setting.

Pneumomyelia Secondary to Interlaminar Cervical Epidural Injection Causing Acute Cord Injury with Transient Quadriparesis.

BACKGROUND: Cervical radiculopathy and cervicalgia are commonly managed with spinal epidural steroid injections in the outpatient setting. Although cervical epidural injections are routinely performed, there is potential for significant complications if proper technique and safety measures are not followed. Spinal cord infarction and stroke following transforaminal injection have been described in the literature, whereas interlaminar injections have been associated with both epidural hematomas and direct cord injury. CASE DESCRIPTION: Here we describe a case of pneumomyelia after cervical interlaminar epidural steroid injection resulting in acute quadriparesis. The patient's symptoms were caused by an inadvertent puncture of the cervical cord and injection of air present in the needle or syringe via an interlaminar approach. The initial computed tomography imaging showed a slit-like lesion at C7-T2 with density consistent with air that migrated rostrally on a follow-up scan. CONCLUSIONS: Epidural steroid injections are often the treatment of choice in management of neck pain and cervical radiculopathy. Devastating complications can ensue if proper safety measures and technique are not used during the procedure regardless of the approach used.

Infection of arachnoid cyst associated with vasospasm and stroke in a pediatric patient: case report.

Arachnoid cysts are relatively common and benign intraarachnoid membrane outpouchings containing CSF-like fluid. The majority of arachnoid cysts remain stable and asymptomatic and do not require intervention in the pediatric population. Here, the authors present the first reported case of an infected arachnoid cyst in a pediatric patient resulting in severe vasospasm of the left terminal internal carotid artery, left A1 segment, and left M1 branches with a left middle cerebral artery infarct. Their experience suggests that close monitoring is warranted for this condition and that the pediatric population may be at higher risk for vasospasm.

Angiotensin II mediates the axonal trafficking of tyrosine hydroxylase and dopamine ß-hydroxylase mRNAs and enhances norepinephrine synthesis in primary sympathetic neurons.

In the sympatho-adrenal system, angiotensin II (Ang II) acts as a key neuromodulatory component. At sympathetic nerve terminals, Ang II influences sympathetic transmission by enhancing norepinephrine (NE) synthesis, facilitating NE release and inhibiting NE uptake. Previously, it was demonstrated that tyrosine hydroxylase (TH) mRNA is trafficked to the distal axons of primary superior cervical ganglia (SCG) neurons, directed by a cis-acting regulatory element (i.e. zipcode) located in the 3'UTR of the transcript. Results of metabolic labeling studies established that the mRNA is locally translated. It was further shown that the axonal trafficking of the mRNA encoding the enzyme plays an important role in mediating dopamine (DA) and NE synthesis and may facilitate the maintenance of axonal catecholamine levels. In the present study, the hypothesis was tested that Ang II induces NE synthesis in rat primary SCG neurons via the modulation of the trafficking of the mRNAs encoding the catecholamine synthesizing enzymes TH and dopamine ß-hydroxylase (DBH). Treatment of SCG neurons with the Ang II receptor type 1 (AT1R) agonist, L-162,313, increases the axonal levels of TH and DBH mRNA and protein and results in elevated NE levels. Conversely, treatment of rat SCG neurons with the AT1R antagonist, Eprosartan, abolished the L-162,313-mediated increase in axonal levels of TH and DBH mRNA and protein. In a first attempt to identify the proteins involved in the Ang II-mediated axonal transport of TH mRNA, we used a biotinylated 50-nucleotide TH RNA zipcode as bait in the affinity purification of TH zipcode-associated proteins. Mass spectrometric analysis of the TH zipcode ribonucleoprotein (RNP) complex immune-purified from SCG neurons led to the identification of 163 somal and 127 axonal proteins functionally involved in binding nucleic acids, the translational machinery or acting as subunits of cytoskeletal and motor proteins. Surprisingly, immune-purification of the TH axonal trafficking complex, results in the acquisition of DBH mRNA, suggesting that these mRNAs maybe transported to the axon together, possibly in the same RNP complex. Taken together, our results point to a novel mechanism by which Ang II participates in the regulation of axonal synthesis of NE by modulating the local trafficking and expression of TH and DBH, two key enzymes involved in the catecholamine biosynthetic pathway.

Kappa Opioid Receptor Distribution and Function in Primary Afferents.

Primary afferents are known to be inhibited by kappa opioid receptor (KOR) signaling. However, the specific types of somatosensory neurons that express KOR remain unclear. Here, using a newly developed KOR-cre knockin allele, viral tracing, single-cell RT-PCR, and ex vivo recordings, we show that KOR is expressed in several populations of primary afferents: a subset of peptidergic sensory neurons, as well as low-threshold mechanoreceptors that form lanceolate or circumferential endings around hair follicles. We find that KOR acts centrally to inhibit excitatory neurotransmission from KOR-cre afferents in laminae I and III, and this effect is likely due to KOR-mediated inhibition of Ca2+ influx, which we observed in sensory neurons from both mouse and human. In the periphery, KOR signaling inhibits neurogenic inflammation and nociceptor sensitization by inflammatory mediators. Finally, peripherally restricted KOR agonists selectively reduce pain and itch behaviors, as well as mechanical hypersensitivity associated with a surgical incision. These experiments provide a rationale for the use of peripherally restricted KOR agonists for therapeutic treatment.

Nuclear-Encoded Mitochondrial mRNAs: A Powerful Force in Axonal Growth and Development.

Axons, their growth cones, and synaptic nerve terminals are neuronal subcompartments that have high energetic needs. As such, they are enriched in mitochondria, which supply the ATP necessary to meet these demands. To date, a heterogeneous population of nuclear-encoded mitochondrial mRNAs has been identified in distal axons and growth cones. Accumulating evidence suggests that the local translation of these mRNAs is required for mitochondrial maintenance and axonal viability. Here, we review evidence that suggests a critical role for axonal translation of nuclear-encoded mitochondrial mRNAs in axonal growth and development. Additionally, we explore the role that site-specific translation at the mitochondria itself may play in this process. Finally, we briefly review the clinical implications of dysregulation of local translation of mitochondrial-related mRNAs in neurodevelopmental disorders.

A heterogeneous population of nuclear-encoded mitochondrial mRNAs is present in the axons of primary sympathetic neurons.

Mitochondria are enriched in subcellular regions of high energy consumption, such as axons and pre-synaptic nerve endings. Accumulating evidence suggests that mitochondrial maintenance in these distal structural/functional domains of the neuron depends on the "in-situ" translation of nuclear-encoded mitochondrial mRNAs. In support of this notion, we recently provided evidence for the axonal targeting of several nuclear-encoded mRNAs, such as cytochrome c oxidase, subunit 4 (COXIV) and ATP synthase, H+ transporting and mitochondrial Fo complex, subunit C1 (ATP5G1). Furthermore, we showed that axonal trafficking and local translation of these mRNAs plays a critical role in the generation of axonal ATP. Using a global gene expression analysis, this study identified a highly diverse population of nuclear-encoded mRNAs that were enriched in the axon and presynaptic nerve terminals. Among this population of mRNAs, fifty seven were found to be at least two-fold more abundant in distal axons, as compared with the parental cell bodies. Gene ontology analysis of the nuclear-encoded mitochondrial mRNAs suggested functions for these gene products in molecular and biological processes, including but not limited to oxidoreductase and electron carrier activity and proton transport. Based on these results, we postulate that local translation of nuclear-encoded mitochondrial mRNAs present in the axons may play an essential role in local energy production and maintenance of mitochondrial function.

Axonal localization and mitochondrial association of precursor microRNA 338.

MicroRNAs (miRNAs) selectively localize to subcompartments of the neuron, such as dendrites, axons, and presynaptic terminals, where they regulate the local protein synthesis of their putative target genes. In addition to mature miRNAs, precursor miRNAs (pre-miRNAs) have also been shown to localize to somatodendritic and axonal compartments. miRNA-338 (miR-338) regulates the local expression of several nuclear-encoded mitochondrial mRNAs within axons of sympathetic neurons. Previous work has shown that precursor miR-338 (pre-miR-338) introduced into the axon can locally be processed into mature miR-338, where it can regulate local ATP synthesis. However, the mechanisms underlying the localization of pre-miRNAs to the axonal compartment remain unknown. In this study, we investigated the axonal localization of pre-miR-338. Using proteomic and biochemical approaches, we provide evidence for the localization of pre-miR-338 to distal neuronal compartments and identify several constituents of the pre-miR-338 ribonucleoprotein complex. Furthermore, we found that pre-miR-338 is associated with the mitochondria in axons of superior cervical ganglion (SCG) neurons. The maintenance of mitochondrial function within axons requires the precise spatiotemporal synthesis of nuclear-encoded mRNAs, some of which are regulated by miR-338. Therefore, the association of pre-miR-338 with axonal mitochondria could serve as a reservoir of mature, biologically active miRNAs, which could coordinate the intra-axonal expression of multiple nuclear-encoded mitochondrial mRNAs.