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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.

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, Aβ 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.

Nerve growth factor (NGF), by binding to TrkA and p75(NTR) 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 NGF(WT) and NGF(R221W) in the heterozygote state formed? And if so, what are the properties of these heterodimeric proteins?How is the processing of proNGF(R221W) to NGF(R221W) affected by the mutation?

Several works reported increased differentiation of neuronal cells grown on graphene; however, the molecular mechanism driving axon elongation on this material has remained elusive. Here, we study the axonal transport of nerve growth factor (NGF), the neurotrophin supporting development of peripheral neurons, as a key player in the time course of axonal elongation of dorsal root ganglion neurons on graphene. We find that graphene drastically reduces the number of retrogradely transported NGF vesicles in favor of a stalled population in the first two days of culture, in which the boost of axon elongation is observed. This correlates with a mutual charge redistribution, observed via Raman spectroscopy and electrophysiological recordings. Furthermore, ultrastructural analysis indicates a reduced microtubule distance and an elongated axonal topology. Thus, both electrophysiological and structural effects can account for graphene action on neuron development. Unraveling the molecular players underneath this interplay may open new avenues for axon regeneration applications. Several works reported increased differentiation of neuronal cells grown on graphene; however, the molecular mechanism driving axon elongation on this material has remained elusive. Here, we study the axonal transport of nerve growth factor (NGF), the neurotrophin supporting development of peripheral neurons, as a key player in the time course of axonal elongation of dorsal root ganglion neurons on graphene. We find that graphene drastically reduces the number of retrogradely transported NGF vesicles in favor of a stalled population in the first 2 days of culture, in which the boost of axon elongation is observed. This correlates with a mutual charge redistribution, observed via Raman spectroscopy and electrophysiological recordings. Furthermore, ultrastructural analysis indicates a reduced microtubule distance and an elongated axonal topology. Thus, both electrophysiological and structural effects can account for graphene action on neuron development. Unraveling the molecular players underneath this interplay may open new avenues for axon regeneration applications.

Alzheimer's disease (AD) is a progressive neurodegenerative disease leading to neuronal dysfunctions with cognitive impairment. AD can affect visual pathways and visual cortex and result in various visual changes and problems. However, how early the visual dysfunctions occur in AD is still a matter of discussion. Here, we used electrophysiological techniques to show the presence of early anomalies in AD visual system. To this aim, we used a familial AD (FAD) model, the 5xFAD transgenic mouse, characterized by severe progressive amyloid pathology and cognitive deficits. We investigated the retina and primary visual cortex responsivity together with behavioral assessment of the visual acuity. Visual tests and recordings were conducted at different ages in 5xFAD mice, corresponding to different phases of neurodegeneration and beta amyloid accumulation. We showed that the visual system is impaired in 5xFAD mice. In particular, we found that the inner retina impairment precedes neuronal disorders in other brain areas and cognitive deficits. Thus, noninvasive retinal electrophysiology can provide a support for assessing early visual dysfunctions in AD.

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 NGF(R100W/wt) mice display impaired nociception. DRG neurons of NGF(R100W/wt) mice are morphologically normal, with no alteration in the different DRG subpopulations, whereas skin innervation is reduced. The NGF(R100W) protein has reduced capability to activate pain-specific signaling, paralleling its reduced ability to induce mechanical allodynia. Surprisingly, however, NGF(R100W/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 (NGF(R100W/wt)). Mutant mice have normal nociceptor populations, which, however, display decreased activation of pain transduction pathways. NGF(R100W) 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.

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. 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 Ngfgene, 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 Ngfgene 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.

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.

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 Aβ 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.

Pain is an unpleasant but necessary sensory experience, which facilitates adaptive behaviours, such as fear. Despite recent advances, the question of how the pain experience influences learning of the fear response is still debated1,2. Genetic disorders rendering patients congenitally unable to feel pain have been described, and are usually explained by defects in peripheral nociceptors3-5. It is not known how growing up without pain affects central emotional and motivational responses to aversive stimuli. The rare autosomal recessive Hereditary Sensory and Autonomic Neuropathy type V (HSAN V) is caused by a mutation in the nerve growth factor (NGF) gene (R100W). HSAN V homozygous patients display a congenital indifference to painful events, with deficits in peripheral nociceptors but without overt cognitive impairment6. In contrast, heterozygous carriers do not present with pain-related deficits and have been identified only through pedigree and genetic screening7,8. We exploited this clinically silent population to dissociate nociceptive from affective features of pain. To address this we investigated both heterozygous knock-in mice bearing the R100W mutation (NGFR100W/m mice) and a cohort of HSAN V heterozygous human carriers. Surprisingly, we found that NGFR100W/m mice, despite normal responses to a noxious conditioning stimulus, show a deficit in learned fear while their social and innate fear responses are normal. The lack of pain-related fear response was linked to a reduced activation of anterior cingulate and motor cortices and of striatum, but not in primary somatosensory cortex. Likewise, human heterozygous R100W carriers, despite perceiving noxious stimuli and reporting subjective pain thresholds, show increased reaction latencies in response to painful stimulation, alongside a decreased subjective urgency to react. Functional magnetic resonance imaging (fMRI) revealed, comparably to the mouse data, an altered processing of painful stimuli in rostral anterior cingulate, medial premotor cortical regions and striatum. These findings from both human and mouse HSAN V carriers uncover behavioural and motivational consequences of a mild genetic pain insensitivity on the establishment of pain-dependent affective responses and memories.

Microglia are the sentinels of the brain but a clear understanding of the factors that modulate their activation in physiological and pathological conditions is still lacking. Here we demonstrate that Nerve Growth Factor (NGF) acts on microglia by steering them toward a neuroprotective and anti-inflammatory phenotype. We show that microglial cells express functional NGF receptors in vitro and ex vivo. Our transcriptomic analysis reveals how, in primary microglia, NGF treatment leads to a modulation of motility, phagocytosis and degradation pathways. At the functional level, NGF induces an increase in membrane dynamics and macropinocytosis and, in vivo, it activates an outward rectifying current that appears to modulate glutamatergic neurotransmission in nearby neurons. Since microglia are supposed to be a major player in Aβ peptide clearance in the brain, we tested the effects of NGF on its phagocytosis. NGF was shown to promote TrkA-mediated engulfment of Aβ by microglia, and to enhance its degradation. Additionally, the proinflammatory activation induced by Aβ treatment is counteracted by the concomitant administration of NGF. Moreover, by acting specifically on microglia, NGF protects neurons from the Aβ-induced loss of dendritic spines and inhibition of long term potentiation. Finally, in an ex-vivo setup of acute brain slices, we observed a similar increase in Aβ engulfment by microglial cells under the influence of NGF. Our work substantiates a role for NGF in the regulation of microglial homeostatic activities and points toward this neurotrophin as a neuroprotective agent in Aβ accumulation pathologies, via its anti-inflammatory activity on microglia. Microglia are the sentinels of the brain but a clear understanding of the factors that modulate their activation in physiological and pathological conditions is still lacking. Here we demonstrate that Nerve Growth Factor (NGF) acts on microglia by steering them toward a neuroprotective and anti-inflammatory phenotype. We show that microglial cells express functional NGF receptors in vitro and ex vivo. Our transcriptomic analysis reveals how, in primary microglia, NGF treatment leads to a modulation of motility, phagocytosis and degradation pathways. At the functional level, NGF induces an increase in membrane dynamics and macropinocytosis and, in vivo, it activates an outward rectifying current that appears to modulate glutamatergic neurotransmission in nearby neurons. Since microglia are supposed to be a major player in Aβ peptide clearance in the brain, we tested the effects of NGF on its phagocytosis. NGF was shown to promote TrkA-mediated engulfment of Aβ by microglia, and to enhance its degradation. Additionally, the proinflammatory activation induced by Aβ treatment is counteracted by the concomitant administration of NGF. Moreover, by acting specifically on microglia, NGF protects neurons from the Aβ-induced loss of dendritic spines and inhibition of long term potentiation. Finally, in an ex-vivo setup of acute brain slices, we observed a similar increase in Aβ engulfment by microglial cells under the influence of NGF. Our work substantiates a role for NGF in the regulation of microglial homeostatic activities and points toward this neurotrophin as a neuroprotective agent in Aβ accumulation pathologies, via its anti-inflammatory activity on microglia.

Hereditary sensory and autonomic neuropathy type V (HSAN V) is an autosomal recessive disorder characterized by the loss of deep pain perception. The anomalous pain and temperature sensations are due to the absence of nociceptive sensory innervation. The neurotrophin nerve growth factor (NGF), by binding to tropomyosin receptor A (TrkA) and p75NTR receptors, is essential for the development and survival of sensory neurons, and for pain perception during adulthood. Recently a homozygous missense mutation (R100W) in the NGF gene has been identified in HSAN V patients. Interestingly, alterations in NGF signalling, due to mutations in the NGF TRKA gene, have also been involved in another congenital insensitivity to pain, HSAN IV, characterized not only by absence of reaction to painful stimuli, but also anhidrosis and mental retardation. These symptoms are absent in HSAN V patients. Unravelling the mechanisms that underlie the differences between HSAN IV and V could assist in better understanding NGF biology. This review highlights the recent key findings in the understanding of HSAN V, including insights into the molecular mechanisms of the disease, derived from genetic studies of patients with this disorder.

ProNGF, the precursor of mature nerve growth factor (NGF), is the most abundant form of NGF in the brain. ProNGF and mature NGF differ significantly in their receptor interaction properties and in their bioactivity. ProNGF increases markedly in the cortex of Alzheimer's disease (AD) brains and proNGF\NGF imbalance has been postulated to play a role in neurodegeneration. However, a direct proof for a causal link between increased proNGF and AD neurodegeneration is lacking. In order to evaluate the consequences of increased levels of proNGF in the postnatal brain, transgenic mice expressing a furin cleavage-resistant form of proNGF, under the control of the neuron-specific mouse Thy1.2 promoter, were derived and characterized. Different transgenic lines displayed a phenotypic gradient of neurodegenerative severity features. We focused the analysis on the two lines TgproNGF#3 and TgproNGF#72, which shared learning and memory impairments in behavioral tests, cholinergic deficit and increased Aβ-peptide immunoreactivity. In addition, TgproNGF#3 mice developed Aβ oligomer immunoreactivity, as well as late diffuse astrocytosis. Both TgproNGF lines also display electrophysiological alterations related to spontaneous epileptic-like events. The results provide direct evidence that alterations in the proNGF/NGF balance in the adult brain can be an upstream driver of neurodegeneration, contributing to a circular loop linking alterations of proNGF/NGF equilibrium to excitatory/inhibitory synaptic imbalance and amyloid precursor protein (APP) dysmetabolism.

Nerve growth factor (NGF) is a therapeutic candidate for Alzheimer’s disease, but must be administered intraparenchymally owing to its pain-inducing activity. Capsoni et al. develop human painless NGF (hNGFp) and show that intranasal administration results in widespread biodistribution in the brains of Alzheimer’s disease mice, with potent anti-amyloidogenic effects., Nerve growth factor is a therapeutic candidate for Alzheimer’s disease. Due to its pain-inducing activity, in current clinical trials nerve growth factor is delivered locally into the brain by neurosurgery, but data on the efficacy of local nerve growth factor delivery in decreasing amyloid-β deposition are not available. To reduce the nerve growth factor pain-inducing side effects, thus avoiding the need for local brain injection, we developed human painless nerve growth factor (hNGFp), inspired by the human genetic disease hereditary sensory and autonomic neuropathy type V. hNGFp has identical neurotrophic potency as wild-type human nerve growth factor, but a 10-fold lower pain sensitizing activity. In this study we first mimicked, in the 5xFAD mouse model, the intraparenchymal delivery of hNGFp used in clinical trials and found it to be ineffective in decreasing amyloid-β plaque load. On the contrary, the same dose of hNGFp delivered intranasally, which was widely biodistributed in the brain and did not induce pain, showed a potent anti-amyloidogenic action and rescued synaptic plasticity and memory deficits. We found that hNGFp acts on glial cells, modulating inflammatory proteins such as the soluble TNFα receptor II and the chemokine CXCL12. We further established that the rescuing effect by hNGFp is mediated by CXCL12, as pharmacological inhibition of CXCL12 receptor CXCR4 occludes most of hNGFp effects. These findings have significant therapeutic implications: (i) we established that a widespread exposure of the brain is required for nerve growth factor to fully exert its neuroprotective actions; and (ii) we have identified a new anti-neurodegenerative pathway as a broad target for new therapeutic opportunities for neurodegenerative diseases.

ProNGF, the precursor of mature Nerve Growth Factor (NGF), is the most abundant NGF form in the brain and increases markedly in the cortex in Alzheimer's Disease (AD), relative to mature NGF. A large body of evidence shows that the actions of ProNGF and mature NGF are often conflicting, depending on the receptors expressed in target cells. TgproNGF#3 mice, expressing furin-cleavage resistant proNGF in CNS neurons, directly reveal consequences of increased proNGF levels on brain homeostasis. Their phenotype clearly indicates that proNGF can be a driver of neurodegeneration, including severe learning and memory behavioral deficits, cholinergic deficits, and diffuse immunoreactivity for A-beta and A-beta-oligomers. In aged TgproNGF#3 mice spontaneous epileptic-like events are detected in entorhinal cortex-hippocampal slices, suggesting occurrence of excitatory/inhibitory (E/I) imbalance. In this paper, we investigate the molecular events linking increased proNGF levels to the epileptiform activity detected in hippocampal slices. The occurrence of spontaneous epileptiform discharges in the hippocampal network in TgproNGF#3 mice suggests an impaired inhibitory interneuron homeostasis. In the present study, we detect the onset of hippocampal epileptiform events at 1-month of age. Later, we observe a regional- and cellular-selective Parvalbumin interneuron and perineuronal net (PNN) depletion in the dentate gyrus (DG), but not in other hippocampal regions of TgproNGF#3 mice. These results demonstrate that, in the hippocampus, the DG is selectively vulnerable to altered proNGF/NGF signaling. Parvalbumin interneuron depletion is also observed in the amygdala, a region strongly connected to the hippocampus and likewise receiving cholinergic afferences. Transcriptome analysis of TgproNGF#3 hippocampus reveals a proNGF signature with broad down-regulation of transcription. The most affected mRNAs modulated at early times belong to synaptic transmission and plasticity and extracellular matrix (ECM) gene families. Moreover, alterations in the expression of selected BDNF splice variants were observed. Our results provide further mechanistic insights into the vicious negative cycle linking proNGF and neurodegeneration, confirming the regulation of E/I homeostasis as a crucial mediating mechanism.

The neurotrophin Nerve Growth Factor (NGF) is essential for the maintenance and differentiation of basal forebrain cholinergic neurons. Since basal forebrain cholinergic neurons represent one major neuronal population affected and progressively degenerating in Alzheimer's disease (AD), interest has grown for NGF as a potential therapeutic agent in neurodegenerative disorders linked to aging, particularly for AD. However, no evidence was available, to link, in a cause-effect manner, deficits in NGF signalling to the broader activation in the Alzheimer's cascade, besides cholinergic deficits. The phenotypic analysis of the AD11 anti-NGF transgenic mouse, obtained by the "neuroantibodies" phenotypic protein knock out strategy, allowed demonstrating a direct causal link between NGF deprivation and AD pathology. Since then, extensive mechanistic studies on the AD11 model provided a new twist to the concept that alterations in NGF transport and signalling play a crucial role in sporadic Alzheimer's neurodegeneration, leading to the hypothesis of "Neurotrophic imbalance" as an upstream driver for sporadic AD. The results obtained with the AD11 anti-NGF mice highlight the fact that the particular mode of NGF neutralization, with an NGF antibody expressed in the brain, selectively interfering with mature NGF versus unprocessed proNGF, plays a major role in the mechanism of neurodegeneration, and could lead to new insights into the mechanisms of human sporadic AD. Here, we will review (1) the renewed neurotrophic imbalance hypothesis for AD and (2) the mechanisms underlying the neurodegenerative phenotype of AD11 anti-NGF mice.

The etiology of Alzheimer's disease (AD) remains elusive. The “amyloid” hypothesis states that toxic action of accumulated β-amyloid peptide (Aβ) on synaptic function causes AD cognitive decline. This hypothesis is supported by analysis of familial AD (FAD)-based transgenic mouse models, where altered amyloid precursor protein (APP) processing leads to Aβ accumulation correlating with hippocampal-dependent memory deficits. Some studies report prominent dentate gyrus (DG) glutamatergic plasticity alterations in these mice, while CA1 plasticity remains relatively unaffected. The “neurotrophic unbalance” hypothesis, on the other hand, states that AD-related loss of cholinergic signaling and altered APP processing are due to alterations in nerve growth factor (NGF) trophic support. This hypothesis is supported by analysis of the AD11 mouse, which exhibits chronic NGF deprivation during adulthood and displays AD-like pathology, including Aβ accumulation and hippocampal-dependent memory deficits. In this study, we analyzed CA1 and DG glutamatergic plasticity in AD11 mice to evaluate whether these mice also share with FAD models a common phenotype in hippocampal synaptic dysfunction. We report that AD11 mice display age-dependent short- and long-term DG plasticity deficits, while CA1 plasticity remains relatively spared. We also report that both structures exhibit enhanced glutamatergic transmission under lower, yet physiological, neurotransmitter release conditions, a defect that should be considered when further evaluating hippocampal synaptic deficits underlying AD pathology. We conclude that severe deficits in DG plasticity represent another common denominator between these two etiologically different types of AD mouse models, independent of the initial insult (overexpression of FAD mutation or NGF deprivation).

We previously showed that anti-nerve growth factor (NGF) antibodies expressed in transgenic mice (AD11) elicit a progressive neurodegeneration, comprising the triad of Alzheimer's disease (AD) hallmarks: cholinergic loss, tau hyperphosphorylation, and amyloid-beta peptide formation. However, since anti-NGF antibodies are produced both in the brain and in peripheral tissues of AD11 mice, the contribution of peripheral neutralization of NGF to the onset of brain neurodegeneration was still unexplored. To address this question, we characterized a line of transgenic mice (AD10) in which anti-NGF antibodies are obligatorily produced only in lymphocytes, being initially found in blood. In AD10 mice, peripheral NGF neutralization elicits shrinkage of superior cervical ganglia (immunosympathectomy) and, as a consequence of this, peripheral anti-NGF antibodies cross the blood brain barrier (BBB) and reach the brain, generating an NGF-dependent neurodegeneration, largely superimposable to that observed in AD11 mice. This demonstrates that peripherally originated anti-NGF antibodies can generate a neurodegeneration in the central nervous system of an animal model. Consistently, peripherally-delivered NGF is effective in preventing the onset of the central cholinergic deficit. These findings could have a direct relevance for some human sporadic AD cases, highlighting the role of the BBB disruption and suggesting a causally relevant role of circulating antibodies in AD pathology.

Alzheimer's disease is a neurodegenerative disorder characterized by neuronal loss associated with a progressive impairment of cognitive functions. Early consequences of Alzheimer's disease include deficit of cholinergic signalling in particular regions controlling memory processes, such as the cortex and hippocampus, and accumulation of beta-amyloid (Abeta) peptide in neuritic plaques. The cholinergic system depends for its integrity and function on nerve growth factor. Chronic nerve growth factor deprivation in transgenic mice (AD11) engineered to produce recombinant neutralizing anti-nerve growth factor antibodies leads to progressive age-dependent Alzheimer's-like neurodegenerative pathology similar to that found in patients with Alzheimer's disease, associated with a selective loss of cholinergic neurones in the basal forebrain. Here we show that in the hippocampus of 6-month-old AD11 mice, Abeta aggregates started appearing in the CA1 region. The accumulation of Abeta was associated with a loss of cholinergic function at CA3-CA1 synapses. Whereas in wild-type mice nicotine induced a persistent increase of synaptic efficacy via alpha7 nicotine acetylcholine receptors, in AD11 mice this alkaloid failed to modify synaptic strength. Moreover, nicotine failed to transiently enhance the frequency of spontaneous miniature glutamatergic currents (miniature excitatory postsynaptic currents) recorded from CA1 but not from CA3 pyramidal neurones of AD11 mice. However, in CA3 principal cells of AD11 mice, the potentiating effect of nicotine on miniature excitatory postsynaptic currents was prevented when Abeta peptide 1-42 was added to the extracellular solution. These data suggest that in AD11 mice, Abeta interferes with nicotine acetylcholine receptors at the level of presynaptic glutamatergic terminals, inhibiting their function possibly through calcium signalling via presynaptic alpha7 nicotine acetylcholine receptors.

1. Alzheimer's disease (AD) is pathologically defined by the deposition of amyloid peptide and neurofibrillary tangles and is characterized by a progressive loss of cognition and memory function, due to marked cortical cholinergic depletion. 2. Cholinergic cortical innervation is provided by basal forebrain cholinergic neurons. The neurotrophin Nerve Growth Factor (NGF) promotes survival and differentiation of basal forebrain cholinergic neurons. 3. This assertion has been at the basis of the hypothesis developed in the last 20 years, whereby NGF deprivation would be one of the factor involved in the etiology of sporadic forms of AD. 4. In this review, we shall summarize data that lead to the production and characterization of a mouse model for AD (AD11 anti-NGF mice), based on the expression of transgenic antibodies neutralizing NGF. The AD-like phenotype of AD11 mice will be discussed on the basis of recent studies that have posed NGF and its precursor pro-NGF back to the stage of AD-like neurodegeneration, showing the involvement of the precursor pro-NGF in one of the cascades leading to AD neurodegeneration.

Cerebral deposition of beta-amyloid (Abeta) is an invariant event of Alzheimer's disease (AD). We recently described that the brain of aged transgenic mice expressing anti-nerve growth factor (NGF) antibodies (AD11 mice) show a dramatic neurodegenerative phenotype, reminiscent of AD, which includes neuronal loss, cholinergic deficit, and tau hyperphosphorylation, associated with neurofibrillary pathology. We now report that brains of aged transgenic mice contain large amounts of beta-amyloid plaques and describe their morphology by a variety of approaches. In conclusion, the chronic deprivation of NGF leads to the formation and deposition of Abeta in AD11 mice, suggesting a direct link between NGF signaling and abnormal processing of amyloid precursor protein.

Deficits in cholinergic systems innervating cerebral cortex are associated with cognitive impairment during senescence and in age-related neurodegenerative pathologies. However, little is known about the role of cholinergic pathways in modulating cortical plasticity. Basal forebrain cholinergic neurons are a major target for nerve-growth factor (NGF). In order to investigate the relationship between cholinergic innervation and cortical synaptic plasticity, we exploited a transgenic mouse model in which the activity of NGF in the adult nervous system is neutralized by the expression of blocking antibodies to NGF itself (anti-NGF mice) [Ruberti, F. et al. (2000). J. Neurosci. 20, 2589-2601]. In 6-month-old anti-NGF mice, we show that the reduction in cholinergic innervation of the cortex is associated with different forms of synaptic plasticity impairment. A local, acute increase in the availability of acetylcholine rescues these synaptic plasticity deficits, thus indicating that a cholinergic system mediates the impairment of cortical plasticity at this early stage of the neurodegenerative process triggered by NGF neutralization. Our results represent an important step in unveiling the pivotal role of cholinergic transmission in modulating adult cortical plasticity.

Brain-derived neurotrophic factor (BDNF) messenger RNA (mRNA) expression in the rat visual cortex of young and postnatal day 90 (P90) animals is developmentally regulated and influenced by visual experience. In the present paper we compared the expression of BDNF mRNA to the actual changes of BDNF protein occurring during postnatal development and verified whether BDNF protein distribution is controlled by visual activity. To achieve this aim we analysed BDNF mRNA and/or BDNF protein cellular distribution in the rat visual cortex at different postnatal ages by using immunohistochemistry and highly sensitive in situ hybridization. We found that before eye opening (P13), in all cortical layers a large number of visual cortical neurons contain BDNF mRNA with no detectable amount of BDNF protein. At later ages (P23 and P90), the number of BDNF-immunostained cells increases; most neurons are double labelled for BDNF mRNA and protein, and a small group of neurons is labelled only for BDNF protein. The cellular increase of BDNF immunolabelling is blocked in animals deprived of visual experience from birth (dark rearing), with a large population of neurons containing BDNF mRNA but not BDNF protein. This is similar to what is observed before eye opening. Exposure of dark-reared rats to a brief period (2 h) of light restores a good match between BDNF mRNA and BDNF protein cellular expression. We propose that visual experience controls the neuronal content of BDNF mRNA and BDNF protein in developing visual cortex.

We have exploited a new monoclonal antibody against the tyrosine kinase A (TrkA) nerve growth factor (NGF) receptor to block the NGF–TrkA interaction in the rat basal forebrain. The monoclonal antibody MNAC13 is a potent antagonist that prevents the binding of NGF to TrkA in a variety of systems. This antibody was used to study the maintenance of the cholinergic phenotype in the rat basal forebrainin vivo, by the implant of antibody-secreting cells. Basal forebrain cholinergic neurons (BFCNs) are greatly affected by the antibody treatment, both in terms of cell number and of cell soma size. When antibody-secreting cells are implanted at postnatal day 2 (P2), the effects observed at P8 are as severe as those obtained with anti-NGF antibodies and, interestingly, are observed also if anti-TrkA cells are implanted at P8, when anti-NGF antibodies, delivered by the same route, are no longer effective (Molnar et al., 1998). The effects induced by anti-TrkA, as those induced by anti-NGF, are reversible, but the time required for recovery and the critical period in the sensitivity of BFCNs to the functional inactivation of TrkA is twice as long than that observed when NGF is intercepted. These results demonstrate that BFCNs are more sensitive to the block of TrkA activation than they are to the block of NGF. The cloning of MNAC13 variable regions and their assembly into a functional polypeptide of reduced size (single chain Fv fragment) will allow its use in gene transfer applications.

It has been suggested that systemic infection, occurring during aging and chronic neurodegenerative diseases, can evoke an immune response that aggravates the progression of neurodegeneration and cognitive decline. It has been shown that the AD11 neurodegeneration mouse model, expressing a recombinant anti-nerve growth factor (NGF) antibody, shows a milder phenotype when housed in murine pathogen-free (MPF) conditions with respect to AD11 mice reared in conventional (CV) housing. AD10 mice, a variant of the anti-NGF AD11 model, expressing only an immunoglobulin light chain for the transgenic anti-NGF antibody, in the absence of the corresponding transgenic antibody chain VH, exhibit a complex neurodegenerative phenotype, similar to that of AD11 mice. Here we show that the AD10 transgenic mice, housed in murine pathogen-free conditions (MPF-AD10 mice), also display a milder behavioral and neurodegenerative phenotype compared to the corresponding mice kept under conventional housing conditions (CV-AD10). As a first step toward the identification of mechanisms underlying this difference, a differential gene expression profiling was performed on brains from CV-AD10 and MPF-AD10 mice, showing a decrease of the immune response and neuroinflammation gene expression in MPF-AD10 mice. Results suggest that the activation of the immune response gene expression in the CV-AD10, in a microbially unprotected environment, might contribute to a more severe and progressive neurodegenerative phenotype, compared to the MPF mice.

Adult neurogenesis is a multistep process regulated by several extrinsic factors, including neurotrophins. Among them, little is known about the role of nerve growth factor (NGF) in the neurogenic niches of the mouse. Here we analyzed the biology of adult neural stem cells (NSCs) from the subventricular zone (SVZ) of AD11 anti-NGF transgenic mice, in which the expression of the recombinant antibody aD11 leads to a chronic postnatal neutralization of endogenous NGF. We showed that AD11-NSCs proliferate 10-fold less, with respect to their control counterparts, and display a significant impairment in their ability to differentiate into β-tubulin positive neurons. We found a considerable reduction in the number of SVZ progenitors and neuroblasts also in vivo, which correlates with a lower number of newborn neurons in the olfactory bulbs of AD11 mice and a severe deficit in the ability of these mice to discriminate between different odors. We also demonstrated that, in AD11 mice, the morphology of both SVZ-resident and neurosphere-derived astrocytes is significantly altered. We were able to reproduce the AD11 phenotype in vitro, by acutely treating wild type NSCs with the anti-NGF antibody, further demonstrating that both the proliferation and the differentiation defects are due to the NGF deprivation. Consistently, the proliferative impairment of AD11 progenitors, as well as the atrophic morphology of AD11 astrocytes, can be partly rescued in vitro and in vivo by exogenous NGF addition. Altogether, our results demonstrate a causal link between NGF signaling and proper proliferation and differentiation of neural stem cells from the SVZ. Adult neurogenesis is a multistep process regulated by several extrinsic factors, including neurotrophins. Among them, little is known about the role of nerve growth factor (NGF) in the neurogenic niches of the mouse. Here we analyzed the biology of adult neural stem cells (NSCs) from the subventricular zone (SVZ) of AD11 anti-NGF transgenic mice, in which the expression of the recombinant antibody aD11 leads to a chronic postnatal neutralization of endogenous NGF. We showed that AD11-NSCs proliferate 10-fold less, with respect to their control counterparts, and display a significant impairment in their ability to differentiate into β-tubulin positive neurons. We found a considerable reduction in the number of SVZ progenitors and neuroblasts also in vivo, which correlates with a lower number of newborn neurons in the olfactory bulbs of AD11 mice and a severe deficit in the ability of these mice to discriminate between different odors. We also demonstrated that, in AD11 mice, the morphology of both SVZ-resident and neurosphere-derived astrocytes is significantly altered. We were able to reproduce the AD11 phenotype in vitro, by acutely treating wild type NSCs with the anti-NGF antibody, further demonstrating that both the proliferation and the differentiation defects are due to the NGF deprivation. Consistently, the proliferative impairment of AD11 progenitors, as well as the atrophic morphology of AD11 astrocytes, can be partly rescued in vitro and in vivo by exogenous NGF addition. Altogether, our results demonstrate a causal link between NGF signaling and proper proliferation and differentiation of neural stem cells from the SVZ.

Autosomal dominant forms of familial Alzheimer's disease are linked to an aberrant processing of the amyloid-β protein precursor, which results in an increased production of amyloid-β (Aβ) peptides that first form oligomers and eventually aggregate in the form of extracellular amyloid plaques in the brain. The accumulation of Aβ peptides oligomers seems to correlate with alterations of synaptic transmission in experimental models of Alzheimer's disease. Whether Aβ aggregation disrupts synaptic function independently of amyloid plaques deposition still needs further research. Here we report an amyloid plaque-independent deficit of neuronal plasticity after short-term sensory deprivation in the visual system of 5XFAD mice.

Sortilin-related receptor with A-type repeats (SorLA, also known as LR11) has been implicated in Alzheimer's disease (AD). Thus, genetic studies associated SorLA gene variants with the risk of sporadic AD. Also, in vitro and in vivo studies showed that SorLA impairs processing of the amyloid-β protein precursor (AβPP) to amyloid-β. In particular, it has been found that loss of SorLA accelerates senile plaque deposition in mouse models overexpressing mutant forms of human AβPP and presenilin 1. Here we tested the possibility that SorLA deficiency also interferes with behavioral and neuropathological endpoints in an alternative murine AD model, the AD10 anti-nerve growth factor (NGF) mouse, in which amyloid-β accumulation derives from the altered processing of endogenous AβPP. In addition to alterations in AβPP processing, AD10 mice also show cholinergic deficit and tau hyperphosphorylation resulting in behavioral deficits in learning and memory paradigms. We found that the loss of SorLA not only exacerbates early amyloid pathology but, at the same time, protects from cholinergic deficit and from early phospho-tau mislocalization. The results show that in the AD10 anti-NGF mouse model the AβPP processing-related aspects of neurodegeneration can be dissociated from those related to tau posttranslational processing and to cholinergic phenotypic maintenance by modulation of SorLA expression. We suggest that SorLA regulates different aspects of neurodegeneration in a complex way, supporting the hypothesis that SorLA expression might be critical not only for amyloid-related pathology but also for other cellular processes altered in AD.

Highlights ► We hypothesize that neurodegeneration in anti-NGF is due to NGF/proNGF imbalance. ► We propose that the proNGF co- receptor sortilin contributes to the neurodegeneration. ► We analyzed anti-NGF mice crossed to sortilin knockout mice. ► Sortilin loss partially protected AD10 anti-NGF mice from neurodegeneration., Sortilin is a member of the family of vacuolar protein sorting 10 protein domain receptors which has emerged as a co-receptor in cell death and neurodegeneration processes mediated by proneurotrophins. Here we tested the possibility that sortilin deficiency interferes with behavioral and neuropathological endpoints in a chronic Nerve Growth factor (NGF)-deprivation model of Alzheimer’s disease (AD), the AD10 anti-NGF mouse. AD10 mice show cholinergic deficit, increased APP processing and tau hyper-phosphorylation, resulting in behavioral deficits in learning and memory paradigms assessed by novel object recognition and Morris water maze tests. Sort1−/− mice were crossed with AD10 anti-NGF mice and the neurodegenerative phenotype was studied. We found that the loss of sortilin partially protected AD10 anti-NGF mice from neurodegeneration. A protective effect was observed on non-spatial memory as assessed by novel object recognition, and histopathologically at the level of Aβ and BFCNs, while the phosphotau increase was unaltered by knocking out sortilin. We suggest that sortilin might be involved in different aspects of neurodegeneration in a complex way, supporting the view that sortilin functions in the CNS are broader than being a co-receptor in proneurotrophin and neurotrophin signaling.

The word "Factor" is misspelled in the article title. The correct title is: Intranasal "painless" Human Nerve Growth Factor Slows Amyloid Neurodegeneration and Prevents Memory Deficits in App X PS1 Mice. The correct citation is: Capsoni S, Marinelli S, Ceci M, Vignone D, Amato G, et al. (2012) Intranasal “painless” Human Nerve Growth Factor Slows Amyloid Neurodegeneration and Prevents Memory Deficits in App X PS1 Mice. PLoS ONE 7(5): e37555. doi:10.1371/journal.pone.0037555

Several studies suggest that systemic infection occurring during aging and chronic neurodegenerative diseases can evoke an exaggerated immune response that contributes to the progression of neurodegeneration and cognitive decline. However, studies directly addressing the relationship between microbial environment and the onset of neurodegeneration in Alzheimer's disease animal models are lacking. Here we show that the onset of neurodegeneration that transgenic mice develop when raised in conventional husbandry slows down when raising anti-nerve growth factor transgenic mice in a murine pathogen free condition.