Cellular and Molecular Mechanisms Underlying Pain Chronicity.

Chronic pain affects a significant amount of the population and is responsible for vast worldwide socio-economic costs [...].

Novel therapeutic approaches to target neurodegeneration.

Ageing is the main risk factor common to most primary neurodegenerative disorders. Indeed, age-related brain alterations have been long considered to predispose to neurodegeneration. Although protein misfolding and the accumulation of toxic protein aggregates have been considered as causative events in neurodegeneration, several other biological pathways affected by brain ageing also contribute to pathogenesis. Here, we discuss the evidence showing the involvement of the mechanisms controlling neuronal structure, gene expression, autophagy, cell metabolism and neuroinflammation in the onset and progression of neurodegenerative disorders. Furthermore, we review the therapeutic strategies currently under development or as future approaches designed to normalize these pathways, which may then increase brain resilience to cope with toxic protein species. In addition to therapies targeting the insoluble protein aggregates specifically associated with each neurodegenerative disorder, these novel pharmacological approaches may be part of combined therapies designed to rescue brain function.

Brain-enriched RagB isoforms regulate the dynamics of mTORC1 activity through GATOR1 inhibition.

Mechanistic target of rapamycin complex 1 (mTORC1) senses nutrient availability to appropriately regulate cellular anabolism and catabolism. During nutrient restriction, different organs in an animal do not respond equally, with vital organs being relatively spared. This raises the possibility that mTORC1 is differentially regulated in different cell types, yet little is known about this mechanistically. The Rag GTPases, RagA or RagB bound to RagC or RagD, tether mTORC1 in a nutrient-dependent manner to lysosomes where mTORC1 becomes activated. Although the RagA and B paralogues were assumed to be functionally equivalent, we find here that the RagB isoforms, which are highly expressed in neurons, impart mTORC1 with resistance to nutrient starvation by inhibiting the RagA/B GTPase-activating protein GATOR1. We further show that high expression of RagB isoforms is observed in some tumours, revealing an alternative strategy by which cancer cells can retain elevated mTORC1 upon low nutrient availability.

Role of Epigenetic Mechanisms in Chronic Pain.

Pain is an unpleasant but essential-to-life sensation, usually resulting from tissue damage. When pain persists long after the injury has resolved, it becomes pathological. The precise molecular and cellular mechanisms causing the transition from acute to chronic pain are not fully understood. A key aspect of pain chronicity is that several plasticity events happen along the neural pathways involved in pain. These long-lasting adaptive changes are enabled by alteration in the expression of relevant genes. Among the different modulators of gene transcription in adaptive processes in the nervous system, epigenetic mechanisms play a pivotal role. In this review, I will first outline the main classes of epigenetic mediators and then discuss their implications in chronic pain.

Protecting against summation of pain.

Temporal summation in the spinal cord is linked to pathological pain. In a translational genetic association study in this issue of Neuron, Trendafilova et al. (2022) identify the sodium-calcium exchanger 3 as a negative regulator of temporal summation and hypersensitivity via its modulation of calcium homeostasis.

Dysregulation of Immune Response Mediators and Pain-Related Ion Channels Is Associated with Pain-like Behavior in the GLA KO Mouse Model of Fabry Disease.

Fabry disease (FD) is a rare life-threatening disorder caused by deficiency of the alpha-galactosidase A (GLA) enzyme with a characteristic pain phenotype. Impaired GLA production or function leads to the accumulation of the cell membrane compound globotriaosylceramide (Gb3) in the neurons of the dorsal root ganglia (DRG) of FD patients. Applying immunohistochemistry (IHC) and quantitative real-time polymerase chain reaction (qRT PCR) analysis on DRG tissue of the GLA knockout (KO) mouse model of FD, we address the question of how Gb3 accumulation may contribute to FD pain and focus on the immune system and pain-associated ion channel gene expression. We show a higher Gb3 load in the DRG of young (<6 months) (p < 0.01) and old (≥12 months) (p < 0.001) GLA KO mice compared to old wildtype (WT) littermates, and an overall suppressed immune response in the DRG of old GLA KO mice, represented by a reduced number of CD206+ macrophages (p < 0.01) and lower gene expression levels of the inflammation-associated targets interleukin(IL)1b (p < 0.05), IL10 (p < 0.001), glial fibrillary acidic protein (GFAP) (p < 0.05), and leucine rich alpha-2-glycoprotein 1 (LRG1) (p < 0.01) in the DRG of old GLA KO mice compared to old WT. Dysregulation of immune-related genes may be linked to lower gene expression levels of the pain-associated ion channels calcium-activated potassium channel 3.1 (KCa3.1) and transient receptor potential ankyrin 1 channel (TRPA1). Ion channel expression might further be disturbed by impaired sphingolipid recruitment mediated via the lipid raft marker flotillin-1 (FLOT1). This impairment is represented by an increased number of FLOT1+ DRG neurons with a membranous expression pattern in old GLA KO mice compared to young GLA KO, young WT, and old WT mice (p < 0.001 each). Further, we provide evidence for aberrant behavior of GLA KO mice, which might be linked to dysregulated ion channel gene expression levels and disturbed FLOT1 distribution patterns. Behavioral testing revealed mechanical hypersensitivity in young (p < 0.01) and old (p < 0.001) GLA KO mice compared to WT, heat hypersensitivity in young GLA KO mice (p < 0.001) compared to WT, age-dependent heat hyposensitivity in old GLA KO mice (p < 0.001) compared to young GLA KO mice, and cold hyposensitivity in young (p < 0.001) and old (p < 0.001) GLA KO mice compared to WT, which well reflects the clinical phenotype observed in FD patients.

Rabphilin-3A Drives Structural Modifications of Dendritic Spines Induced by Long-Term Potentiation.

The interaction of Rabphilin-3A (Rph3A) with the NMDA receptor (NMDAR) in hippocampal neurons plays a pivotal role in the synaptic retention of this receptor. The formation of a Rph3A/NMDAR complex is needed for the induction of long-term potentiation and NMDAR-dependent hippocampal behaviors, such as spatial learning. Moreover, Rph3A can also interact with AMPA receptors (AMPARs) through the formation of a complex with myosin Va. Here, we used a confocal imaging approach to show that Rph3A overexpression in primary hippocampal neuronal cultures is sufficient to promote increased dendritic spine density. This morphological event is correlated with an increase in GluN2A-containing NMDARs at synaptic membranes and a decrease in the surface levels of GluA1-containing AMPARs. These molecular and morphological modifications of dendritic spines are sufficient to occlude the spine formation induced by long-term potentiation, but do not prevent the spine loss induced by long-term depression. Overall, our results demonstrate a key role for Rph3A in the modulation of structural synaptic plasticity at hippocampal synapses that correlates with its interactions with both NMDARs and AMPARs.

Organic anion transporter 1 is an HDAC4-regulated mediator of nociceptive hypersensitivity in mice.

Persistent pain is sustained by maladaptive changes in gene transcription resulting in altered function of the relevant circuits; therapies are still unsatisfactory. The epigenetic mechanisms and affected genes linking nociceptive activity to transcriptional changes and pathological sensitivity are unclear. Here, we found that, among several histone deacetylases (HDACs), synaptic activity specifically affects HDAC4 in murine spinal cord dorsal horn neurons. Noxious stimuli that induce long-lasting inflammatory hypersensitivity cause nuclear export and inactivation of HDAC4. The development of inflammation-associated mechanical hypersensitivity, but neither acute nor basal sensitivity, is impaired by the expression of a constitutively nuclear localized HDAC4 mutant. Next generation RNA-sequencing revealed an HDAC4-regulated gene program comprising mediators of sensitization including the organic anion transporter OAT1, known for its renal transport function. Using pharmacological and molecular tools to modulate OAT1 activity or expression, we causally link OAT1 to persistent inflammatory hypersensitivity in mice. Thus, HDAC4 is a key epigenetic regulator that translates nociceptive activity into sensitization by regulating OAT1, which is a potential target for pain-relieving therapies.

The impact of Semaphorin 4C/Plexin-B2 signaling on fear memory via remodeling of neuronal and synaptic morphology.

Aberrant fear is a cornerstone of several psychiatric disorders. Consequently, there is large interest in elucidation of signaling mechanisms that link extracellular cues to changes in neuronal function and structure in brain pathways that are important in the generation and maintenance of fear memory and its behavioral expression. Members of the Plexin-B family of receptors for class 4 semaphorins play important roles in developmental plasticity of neurons, and their expression persists in some areas of the adult nervous system. Here, we aimed to elucidate the role of Semaphorin 4C (Sema4C) and its cognate receptor, Plexin-B2, in the expression of contextual and cued fear memory, setting a mechanistic focus on structural plasticity and exploration of contributing signaling pathways. We observed that Plexin-B2 and Sema4C are expressed in forebrain areas related to fear memory, such as the anterior cingulate cortex, amygdala and the hippocampus, and their expression is regulated by aversive stimuli that induce fear memory. By generating forebrain-specific Plexin-B2 knockout mice and analyzing fear-related behaviors, we demonstrate that Sema4C-PlexinB2 signaling plays a crucial functional role in the recent and remote recall of fear memory. Detailed neuronal morphological analyses revealed that Sema4C-PlexinB2 signaling largely mediates fear-induced structural plasticity by enhancing dendritic ramifications and modulating synaptic density in the adult hippocampus. Analyses on signaling-related mutant mice showed that these functions are mediated by PlexinB2-dependent RhoA activation. These results deliver important insights into the mechanistic understanding of maladaptive plasticity in fear circuits and have implications for novel therapeutic strategies against fear-related disorders.

Cyclase-associated protein 2 dimerization regulates cofilin in synaptic plasticity and Alzheimer's disease.

Regulation of actin cytoskeleton dynamics in dendritic spines is crucial for learning and memory formation. Hence, defects in the actin cytoskeleton pathways are a biological trait of several brain diseases, including Alzheimer's disease. Here, we describe a novel synaptic mechanism governed by the cyclase-associated protein 2, which is required for structural plasticity phenomena and completely disrupted in Alzheimer's disease. We report that the formation of cyclase-associated protein 2 dimers through its Cys32 is important for cyclase-associated protein 2 binding to cofilin and for actin turnover. The Cys32-dependent cyclase-associated protein 2 homodimerization and association to cofilin are triggered by long-term potentiation and are required for long-term potentiation-induced cofilin translocation into spines, spine remodelling and the potentiation of synaptic transmission. This mechanism is specifically affected in the hippocampus, but not in the superior frontal gyrus, of both Alzheimer's disease patients and APP/PS1 mice, where cyclase-associated protein 2 is down-regulated and cyclase-associated protein 2 dimer synaptic levels are reduced. Notably, cyclase-associated protein 2 levels in the cerebrospinal fluid are significantly increased in Alzheimer's disease patients but not in subjects affected by frontotemporal dementia. In Alzheimer's disease hippocampi, cofilin association to cyclase-associated protein 2 dimer/monomer is altered and cofilin is aberrantly localized in spines. Taken together, these results provide novel insights into structural plasticity mechanisms that are defective in Alzheimer's disease.

VEGF-D Downregulation in CA1 Pyramidal Neurons Exerts Asymmetric Changes of Dendritic Morphology without Correlated Electrophysiological Alterations.

The morphology of dendritic arbors determines the location, strength and interaction of synaptic inputs. It is therefore important to understand the factors regulating dendritic arborization both during development and in situations of physiological or pathological plasticity. We have recently shown that VEGF-D (Vascular Endothelial Growth Factor D) is required to maintain length and complexity of basal dendrites in mouse hippocampal pyramidal cells. Lack of VEGF-D resulted in long-term memory deficits, suggesting a link between dendritic morphology and cognitive function. Here, we compared the effect of VEGF-D expression on basal versus apical dendrites of CA1 pyramidal cells, as well as its importance for synaptic processing of network oscillations. We report opposing, layer-specific effects of VEGF-D knockdown which resulted in shrinkage of basal and increased complexity of apical dendrites. Synaptic potentials and layer-specific voltage gradients during network oscillations remained, however, unaltered. These findings reveal a high spatial selectivity of VEGF-D effects at the sub-cellular level, and strong homeostatic mechanisms which keep spatially segregated synaptic inputs in a balance.

Nasally delivered VEGFD mimetics mitigate stroke-induced dendrite loss and brain damage.

In the adult brain, vascular endothelial growth factor D (VEGFD) is required for structural integrity of dendrites and cognitive abilities. Alterations of dendritic architectures are hallmarks of many neurologic disorders, including stroke-induced damage caused by toxic extrasynaptic NMDA receptor (eNMDAR) signaling. Here we show that stimulation of eNMDARs causes a rapid shutoff of VEGFD expression, leading to a dramatic loss of dendritic structures. Using the mouse middle cerebral artery occlusion (MCAO) stroke model, we have established the therapeutic potential of recombinant mouse VEGFD delivered intraventricularly to preserve dendritic architecture, reduce stroke-induced brain damage, and facilitate functional recovery. An easy-to-use therapeutic intervention for stroke was developed that uses a new class of VEGFD-derived peptide mimetics and postinjury nose-to-brain delivery.

VEGFD Protects Retinal Ganglion Cells and, consequently, Capillaries against Excitotoxic Injury.

In the central nervous system, neurons and the vasculature influence each other. While it is well described that a functional vascular system is trophic to neurons and that vascular damage contributes to neurodegeneration, the opposite scenario in which neural damage might impact the microvasculature is less defined. In this study, using an in vivo excitotoxic approach in adult mice as a tool to cause specific damage to retinal ganglion cells, we detected subsequent damage to endothelial cells in retinal capillaries. Furthermore, we detected decreased expression of vascular endothelial growth factor D (VEGFD) in retinal ganglion cells. In vivo VEGFD supplementation via neuronal-specific viral-mediated expression or acute intravitreal delivery of the mature protein preserved the structural and functional integrity of retinal ganglion cells against excitotoxicity and, additionally, spared endothelial cells from degeneration. Viral-mediated suppression of expression of the VEGFD-binding receptor VEGFR3 in retinal ganglion cells revealed that VEGFD exerts its protective capacity directly on retinal ganglion cells, while protection of endothelial cells is the result of upheld neuronal integrity. These findings suggest that VEGFD supplementation might be a novel, clinically applicable approach for neuronal and vascular protection.

Dynamic Regulation of Synaptopodin and the Axon Initial Segment in Retinal Ganglion Cells During Postnatal Development.

A key component allowing a neuron to function properly within its dynamic environment is the axon initial segment (AIS), the site of action potential generation. In visual cortex, AIS of pyramidal neurons undergo periods of activity-dependent structural plasticity during development. However, it remains unknown how AIS morphology is organized during development for downstream cells in the visual pathway (retinal ganglion cells; RGCs) and whether AIS retain the ability to dynamically adjust to changes in network state. Here, we investigated the maturation of AIS in RGCs during mouse retinal development, and tested putative activity-dependent mechanisms by applying visual deprivation with a focus on the AIS-specific cisternal organelle (CO), a presumed Ca2+-store. Whole-mount retinae from wildtype and Thy1-GFP transgenic mice were processed for multi-channel immunofluorescence using antibodies against AIS scaffolding proteins ankyrin-G, ßIV-spectrin and the CO marker synaptopodin (synpo). Confocal microscopy in combination with morphometrical analysis of AIS length and position as well as synpo cluster size was performed. Data indicated that a subset of RGC AIS contains synpo clusters and that these show significant dynamic regulation in size during development as well as after visual deprivation. Using super resolution microscopy, we addressed the subcellular localization of synpo in RGC axons. Similar to cortical neurons, RGCs show a periodic distribution of AIS scaffolding proteins. A previously reported scaffold-deficient nanodomain correlating with synpo localization is not evident in all RGC AIS. In summary, our work demonstrates a dynamic regulation of both the AIS and synpo in RGCs during retinal development and after visual deprivation, providing first evidence that the AIS and CO in RGCs can undergo structural plasticity in response to changes in network activity.

Histone Deacetylases Contribute to Excitotoxicity-Triggered Degeneration of Retinal Ganglion Cells In Vivo.

Excitotoxicity is known to modulate the nuclear accumulation, and thus activity state, of histone deacetylases (HDACs) in pyramidal neurons. In the retina, deregulation in activity and expression of different HDACs has been linked to pathological conditions such as retinitis pigmentosa, retinal ischemia, glaucoma, and acute optic nerve injury. Up to now, however, the effects of in vivo excitotoxicity on the different HDACs in retinal ganglion cells (RGCs) have not been thoroughly investigated. Here, we injected adult mice intravitreally with N-methyl-D-aspartate (NMDA) as a mean to trigger excitotoxicity-mediated RGC degeneration and we detected time-dependent loss of RGCs at 1 and 7 days after the insult. Further, we characterized the subcellular localization of HDACs belonging to class I (HDAC1, HDAC3), IIa (HDAC4, HDAC5, HDAC7, HDAC9), IIb (HDAC6, HDAC10), and IV (HDAC11) in RGCs. Our analyses revealed a differential pattern of HDACs nuclear distribution in RGCs following excitotoxicity. After 1 day, HDAC3, HDAC5, HDAC6, HDAC7, and HDAC11 showed altered subcellular localization in RGCs while 7 days after the excitotoxic insult, HDAC4 and HDAC9 were the only HDACs displaying changes in their subcellular distribution. Moreover, we found that in vivo selective inhibition of HDAC1/3 or HDAC4/5 via MS-275 (entinostat) or LMK-235, respectively, could prevent ongoing RGC degeneration. In conclusion, our results point towards a role of HDACs in RGC degeneration and identify HDAC1/3 and HDAC4/5 as potential therapeutic targets to treat degenerative retinal diseases.

The Synaptonuclear Messenger RNF10 Acts as an Architect of Neuronal Morphology.

The Ring Finger Protein 10 [RNF10] is a novel synapse-to-nucleus signaling protein that specifically links activation of synaptic NMDA receptors to modulation of gene expression. RNF10 dissociation from the GluN2A subunit of the NMDA receptor represents the first step of its synaptonuclear transport and it is followed by an importin-dependent translocation into the nucleus. Here, we have identified protein kinase C [PKC]-dependent phosphorylation of RNF10 Ser31 as a key step for RNF10 detachment from NMDA receptor and its subsequent trafficking to the nucleus. We show that pSer31-RNF10 plays a role both in synaptonuclear signaling and in neuronal morphology. In particular, the prevention of Ser31 RNF10 phosphorylation induces a decrease in spine density, neuronal branching, and CREB signaling, while opposite effects are obtained by mimicking a stable RNF10 phosphorylation at Ser31. Overall, these results add novel information about the functional and structural role of synaptonuclear protein messengers in shaping dendritic architecture in hippocampal neurons.

Epigenetic control of hypersensitivity in chronic inflammatory pain by the de novo DNA methyltransferase Dnmt3a2.

Chronic pain is a pathological manifestation of neuronal plasticity supported by altered gene transcription in spinal cord neurons that results in long-lasting hypersensitivity. Recently, the concept that epigenetic regulators might be important in pathological pain has emerged, but a clear understanding of the molecular players involved in the process is still lacking. In this study, we linked Dnmt3a2, a synaptic activity-regulated de novo DNA methyltransferase, to chronic inflammatory pain. We observed that Dnmt3a2 levels are increased in the spinal cord of adult mice following plantar injection of Complete Freund's Adjuvant, an in vivo model of chronic inflammatory pain. In vivo knockdown of Dnmt3a2 expression in dorsal horn neurons blunted the induction of genes triggered by Complete Freund's Adjuvant injection. Among the genes whose transcription was found to be influenced by Dnmt3a2 expression in the spinal cord is Ptgs2, encoding for Cox-2, a prime mediator of pain processing. Lowering the levels of Dnmt3a2 prevented the establishment of long-lasting inflammatory hypersensitivity. These results identify Dnmt3a2 as an important epigenetic regulator needed for the establishment of central sensitization. Targeting expression or function of Dnmt3a2 may be suitable for the treatment of chronic pain.

Histone deacetylase 4 shapes neuronal morphology via a mechanism involving regulation of expression of vascular endothelial growth factor D.

Nucleo-cytoplasmic shuttling of class IIa histone deacetylases (i.e HDAC4, -5, -7, and -9) is a synaptic activity- and nuclear calcium-dependent mechanism important for epigenetic regulation of signal-regulated gene expression in hippocampal neurons. HDAC4 in particular has been linked to the regulation of genes important for both synaptic structure and plasticity. Here, using a constitutively nuclear-localized, dominant-active variant of HDAC4 (HDAC4 3SA), we demonstrate that HDAC4 accumulation in the nucleus severely reduces both the length and complexity of dendrites of cultured mature hippocampal neurons, but does not affect the number of dendritic spines. This phenomenon appeared to be specific to HDAC4, as increasing the expression of HDAC3 or HDAC11, belonging to class I and class IV HDACs, respectively, did not alter dendritic architecture. We also show that HDAC4 3SA decreases the expression of vascular endothelial growth factor D (VEGFD), a key protein required for the maintenance of dendritic arbors. The expression of other members of the VEGF family and their receptors was not affected by the nuclear accumulation of HDAC4. VEGFD overexpression or administration of recombinant VEGFD, but not VEGFC, the closest VEGFD homologue, rescued the impaired dendritic architecture caused by the nuclear-localized HDAC4 variant. These results identify HDAC4 as an epigenetic regulator of neuronal morphology that controls dendritic arborization via the expression of VEGFD.

Neuroprotective effect of acute ethanol intoxication in TBI is associated to the hierarchical modulation of early transcriptional responses.

Ethanol intoxication is a risk factor for traumatic brain injury (TBI) but clinical evidence suggests that it may actually improve the prognosis of intoxicated TBI patients. We have employed a closed, weight-drop TBI model of different severity (2cm or 3cm falling height), preceded (-30min) or followed (+20min) by ethanol administration (5g/Kg). This protocol allows us to study the interaction of binge ethanol intoxication in TBI, monitoring behavioral changes, histological responses and the transcriptional regulation of a series of activity-regulated genes (immediate early genes, IEGs). We demonstrate that ethanol pretreatment before moderate TBI (2cm) significantly reduces neurological impairment and accelerates recovery. In addition, better preservation of neuronal numbers and cFos+cells was observed 7days after TBI. At transcriptional level, ethanol reduced the upregulation of a subset of IEGs encoding for transcription factors such as Atf3, c-Fos, FosB, Egr1, Egr3 and Npas4 but did not affect the upregulation of others (e.g. Gadd45b and Gadd45c). While a subset of IEGs encoding for effector proteins (such as Bdnf, InhbA and Dusp5) were downregulated by ethanol, others (such as Il-6) were unaffected. Notably, the majority of genes were sensitive to ethanol only when administered before TBI and not afterwards (the exceptions being c-Fos, Egr1 and Dusp5). Furthermore, while severe TBI (3cm) induced a qualitatively similar (but quantitatively larger) transcriptional response to moderate TBI, it was no longer sensitive to ethanol pretreatment. Thus, we have shown that a subset of the TBI-induced transcriptional responses were sensitive to ethanol intoxication at the instance of trauma (ultimately resulting in beneficial outcomes) and that the effect of ethanol was restricted to a certain time window (pre TBI treatment) and to TBI severity (moderate). This information could be critical for the translational value of ethanol in TBI and for the design of clinical studies aimed at disentangling the role of ethanol intoxication in TBI.

Nuclear Calcium Buffering Capacity Shapes Neuronal Architecture.

Calcium-binding proteins (CaBPs) such as parvalbumin are part of the cellular calcium buffering system that determines intracellular calcium diffusion and influences the spatiotemporal dynamics of calcium signals. In neurons, CaBPs are primarily localized to the cytosol and function, for example, in nerve terminals in short-term synaptic plasticity. However, CaBPs are also expressed in the cell nucleus, suggesting that they modulate nuclear calcium signals, which are key regulators of neuronal gene expression. Here we show that the calcium buffering capacity of the cell nucleus in mouse hippocampal neurons regulates neuronal architecture by modulating the expression levels of VEGFD and the complement factor C1q-c, two nuclear calcium-regulated genes that control dendrite geometry and spine density, respectively. Increasing the levels of nuclear calcium buffers by means of expression of a nuclearly targeted form of parvalbumin fused to mCherry (PV.NLS-mC) led to a reduction in VEGFD expression and, as a result, to a decrease in total dendritic length and complexity. In contrast, mRNA levels of the synapse pruning factor C1q-c were increased in neurons expressing PV.NLS-mC, causing a reduction in the density and size of dendritic spines. Our results establish a close link between nuclear calcium buffering capacity and the transcription of genes that determine neuronal structure. They suggest that the development of cognitive deficits observed in neurological conditions associated with CaBP deregulation may reflect the loss of necessary structural features of dendrites and spines.