Carboxypeptidase E Independently Changes Microtubule Glutamylation, Dendritic Branching, and Neuronal Migration

Neuronal migration and dendritogenesis are dependent on dynamic changes to the microtubule (MT) network. Among various factors that regulate MT dynamics and stability, post-translational modifications (PTMs) of MTs play a critical role in conferring specificity of regulatory protein binding to MTs. Thus, it is important to understand the regulation of PTMs during brain development as multiple developmental processes are dependent on MTs. In this study, we identified that carboxypeptidase E (CPE) changes tubulin polyglutamylation, a major PTM in the brain, and we examine the impact of CPE-mediated changes to polyglutamylation on cortical neuron migration and dendrite morphology. We show, for the first time, that overexpression of CPE increases the level of polyglutamylated α-tubulin while knockdown decreases the level of polyglutamylation. We also demonstrate that CPE-mediated changes to polyglutamylation are dependent on the CPE zinc-binding motif and that this motif is necessary for CPE action on p150Glued localization. However, overexpression of a CPE mutant that does not increase MT glutamylation mimics the effects of overexpression of wild type CPE on dendrite branching. Furthermore, although overexpression of wild type CPE does not alter cortical neuron migration, overexpression of the mutant may act in a dominant-negative manner as it decreases the number of neurons that reach the cortical plate (CP), as we previously reported for CPE knockdown. Overall, our data suggest that CPE changes MT glutamylation and redistribution of p150Glued and that this function of CPE is independent of its role in shaping dendrite development but plays a partial role in regulating cortical neuron migration.

Dendritic Polyglycerol Amine: An Enhanced Substrate to Support Long-Term Neural Cell Culture

Long-term stable cell culture is a critical tool to better understand cell function. Most adherent cell culture models require a polymer substrate coating of poly-lysine or poly-ornithine for the cells to adhere and survive. However, polypeptide-based substrates are degraded by proteolysis and it remains a challenge to maintain healthy cell cultures for extended periods of time. Here, we report the development of an enhanced cell culture substrate based on a coating of dendritic polyglycerol amine (dPGA), a non-protein macromolecular biomimetic of poly-lysine, to promote the adhesion and survival of neurons in cell culture. We show that this new polymer coating provides enhanced survival, differentiation and long-term stability for cultures of primary neurons or neurons derived from human induced pluripotent stem cells (hiPSCs). Atomic force microscopy analysis provides evidence that greater nanoscale roughness contributes to the enhanced capacity of dPGA-coated surfaces to support cells in culture. We conclude that dPGA is a cytocompatible, functionally superior, easy to use, low cost and highly stable alternative to poly-cationic polymer cell culture substrate coatings such as poly-lysine and poly-ornithine. Summary statement Here, we describe a novel dendritic polyglycerol amine-based substrate coating, demonstrating superior performance compared to current polymer coatings for long-term culture of primary neurons and neurons derived from induced pluripotent stem cells.

Optimization of Long-Term Human iPSC-Derived Spinal Motor Neuron Culture Using a Dendritic Polyglycerol Amine-Based Substrate

Human induced pluripotent stem cells (hiPSCs) derived from healthy and diseased individuals can give rise to many cell types, facilitating the study of mechanisms of development, human disease modeling, and early drug target validation. In this context, experimental model systems based on hiPSC-derived motor neurons (MNs) have been used to study MN diseases such as spinal muscular atrophy and amyotrophic lateral sclerosis. Modeling MN disease using hiPSC-based approaches requires culture conditions that can recapitulate in a dish the events underlying differentiation, maturation, aging, and death of MNs. Current hiPSC-derived MN-based applications are often hampered by limitations in our ability to monitor MN morphology, survival, and other functional properties over a prolonged timeframe, underscoring the need for improved long-term culture conditions. Here we describe a cytocompatible dendritic polyglycerol amine (dPGA) substrate-based method for prolonged culture of hiPSC-derived MNs. We provide evidence that MNs cultured on dPGA-coated dishes are more amenable to long-term study of cell viability, molecular identity, and spontaneous network electrophysiological activity. The present study has the potential to improve hiPSC-based studies of human MN biology and disease. We describe the use of a new coating substrate providing improved conditions for long-term cultures of human iPSC-derived motor neurons, thus allowing evaluation of cell viability, molecular identity, spontaneous network electrophysiological activity, and single-cell RNA sequencing of mature motor neurons.

Oxytocin Modulation of Maternal Behavior and Its Association With Immunological Activity in Rats With Cesarean Delivery

Oxytocin (OT), a neuropeptide produced in the supraoptic (SON) and paraventricular (PVN) nuclei, is not only essential for lactation and maternal behavior but also for normal immunological activity. However, mechanisms underlying OT regulation of maternal behavior and its association with immunity around parturition, particularly under mental and physical stress, remain unclear. Here, we observed effects of OT on maternal behavior in association with immunological activity in rats after cesarean delivery (CD), a model of reproductive stress. CD significantly reduced maternal interests to the pups throughout postpartum day 1-8. On postpartum day 5, CD decreased plasma OT levels and thymic index but increased vasopressin, interleukin (IL)-1β, IL-6 and IL-10 levels. CD had no significant effect on plasma adrenocorticotropic hormone and corticosterone levels. In the hypothalamus, CD decreased corticotropin-releasing hormone contents in the PVN but increased OT contents in the PVN and SON and OT release from hypothalamic implants. CD also increased c-Fos expression, particularly in the cytoplasm of OT neurons. Lastly, CD depolarized resting membrane potential and increased spike width while increasing the variability of the firing rate of OT neurons in brain slices. Thus, CD can increase hypothalamic OT contents and release but reduce pituitary release of OT into the blood, which is associated with depressive-like maternal behavior, increased inflammatory cytokine release and decreased relative weight of the thymus.

Restoration of Noradrenergic Function in Parkinson’s Disease Model Mice

Dysfunction of the central noradrenergic and dopaminergic systems is the primary neurobiological characteristic of Parkinson’s disease (PD). Importantly, neuronal loss in the locus coeruleus (LC) that occurs in early stages of PD may accelerate progressive loss of dopaminergic neurons. Therefore, restoring the activity and function of the deficient noradrenergic system may be an important therapeutic strategy for early PD. In the present study, the lentiviral constructions of transcription factors Phox2a/2b, Hand2 and Gata3, either alone or in combination, were microinjected into the LC region of the PD model VMAT2 Lo mice at 12 and 18 month age. Biochemical analysis showed that microinjection of lentiviral expression cassettes into the LC significantly increased mRNA levels of Phox2a, and Phox2b, which were accompanied by parallel increases of mRNA and proteins of dopamine β-hydroxylase (DBH) and tyrosine hydroxylase (TH) in the LC. Furthermore, there was considerable enhancement of DBH protein levels in the frontal cortex and hippocampus, as well as enhanced TH protein levels in the striatum and substantia nigra. Moreover, these manipulations profoundly increased norepinephrine and dopamine concentrations in the striatum, which was followed by a remarkable improvement of the spatial memory and locomotor behavior. These results reveal that over-expression of these transcription factors in the LC improves noradrenergic and dopaminergic activities and functions in this rodent model of PD. It provides the necessary groundwork for the development of gene therapies of PD, and expands our understanding of the link between the LC-norepinephrine and dopamine systems during the progression of PD.

The Role of NLRP3 Inflammasome in Cerebrovascular Diseases Pathology and Possible Therapeutic Targets

Cerebrovascular diseases are pathological conditions involving impaired blood flow in the brain, primarily including ischaemic stroke, intracranial haemorrhage, and subarachnoid haemorrhage. The nucleotide-binding and oligomerisation (NOD) domain-like receptor (NLR) family pyrin domain (PYD)-containing 3 (NLRP3) inflammasome is a protein complex and a vital component of the immune system. Emerging evidence has indicated that the NLRP3 inflammasome plays an important role in cerebrovascular diseases. The function of the NLRP3 inflammasome in the pathogenesis of cerebrovascular diseases remains an interesting field of research. In this review, we first summarised the pathological mechanism of cerebrovascular diseases and the pathological mechanism of the NLRP3 inflammasome in aggravating atherosclerosis and cerebrovascular diseases. Second, we outlined signalling pathways through which the NLRP3 inflammasome participates in aggravating or mitigating cerebrovascular diseases. Reactive oxygen species (ROS)/nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB), ROS/thioredoxin-interacting protein (TXNIP) and purinergic receptor-7 (P2X7R) signalling pathways can activate the NLRP3 inflammasome; activation of the NLRP3 inflammasome can aggravate cerebrovascular diseases by mediating apoptosis and pyroptosis. Autophagy/mitochondrial autophagy, nuclear factor E2-related factor-2 (Nrf2), interferon (IFN)-β, sirtuin (SIRT), and phosphoinositide 3-kinase (PI3K)/protein kinase B (AKT) reportedly alleviate cerebrovascular diseases by inhibiting NLRP3 inflammasome activation. Finally, we explored specific inhibitors of the NLRP3 inflammasome based on the two-step activation of the NLRP3 inflammasome, which can be developed as new drugs to treat cerebrovascular diseases.

Microglial- and Astrocyte-Specific Expression of Purinergic Signaling Components and Inflammatory Mediators in the Rat Hippocampus During Trimethyltin-Induced Neurodegeneration

The present study examined the involvement of purinergic signaling components in the rat model of hippocampal degeneration induced by trimethyltin (TMT) intoxication (8 mg/kg, single intraperitoneal injection), which results in behavioral and neurological dysfunction similar to neurodegenerative disorders. We investigated spatial and temporal patterns of ecto-nucleoside triphosphate diphosphohydrolase 1 (NTPDase1/CD39) and ecto-5′ nucleotidase (eN/CD73) activity, their cell-specific localization, and analyzed gene expression pattern and/or cellular localization of purinoreceptors and proinflammatory mediators associated with reactive glial cells. Our study demonstrated that all Iba1+ cells at the injured area, irrespective of their morphology, upregulated NTPDase1/CD39, while induction of eN/CD73 has been observed at amoeboid Iba1+ cells localized within the hippocampal neuronal layers with pronounced cell death. Marked induction of P2Y12R, P2Y6R, and P2X4-messenger RNA at the early stage of TMT-induced neurodegeneration might reflect the functional properties, migration, and chemotaxis of microglia, while induction of P2X7R at amoeboid cells probably modulates their phagocytic role. Reactive astrocytes expressed adenosine A1, A2A, and P2Y1 receptors, revealed induction of complement component C3, inducible nitric oxide synthase, nuclear factor-kB, and proinflammatory cytokines at the late stage of TMT-induced neurodegeneration. An increased set of purinergic system components on activated microglia (NTPDase1/CD39, eN/CD73, and P2X7) and astrocytes (A1R, A2AR, and P2Y1), and loss of homeostatic glial and neuronal purinergic pathways (P2Y12 and A1R) may shift purinergic signaling balance toward excitotoxicity and inflammation, thus favoring progression of pathological events. These findings may contribute to a better understanding of the involvement of purinergic signaling components in the progression of neurodegenerative disorders that could be target molecules for the development of novel therapies.

17β-Estradiol Attenuates Intracerebral Hemorrhage-Induced Blood–Brain Barrier Injury and Oxidative Stress Through SRC3-Mediated PI3K/Akt Signaling Pathway in a Mouse Model

Estrogen is neuroprotective in brain injury models, and steroid receptor cofactor 3 (SRC3) mediates estrogen signaling. We aimed to investigate whether and how SRC3 is involved in the neuroprotective effects of 17ß-estradiol (E2) in a mouse model of intracerebral hemorrhage (ICH). Ovariectomized female mice were treated with E2 after autologous blood injection-induced ICH. Brain damage was assessed by neurological deficit score, brain water content, and oxidative stress levels. Blood–brain barrier (BBB) integrity was evaluated by Evan's blue extravasation and claudin-5, ZO-1, and occludin levels. SRC3 expression and PI3K/Akt signaling pathway were examined in ICH mice treated with E2. The effect of SRC3 on E2-mediated neuroprotection was determined by examining neurological outcomes in SRC3-deficient mice undergone ICH and E2 treatment. We found that E2 alleviated ICH-induced brain edema and neurological deficits, protected BBB integrity, and suppressed oxidative stress. E2 enhanced SRC3 expression and PI3K-/Akt signaling pathway. SRC3 deficiency abolished the protective effects of E2 on ICH-induced neurological deficits, brain edema, and BBB integrity. Our results suggest that E2 suppresses ICH-induced brain injury and SRC3 plays a critical role in E2-mediated neuroprotection.

Identification of miRNAs That Mediate Protective Functions of Anti-Cancer Drugs During White Matter Ischemic Injury

We have previously shown that two anti-cancer drugs, CX-4945 and MS-275, protect and preserve white matter (WM) architecture and improve functional recovery in a model of WM ischemic injury. While both compounds promote recovery, CX-4945 is a selective Casein kinase 2 (CK2) inhibitor and MS-275 is a selective Class I histone deacetylase (HDAC) inhibitor. Alterations in microRNAs (miRNAs) mediate some of the protective actions of these drugs. In this study, we aimed to (1) identify miRNAs expressed in mouse optic nerves (MONs); (2) determine which miRNAs are regulated by oxygen glucose deprivation (OGD); and (3) determine the effects of CX-4945 and MS-275 treatment on miRNA expression. RNA isolated from MONs from control and OGD-treated animals with and without CX-4945 or MS-275 treatment were quantified using NanoString nCounter® miRNA expression profiling. Comparative analysis of experimental groups revealed that 12 miRNAs were expressed at high levels in MONs. OGD upregulated five miRNAs (miR-1959, miR-501-3p, miR-146b, miR-201, and miR-335-3p) and downregulated two miRNAs (miR-1937a and miR-1937b) compared to controls. OGD with CX-4945 upregulated miR-1937a and miR-1937b, and downregulated miR-501-3p, miR-200a, miR-1959, and miR-654-3p compared to OGD alone. OGD with MS-275 upregulated miR-2134, miR-2141, miR-2133, miR-34b-5p, miR-153, miR-487b, miR-376b, and downregulated miR-717, miR-190, miR-27a, miR-1959, miR-200a, miR-501-3p, and miR-200c compared to OGD alone. Interestingly, miR-501-3p and miR-1959 were the only miRNAs upregulated by OGD, and downregulated by OGD plus CX-4945 and MS-275. Therefore, we suggest that protective functions of CX-4945 or MS-275 against WM injury maybe mediated, in part, through miRNA expression.