Human TrkAR649W mutation impairs nociception, sweating and cognitive abilities: a mouse model of HSAN IV.

A functional nerve growth factor NGF-Tropomyosin Receptor kinase A (TrkA) system is an essential requisite for the generation and maintenance of long-lasting thermal and mechanical hyperalgesia in adult mammals. Indeed, mutations in the gene encoding for TrkA are responsible for a rare condition, named Hereditary Sensory and Autonomic Neuropathy type IV (HSAN IV), characterized by the loss of response to noxious stimuli, anhidrosis and cognitive impairment. However, to date, there is no available mouse model to properly understand how the NGF-TrkA system can lead to pathological phenotypes that are distinctive of HSAN IV. Here, we report the generation of a knock-in mouse line carrying the HSAN IV TrkAR649W mutation. First, by in vitro biochemical and biophysical analyses, we show that the pathological R649W mutation leads to kinase-inactive TrkA also affecting its membrane dynamics and trafficking. In agreement with the HSAN IV human phenotype, TrkAR649W/m mice display a lower response to thermal and chemical noxious stimuli, correlating with reduced skin innervation, in addition to decreased sweating in comparison to TrkAh/m controls. Moreover, the R649W mutation decreases anxiety-like behavior and compromises cognitive abilities, by impairing spatial-working and social memory. Our results further uncover unexplored roles of TrkA in thermoregulation and sociability. In addition to accurately recapitulating the clinical manifestations of HSAN IV patients, our findings contribute to clarifying the involvement of the NGF-TrkA system in pain sensation.

Reversal of neurological deficits by painless nerve growth factor in a mouse model of Rett syndrome.

Rett syndrome is a rare genetic neurodevelopmental disease, affecting 1 in over 10 000 females born worldwide, caused by de novo mutations in the X-chromosome-located methyl-CpG-binding protein 2 (MeCP2) gene. Despite the great effort put forth by the scientific community, a therapy for this devastating disease is still needed. Here, we tested the therapeutic effects of a painless mutein of the nerve growth factor (NGF), called human NGF painless (hNGFp), via a non-invasive intranasal delivery in female MeCP2+/- mice. Of note, previous work had demonstrated a broad biodistribution of hNGFp in the mouse brain by the nasal delivery route. We report that (i) the long-term lifelong treatment of MeCP2+/- mice with hNGFp, starting at 2 months of age, increased the chance of survival while also greatly improving behavioural parameters. Furthermore, when we assessed the phenotypic changes brought forth by (ii) a short-term 1-month-long hNGFp-treatment, starting at 3 months of age (right after the initial presentation of symptoms), we observed the rescue of a well known neuronal target population of NGF, cholinergic neurons in the medial septum. Moreover, we reveal a deficit in microglial morphology in MeCP2+/- mice, completely reversed in treated animals. This effect on microglia is in line with reports showing microglia to be a TrkA-dependent non-neuronal target cell population of NGF in the brain. To understand the immunomodulatory activity of hNGFp, we analysed the cytokine profile after hNGFp treatment in MeCP2+/- mice, to discover that the treatment recovered the altered expression of key neuroimmune-communication molecules, such as fractalkine. The overall conclusion is that hNGFp delivered intranasally can ameliorate symptoms in the MeCP2+/- model of Rett syndrome, by exerting strong neuroprotection with a dual mechanism of action: directly on target neurons and indirectly via microglia.

Exploring miRNAs' Based Modeling Approach for Predicting PIRA in Multiple Sclerosis: A Comprehensive Analysis.

The current hypothesis on the pathophysiology of multiple sclerosis (MS) suggests the involvement of both inflammatory and neurodegenerative mechanisms. Disease Modifying Therapies (DMTs) effectively decrease relapse rates, thus reducing relapse-associated disability in people with MS. In some patients, disability progression, however, is not solely linked to new lesions and clinical relapses but can manifest independently. Progression Independent of Relapse Activity (PIRA) significantly contributes to long-term disability, stressing the urge to unveil biomarkers to forecast disease progression. Twenty-five adult patients with relapsing-remitting multiple sclerosis (RRMS) were enrolled in a cohort study, according to the latest McDonald criteria, and tested before and after high-efficacy Disease Modifying Therapies (DMTs) (6-24 months). Through Agilent microarrays, we analyzed miRNA profiles from peripheral blood mononuclear cells. Multivariate logistic and linear models with interactions were generated. Robustness was assessed by randomization tests in R. A subset of miRNAs, correlated with PIRA, and the Expanded Disability Status Scale (EDSS), was selected. To refine the patient stratification connected to the disease trajectory, we computed a robust logistic classification model derived from baseline miRNA expression to predict PIRA status (AUC = 0.971). We built an optimal multilinear model by selecting four other miRNA predictors to describe EDSS changes compared to baseline. Multivariate modeling offers a promising avenue to uncover potential biomarkers essential for accurate prediction of disability progression in early MS stages. These models can provide valuable insights into developing personalized and effective treatment strategies.

Retinal pathological features and proteome signatures of Alzheimer's disease.

Alzheimer's disease (AD) pathologies were discovered in the accessible neurosensory retina. However, their exact nature and topographical distribution, particularly in the early stages of functional impairment, and how they relate to disease progression in the brain remain largely unknown. To better understand the pathological features of AD in the retina, we conducted an extensive histopathological and biochemical investigation of postmortem retina and brain tissues from 86 human donors. Quantitative examination of superior and inferior temporal retinas from mild cognitive impairment (MCI) and AD patients compared to those with normal cognition (NC) revealed significant increases in amyloid ß-protein (Aß42) forms and novel intraneuronal Aß oligomers (AßOi), which were closely associated with exacerbated retinal macrogliosis, microgliosis, and tissue atrophy. These pathologies were unevenly distributed across retinal layers and geometrical areas, with the inner layers and peripheral subregions exhibiting most pronounced accumulations in the MCI and AD versus NC retinas. While microgliosis was increased in the retina of these patients, the proportion of microglial cells engaging in Aß uptake was reduced. Female AD patients exhibited higher levels of retinal microgliosis than males. Notably, retinal Aß42, S100 calcium-binding protein B+ macrogliosis, and atrophy correlated with severity of brain Aß pathology, tauopathy, and atrophy, and most retinal pathologies reflected Braak staging. All retinal biomarkers correlated with the cognitive scores, with retinal Aß42, far-peripheral AßOi and microgliosis displaying the strongest correlations. Proteomic analysis of AD retinas revealed activation of specific inflammatory and neurodegenerative processes and inhibition of oxidative phosphorylation/mitochondrial, and photoreceptor-related pathways. This study identifies and maps retinopathy in MCI and AD patients, demonstrating the quantitative relationship with brain pathology and cognition, and may lead to reliable retinal biomarkers for noninvasive retinal screening and monitoring of AD.

Disentangling the signaling complexity of nerve growth factor receptors by CRISPR/Cas9.

The binding of nerve growth factor (NGF) to the tropomyosin-related kinase A (TrkA) and p75NTR receptors activates a large variety of pathways regulating critical processes as diverse as proliferation, differentiation, membrane potential, synaptic plasticity, and pain. To ascertain the details of TrkA-p75NTR interaction and cooperation, a plethora of experiments, mostly based on receptor overexpression or downregulation, have been performed. Among the heterogeneous cellular systems used for studying NGF signaling, the PC12 pheochromocytoma-derived cell line is a widely used model. By means of CRISPR/Cas9 genome editing, we created PC12 cells lacking TrkA, p75NTR , or both. We found that TrkA-null cells become unresponsive to NGF. Conversely, the absence of p75NTR enhances the phosphorylation of TrkA and its effectors. Using a patch-clamp, we demonstrated that the individual activation of TrkA and p75NTR by NGF results in antagonizing effects on the membrane potential. These newly developed PC12 cell lines can be used to investigate the specific roles of TrkA and p75NTR in a genetically defined cellular model, thus providing a useful platform for future studies and further gene editing.

A Vicious NGF-p75NTR Positive Feedback Loop Exacerbates the Toxic Effects of Oxidative Damage in the Human Retinal Epithelial Cell Line ARPE-19.

In spite of its variety of biological activities, the clinical exploitation of human NGF (hNGF) is currently limited to ocular pathologies. It is therefore interesting to test the effects of hNGF in preclinical models that may predict their efficacy and safety in the clinical setting of ocular disorders and compare the effects of hNGF with those of its analogs. We used a human retinal pigment cell line, ARPE-19 cells, to investigate the effects of hNGF and its analogs, mouse NGF (mNGF) and painless NGF (pNGF), on cell viability under basal conditions and after exposure to oxidative stimuli, i.e., hydrogen peroxide (H2O2) and ultraviolet (UV)-A rays. The effects of hNGF and pNGF were also tested on the gene expression and protein synthesis of the two NGF receptor subtypes, p75 neurotrophic receptors (p75NTR) and tyrosine kinase A (TrkA) receptors. We drew the following conclusions: (i) the exposure of ARPE-19 cells to H2O2 or UV-A causes a dose-dependent decrease in the number of viable cells; (ii) under baseline conditions, hNGF, but not pNGF, causes a concentration-dependent decrease in cell viability in the range of doses 1-100 ng/mL; (iii) hNGF, but not pNGF, significantly potentiates the toxic effects of H2O2 or of UV-A on ARPE-19 cells in the range of doses 1-100 ng/mL, while mNGF at the same doses presents an intermediate behavior; (iv) 100 ng/mL of hNGF triggers an increase in p75NTR expression in H2O2-treated ARPE-19 cells, while pNGF at the same dose does not; (v) pNGF, but not hNGF (both given at 100 ng/mL), increases the total cell fluorescence intensity for TrkA receptors in H2O2-treated ARPE-19 cells. The present findings suggest a vicious positive feedback loop through which NGF-mediated upregulation of p75NTR contributes to worsening the toxic effects of oxidative damage in the human retinal epithelial cell line ARPE-19. Looking at the possible clinical relevance of these findings, one can postulate that pNGF might show a better benefit/risk ratio than hNGF in the treatment of ocular disorders.

Fast-diffusing p75NTR monomers support apoptosis and growth cone collapse by neurotrophin ligands.

The p75 neurotrophin (NT) receptor (p75NTR) plays a crucial role in balancing survival-versus-death decisions in the nervous system. Yet, despite 2 decades of structural and biochemical studies, a comprehensive, accepted model for p75NTR activation by NT ligands is still missing. Here, we present a single-molecule study of membrane p75NTR in living cells, demonstrating that the vast majority of receptors are monomers before and after NT activation. Interestingly, the stoichiometry and diffusion properties of the wild-type (wt) p75NTR are almost identical to those of a receptor mutant lacking residues previously believed to induce oligomerization. The wt p75NTR and mutated (mut) p75NTR differ in their partitioning in cholesterol-rich membrane regions upon nerve growth factor (NGF) stimulation: We argue that this is the origin of the ability of wt p75NTR , but not of mut p75NTR, to mediate immature NT (proNT)-induced apoptosis. Both p75NTR forms support proNT-induced growth cone retraction: We show that receptor surface accumulation is the driving force for cone collapse. Overall, our data unveil the multifaceted activity of the p75NTR monomer and let us provide a coherent interpretative frame of existing conflicting data in the literature.

Untangling the Conformational Plasticity of V66M Human proBDNF Polymorphism as a Modifier of Psychiatric Disorder Susceptibility.

The human genetic variant BDNF (V66M) represents the first example of neurotrophin family member that has been linked to psychiatric disorders. In order to elucidate structural differences that account for the effects in cognitive function, this hproBDNF polymorph was expressed, refolded, purified, and compared directly to the WT variant for the first time for differences in their 3D structures by DSF, limited proteolysis, FT-IR, and SAXS measurements in solution. Our complementary studies revealed a deep impact of V66M polymorphism on hproBDNF conformations in solution. Although the mean conformation in solution appears to be more compact in the V66M variant, overall, we demonstrated a large increase in flexibility in solution upon V66M mutation. Thus, considering that plasticity in IDR is crucial for protein function, the observed alterations may be related to the functional alterations in hproBDNF binding to its receptors p75NTR, sortilin, HAP1, and SorCS2. These effects can provoke altered intracellular neuronal trafficking and/or affect proBDNF physiological functions, leading to many brain-associated diseases and conditions such as cognitive impairment and anxiety. The structural alterations highlighted in the present study may pave the way to the development of drug discovery strategies to provide greater therapeutic responses and of novel pharmacologic strategy in human populations with this common polymorphism, ultimately guiding personalized medicine for neuropsychiatric disorders.

Acetylation-Specific Interference by Anti-Histone H3K9ac Intrabody Results in Precise Modulation of Gene Expression.

Among Histone post-translational modifications (PTMs), lysine acetylation plays a pivotal role in the epigenetic regulation of gene expression, mediated by chromatin modifying enzymes. Due to their activity in physiology and pathology, several chemical compounds have been developed to inhibit the function of these proteins. However, the pleiotropy of these classes of proteins represents a weakness of epigenetic drugs. Ideally, a new generation of epigenetic drugs should target with molecular precision individual acetylated lysines on the target protein. We exploit a PTM-directed interference, based on an intrabody (scFv-58F) that selectively binds acetylated lysine 9 of histone H3 (H3K9ac), to test the hypothesis that targeting H3K9ac yields more specific effects than inhibiting the corresponding HAT enzyme that installs that PTM. In yeast scFv-58F modulates, gene expression in a more specific way, compared to two well-established HAT inhibitors. This PTM-specific interference modulated expression of genes involved in ribosome biogenesis and function. In mammalian cells, the scFv-58F induces exclusive changes in the H3K9ac-dependent expression of specific genes. These results suggest the H3K9ac-specific intrabody as the founder of a new class of molecules to directly target histone PTMs, inverting the paradigm from inhibiting the writer enzyme to acting on the PTM.

Protein Structural Information and Evolutionary Landscape by In Vitro Evolution.

Protein structure is tightly intertwined with function according to the laws of evolution. Understanding how structure determines function has been the aim of structural biology for decades. Here, we have wondered instead whether it is possible to exploit the function for which a protein was evolutionary selected to gain information on protein structure and on the landscape explored during the early stages of molecular and natural evolution. To answer to this question, we developed a new methodology, which we named CAMELS (Coupling Analysis by Molecular Evolution Library Sequencing), that is able to obtain the in vitro evolution of a protein from an artificial selection based on function. We were able to observe with CAMELS many features of the TEM-1 beta-lactamase local fold exclusively by generating and sequencing large libraries of mutational variants. We demonstrated that we can, whenever a functional phenotypic selection of a protein is available, sketch the structural and evolutionary landscape of a protein without utilizing purified proteins, collecting physical measurements, or relying on the pool of natural protein variants.

Understanding pain perception through genetic painlessness diseases: The role of NGF and proNGF.

Nerve growth factor (NGF), by binding to TrkA and p75NTR receptors, regulates the survival and differentiation of sensory neurons during development and mediates pain transmission and perception during adulthood, by acting at different levels of the nervous system. Key to understanding the role of NGF as a pain mediator is the finding that mutations (namely, R121W, V232fs and R221W) in the NGF gene cause painlessness disease Hereditary Sensory and Autonomic Neuropathy type V (HSAN V). Here we shall review the consequences of these NGF mutations, each of which results in specific clinical signs: R221W determines congenital pain insensitivity with no overt cognitive disabilities, whereas V232fs and R121W also result in intellectual disability, thus showing similarities to HSAN IV, which is caused by mutations in TrkA, rather than to HSAN V. Comparing the cellular, biochemical and clinical findings of these mutations could help in better understanding not only the possible mechanisms underlying HSAN V, but also mechanisms of NGF signalling and roles. These mutations alter the balance between NGF and proNGF in favour of an accumulation of the latter, suggesting a possible role of proNGF as a molecule with an analgesic role. Furthermore, the neurotrophic and pronociceptive functions of NGF are split by the R221W mutation, making NGF variants based on this mutation interesting for designing therapeutic applications for many diseases. This review emphasizes the possibility of using the mutations involved in "painlessness" clinical disorders as an innovative approach to identify new proteins and pathways involved in pain transmission and perception. OUTSTANDING QUESTIONS: Why do homozygous HSAN V die postnatally? What is the cause of this early postnatal lethality? Is the development of a mouse or a human feeling less pain affecting higher cognitive and perceptual functions? What is the consequence of the HSAN V mutation on the development of joints and bones? Are the multiple fractures observed in HSAN V patients due exclusively to the carelessness consequent to not feeling pain, or also to an intrinsic frailty of their bones? Are heterodimers of NGFWT and NGFR221W in the heterozygote state formed? And if so, what are the properties of these heterodimeric proteins? How is the processing of proNGFR221W to NGFR221W affected by the mutation?

Intranasal delivery of BDNF rescues memory deficits in AD11 mice and reduces brain microgliosis.

A decrease in brain-derived neurotrophic factor (BDNF), a neurotrophin essential for synaptic function, plasticity and neuronal survival, is evident early in the progression of Alzheimer's disease (AD), being apparent in subjects with mild cognitive impairment or mild AD, and both proBDNF and mature BDNF levels are positively correlated with cognitive measures. BDNF delivery is, therefore, considered of great interest as a potentially useful therapeutic strategy to contrast AD. Invasive BDNF administration has indeed been recently used in animal models of AD with promising results in rescuing memory deficits, synaptic density and cell loss. Here, we tested whether non-invasive intranasal administration of different BDNF concentrations after the onset of cognitive and anatomical deficits (6 months of age) could rescue neuropathological and memory deficits in AD11 mice, a model of NGF deprivation-induced neurodegeneration. In addition to AD hallmarks, we investigated BDNF effects on microglia presence in the brain of AD11 mice, since alterations in microglia activation have been associated with ageing-related cognitive decline and with the progression of neurodegenerative diseases, including AD. We found that intranasal delivery of 42 pmol BDNF (1 µM), but not PBS, was sufficient to completely rescue performance of AD11 mice both in the object recognition test and in the object context test. No further improvement was obtained with 420 pmol (10 µM) BDNF dose. The strong improvement in memory performance in BDNF-treated mice was not accompanied by an amelioration of AD-like pathology, Aß burden, tau hyperphosphorylation and cholinergic deficit, but there was a dramatic decrease of CD11b immunoreactive brain microglia. These results reinforce the potential therapeutic uses of BDNF in AD and the non-invasive intranasal route as an effective delivery strategy of BDNF to the brain. They also strengthen the connection between neuroinflammation and neurodegenerative dementia and suggest microglia as a possible mediator of BDNF therapeutic actions in the brain.

Non-Canonical Roles of Tau and Their Contribution to Synaptic Dysfunction.

Tau plays a central role in a group of neurodegenerative disorders collectively named tauopathies. Despite the wide range of diverse symptoms at the onset and during the progression of the pathology, all tauopathies share two common hallmarks, namely the misfolding and aggregation of Tau protein and progressive synaptic dysfunctions. Tau aggregation correlates with cognitive decline and behavioural impairment. The mechanistic link between Tau misfolding and the synaptic dysfunction is still unknown, but this correlation is well established in the human brain and also in tauopathy mouse models. At the onset of the pathology, Tau undergoes post-translational modifications (PTMs) inducing the detachment from the cytoskeleton and its release in the cytoplasm as a soluble monomer. In this condition, the physiological enrichment in the axon is definitely disrupted, resulting in Tau relocalization in the cell soma and in dendrites. Subsequently, Tau aggregates into toxic oligomers and amyloidogenic forms that disrupt synaptic homeostasis and function, resulting in neuronal degeneration. The involvement of Tau in synaptic transmission alteration in tauopathies has been extensively reviewed. Here, we will focus on non-canonical Tau functions mediating synapse dysfunction.

The NGFR100W Mutation Specifically Impairs Nociception without Affecting Cognitive Performance in a Mouse Model of Hereditary Sensory and Autonomic Neuropathy Type V.

Nerve growth factor (NGF) is a key mediator of nociception, acting during the development and differentiation of dorsal root ganglion (DRG) neurons, and on adult DRG neuron sensitization to painful stimuli. NGF also has central actions in the brain, where it regulates the phenotypic maintenance of cholinergic neurons. The physiological function of NGF as a pain mediator is altered in patients with Hereditary Sensory and Autonomic Neuropathy type V (HSAN V), caused by the 661C>T transition in the Ngf gene, resulting in the R100W missense mutation in mature NGF. Homozygous HSAN V patients present with congenital pain insensitivity, but are cognitively normal. This led us to hypothesize that the R100W mutation may differentially affect the central and peripheral actions of NGF. To test this hypothesis and provide a mechanistic basis to the HSAN V phenotype, we generated transgenic mice harboring the human 661C>T mutation in the Ngf gene and studied both males and females. We demonstrate that heterozygous NGFR100W/wt mice display impaired nociception. DRG neurons of NGFR100W/wt mice are morphologically normal, with no alteration in the different DRG subpopulations, whereas skin innervation is reduced. The NGFR100W protein has reduced capability to activate pain-specific signaling, paralleling its reduced ability to induce mechanical allodynia. Surprisingly, however, NGFR100W/wt mice, unlike heterozygous mNGF+/- mice, show no learning or memory deficits, despite a reduction in secretion and brain levels of NGF. The results exclude haploinsufficiency of NGF as a mechanistic cause for heterozygous HSAN V mice and demonstrate a specific effect of the R100W mutation on nociception.SIGNIFICANCE STATEMENT The R100W mutation in nerve growth factor (NGF) causes Hereditary Sensory and Autonomic Neuropathy type V, a rare disease characterized by impaired nociception, even in apparently clinically silent heterozygotes. For the first time, we generated and characterized heterozygous knock-in mice carrying the human R100W-mutated allele (NGFR100W/wt). Mutant mice have normal nociceptor populations, which, however, display decreased activation of pain transduction pathways. NGFR100W interferes with peripheral and central NGF bioavailability, but this does not impact on CNS function, as demonstrated by normal learning and memory, in contrast with heterozygous NGF knock-out mice. Thus, a point mutation allows neurotrophic and pronociceptive functions of NGF to be split, with interesting implications for the treatment of chronic pain.

Post-translational selective intracellular silencing of acetylated proteins with de novo selected intrabodies.

The ability to selectively interfere with post-translationally modified proteins would have many biological and therapeutic applications. However, post-translational modifications cannot be selectively targeted by nucleic-acid-based interference approaches. Here we describe post-translational intracellular silencing antibody technology (PISA), a method for selecting intrabodies against post-translationally modified proteins. We demonstrate our method by generating intrabodies against native acetylated proteins and showing functional interference in living cells.

ProNGF Is a Cell-Type-Specific Mitogen for Adult Hippocampal and for Induced Neural Stem Cells.

The role of proNGF, the precursor of nerve growth factor (NGF), in the biology of adult neural stem cells (aNSCs) is still unclear. Here, we analyzed adult hippocampal neurogenesis in AD11 transgenic mice, in which the constitutive expression of anti-NGF antibody leads to an imbalance of proNGF over mature NGF. We found increased proliferation of progenitors but a reduced neurogenesis in the AD11 dentate gyrus (DG)-hippocampus (HP). Also in vitro, AD11 hippocampal neural stem cells (NSCs) proliferated more, but were unable to differentiate into morphologically mature neurons. By treating wild-type hippocampal progenitors with the uncleavable form of proNGF (proNGF-KR), we demonstrated that proNGF acts as mitogen on aNSCs at low concentration. The mitogenic effect of proNGF was specifically addressed to the radial glia-like (RGL) stem cells through the induction of cyclin D1 expression. These cells express high levels of p75NTR , as demonstrated by immunofluorescence analyses performed ex vivo on RGL cells isolated from freshly dissociated HP-DG or selected in vitro from NSCs by leukemia inhibitory factor. Clonogenic assay performed in the absence of mitogens showed that RGLs respond to proNGF-KR by reactivating their proliferation and thus leading to neurospheres formation. The mitogenic effect of proNGF was further exploited in the expansion of mouse-induced neural stem cells (iNSCs). Chronic exposure of iNSCs to proNGF-KR increased their proliferation. Altogether, we demonstrated that proNGF acts as mitogen on hippocampal and iNSCs. Stem Cells 2019;37:1223-1237.

Increased cytoplasmic TDP-43 reduces global protein synthesis by interacting with RACK1 on polyribosomes.

TDP-43 is a well known RNA binding protein involved in the pathogenesis of Amyotrophic Lateral Sclerosis (ALS) and Frontotemporal Lobar Dementia (FTLD). In physiological conditions, TDP-43 mainly localizes in the nucleus and shuttles, at least in neurons, to the cytoplasm to form TDP-43 RNA granules. In the nucleus, TDP-43 participates to the expression and splicing of RNAs, while in the cytoplasm its functions range from transport to translation of specific mRNAs. However, if loss or gain of these TDP-43 functions are affected in ALS/FTLD pathogenesis is not clear. Here, we report that TDP-43 localizes on ribosomes not only in primary neurons but also in SH-SY5Y human neuroblastoma cells. We find that binding of TDP-43 to the translational machinery is mediated by an interaction with a specific ribosomal protein, RACK1, and that an increase in cytoplasmic TDP-43 represses global protein synthesis, an effect which is rescued by overexpression of RACK1. Ribosomal loss of RACK1, which excludes TDP-43 from the translational machinery, remarkably reduces formation of TDP-43 cytoplasmic inclusions in neuroblastoma cells. Finally, we corroborate the interaction between TDP-43 and RACK1 on polyribosomes of neuroblastoma cells with mis-localization of RACK1 on TDP-43 positive cytoplasmic inclusions in motor neurons of ALS patients. In conclusions, results from this study suggest that TDP-43 represents a translational repressor not only for specific mRNAs but for overall translation and that its binding to polyribosomes through RACK1 may promote, under conditions inducing ALS pathogenesis, the formation of cytoplasmic inclusions.

Cholinergic striatal neurons are increased in HSAN V homozygous mice despite reduced NGF bioavailability.

The neurotrophin Nerve growth factor (NGF) plays a critical role in the mature and developing nervous system. A point mutation (R100W) in the NGFB gene was found in patients with Hereditary Sensory and Autonomic Neuropathy type V (HSAN V), which leads to pain insensitivity. In a previous work it has been shown that the mutation provokes a reduced secretion of mature NGF. In this study we generated and analyzed homozygous NGFR100W/R100W mice to understand whether the reduced NGF bioavailability can contribute to the clinical phenotype of the homozygous condition. We found that the majority of NGFR100W/R100W mice were born normal but failed to reach the first month of age. This early lethality was rescued by daily treatment with wild type NGF. In addition, we found that the density of cholinergic neurons of homozygous mice was unaffected in the medial septum and in the nucleus basalis of Meynert, whereas, suprisingly, it was increased specifically in the striatum. Due to the known action of the striatal cholinergic tone in modulating pain, our findings support the hypothesis that a central mechanism, linked to the NGFR100W-dependent increase of the striatal cholinergic tone, can contribute to the pain insensitivity observed in HSAN V patients.

Painless Nerve Growth Factor: A TrkA biased agonist mediating a broad neuroprotection via its actions on microglia cells.

Nerve Growth Factor (NGF) is a therapeutic candidate for Alzheimer's disease, based on its well known actions on basal forebrain cholinergic neurons. However, because of its pro-nociceptive activity, in current clinical trials NGF has to be administered intraparenchymally into the brain by neurosurgery via cell or gene therapy approaches. To prevent the NGF pain-inducing collateral effects, thus avoiding the necessity for local brain injection, we developed painless NGF (hNGFp), based on the human genetic disease Hereditary Sensory and Autonomic Neuropathy type V (HSAN V). hNGFp has similar neurotrophic activity as wild type human NGF, but its pain sensitizing activity is tenfold lower. Pharmacologically, hNGFp is a biased receptor agonist of NGF TrkA receptor. The results of recent studies shed new light on the neuroprotective mechanism by hNGFp and are highly relevant for the planning of NGF-based clinical trials. The intraparenchymal delivery of hNGFp, as used in clinical trials, was simulated in the 5xFAD mouse model and found to be inefficacious in reducing Aß plaque load. On the contrary, the same dose of hNGFp administered intranasally, which was rather widely biodistributed in the brain and did not induce pain sensitization, blocked APP processing into amyloid and restored synaptic plasticity and memory in this aggressive neurodegeneration model. This potent and broad neuroprotection by hNGFp was found to be mediated by hNGFp actions on glial cells. hNGFp increases inflammatory proteins such as the soluble TNFα receptor II and the chemokine CXCL12. Independent work has shown that NGF has a potent anti-inflammatory action on microglia and steers them towards a neuroprotective phenotype. These studies demonstrate that microglia cells are a new target cell of NGF in the brain and have therapeutic significance: i) they establish that the neuroprotective actions of hNGFp relies on a widespread exposure of the brain, ii) they identify a new anti-neurodegenerative pathway, linking hNGFp to inflammatory chemokines and cytokines via microglia, a common target for new therapeutic opportunities for neurodegenerative diseases, iii) they extend the neuroprotective potential of hNGFp beyond its classical cholinergic target, thereby widening the range of neurological diseases for which this neurotrophic factor might be used therapeutically, iv) they help interpreting the results of current NGF clinical trials in AD and the design of future trials with this new potent therapeutic candidate.

Cortical Seizures in FoxG1+/- Mice are Accompanied by Akt/S6 Overactivation, Excitation/Inhibition Imbalance and Impaired Synaptic Transmission.

The correct morphofunctional shaping of the cerebral cortex requires a continuous interaction between intrinsic (genes/molecules expressed within the tissue) and extrinsic (e.g., neural activity) factors at all developmental stages. Forkhead Box G1 (FOXG1) is an evolutionarily conserved transcription factor, essential for the cerebral cortex patterning and layering. FOXG1-related disorders, including the congenital form of Rett syndrome, can be caused by deletions, intragenic mutations or duplications. These genetic alterations are associated with a complex phenotypic spectrum, spanning from intellectual disability, microcephaly, to autistic features, and epilepsy. We investigated the functional correlates of dysregulated gene expression by performing electrophysiological assays on FoxG1+/- mice. Local Field Potential (LFP) recordings on freely moving animals detected cortical hyperexcitability. On the other hand, patch-clamp recordings showed a downregulation of spontaneous glutamatergic transmission. These findings were accompanied by overactivation of Akt/S6 signaling. Furthermore, the expression of vesicular glutamate transporter 2 (vGluT2) was increased, whereas the level of the potassium/chloride cotransporter KCC2 was reduced, thus indicating a higher excitation/inhibition ratio. Our findings provide evidence that altered expression of a key gene for cortical development can result in specific alterations in neural circuit function at the macro- and micro-scale, along with dysregulated intracellular signaling and expression of proteins controlling circuit excitability.