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.

Regulation of BACE1 expression after injury is linked to the p75 neurotrophin receptor.

BACE1 is a transmembrane aspartic protease that cleaves various substrates and it is required for normal brain function. BACE1 expression is high during early development, but it is reduced in adulthood. Under conditions of stress and injury, BACE1 levels are increased; however, the underlying mechanisms that drive BACE1 elevation are not well understood. One mechanism associated with brain injury is the activation of injurious p75 neurotrophin receptor (p75), which can trigger pathological signals. Here we report that within 72 h after controlled cortical impact (CCI) or laser injury, BACE1 and p75 are increased and tightly co-expressed in cortical neurons of mouse brain. Additionally, BACE1 is not up-regulated in p75 null mice in response to focal cortical injury, while p75 over-expression results in BACE1 augmentation in HEK-293 and SY5Y cell lines. A luciferase assay conducted in SY5Y cell line revealed that BACE1 expression is regulated at the transcriptional level in response to p75 transfection. Interestingly, this effect does not appear to be dependent upon p75 ligands including mature and pro-neurotrophins. In addition, BACE1 activity on amyloid precursor protein (APP) is enhanced in SY5Y-APP cells transfected with a p75 construct. Lastly, we found that the activation of c-jun n-terminal kinase (JNK) by p75 contributes to BACE1 up-regulation. This study explores how two injury-induced molecules are intimately connected and suggests a potential link between p75 signaling and the expression of BACE1 after brain injury.

NGF steers microglia toward a neuroprotective phenotype.

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 Aß peptide clearance in the brain, we tested the effects of NGF on its phagocytosis. NGF was shown to promote TrkA-mediated engulfment of Aß by microglia, and to enhance its degradation. Additionally, the proinflammatory activation induced by Aß treatment is counteracted by the concomitant administration of NGF. Moreover, by acting specifically on microglia, NGF protects neurons from the Aß-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 Aß 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 Aß accumulation pathologies, via its anti-inflammatory activity on microglia.

Reduced levels of NGF shift astrocytes toward a neurotoxic phenotype.

Nerve growth factor (NGF) is critical for neuronal physiology during development and adulthood. Despite the well-recognized effect of NGF on neurons, less is known about whether NGF can actually affect other cell types in the central nervous system (CNS). In this work, we show that astrocytes are susceptible to changes in ambient levels of NGF. First, we observe that interfering with NGF signaling in vivo via the constitutive expression of an antiNGF antibody induces astrocytic atrophy. A similar asthenic phenotype is encountered in an uncleavable proNGF transgenic mouse model (TgproNGF#72), effectively increasing the brain proNGF levels. To examine whether this effect on astrocytes is cell-autonomous, we cultured wild-type primary astrocytes in the presence of antiNGF antibodies, uncovering that a short incubation period is sufficient to potently and rapidly trigger calcium oscillations. Acute induction of calcium oscillations by antiNGF antibodies is followed by progressive morphological changes similar to those observed in antiNGF AD11 mice. Conversely, incubation with mature NGF has no effect on either calcium activity nor on astrocytic morphology. At longer timescales, transcriptomic analysis revealed that NGF-deprived astrocytes acquire a proinflammatory profile. In particular, antiNGF-treated astrocytes show upregulation of neurotoxic transcripts and downregulation of neuroprotective mRNAs. Consistent with that data, culturing wild-type neurons in the presence of NGF-deprived astrocytes leads to neuronal cell death. Finally, we report that in both awake and anesthetized mice, astrocytes in layer I of the motor cortex respond with an increase in calcium activity to acute NGF inhibition using either NGF-neutralizing antibodies or a TrkA-Fc NGF scavenger. Moreover, in vivo calcium imaging in the cortex of the 5xFAD neurodegeneration mouse model shows an increased level of spontaneous calcium activity in astrocytes, which is significantly reduced after acute administration of NGF. In conclusion, we unveil a novel neurotoxic mechanism driven by astrocytes, triggered by their sensing and reacting to changes in the levels of ambient NGF.

A Microglial Function for the Nerve Growth Factor: Predictions of the Unpredictable.

Microglia are the only immune cell population present in the brain parenchyma. Their vantage position in the central nervous system (CNS) enables these myeloid cells to perform the most disparate of tasks: from the classical immune functions of fighting infections and surveilling the extracellular space for pathogens and damage, to sculpting the neuronal circuitry by pruning unnecessary synapses and assisting neurons in spine formation, aiding in the maintenance of brain homeostasis. The neurotrophin field has always been dominated by the neurocentric view that the primary target of these molecules must be neurons: this holds true even for the Nerve Growth Factor (NGF), which owes its popularity in the neuroscience community to its trophic and tropic activity towards sensory and sympathetic neurons in the peripheral nervous system, and cholinergic neurons in the CNS. The increasing evidence that microglia are an integral part of neuronal computation calls for a closer look as to whether these glial cells are capable of responding directly to NGF. In this review, we will first outline evidence in support of a role for NGF as a molecule mediating neuroimmune communication. Then, we will illustrate some of those non-immune features that have made microglial cells one of the hottest topics of this last decade. In conclusion, we will discuss evidence in support of a microglial function for NGF.