Neurotrophin receptors in the pathogenesis, diagnosis and therapy of neurodegenerative diseases

Accepted Manuscript Title: Neurotrophin Receptors in the Pathogenesis, Diagnosis and Therapy of Neurodegenerative Diseases Author: Jacopo Meldolesi PII: DOI: Reference:

S1043-6618(17)30484-X http://dx.doi.org/doi:10.1016/j.phrs.2017.04.024 YPHRS 3575

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Pharmacological Research

Please cite this article as: Meldolesi Jacopo.Neurotrophin Receptors in the Pathogenesis, Diagnosis and Therapy of Neurodegenerative Diseases.Pharmacological Research http://dx.doi.org/10.1016/j.phrs.2017.04.024 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Neurotrophin Receptors in the Pathogenesis, Diagnosis and Therapy of Neurodegenerative Diseases.

Jacopo Meldolesi, Vita-Salute San Raffaele University and Scientific Institute San Raffaele via Olgettina 58, 20132 Milan, Italy.

Address all correspondence to: Jacopo Meldolesi, Department of Neuroscience, Vita-Salute San Raffaele University and Scientific Institute San Raffaele, via Olgettina 58, 20132, Milan, Italy. Email: [email protected]

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Graphical Abstract

Properties of Neurotrophin (NT) receptors. Receptors

Mol. wt.

agonists1 NT binding major brain affinity, Kd

distribution2

extra-neuron localization

_____________________________________________________________________________________________

p75NTR

75 kD (45 kD3)

all NTs

10-9

TrkA

145 kD (87 kD)

NGF (NT34)

higher

TrkB

150 kD (92 kD)

BDNF (NT4)

higher

widespread

TrkC

110 kD

NT3

higher

widespread

1. 2. 3. 4.

widespread

many cell types

basal forebrain: heart fibers, limbus, brainstem macrophages, caudate/putamen others limited

limited

In addition to mature NTs, this column should include also the corresponding precursors proNTs. This column includes general data, not detailed examples. In this column, the values in parenthesis are calculated weights. The NTs shown in parentheses are agonists less prominent than those without parentheses.

Abstract In the last few years, exciting properties have emerged regarding the activation, signaling, mechanisms of action, and therapeutic targeting of the two types of neurotrophin receptors: the p75NTR with its intracellular and extracellular

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peptides, the Trks, their precursors and their complexes. This review summarizes these new developments, with particular focus on neurodegenerative diseases. Based on the evolving knowledge, innovative concepts have been formulated regarding the pathogenesis of these diseases, especially the Alzheimer’s and two other, the Parkinson’s and Huntington’s diseases. The medical progresses include original procedures of diagnosis, started from studies in mice and now investigated for human application, based on innovative classes of receptor agonists and blockers. In parallel, comprehensive studies have been and are being carried out for the development of drugs. The relevance of these studies is based on the limitations of the therapies employed until recently, especially for the treatment of Alzheimer’s patients. Starting from well known drugs, previously employed for nonneurodegenerative diseases, the ongoing progress has lead to the development of small molecules that cross rapidly the blood-brain barrier. Among these molecules the most promising are specific blockers of the p75NTR receptor. Additional drugs, that activate Trk receptors, were shown effective against synaptic loss and memory deficits. In the near future such approaches, coordinated with treatments with monoclonal antibodies and with developments in the microRNA field, are expected to improve the therapy of neurodegenerative diseases, and may be relevant also for other human disease conditions.

Abbreviations

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As, A 1-40 and A 1-42: Amyloid  peptides. AD, Alzheimer’s disease. APP: trans-membrane Amyloid Protein Precursor. BDNF: Brain-Derived Neural Factor, the neurotrophin agonist of p75NTR and TrkB receptors. Calpains: Ca2+dependent intracellular cysteine proteases that cleave Trks. Caspases: intracellular proteases. CNS: Central Nervous System. CSF: Cerebro-Spinal Fluid. C57BL/6: a Parkinson’s Disease murine model. HD: Huntington’s Disease. HdhQ111/111, N171-82Q and R6/1: mouse models of Huntington’s Disease.. K252a: a blocker of TrkB. LINGO-1: Leucine-rich repeat and Ig domaincontaining 1, associated to p75NTR and NgR, inhibits axonal outgrowth. LM11A: family of small molecule drugs targeted to individual receptors, such as TrkA, TrkB, TrkC, p75NTR or sortilin, MPTP: 1-Methyl-4-Phenyl-1,2,3,6TetrahydroPyridine, a molecule that triggers Parkinson’s Disease in mice and men. NGF: Nerve Growth Factor, agonist of p75NTR and TrkA receptors. NgR: also known as Nogo-66, a receptor that associates with p75NTR and LINGO-1, inhibits axonal outgrowth. NTs: NeuroTrophins. NTRK1 and NTRK2: the genes coding for TrkA and TrkB. nu/nu athymic xenograft: a mouse model employed for cancer studies. PC12: a cell line from a rat pheohromocytoma. PD90780: drug blocker of p75NTR. PNS: Peripheral Nervous System. p75NTR: a non-enzymatic, pan-neurotrophin receptor of the tumor necrosis receptor family. p75-ECD and p75-ICD: the ExtraCellular and IntraCellular domains, peptides cleaved from p75NTR. PD: Parkinson’s Disease. proNT, proNGF, proBDNF: Precursors of NTs. SALL2: transcription factor transferred to the nucleus upon Trk activation.SH2B-1: adaptor protein controlling vesicle traffic and neurite outgrowth. Sortilin: a neurotensin-like, non-G protein-coupled stimulatory receptor, complexed to various NT receptors to induce peculiar effects. Tg2576, APPL/S, CaM/Tet-DTA, 4

and APP-PS1: murine models of AD. TrkA, TrkB and TrkC: Tropomysin-related kinases A, B and C, the three tyrosine kinase receptors of NTs. TRPV1: TRansient Potential Vanilloid 1 receptor, controlled by TrkA, governs pain expression.

Keywords Neurotrophin receptors, p75NTR, intra and extracellular peptides, Trk, receptor complexes, Alzheimer’s disease, neurodegeneration disease diagnosis, neurodegeneration disease therapy, p75NTR inhibitors, Trk stimulatory drugs.

Keywords: p75NTR and Trk receptors; Alzheimer’s disease; other neurodegenerative diseases; diagnoses; new therapies.

Introduction (For classical data sia Box 1) Neurotrophins (NTs), a group of specific factors, operate through the activation of two distinct types of receptor (Fig. 1) inducing different signals and effects in neurons and other cell types, such as astrocytes, oligodendrocytes, macrophages, pancreatic  cells, smooth/striated muscle fibers 8, 70. The 75 kDa receptor (p75NTR), a receptor activated by all NTs, is a non-enzymatic, trans-membrane protein of the tumor necrosis receptor (TNFR) family. The other types of receptor, the tyrosine kinase receptors (Trks) A, B, and C, are activated, specifically, and with high affinity, by nerve growth factor (NGF) (TrkA), brain derived growth factor and neurotrophin 4 (BDNF and NT4) (TrkB), and

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neuroptrophin 3 (NT3) (TrkC). Our Graphical Abstract summarizes properties of all NT receptors. Based on the extensive studies in the last few years, our knowledge on NT receptors has been expanded. Given the complexity of their signaling pathways, they are now known to play numerous roles relevant in cell physiology. Moreover, the results obtained are critical for the understanding of various diseases. In the central and peripheral nervous system (CNS and PNS), NT receptor-regulated processes include neuronal differentiation, survival and death, axonal outgrowth, synapse generation and plasticity. These processes have been particularly investigated during various neurodegenerative diseases 8,70. Concomitantly, the relevance of NT receptors has also been demonstrated in the pathogenesis of other diseases known to affect large populations of patients 8, 11,70, including various neural disorders (Box 2), a few non-neural diseases, such as cardiovascular diseases and diabetes (Box 3), and a number of cancers such as breast, lung and colon-rectum cancers, myelomas, lymphoid tumors and gliomas (Box 4). Here, we summarize the current state of knowledge on the properties of p75NTR and Trk receptors. The review is focused especially on new information about the pathogenesis of neurodegenerative diseases, revealed by recent in vitro and in vivo studies in rodents and humans. Our major interest is on the discussion of diagnosis and therapy. In particular, the latter is highly promising and therefore at the moment is attracting great interest. In addition, NTs and their receptors are envisaged as novel candidate targets to treat not only neurodegenerative but also a variety of other diseases, specified in the Boxes 24, of this review. 6

NT receptors and the Nervous System p75NTR, a Multifunction, pan-NT Receptor (Fig. 1) is expressed at high level in neurons of the CNS and PNS and in cancer cells (Box 4) of rodents and humans. In addition, p75NTR is present in many, non-neural cell types already mentioned in the Introduction (see also Box 3) 8, 11, 70. Upon NT binding, p75NTR undergoes oligomerization with other p75NTR or homologous receptors 1, 90. The process reinforces the association of the receptor to its signaling molecules, such as Jun kinase, small GTPases, as well as the transcription factor NF-kB 1, 90. Oligomerized p75NTR receptors can be cleaved by - and -secretases (Fig. 1), with release to the cytoplasm of the peptide p75-intracellular domain (p75-ICD) 41, 61. Upon transfer to the nucleus, p75-ICD participates in the regulated transcription of various genes, such as hypoxia-induced HIF-1 and CycE1 41, 61. Moreover, convincing results in mice have shown that another peptide, the p75-extracellular domain (p75-ECD), results from the p75NTR cleavage by surface metalloproteinases. Such peptide, a protector of neurons, attenuates the toxicity and cognitive deficits of the Alzheimer’s disease (AD) 92, 99. Confirmed by recent evidence in both normal and pathological cells, a key role of p75NTR consists in the inhibition of cellular functions. For example, in vitro results in primary cultures of mice neurons, in the rat neural cell line PC12, and in vivo experiments in mice, carried out by various types of techniques such as immunofluorescence microscopy, morphometric analyses, patch clamp electrophysiology and Ca2+ imaging, have shown p75NTR to inhibit processes such

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as Na+ currents, cytosolic Ca2+ responses, excitability, persistent firing, and neurite outgrowth 26,46, 84. Additional effects, both in vitro and in vivo in mouse hippocampal neurons, induced by p75NTR associated with sortilin and activated with high affinity by NT precursors (ProNTs), result in inhibition and even neuronal death 98. The latter effect, which is competed by p75-ICD 50, is further discussed in the subsequent section of this review dealing with the effects of the p75NTR-sortilin complex activation. However, other studies have shown that, for p75NTR, inhibition of cell function does not necessarily occur. In primary cultures of mouse hippocampal neurons, immunofluorescence microscopy has in fact revealed that neurotrophin activation of p75NTR stimulates two critical processes of neuronal differentiation, the conversion of dendrites into axons and the establishment of neuronal polarity, necessary for the expansion of neuronal circuits 109.

Trks, the Cell Survival Receptors (Fig. 1), are variedly distributed in the CNS, PNS, and also non-neural organs. At resting state, Trks diffuse laterally at the cell surface. Upon binding their specific NTs or other activation agents (EGF, L1CAM and others) 12, 67, Trks dimerize and undergo autophosphorylation (Fig. 1). The effects of their activation, including neuronal survival, proliferation, and differentiation, are lasting longer than the effects of growth factor receptors 8, 70. These effects are potentiated when the receptors are internalized and undergo intracellular trafficking 8, 63, 70, 81. Signaling of all Trks include activation of ERK, AKT, and PLC signaling cascades 10, 52, 70 which can, however, trigger differential effects in various neurons. For instance, in mice Purkinje neurons, neurite outgrowth and spine

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density depend primarily on TrkC 39, whereas in pyramidal CA1 hippocampal neurons they depend on TrkB 91,97. In hippocampal and other neuron synapses, TrkB regulates also the prolonged reinforcement of synaptic activity (long-term potentiation, LTP) 29, 42, 60. However, at layer 2/3 cortical synapses, the activation of TrkB does not result on synaptic reinforcement but rather on prolonged weakening (long-term depression, LTD). Results obtained with various types of receptor inhibitors have shown this BDNF effect to be induced indirectly, via the BDNF-induced release of endocannabinoids 104. Thus, even if Trks can act as stimulatory receptors, their final effects can be inhibitory due to cross-talk to other receptors (in this case the type-1 cannabinoid receptor 104) . In wild-type mice, among the effects induced by TrkB is the increased gene translation with increased protein expression 101. These effects are relevant for synaptic vesicle trafficking 108. In addition, TrkB induces the induction or repression of several gene transcription, such as those encoding the adaptor protein SH2-B-1, that contributes to vesicle traffic and neurite outgrowth, and the transcription factor SALL2 64, 73. Indeed, experiments in wild-type mice neurons have revealed the increased transcription of these and other genes involved in synaptic function 49, suggesting that Trks participate in the establishment and turnover of brain circuits. The ensuing processes, essential for the dynamics of important brain functions, include taste, fear, and establishment and maintenance of memory 9, 19, 30, 62. In conclusion, the two types of NT receptors have complex, often opposite roles (Fig. 1 and 2) in a variety of brain functions. In these functions the

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receptors play defined, but not unique roles. Because of their multiple functions, the relevance of the two type NT receptors in several diseases is highly relevant.

NT Receptor Complexes. The p75NTR/Trk Complexes (Fig. 1). A main effect of the p75NTR /Trk complexes is the reinforcements of Trk signaling. However, studies in mouse sympathetic neurons have shown such reinforcement to be reduced by the internalization of the two receptors into distinct endocytic vesicles, where separate signaling occurs 31. Recently, however, other complexes have been identified where Trk is not associated to p75NTR, but rather to p75-ICD. The iuxtamembrane sequence of this peptide binds the intracellular domain of Trks. (Fig. 1) This leads to reinforced signaling, as shown in primary neurons, PC12 and glioma cell cultures 7, 23. Upon its establishment, signaling via Trk and p75-ICD is stronger and can persist longer than via Trk and p75NTR 7, 23.

NT Receptor/Sortilin Complexes. Sortilin is a multifarious neurotensin-like, non-G-protein-coupled stimulatory receptor abundant in neurons 57. Upon its activation by various factors, sortilin induces activation of ERK and AKT signaling cascades together with the expression of various genes, modulating proteins transport and cell migrations 57. However, the most important effects of sortilin depend on its complex formation with NT receptors, as revealed in neurons and nerve cell cultures from various species 18, 20, 57. Extensive in vitro and in vivo studies in mice neurons have shown that, upon activation by proNTs, the sortilin/p75NTR complex 20, 80 induces the activation of various types of intracellular proteases, the caspases 57. The

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main effects of this signaling are the block of synaptic plasticity and neuronal remodeling, and the stimulation of apoptosis 8, 57, 70, 80. By contrast, when sortilin complexes to p75-ICD, their signaling induces neuronal survival and neurite outgrowth 20. When sortilin complexes to various Trks, activation by proNTs in murine neurons does not lead to apoptosis. Instead, the sortilin/Trk complexes induce anti-apoptotic, transport and differentiation effects similar to those of the sortilin /p75-ICD complex 14, 87.

p75NTR/NgR/LINGO-1 Complexes. Separate or complexed together, the NgR and LINGO-1 receptors have roles in several, mostly inhibitory processes of the brain. In murine motoneurons, however, NgR and/or LINGO-1, together with myelin-associated glycoproteins, are complexed to p75NTR. Activation of these complexes occurs upon brain traumatic injury, where the risk of axon degeneration is high 59. This results in the activation of ROCK, a Rho kinase, with an ensuing block in axon growth and protection to neurodegeneration. When the pathological conditions ameliorate, ROCK deactivation can result in the activation of axon outgrow and function 59. In conclusion, numerous complexes are established by the two types NT receptors, associated to each other and to different receptors. The properties of these complexes reside in the specificity of their components and in their signaling pathways. The results obtained have revealed new information about the functioning of p75NTR and Trks, and expanded our understanding of the mechanisms involved.

NT Receptors in Disease Pathogenesis.

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Studies on the role of NT receptors in CNS diseases started soon after their discovery. During the last few years these studies have been extended, with major progress. Here we report about neurodegenerative diseases. Data about other nervous diseases (including pain, stroke, epilepsy and certain psychiatric diseases), non-nervous diseases (including cardiovascular diseases and diabetes), and cancers, pathologies in which important roles are also played by NT receptors, are summarized in Boxes 2, 3 and 4.

Alzheimer’s Disease. AD, the prevalent form of neurodegeneration, emerges during aging in a considerable fraction of the population. The symptoms include progressive worsening of memory, brain confusion and neuropsychiatric defects. Decades ago, the disease was shown to depend on the altered cleavage of the amyloid precursor glycoprotein (APP), a widely expressed, trans-membrane surface protein. Extracellular β-amyloid peptides Aβ 1-40 and 1-42, generated by APP cleavage, assemble into filaments and plaques which affect the neuronal function and lead to AD. The process remained unclear until murine studies revealed that AD pathogenesis could be sustained by both p75NTR trimers and monomers 95. Recent development of these findings in basal forebrain and hippocampal neurons, from mice as well as in cortical neurons from human patients, lead to the identification of p75NTR/sortilin/proNT as a complex inducing neurodegeneration, acting via direct binding to Aβs and activation of death programs 32, 53, 54 (Fig. 2). Further evidence was obtained by results in the brain cortex of two AD mouse models, Tg2576 and APP-PS1, where high p75NTR combined to low p75-ECD (the neuron-protective, extracellularly released peptide 53, 54, 92, 99) were instrumental to the development of the disease 12

99. In fact, reduction of p75NTR and restoration of p75-ECD in mouse brain models reversed the increased Aβ levels and ameliorated the symptoms of the disease, including the defects in learning and memory 53, 54, 92, 99 (Fig. 2). The validity of the altered p75NTR/p75-ECD ratios as markers of AD was documented also to human patients by analyses of cerebrospinal fluid (CSF), blood and brain 38, 99. The data presented so far have documented the instrumental role of p75NTR in the pathogenesis of AD. The Trks (the other NT receptors) appear to play a protective role, opposite to that of p75NTR. In mouse models of AD the levels of Trks are low, due to the A-induced stimulation of a number of critical processes affecting their turn-over and signaling. These processes include: deubiquitination of Trks and de-phosphorylation of Trk signaling proteins; blocking of intracellular traffiking of Trk-loaded vesicles; increase of cytosolic Ca2+ with activation of calpains, the Ca2+-regulated cysteine proteases that cleave the Trks 35, 45, 105 (Fig. 2). Moreover, when TrkB is activated by BDNF, its role on neurons is protective 45. However, when activation is by proBDNF, TrkB exhibits a toxic synergy with As 45. In conclusion, the contradictory actions of NT receptors and p75 peptides on AD neurons depend largely on the levels, high for p75NTR and low for p75ECD, which appear likely governed by As. The role of these properties dependent of p75NTR is strengthened also by the concomitant decrease of the Trk receptors (Fig. 2). The progressive deterioration of these values could contribute substantially to the increasing severity of the disease 35, 38.

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Other Neurodegenerative Diseases. The various neurodegenerative diseases are due on processes that affect function and survival of neurons distributed in distinct areas of the brain. Because of their distinct properties and differential distribution, the diseases can exhibit largely different properties. However, NT receptors are certainly involved in the pathogenesis of many such diseases. In view of this property, which is common to AD, we have decided to include in this review two other, highly important neurodegenerative diseases, Parkinson’s (PD) and Huntington’s (HD) diseases. Defects on nigral dopaminergic neurons, typical of PD, as well as its well known symptoms (muscle shaking, stiffness, slowness of movements; progressive cognitive impairment) can be induced by treatment with toxic molecules such as 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) and 6hydroxydopamine (6-OHDA). In mice, these insults are established via the proNGF-p75NTR-sortilin complex, whereas both TrkB and TrkC are protective 15, 93. The critical role of NT receptors has been confirmed by the use of a specific drug that activates TrkB purified from an Indian neem tree, protects human and rodent nigrostriatal dopaminergic neurons from cell death induced by MPTP 56. In contrast K252a, a blocker of TrkB, was shown to prevent the beneficial effects of physical exercise in a rat unilateral model of the PD induced by striatal injection of 6-OHDA 69. HD is an autosomal dominant neurodegenerative disorder sustained by mutations of huntingtin exhibiting abnormal expansions of the glutamine stretch at its N-terminal sequence. The defects are concentrated in basal ganglia, frontostriatal transmission and hippocampus. Brain investigation of mice models HdhQ111/111 and R6/1, and of monkeys and humans, has found the disease to 14

exhibit enhanced p75NTR signaling with antagonism by TrkB. In HD patients. However, TrkB receptors have been shown to induce a defect of postsynaptic signaling at cortico-striatal synapses. This defect alters the functioning of both cortical and basal neurons, with induction of typical symptoms of the disease, debilitating choreic movements and cognitive impairments 5, 65, 66.

NT Receptors in Medical Practice. During the last few years, the interest in NT receptors has increased considerably. In many cases they have been recognized as tools useful to identifying therapies for a variety of neurodegenerative diseases. NT receptors might well inform decisions regarding diagnosis, choice of therapies, and provision of patient clinical outcomes 75. Two examples of such processes – diagnosis and therapy—are described here.

Diagnosis. Diagnosis procedures have been developed to reveal the nature and the state of the diseases dependent on, or at least influenced by NT receptors. For example, useful results emerged from the study examining the ratio of BDNF relative to its precursor, proBDNF. In the brain of patients with AD, such a ratio has been found up to 30 fold lower than in controls 44. This result is useful because, in the same patients, low BDNF/proBDNF ratios have been detected in fluids that are easy to collect, urine, blood, and CSF 38, 99. Even more conclusive results have been obtained with p75-ICD collected from the same fluids, higher in human patients with malignant gliomas relative to controls 23.

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Another p75NTR peptide employed for diagnosis is p75-ECDs. In the CSF and blood of human AD patient, significantly lower levels have been found at early stages of the disease, with further decrease during disease progression. Therefore, the analysis of p75-ECD in human fluids can help to identify the disease and monitor its progression 38. In contrast, the levels of p75-ECD in the urine of patients with amyotrophic lateral sclerosis are higher than in controls 72. Of note, a problem in the diagnosis of neurodegenerative diseases lies on the distinction of AD (at early stages) and PD patients. This problem has been solved by microRNA analysis of extracellular vesicles isolated from human CSF 28. These vesicles are interesting because they contain, in addition to proteins and mRNAs, also several microRNAs, some of which (for example, miR-153, miR409-3p) are significantly over-expressed in PD, whereas others (for example, mir-151 and mir-19b-3p) are more abundant in AD 28. The CSF vesicle microRNAs appear therefore as biomarkers reliable, in regard to specificity and sensitivity, to distinguish patients from the two diseases. At the moment, assays analogous to those employed for neurodegenerative diseases are being investigated in patients affected by other diseases such as certain cancers.

Therapy. Most therapeutic studies have been dedicated to diseases where NT receptors have been implicated. But the relevance of these therapies has been variable. In diseases where the success of present pharmacology is limited, as in the case of some neurodegenerative diseases, anti-p75NTR drugs and Trk agonists are promising for treatment. In diseases, as the case of cancers, which benefit

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from efficient drugs, anti-Trk drugs might also be administered in combination, to synergize with the effects, stemming from anti-tumoral and anti-Trk drugs 13; see Box 4.

Neurodegenerative Diseases. Except for the levodopa treatments, which are widely employed to treat PD patients, the pharmacological development has gained only limited success in this area. New therapeutic perspectives targeting NT receptor drugs, in particular by the inhibition of p75NTR and activation of Trks, might therefore be promising. A few drugs, such as cerebrolysin, memantine and zonisamide, developed 4-5 decades ago and acting on various targets, have also been shown to modulate the levels of NT receptors. For instance, cerebrolysin, a peptide mixture, has been shown to act on the NGF/proNGF balance via multiple mechanisms including the control of A generation and NT protein expression 86. Memantine and zonisamide, known as anti-epileptic/anti-protein kinase drugs, have also been employed to improve the cognitive impairment and induce nerve regeneration of AD and PK patients. When injected into the PD murine model C57BL/6, these drugs have been found to induce several responses, including nerve regeneration, transcription of genes including NTRK1 and NTRK2 (the genes of TrkA and B), improvement of the Trk/p75NTR imbalance triggered by injection of MPTP 96, 106. In human patients, however, in spite of their wide employment, the therapeutic effects of these drugs have remained only moderate 2, 24. A new strategy, developed to overcome the limitations of neurodegeneration therapy, is to target individual neurotrophin receptors, such

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as TrkA, TrkB, TrkC, the p75NTR or sortilin, with small-molecule ligands that cross the blood-brain-barrier rapidly 47. The task expected for these drugs include to promote survival-related signaling and to reduce neuron. Moreover, such molecules could modulate various aspects of the signalling pathways in ways different from those triggered by native NTs 47. Many drugs of this class, the LM11A drugs, operate in mice as blockers of p75NTR. For instance, they have proven to be effective in the APPL/S and Tg2576 AD murine models, where LM11A-31 and LM11A-24 were shown to prevent the progression of the disease 55, 76. Interestingly, the effect of LM drugs are not limited to AD but protect rodent neurons also from other classical neurodegenerative diseases, such as HD 77, and against diseases induced by viruses, such as HIV, that establish a persistent infection of macrophages and microglia 51. Additional protective anti-AD results, similar to those observed with the LM11-A drugs, were obtained with another p75NTR blocker, PD90780 74. Analogous to the effects found using of p75NTR blockers has been observed also with administration of Trk agonists. One such drug, 7, 8dihydroxyflavone, has been investigated in several diseases. In the AD murine model CaM/Tet-DTA, administration of this drug prevented synaptic loss and memory deficits 6; in the HD mouse model N171-82Q, the drug extended neuron survival 37. Neuronal protection has also been achieved upon administration of small, non-peptide TrkB agonists (LM11A-3, LM11A-4 and others) (Fig. 2). These findings were confirmed by comparing the effects of drugs with those obtained using a monoclonal TrkB agonist antibody 83. Furthermore, as described earlier, human PD dopaminergic neurons have been

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found protected when a TrkB agonist drug of vegetal origin was administered 56. Of note, some of the above BDNF agonists might operate not only directly but also by reducing neuroinflammation, a process favoring the subsequent progression of neurodegeneration 79.

Conclusive Remarks. During the last few years, increasing knowledge on NT receptors has introduced new concepts regarding their involvement and function in the mammalian nervous systems. p75NTR and Trks are now recognized not only for their main effects but also for their functional heterogeneity. p75NTR mostly inhibits while Trks stimulate cell survival. An example of the present novelty in the field is the existence and unexpected functions of that the two cleaved p75NTR peptides, the intracellular domain p75-ICD, relevant especially in signaling and gene expression, and the extracellular domain p75-ECD, a protector of neurons. The success of receptor studies in basic research has contributed significantly to the progress in our understanding of neurodegenerative diseases. In the field, imbalances of proNTs and mature NTs, of p75NTR and its cleaved peptides, as well as of p75NTR and Trks, play important roles in the pathogenesis. The recent interest in NT receptors in medicine is not limited to their role in pathology but includes for purposes both diagnosis and therapy. Diagnosis, aimed at the identification of diseases even at early stages of their development, has become feasible based on the innovative analysis of NTs and NT receptors in human fluids. Therapy, based on small molecules that activate or block NT receptors, has been mostly developed in mice. Ongoing work is exploring the possibility of using drugs targeting NT receptors in humans, either alone, or in 19

combination with other drugs or factors already available to treat various diseases. In conclusion, the future development of NT receptors appears promising. The discovery of new properties will presumably continue at rates similar to those of previous years. In addition to the already mentioned neurodegenerative diseases investigated from various points of view, relevant studies will be dedicated to other, highly relevant brain processes and diseases such as pain, epilepsy and psychiatric diseases (Box 2); to non-neural diseases, such as cardiovascular diseases and diabetes (Box 3); and to various types of cancer (Box 4), all regulated to significant extent by NT receptors. Finally, given the already demonstrated relevance of NT receptors in various physiological processes, interest in them is expected to increase, with possible expansion to additional physiologies and pathologies that up to now have been overlooked.

Acknowledgments I am grateful to the young colleagues, Rosalba D’Alessandro and Sara Negrini. Working in my lab they introduced new approaches/data in the study of NT receptors. Support for our work included in this review came from a grant by Telethon (GGGP09066) and support from the San Raffaele Institute.

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Box 1. Historical perspective of NT receptors. The study of NT receptors, initiated shortly after their discovery, led to a large accumulation of knowledge 8. 70. Binding of NTs was initially shown to occur by two processes, one slow and the other fast, with dissociation constant (Kd) of 10-9 and 10-11, respectively. The first, lower affinity value, due to p75NTR, occurs with all NTs, which however bind at non-identical sites of the receptor. Binding of p75NTR to proNTs occurs in all cases at higher affinity. Among the

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problems solved within the first two decades in the p75NTR field were the identification of the receptor signaling pathways and the multiplicity of effects induced by receptor activation on neurons and other cell types. Signaling of the three Trk receptors is known since the early 90s. Also established has been the intracellular traffic of activated p75NTR and Trk receptors initiated by their internalization into distinct endocytic vesicles. Many of the critical roles of NT receptors in the regulation of important functions of neurons and other cell types, were also established during the first two decades of investigation. Concomitantly, the relevance of NT receptors in various, neural and non-neural diseases and cancers, was also demonstrated. Recent developments of NT receptor studies, highly relevant in cell physiology and diseases (diagnosis and therapy), are illustrated in the present review.

Box 2. NT Receptors and Neural Disorders. Pain, due to heat, inflammation, or neuropathic hyperalgesia, can depend on the transient receptor potential vanilloid 1 (TRPV1), a non-NT receptor expressed by afferent neurons, ganglia and spinal cord 16, 17, 22. TRPV1, however, is sensitized by NGF activation of TrkA via direct phosphorylation and membrane trafficking 16, 17. Thus TrkA plays a key role in pain. NT receptors are also critical in epilepsy and ischemic injury. For example, kainite, the agonist of a glutamate receptor-channel, induces in mouse hippocampal neurons an increase in the levels of microRNA 1-32, which in turn induces alteration of NT receptors: increase of proBDNF and p75NTR levels and suppressed TrkB signaling, together with enhanced activity of voltage-gated Ca2+

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channels, with ensuing appearance of epileptiform discharges 94. Likewise, in an in vivo mouse model of brain local ischemia, the microRNA 592 induces increases of both pro-NGF and p75NTR 33. However, a concomitant overexpression of the enzyme heme oxygenase activates TrkB signaling, with attenuation of the local neuronal injury 68. It appears therefore that, in epileptic and ischemic injuries, Trk and p75NTR receptors play protective and deleterious roles, respectively, analogous to those observed in certain neurodegenerative diseases. Involvement of NGF, BDNF, and Trks has also been recently reported for psychiatric diseases For example, BDNF and TrkB appear to participate in the induction of social defeat stress and anxiety by acting in human neurons of the nucleus accumbens, hippocampus, and other brain areas 27. In contrast, results of depression disorders in mouse models suggest such diseases to be sustained by loss of functional BDNF and TrkB 85. Analogous defects of TrkA and TrkB in patients affected by schizophrenia may depend on altered plasticity of synapses 100.

Box 3. NT receptors in cardiovascular diseases and diabetes. NTs and their receptors are expressed also by cells other than neurons. Not surprisingly, therefore, they may be involved in the pathogenesis of diseases affecting non-cerebral organs. Under physiological conditions, BDNF and TrkB have been shown to function in parallel to the -adrenergic receptor system to sustain the contraction/relaxation performance of the heart 21. NTs, however, do not always favor contraction of the heart. A study comparing mice, normal

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and lacking p75NTR, revealed that, upon infarction of the myocardium, increases of contractility were induced by a transient receptor denervation in the surrounding area 48. The sympathetic hyperinnervation that develops upon reperfusion in the same area is due to TrkB increase 48. In addition, a casecontrol human study revealed that TrkB also promotes the integrity of endothelia and therefore protects against coronary artery diseases 36. Similar to results reported in the brain, the differential expression of the two types of NT receptor might offer the heart and blood vessels some mechanisms to recover from lesions induced by the diseases. In pancreatic islets, regulation of -cell survival and fine-tuning of insulin secretion are known to depend on NGF and TrkA. p75NTR plays a key role in the start of diabetes by inducing denervation of islets accompanied by injury to cells and vasculature 82. The ensuing irreversible defect of insulin secretion gives rise to many secondary lesions. Among these is retinal damage which depends on neuronal defect 89. The study of a streptozotocyn-induced mouse model of diabetes has documented in retinal neurons the up-regulation of proNTs and p75NTR accompanied by defects of NGF and TrkA, followed by retinal inflammation, neurodegeneration and vascular dysfunction 4. The identification of such receptor imbalance has opened the way to a therapy delivered to human patients in parallel to the insulin administration, by which retinopathy progression and ensuing blindness can be prevented 40.

Box 4. NT Receptors and Malignancies.

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Since many years, TrkA and TrkB are known to operate as protooncogenes contributing to cell migration and invasion as well as neovascularization in various types of cancer (of breast, lung, colon-rectum, and liver), in myelomas, lymphoid tumors, gliomas, and neuroblastomas 88. In some these cancers, Trks can also promote the formation of metastases 78. Mutations, fusions, and other alterations of Trk genes can result in their longterm activation, with reinforcement of the receptor effects 3. For example, stimulation of human breast cancer cells can be due to the activation of Trks by sortilin and proNTs 14, two agents that, in most non-cancer cells, activate in contrast the death program by their association with p75NTR 57, 80. In human cell lines from malignant gliomas, the co-operation of Trks with p75-ICD triggers reinforcement of cancer development 23. Interestingly, the function of p75NTR alone is not in favor, but often against cancer. This can happen also with Trks. In human cells from neuroblastoma, a nerve cancer, spontaneous regression has been reported to depend indirectly on down-regulation of TrkA, with ensuing generation of peptides that activate transcription of pro-apoptotic target genes 107. Repression in human colon and breast cancers can be induced by TrkC 25, 103. Interest in new, NT receptor-targeted drugs has stemmed from the identification of excess Trk levels in various cancers such as pancreatic, breast, lung, neuroblastoma, and neuroendocrine tumors. For example, blockers of TrkA and, especially, of TrkB, such as entrectinib and GNF-4256, have prevented the development, or impeded the efficacy of cancer growth and aggressiveness 13, 34, 58, 71. In most malignancies, however, established regimens already exist in

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which chemotherapy drugs are administered according to established criteria. In many such cases, anti-tumor chemotherapeutic drugs can be combined with Trk inhibitors, with significant increase of therapeutic regimen success 13, 43, 102. Whether the anti-Trk receptor drugs are effective also when administered in combination with newer generations of immunotherapeutic agents, such as monoclonal antibodies, vaccines and other agents, remains however to be confirmed.

Figure Legends Fig. 1. NT Receptor Signaling. On the left, NT receptors of the two types, p75NTR (a single transmembrane protein shown as a dimer, stained green) and Trk (two associated trans-membrane tyrosine kinases in open configuration, stained magenta) are activated by NTs (stained yellow). Of the intracellular repeats of Trk (small circles), one appears phosphorylated (red dot). On the right, p75NTR undergoes cleavage by a secretase (scissors), with ensuing shedding to the cytoplasm of the intracellular domain, p75-ICD, which complexes with the iuxtamembrane intracellular sequence of Trk. The intracellular complex, composed of two Trks and two p75-ICDs, triggers a change in the Trk extracellular domain, converting it from open (left) to close (right). Compared to the single Trk, the change accounts for the increased potency of Trk signaling. The increased Trk signaling, marked by four (right) compared to one (left) red dots (phosphorylated sites), is also illustrated by the white boxes shown below, small to the left and larger to the right.

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Fig. 2. The A dependence of Alzheimer’s disease occurs via NT receptors. p75NTR and the Trks are known to play distinct roles in the pathogenesis of AD. The disease depends on excess A, which strongly stimulates an increased expression of p75NTR (left). High p75NTR, by interacting with sortilin and upon

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activation by proNT, activates the cell death program leading to the apoptosis of neurons 41. Concomitantly, excess A activates three processes (decreased traffic of Trk-positive endocytic vesicles, increased de-ubiquitination of Trk, and increased cytosolic Ca2+ with ensuing Trk proteolysis 54,56,57) that reduce the level of Trk (right) and thus decrease its anti-AD protection. However, the effects of p75NTR and Trks on AD are not always negative. p75-ECD (p75 extracellular domain peptide, down/left), derived from the cleavage of p75NTR by surface metalloproteinases, is shown to reduce the severity of the disease 8,9,52,53; two types of drug, p75NTR inhibitors 74,75,78 and Trk activators 79,80 (down/right), operate against the disease.

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