AraC interacts with p75NTR transmembrane domain to induce cell death of mature neurons.

Cytosine arabinoside (AraC) is one of the main therapeutic treatments for several types of cancer, including acute myeloid leukaemia. However, after a high-dose AraC chemotherapy regime, patients develop severe neurotoxicity and cell death in the central nervous system leading to cerebellar ataxia, dysarthria, nystagmus, somnolence and drowsiness. AraC induces apoptosis in dividing cells. However, the mechanism by which it leads to neurite degeneration and cell death in mature neurons remains unclear. We hypothesise that the upregulation of the death receptor p75NTR is responsible for AraC-mediated neurodegeneration and cell death in leukaemia patients undergoing AraC treatment. To determine the role of AraC-p75NTR signalling in the cell death of mature neurons, we used mature cerebellar granule neurons' primary cultures from p75NTR knockout and p75NTRCys259 mice. Evaluation of neurite degeneration, cell death and p75NTR signalling was done by immunohistochemistry and immunoblotting. To assess the interaction between AraC and p75NTR, we performed cellular thermal shift and AraTM assays as well as Homo-FRET anisotropy imaging. We show that AraC induces neurite degeneration and programmed cell death of mature cerebellar granule neurons in a p75NTR-dependent manner. Mechanistically, Proline 252 and Cysteine 256 residues facilitate AraC interaction with the transmembrane domain of p75NTR resulting in uncoupling of p75NTR from the NFκB survival pathway. This, in turn, exacerbates the activation of the cell death/JNK pathway by recruitment of TRAF6 to p75NTR. Our findings identify p75NTR as a novel molecular target to develop treatments for counteract AraC-mediated cell death of mature neurons.

Convergent dopamine and ALK4 signaling to PCBP1 controls FosB alternative splicing and cocaine behavioral sensitization.

ΔfosB is an alternatively spliced product of the FosB gene that is essential for dopamine-induced reward pathways and that acts as a master switch for addiction. However, the molecular mechanisms of its generation and regulation by dopamine signaling are unknown. Here, we report that dopamine D1 receptor signaling synergizes with the activin/ALK4/Smad3 pathway to potentiate the generation of ΔFosB mRNA in medium spiny neurons (MSNs) of the nucleus accumbens (NAc) via activation of the RNA-binding protein PCBP1, a regulator of mRNA splicing. Concurrent activation of PCBP1 and Smad3 by D1 and ALK4 signaling induced their interaction, nuclear translocation, and binding to sequences in exon-4 and intron-4 of FosB mRNA. Ablation of either ALK4 or PCBP1 in MSNs impaired ΔFosB mRNA induction and nuclear translocation of ΔFosB protein in response to repeated co-stimulation of D1 and ALK4 receptors. Finally, ALK4 is required in NAc MSNs of adult mice for behavioral sensitization to cocaine. These findings uncover an unexpected mechanism for ΔFosB generation and drug-induced sensitization through convergent dopamine and ALK4 signaling.

Adult medial habenula neurons require GDNF receptor GFRα1 for synaptic stability and function.

The medial habenula (mHb) is an understudied small brain nucleus linking forebrain and midbrain structures controlling anxiety and fear behaviors. The mechanisms that maintain the structural and functional integrity of mHb neurons and their synapses remain unknown. Using spatiotemporally controlled Cre-mediated recombination in adult mice, we found that the glial cell-derived neurotrophic factor receptor alpha 1 (GFRα1) is required in adult mHb neurons for synaptic stability and function. mHb neurons express some of the highest levels of GFRα1 in the mouse brain, and acute ablation of GFRα1 results in loss of septohabenular and habenulointerpeduncular glutamatergic synapses, with the remaining synapses displaying reduced numbers of presynaptic vesicles. Chemo- and optogenetic studies in mice lacking GFRα1 revealed impaired circuit connectivity, reduced AMPA receptor postsynaptic currents, and abnormally low rectification index (R.I.) of AMPARs, suggesting reduced Ca2+ permeability. Further biochemical and proximity ligation assay (PLA) studies defined the presence of GluA1/GluA2 (Ca2+ impermeable) as well as GluA1/GluA4 (Ca2+ permeable) AMPAR complexes in mHb neurons, as well as clear differences in the levels and association of AMPAR subunits with mHb neurons lacking GFRα1. Finally, acute loss of GFRα1 in adult mHb neurons reduced anxiety-like behavior and potentiated context-based fear responses, phenocopying the effects of lesions to septal projections to the mHb. These results uncover an unexpected function for GFRα1 in the maintenance and function of adult glutamatergic synapses and reveal a potential new mechanism for regulating synaptic plasticity in the septohabenulointerpeduncular pathway and attuning of anxiety and fear behaviors.

ALK4 coordinates extracellular and intrinsic signals to regulate development of cortical somatostatin interneurons.

Although the role of transcription factors in fate specification of cortical interneurons is well established, how these interact with extracellular signals to regulate interneuron development is poorly understood. Here we show that the activin receptor ALK4 is a key regulator of the specification of somatostatin interneurons. Mice lacking ALK4 in GABAergic neurons of the medial ganglionic eminence (MGE) showed marked deficits in distinct subpopulations of somatostatin interneurons from early postnatal stages of cortical development. Specific losses were observed among distinct subtypes of somatostatin+/Reelin+ double-positive cells, including Hpse+ layer IV cells targeting parvalbumin+ interneurons, leading to quantitative alterations in the inhibitory circuitry of this layer. Activin-mediated ALK4 signaling in MGE cells induced interaction of Smad2 with SATB1, a transcription factor critical for somatostatin interneuron development, and promoted SATB1 nuclear translocation and repositioning within the somatostatin gene promoter. These results indicate that intrinsic transcriptional programs interact with extracellular signals present in the environment of MGE cells to regulate cortical interneuron specification.

Correction: MAG induces apoptosis in cerebellar granule neurons through p75NTR demarcating granule layer/white matter boundary.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

MAG induces apoptosis in cerebellar granule neurons through p75NTR demarcating granule layer/white matter boundary.

MAG (Myelin-associated glycoprotein) is a type I transmembrane glycoprotein expressed by Schwann cells and oligodendrocytes, that has been implicated in the control of axonal growth in many neuronal populations including cerebellar granule neurons (CGNs). However, it is unclear whether MAG has other functions in central nervous system, in particular, in cerebellar development and patterning. We find that MAG expression in the cerebellum is compartmentalised resulting in increased MAG protein levels in the cerebellar white matter. MAG induces apoptosis in developing CGNs through p75NTR signalling. Deletion of p75NTR in vivo reduced the number of apoptotic neurons in cerebellar white matter during development leading to reduction in the size of white matter in the adulthood. Furthermore, we show that MAG impairs CGNs neurite outgrowth as consequence of MAG-induced apoptosis in CGNs. Mechanistically, we find that MAG/NgR1-induced cell death is dependent of p75NTR-mediated activation of JNK/cell death signalling pathway. Together, these findings identify the mechanisms by which MAG induces CGNs apoptotic activity, a crucial event that facilitates cerebellar layer refinement during development.

RIP2 Gates TRAF6 Interaction with Death Receptor p75NTR to Regulate Cerebellar Granule Neuron Survival.

Cerebellar granule neurons (CGNs) undergo programmed cell death during the first postnatal week of mouse development, coincident with sustained expression of the death receptor p75NTR. Although ablation of p75NTR does not affect CGN cell death, deletion of the downstream effector RIP2 significantly increases CGN apoptosis, resulting in reduced adult CGN number and impaired behaviors associated with cerebellar function. Remarkably, CGN death is restored to basal levels when p75NTR is deleted in RIP2-deficient mice. We find that RIP2 gates the signaling output of p75NTR by competing with TRAF6 for binding to the receptor intracellular domain. In CGNs lacking RIP2, more TRAF6 is associated with p75NTR, leading to increased JNK-dependent apoptosis. In agreement with this, pharmacological inhibition or genetic ablation of TRAF6 restores cell death levels in CGNs lacking RIP2. These results reveal an unexpected mechanism controlling CGN number and highlight how competitive interactions govern the logic of death receptor function.

Cell-autonomous role of GFRα1 in the development of olfactory bulb GABAergic interneurons.

GFRα1, a receptor for glial cell line-derived neurotrophic factor (GDNF), is critical for the development of the main olfactory system. The olfactory bulb (OB) of Gfra1 knockout mice shows significant reductions in the number of olfactory sensory neurons, mitral and tufted cells, as well as all major classes of OB GABAergic interneurons. However, the latter do not express significant levels of GFRα1, leaving the mechanism of action of GFRα1 in OB interneuron development unexplained. Here we report that GFRα1 is highly expressed in the precursor cells that give rise to all major classes of OB interneurons, but is downregulated as these neurons mature. Conditional ablation of GFRα1 in embryonic GABAergic cells recapitulated the cell losses observed in global Gfra1 knockouts at birth. GFRα1 was also required for the sustained generation and allocation of OB interneurons in adulthood. Conditional loss of GFRα1 altered the migratory behaviour of neuroblasts along the rostral migratory stream (RMS) as well as RMS glial tunnel formation. Together, these data indicate that GFRα1 functions cell-autonomously in subpopulations of OB interneuron precursors to regulate their generation and allocation in the mammalian OB.

GPR55: A therapeutic target for Parkinson's disease?

The GPR55 receptor is expressed abundantly in the brain, especially in the striatum, suggesting it might fulfill a role in motor function. Indeed, motor behavior is impaired in mice lacking GPR55, which also display dampened inflammatory responses. Abnormal-cannabidiol (Abn-CBD), a synthetic cannabidiol (CBD) isomer, is a GPR55 agonist that may serve as a therapeutic agent in the treatment of inflammatory diseases. In this study, we explored whether modulating GPR55 could also represent a therapeutic approach for the treatment of Parkinson's disease (PD). The distribution of GPR55 mRNA was first analyzed by in situ hybridization, localizing GPR55 transcripts to neurons in brain nuclei related to movement control, striatum, globus pallidus, subthalamic nucleus, substantia nigra and cortex. Striatal expression of GPR55 was downregulated in parkinsonian conditions. When Abn-CBD and CBD (5 mg/kg) were chronically administered to mice treated over 5 weeks with 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine and probenecid (MPTPp), Abn-CBD but not CBD prevented MPTPp induced motor impairment. Although Abn-CBD protected dopaminergic cell bodies, it failed to prevent degeneration of the terminals or preserve dopamine levels in the striatum. Both compounds induced morphological changes in microglia that were compatible with an anti-inflammatory phenotype that did not correlate with a neuroprotective activity. The symptomatic relief of Abn-CBD was further studied in the haloperidol-induced catalepsy mouse model. Abn-CBD had an anti-cataleptic effect that was reversed by CBD and PSB1216, a newly synthesized GPR55 antagonist, and indeed, two other GPR55 agonists also displayed anti-cataleptic effects (CID1792197 and CID2440433). These results demonstrate for the first time that activation of GPR55 might be beneficial in combating PD.

Fatty acid amide hydrolase inhibition for the symptomatic relief of Parkinson's disease.

Elements of the endocannabinoid system are strongly expressed in the basal ganglia where they suffer profound rearrangements after dopamine depletion. Modulation of the levels of the endocannabinoid 2-arachidonoyl-glycerol by inhibiting monoacylglycerol lipase alters glial phenotypes and provides neuroprotection in a mouse model of Parkinson's disease. In this study, we assessed whether inhibiting fatty acid amide hydrolase could also provide beneficial effects on the time course of this disease. The fatty acid amide hydrolase inhibitor, URB597, was administered chronically to mice treated with 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine and probenecid (MPTPp) over 5weeks. URB597 (1mg/kg) prevented MPTPp induced motor impairment but it did not preserve the dopamine levels in the nigrostriatal pathway or regulate glial cell activation. The symptomatic relief of URB597 was confirmed in haloperidol-induced catalepsy assays, where its anti-cataleptic effects were both blocked by antagonists of the two cannabinoid receptors (CB1 and CB2), and abolished in animals deficient in these receptors. Other fatty acid amide hydrolase inhibitors, JNJ1661010 and TCF2, also had anti-cataleptic properties. Together, these results demonstrate an effect of fatty acid amide hydrolase inhibition on the motor symptoms of Parkinson's disease in two distinct experimental models that is mediated by cannabinoid receptors.

Alternatively activated microglia and macrophages in the central nervous system.

Macrophages are important players in the fight against viral, bacterial, fungal and parasitic infections. From a resting state they may undertake two activation pathways, the classical known as M1, or the alternative known as M2. M1 markers are mostly mediators of pro-inflammatory responses whereas M2 markers emerge for resolution and cleanup. Microglia exerts in the central nervous system (CNS) a function similar to that of macrophages in the periphery. Microglia activation and proliferation occurs in almost any single pathology affecting the CNS. Often microglia activation has been considered detrimental and drugs able to stop microglia activation were considered for the treatment of a variety of diseases. Cumulative evidence shows that microglia may undergo the alternative activation pathway, express M2-type markers and contribute to neuroprotection. This review focuses on details about the role of M2 microglia and in the approaches available for its identification. Approaches to drive the M2 phenotype and data on its potential in CNS diseases are also reviewed.

Monoacylglycerol lipase inhibitor JZL184 is neuroprotective and alters glial cell phenotype in the chronic MPTP mouse model.

Changes in cannabinoid receptor expression and concentration of endocannabinoids have been described in Parkinson's disease; however, it remains unclear whether they contribute to, or result from, the disease process. To evaluate whether targeting the endocannabinoid system could provide potential benefits in the treatment of the disease, the effect of a monoacylglycerol lipase inhibitor that prevents degradation of 2-arachidonyl-glycerol was tested in mice treated chronically with probenecid and 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTPp). Chronic administration of the compound, JZL184 (8 mg/kg), prevented MPTPp-induced motor impairment and preserved the nigrostriatal pathway. Furthermore, none of the hypokinetic effects associated with cannabinoid receptor agonism were observed. In the striatum and substantia nigra pars compacta, MPTPp animals treated with JZL184 exhibited astroglial and microglial phenotypic changes that were accompanied by increases in TGFß messenger RNA expression and in glial cell-derived neurotrophic factor messenger RNA and protein levels. JZL184 induced an increase in ß-catenin translocation to the nucleus, implicating the Wnt/catenin pathway. Together, these results demonstrate a potent neuroprotective effect of JZL184 on the nigrostriatal pathway of parkinsonian animals, likely involving restorative astroglia and microglia activation and the release of neuroprotective and antiinflammatory molecules.

Loss of parvalbumin-positive neurons from the globus pallidus in animal models of Parkinson disease.

The external segment of the globus pallidus (GPe) in humans and the equivalent structure in rodents, the globus pallidus (GP), influence signal processing in the basal ganglia under normal and pathological conditions. Parvalbumin (PV) immunoreactivity defines 2 main neuronal subpopulations in the GP/GPe: PV-immunopositive cells that project mainly to the subthalamic nucleus and the internal segment of the GP and PV-negative cells that mainly project to the striatum. We evaluated the number of neurons in the GP/GPe in animal models of Parkinson disease. In rats, dopaminergic denervation with 6-hydroxydopamine (6-OHDA) provoked a significant decrease in the number of GP neurons (12% ± 4%, p < 0.05), which specifically affected the PV subpopulation. A similar trend was observed in 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-treated monkeys. Markers of GABAergic activity (GAD65 and GAD67 mRNA) were not different from those of controls in 6-OHDA-lesioned rats. Taken together, these findings provide evidence for nondopaminergic neuronal cell loss in the basal ganglia of 6-OHDA-lesioned rats and suggest that a similar loss may occur in the MPTP monkey. These data suggest that in patients with Parkinson disease, the loss of GABAergic neurons projecting to the subthalamic nucleus may contribute to the hyperactivity of this nucleus despite the absence of gross alterations in GAD mRNA expression.