scholarly journals Inhibition of the autophagic protein ULK1 attenuates axonal degeneration in vitro and in vivo, enhances translation, and modulates splicing

2020 ◽  
Vol 27 (10) ◽  
pp. 2810-2827 ◽  
Author(s):  
Björn Friedhelm Vahsen ◽  
Vinicius Toledo Ribas ◽  
Jonas Sundermeyer ◽  
Alexander Boecker ◽  
Vivian Dambeck ◽  
...  
Keyword(s):  
Cortical Neurons ◽  
Axonal Degeneration ◽  
Dominant Negative ◽  
Focal Lesion ◽  
Pathological Feature ◽  
Nerve Crush ◽  
Cord Injury ◽  
Rat Cortical Neurons

Abstract Axonal degeneration is a key and early pathological feature in traumatic and neurodegenerative disorders of the CNS. Following a focal lesion to axons, extended axonal disintegration by acute axonal degeneration (AAD) occurs within several hours. During AAD, the accumulation of autophagic proteins including Unc-51 like autophagy activating kinase 1 (ULK1) has been demonstrated, but its role is incompletely understood. Here, we study the effect of ULK1 inhibition in different models of lesion-induced axonal degeneration in vitro and in vivo. Overexpression of a dominant negative of ULK1 (ULK1.DN) in primary rat cortical neurons attenuates axotomy-induced AAD in vitro. Both ULK1.DN and the ULK1 inhibitor SBI-0206965 protect against AAD after rat optic nerve crush in vivo. ULK1.DN additionally attenuates long-term axonal degeneration after rat spinal cord injury in vivo. Mechanistically, ULK1.DN decreases autophagy and leads to an mTOR-mediated increase in translational proteins. Consistently, treatment with SBI-0206965 results in enhanced mTOR activation. ULK1.DN additionally modulates the differential splicing of the degeneration-associated genes Kif1b and Ddit3. These findings uncover ULK1 as an important mediator of axonal degeneration in vitro and in vivo, and elucidate its function in splicing, defining it as a putative therapeutic target.

scholarly journals Cyclin-dependent Kinases Participate in Death of Neurons Evoked by DNA-damaging Agents

1998 ◽  
Vol 143 (2) ◽  
pp. 457-467 ◽  
Author(s):  
David S. Park ◽  
Erick J. Morris ◽  
Jaya Padmanabhan ◽  
Michael L. Shelanski ◽  
Herbert M. Geller ◽  
...  
Keyword(s):  
Cortical Neurons ◽  
Sindbis Virus ◽  
Kinase Inhibitors ◽  
Sympathetic Neurons ◽  
Dominant Negative ◽  
Cyclin Dependent Kinase ◽  
Cyclin Dependent Kinases ◽  
Dna Damaging Agents

Previous reports have indicated that DNA-damaging treatments including certain anticancer therapeutics cause death of postmitotic nerve cells both in vitro and in vivo. Accordingly, it has become important to understand the signaling events that control this process. We recently hypothesized that certain cell cycle molecules may play an important role in neuronal death signaling evoked by DNA damage. Consequently, we examined whether cyclin-dependent kinase inhibitors (CKIs) and dominant-negative (DN) cyclin-dependent kinases (CDK) protect sympathetic and cortical neurons against DNA-damaging conditions. We show that Sindbis virus–induced expression of CKIs p16ink4, p21waf/cip1, and p27kip1, as well as DN-Cdk4 and 6, but not DN-Cdk2 or 3, protect sympathetic neurons against UV irradiation– and AraC-induced death. We also demonstrate that the CKIs p16 and p27 as well as DN-Cdk4 and 6 but not DN-Cdk2 or 3 protect cortical neurons from the DNA damaging agent camptothecin. Finally, in consonance with our hypothesis and these results, cyclin D1–associated kinase activity is rapidly and highly elevated in cortical neurons upon camptothecin treatment. These results suggest that postmitotic neurons may utilize Cdk4 and 6, signals that normally control proliferation, to mediate death signaling resulting from DNA-damaging conditions.

scholarly journals Dopamine modulates the integrity of the perisynaptic extracellular matrix at excitatory synapses

2019 ◽  
Author(s):  
Jessica Mitlöhner ◽  
Rahul Kaushik ◽  
Hartmut Niekisch ◽  
Armand Blondiaux ◽  
Christine E. Gee ◽  
...  
Keyword(s):  
Extracellular Matrix ◽  
Cortical Neurons ◽  
Receptor Activation ◽  
Synaptic Activity ◽  
Excitatory Synapses ◽  
Intracellular Calcium Signaling ◽  
Rat Cortical Neurons ◽  
Synaptic Receptors

SummaryIn the brain, Hebbian-type and homeostatic forms of plasticity are affected by neuromodulators like dopamine (DA). Modifications of the perisynaptic extracellular matrix (ECM), controlling functions and mobility of synaptic receptors as well as diffusion of transmitters and neuromodulators in the extracellular space, are crucial for the manifestation of plasticity. Mechanistic links between synaptic activation and ECM modifications are largely unknown. Here, we report that neuromodulation via D1-type DA receptors can induce targeted ECM proteolysis specifically at excitatory synapses of rat cortical neurons via proteases ADAMTS-4 and -5. We show that receptor activation induces increased proteolysis of brevican (BC) and aggrecan, two major constituents of the adult ECM, in vivo and in vitro. ADAMTS immunoreactivity is detected near synapses, and shRNA-mediated knockdown reduced BC cleavage. We outline a molecular scenario how synaptic activity and neuromodulation are linked to ECM rearrangements via increased cAMP levels, NMDA receptor activation, and intracellular calcium signaling.

scholarly journals Nafamostat and sepimostat identified as novel neuroprotective agents via NR2B N-methyl-D-aspartate receptor antagonism using a rat retinal excitotoxicity model

2019 ◽  
Vol 9 (1) ◽  
Author(s):  
Masahiro Fuwa ◽  
Masaaki Kageyama ◽  
Koji Ohashi ◽  
Masaaki Sasaoka ◽  
Ryuichi Sato ◽  
...  
Keyword(s):  
Cortical Neurons ◽  
Nr2b Subunit ◽  
Neuroprotective Effects ◽  
Receptor Antagonism ◽  
Rat Brain Membranes ◽  
Rat Cortical Neurons ◽  
Orally Active ◽  
Related Compounds

AbstractIn addition to its role in the treatment of pancreatitis, the serine protease inhibitor nafamostat exhibits a retinal protective effect. However, the exact mechanisms underlying this effect are unknown. In this study, the neuroprotective effects of nafamostat and its orally active derivative sepimostat against excitotoxicity were further characterised in vitro and in vivo. In primary rat cortical neurons, nafamostat completely suppressed N-methyl-D-aspartate (NMDA)-induced cell death. Intravitreal injection of nafamostat and sepimostat protected the rat retina against NMDA-induced degeneration, whereas the structurally related compounds, gabexate and camostat, did not. The neuroprotective effects of nafamostat and the NR2B antagonist ifenprodil were remarkably suppressed by spermidine, a naturally occurring polyamine that modulates the NR2B subunit. Both nafamostat and sepimostat inhibited [3H]ifenprodil binding to fractionated rat brain membranes. Thus, nafamostat and sepimostat may exert neuroprotective effects against excitotoxic retinal degeneration through NMDA receptor antagonism at the ifenprodil-binding site of the NR2B subunit.

scholarly journals The necroptosis machinery mediates axonal degeneration in a model of Parkinson disease

2019 ◽  
Author(s):  
Maritza Oñate ◽  
Alejandra Catenaccio ◽  
Natalia Salvadores ◽  
Cristian Saquel ◽  
Alexis Martinez ◽  
...  
Keyword(s):  
Cortical Neurons ◽  
Axonal Degeneration ◽  
Neuronal Loss ◽  
Postmortem Brain ◽  
Death Process ◽  
Early Event ◽  
Genetic Ablation ◽  
Postmortem Brain Tissue

AbstractParkinson’s disease (PD) is the second most common neurodegenerative condition, characterized by motor impairment due to the progressive degeneration of dopaminergic neurons in the substantia nigra and depletion of dopamine release in the striatum. Accumulating evidence suggest that degeneration of axons is an early event in the disease, involving destruction programs that are independent of the survival of the cell soma. Necroptosis, a programmed cell death process, is emerging as a mediator of neuronal loss in models of neurodegenerative diseases. Here, we demonstrate activation of necroptosis in postmortem brain tissue from PD patients and in a toxin-based mouse model of the disease. Inhibition of key components of the necroptotic pathway resulted in a significant delay of 6-hydroxydopamine dependent axonal degeneration of dopaminergic and cortical neurons in vitro. Genetic ablation of necroptosis mediators MLKL and RIPK3, as well as pharmacological inhibition of RIPK1 in vivo, decreased dopaminergic neuron degeneration, improving motor performance. Together, these findings suggest that axonal degeneration in PD is mediated by the necroptosis machinery, a process here referred to as necroaxoptosis, a druggable pathway to target dopaminergic neuronal loss.

scholarly journals AAV-mediated inhibition of ULK1 promotes axonal regeneration in the central nervous system in vitro and in vivo

2021 ◽  
Vol 12 (2) ◽  
Author(s):  
Vinicius Toledo Ribas ◽  
Björn Friedhelm Vahsen ◽  
Lars Tatenhorst ◽  
Veronica Estrada ◽  
Vivian Dambeck ◽  
...  
Keyword(s):  
Central Nervous System ◽  
Nervous System ◽  
Axonal Regeneration ◽  
Dominant Negative ◽  
Neurological Deficits ◽  
Nerve Lesion ◽  
Cord Injury ◽  
The Central Nervous System

AbstractAxonal damage is an early step in traumatic and neurodegenerative disorders of the central nervous system (CNS). Damaged axons are not able to regenerate sufficiently in the adult mammalian CNS, leading to permanent neurological deficits. Recently, we showed that inhibition of the autophagic protein ULK1 promotes neuroprotection in different models of neurodegeneration. Moreover, we demonstrated previously that axonal protection improves regeneration of lesioned axons. However, whether axonal protection mediated by ULK1 inhibition could also improve axonal regeneration is unknown. Here, we used an adeno-associated viral (AAV) vector to express a dominant-negative form of ULK1 (AAV.ULK1.DN) and investigated its effects on axonal regeneration in the CNS. We show that AAV.ULK1.DN fosters axonal regeneration and enhances neurite outgrowth in vitro. In addition, AAV.ULK1.DN increases neuronal survival and enhances axonal regeneration after optic nerve lesion, and promotes long-term axonal protection after spinal cord injury (SCI) in vivo. Interestingly, AAV.ULK1.DN also increases serotonergic and dopaminergic axon sprouting after SCI. Mechanistically, AAV.ULK1.DN leads to increased ERK1 activation and reduced expression of RhoA and ROCK2. Our findings outline ULK1 as a key regulator of axonal degeneration and regeneration, and define ULK1 as a promising target to promote neuroprotection and regeneration in the CNS.

scholarly journals High current optogenetic channels for stimulation and inhibition of primary rat cortical neurons

2017 ◽  
Author(s):  
Lei Jin ◽  
Eike Frank Joest ◽  
Wenfang Li ◽  
Shiqiang Gao ◽  
Andreas Offenhäusser ◽  
...  
Keyword(s):  
Neural Development ◽  
Cortical Neurons ◽  
Single Channel ◽  
Chloride Concentration ◽  
Neuronal Firing ◽  
High Current ◽  
Neuronal Inhibition ◽  
Rat Cortical Neurons

AbstractChR2-XXL and GtACR1 are currently the cation and anion ends of the optogenetic single channel current range. These were used in primary rat cortical neurons in vitro to manipulate neuronal firing patterns. ChR2-XXL provides high cation currents via elevated light sensitivity and a prolonged open state. Stimulating ChR2-XXL expressing putative presynaptic neurons induced neurotransmission. Moreover, stable depolarisation block could be generated in single neurons using ChR2-XXL, proving that ChR2-XXL is a promising candidate for in vivo applications of optogenetics, for example to treat peripheral neuropathic pain. We also addressed an anion channelrhodopsin (GtACR1) for the next generation of optogenetic neuronal inhibition in primary rat cortical neurons. GtACR1‘s light-gated chloride conduction was verified in primary neurons and the efficient photoinhibition of action potentials, including spontaneous activity, was shown. Our data also implies that the chloride concentration in neurons decreases during neural development. In both cases, we find surprising applications of these high current channels. For ChR2-XXL inhibition and stimulation are possible, while for GtACR1 the role of Cl−during neural development becomes a new optogenetic target.

scholarly journals New Treatment for Alzheimer’s Disease, Kamikihito, Reverses Amyloid-β-Induced Progression of Tau Phosphorylation and Axonal Atrophy

2014 ◽  
Vol 2014 ◽  
pp. 1-10 ◽  
Author(s):  
Hidetoshi Watari ◽  
Yutaka Shimada ◽  
Chihiro Tohda
Keyword(s):  
Alzheimer’S Disease ◽  
Alzheimer's Disease ◽  
Cortical Neurons ◽  
Axonal Degeneration ◽  
Amyloid Β ◽  
Tau Phosphorylation ◽  
Axonal Atrophy ◽  
Pp2a Activity

Aims.We previously reported that kamikihito (KKT), a traditional Japanese medicine, improved memory impairment and reversed the degeneration of axons in the 5XFAD mouse model of Alzheimer’s disease (AD). However, the mechanism underlying the effects of KKT remained unknown. The aim of the present study was to investigate the mechanism by which KKT reverses the progression of axonal degeneration.Methods.Primary cultured cortical neurons were treated with amyloid beta (Aβ) fragment comprising amino acid residues (25–35) (10 μM) in anin vitroAD model. KKT (10 μg/mL) was administered to the cells before or after Aβtreatment. The effects of KKT on Aβ-induced tau phosphorylation, axonal atrophy, and protein phosphatase 2A (PP2A) activity were investigated. We also performed anin vivoassay in which KKT (500 mg/kg/day) was administered to 5XFAD mice once a day for 15 days. Cerebral cortex homogenates were used to measure PP2A activity.Results.KKT improved Aβ-induced tau phosphorylation and axonal atrophy after they had already progressed. In addition, KKT increased PP2A activityin vitroandin vivo.Conclusions.KKT reversed the progression of Aβ-induced axonal degeneration. KKT reversed axonal degeneration at least in part through its role as an exogenous PP2A stimulator.

scholarly journals Dopamine Receptor Activation Modulates the Integrity of the Perisynaptic Extracellular Matrix at Excitatory Synapses

Cells ◽  
2020 ◽  
Vol 9 (2) ◽  
pp. 260 ◽  
Author(s):  
Jessica Mitlöhner ◽  
Rahul Kaushik ◽  
Hartmut Niekisch ◽  
Armand Blondiaux ◽  
Christine E. Gee ◽  
...  
Keyword(s):  
Extracellular Matrix ◽  
Cortical Neurons ◽  
Receptor Activation ◽  
Synaptic Activity ◽  
Excitatory Synapses ◽  
Intracellular Calcium Signaling ◽  
Rat Cortical Neurons ◽  
Synaptic Receptors

In the brain, Hebbian-type and homeostatic forms of plasticity are affected by neuromodulators like dopamine (DA). Modifications of the perisynaptic extracellular matrix (ECM), which control the functions and mobility of synaptic receptors as well as the diffusion of transmitters and neuromodulators in the extracellular space, are crucial for the manifestation of plasticity. Mechanistic links between synaptic activation and ECM modifications are largely unknown. Here, we report that neuromodulation via D1-type DA receptors can induce targeted ECM proteolysis specifically at excitatory synapses of rat cortical neurons via proteases ADAMTS-4 and -5. We showed that receptor activation induces increased proteolysis of brevican (BC) and aggrecan, two major constituents of the adult ECM both in vivo and in vitro. ADAMTS immunoreactivity was detected near synapses, and shRNA-mediated knockdown reduced BC cleavage. We have outlined a molecular scenario of how synaptic activity and neuromodulation are linked to ECM rearrangements via increased cAMP levels, NMDA receptor activation, and intracellular calcium signaling.

scholarly journals Ligand-Induced GPR110 Activation Facilitates Axon Growth after Injury

2021 ◽  
Vol 22 (7) ◽  
pp. 3386
Author(s):  
Heungsun Kwon ◽  
Karl Kevala ◽  
Hu Xin ◽  
Samarjit Patnaik ◽  
Juan Marugan ◽  
...  
Keyword(s):  
Optic Nerve ◽  
Neurite Outgrowth ◽  
Nerve Injury ◽  
Cortical Neurons ◽  
Axon Growth ◽  
Neurite Growth ◽  
Nerve Crush ◽  
Developing Neurons

Recovery from axonal injury is extremely difficult, especially for adult neurons. Here, we demonstrate that the activation of G-protein coupled receptor 110 (GPR110, ADGRF1) is a mechanism to stimulate axon growth after injury. N-docosahexaenoylethanolamine (synaptamide), an endogenous ligand of GPR110 that promotes neurite outgrowth and synaptogenesis in developing neurons, and a synthetic GPR110 ligand stimulated neurite growth in axotomized cortical neurons and in retinal explant cultures. Intravitreal injection of GPR110 ligands following optic nerve crush injury promoted axon extension in adult wild-type, but not in gpr110 knockout, mice. In vitro axotomy or in vivo optic nerve injury rapidly induced the neuronal expression of gpr110. Activating the developmental mechanism of neurite outgrowth by specifically targeting GPR110 that is upregulated upon injury may provide a novel strategy for stimulating axon growth after nerve injury in adults.

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