CHARACTERIZATION OF MICROGLIA DURING NGF DEPRIVATION IN VITRO AND IN A MOUSE MODEL OF ALZHEIMER’S DISEASE

Microglia, the guardians of the CNS, are the resident tissue macrophages and constitute –10% of the total cells in the adult brain. They perform homeostatic activity in the healthy brain, a function associated with the high mobility of their processes that scan surrounding area constantly and phagocytic clearance of extracellular debris. During brain injury and disease they are capable of responding quickly to disturbances in tissue homeostasis by altering their morphology and acquiring the so called “activated state”. Morphologically activated microglia exists as many different phenotypes depending on the insult corresponding to a vast array of specific responses. Whether microglia play a harmful or a beneficial role during neurodegenerative disorders such as Alzheimer disease(AD) has been a matter of discussion in the last few decades. Recent advances in understanding the pathogenesis of AD characterized by accumulation of fibrillar β-amyloid, highlight the importance of these professional phagocytes as key players in the genesis and modulation of the disease. Efficient clearance of Aβ is essential in maintaining an healthy brain and in the light of this notion, enhancing microglia phagocytic activity might constitute a possible therapeutic target. The road of harnessing the immune system as a way of lowering Aβ burden in diseased brain has already been taken in the past through Aβ immunization, both in mouse models and in humans, though results have been widely inconclusive. Further investigation is needed to fully comprehend the role of microglia and neuroinflammation in disease pathogenesis and thus applying the right course of action in developing a treatment. My thesis research in the lab of the Scuola Normale Superiore focuses on the relationship between microglia, NGF and Alzheimer’s Disease. Our lab uses a particular AD mouse model, AD11, which expresses an antibody against NGF(αD11). This model presents Aβ plaques and cognitive impairment at an early age and thus constitutes a good model for AD. In previous studies on this model, various changes in the inflammatory landscape have been identified, suggesting the possible role of microglia as the primary effector of NGF deprivation. My work consists primarily in identifying specific changes in this cell type under the action of αD11 to assess whether microglial activation might be responsible for AD11 phenotype. The main points of my research involve: • Characterization morphology and activation of microglia in the AD11 brains; • In vitro studies: treatment with αD11 on primary microglia to investigate the inflammatory state induced by NGF deprivation. This project entails the use of IHC, ICC, confocal imaging, RT PCR, WB

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

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, 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 (1) 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 behavioral parameters. Furthermore, when we assessed the phenotypic changes brought forth by (2) 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 analyzed 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

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

A functional nerve growth factor (NGF)-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 clarify the involvement of the NGF-TrkA system in pain sensation

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

Table1_Reduced levels of NGF shift astrocytes toward a neurotoxic phenotype.XLSX

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.</p

Image1_Reduced levels of NGF shift astrocytes toward a neurotoxic phenotype.PDF

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.</p