NGF and the Amyloid Precursor Protein in Alzheimer's Disease: From Molecular Players to Neuronal Circuits.

Alzheimer's disease (AD), one of the most common causes of dementia in elderly people, is characterized by progressive impairment in cognitive function, early degeneration of basal forebrain cholinergic neurons (BFCNs), abnormal metabolism of the amyloid precursor protein (APP), amyloid beta-peptide (Aß) depositions, and neurofibrillary tangles. According to the cholinergic hypothesis, dysfunction of acetylcholine-containing neurons in the basal forebrain contributes markedly to the cognitive decline observed in AD. In addition, the neurotrophic factor hypothesis posits that the loss nerve growth factor (NGF) signalling in AD may account for the vulnerability to atrophy of BFCNs and consequent impairment of cholinergic functions. Though acetylcholinesterase inhibitors provide only partial and symptomatic relief to AD patients, emerging data from in vivo magnetic resonance imaging (MRI) and positron emission tomography (PET) studies in mild cognitive impairment (MCI) and AD patients highlight the early involvement of BFCNs in MCI and the early phase of AD. These data support the cholinergic and neurotrophic hypotheses of AD and suggest new targets for AD therapy.Different mechanisms account for selective vulnerability of BFCNs to AD pathology, with regard to altered metabolism of APP and tau. In this review, we provide a general overview of the current knowledge of NGF and APP interplay, focusing on the role of APP in regulating NGF receptors trafficking/signalling and on the involvement of NGF in modulating phosphorylation of APP, which in turn controls APP intracellular trafficking and processing. Moreover, we highlight the consequences of APP interaction with p75NTR and TrkA receptor, which share the same binding site within the APP juxta-membrane domain. We underline the importance of insulin dysmetabolism in AD pathology, in the light of our recent data showing that overlapping intracellular signalling pathways stimulated by NGF or insulin can be compensatory. In particular, NGF-based signalling is able to ameliorates deficiencies in insulin signalling in the medial septum of 3×Tg-AD mice. Finally, we present an overview of NGF-regulated microRNAs (miRNAs). These small non-coding RNAs are involved in post-transcriptional regulation of gene expression , and we focus on a subset that are specifically deregulated in AD and thus potentially contribute to its pathology.

SMN affects membrane remodelling and anchoring of the protein synthesis machinery.

Disconnection between membrane signalling and actin networks can have catastrophic effects depending on cell size and polarity. The survival motor neuron (SMN) protein is ubiquitously involved in assembly of spliceosomal small nuclear ribonucleoprotein particles. Other SMN functions could, however, affect cellular activities driving asymmetrical cell surface expansions. Genes able to mitigate SMN deficiency operate within pathways in which SMN can act, such as mRNA translation, actin network and endocytosis. Here, we found that SMN accumulates at membrane protrusions during the dynamic rearrangement of the actin filaments. In addition to localization data, we show that SMN interacts with caveolin-1, which mediates anchoring of translation machinery components. Importantly, SMN deficiency depletes the plasma membrane of ribosomes, and this correlates with the failure of fibroblasts to extend membrane protrusions. These findings strongly support a relationship between SMN and membrane dynamics. We propose that SMN could assembly translational platforms associated with and governed by the plasma membrane. This activity could be crucial in cells that have an exacerbated interdependence of membrane remodelling and local protein synthesis.

Membrane cholesterol depletion in cortical neurons highlights altered NMDA receptor functionality in a mouse model of amyotrophic lateral sclerosis.

Amyotrophic Lateral Sclerosis (ALS) is a chronic neurodegenerative disease affecting upper and lower motor neurons, with unknown aetiology. Lipid rafts, cholesterol enriched microdomains of the plasma membrane, have been linked to neurodegenerative disorders like ALS. The NMDA-receptor subcellular localization in lipid rafts is known to play many roles, from modulating memory strength to neurotoxicity. In this study, performed on the widely used G93A mouse model of ALS, we have shown an equal content of total membrane cholesterol in Control and G93A cortical cultures. Moreover, by electrophysiological studies, we have recorded NMDA- and AMPA-evoked currents which were not significantly different between the two neuronal populations. To study the role of membrane cholesterol on glutamate receptor functionality, we have analysed NMDA and AMPA receptors following cholesterol membrane depletion by methyl-ß-cyclodextrin (MßCD). Interestingly, MßCD chronic treatment has provoked a significant reduction of NMDA-evoked currents in both cellular populations which was dose- and time-dependent but significantly higher in ALS neurons compared to Control. The different MßCD effect on NMDA-evoked currents was not due to a different membrane receptor subunit composition but seemed to cause in both neuronal populations a NMDA receptor membrane redistribution. MßCD treatment effect was receptor-specific since no alterations in the two neuronal populations were detected on AMPA receptors. These results lead us to speculate for an altered proteomic composition of lipid rafts in cortical mutated neurons and suggest the need for further studies on the lipid rafts composition and on their interaction with membrane receptors in ALS cortices.

The Intersection of NGF/TrkA Signaling and Amyloid Precursor Protein Processing in Alzheimer's Disease Neuropathology.

Dysfunction of nerve growth factor (NGF) and its high-affinity Tropomyosin receptor kinase A (TrkA) receptor has been suggested to contribute to the selective degeneration of basal forebrain cholinergic neurons (BFCN) associated with the progressive cognitive decline in Alzheimer's disease (AD). The aim of this review is to describe our progress in elucidating the molecular mechanisms underlying the dynamic interplay between NGF/TrkA signaling and amyloid precursor protein (APP) metabolism within the context of AD neuropathology. This is mainly based on the finding that TrkA receptor binding to APP depends on a minimal stretch of ~20 amino acids located in the juxtamembrane/extracellular domain of APP that carries the α- and ß-secretase cleavage sites. Here, we provide evidence that: (i) NGF could be one of the "routing" proteins responsible for modulating the metabolism of APP from amyloidogenic towards non-amyloidogenic processing via binding to the TrkA receptor; (ii) the loss of NGF/TrkA signaling could be linked to sporadic AD contributing to the classical hallmarks of the neuropathology, such as synaptic loss, ß-amyloid peptide (Aß) deposition and tau abnormalities. These findings will hopefully help to design therapeutic strategies for AD treatment aimed at preserving cholinergic function and anti-amyloidogenic activity of the physiological NGF/TrkA pathway in the septo-hippocampal system.

Correction: Iannuzzi et al. Fyn Tyrosine Kinase Elicits Amyloid Precursor Protein Tyr682 Phosphorylation in Neurons from Alzheimer's Disease Patients. Cells 2020, 9, 1807.

Error in Figure [...].

Insulin and serine metabolism as sex-specific hallmarks of Alzheimer's disease in the human hippocampus.

Healthy aging is an ambitious aspiration for humans, but neurodegenerative disorders, such as Alzheimer's disease (AD), strongly affect quality of life. Using an integrated omics approach, we investigate alterations in the molecular composition of postmortem hippocampus samples of healthy persons and individuals with AD. Profound differences are apparent between control and AD male and female cohorts in terms of up- and downregulated metabolic pathways. A decrease in the insulin response is evident in AD when comparing the female with the male group. The serine metabolism (linked to the glycolytic pathway and generating the N-methyl-D-aspartate [NMDA] receptor coagonist D-serine) is also significantly modulated: the D-Ser/total serine ratio represents a way to counteract age-related cognitive decline in healthy men and during AD onset in women. These results show how AD changes and, in certain respects, almost reverses sex-specific proteomic and metabolomic profiles, highlighting how different pathophysiological mechanisms are active in men and women.

New insights on the influence of free d-aspartate metabolism in the mammalian brain during prenatal and postnatal life.

Free d-aspartate is abundant in the mammalian embryonic brain. However, following the postnatal onset of the catabolic d-aspartate oxidase (DDO) activity, cerebral d-aspartate levels drastically decrease, remaining constantly low throughout life. d-Aspartate stimulates both glutamatergic NMDA receptors (NMDARs) and metabotropic Glu5 receptors. In rodents, short-term d-aspartate exposure increases spine density and synaptic plasticity, and improves cognition. Conversely, persistently high d-Asp levels produce NMDAR-dependent neurotoxic effects, leading to precocious neuroinflammation and cell death. These pieces of evidence highlight the dichotomous impact of d-aspartate signaling on NMDAR-dependent processes and, in turn, unveil a neuroprotective role for DDO in preventing the detrimental effects of excessive d-aspartate stimulation during aging. Here, we will focus on the in vivo influence of altered d-aspartate metabolism on the modulation of glutamatergic functions and its involvement in translational studies. Finally, preliminary data on the role of embryonic d-aspartate in the mouse brain will also be reviewed.

Fyn Tyrosine Kinase Elicits Amyloid Precursor Protein Tyr682 Phosphorylation in Neurons from Alzheimer's Disease Patients.

Alzheimer's disease (AD) is an incurable neurodegenerative disorder with a few early detection strategies. We previously proposed the amyloid precursor protein (APP) tyrosine 682 (Tyr682) residue as a valuable target for the development of new innovative pharmacologic or diagnostic interventions in AD. Indeed, when APP is phosphorylated at Tyr682, it is forced into acidic neuronal compartments where it is processed to generate neurotoxic amyloid ß peptides. Of interest, Fyn tyrosine kinase (TK) interaction with APP Tyr682 residue increases in AD neurons. Here we proved that when Fyn TK was overexpressed it elicited APP Tyr682 phosphorylation in neurons from healthy donors and promoted the amyloidogenic APP processing with Aß peptides accumulation and neuronal death. Phosphorylation of APP at Tyr (pAPP-Tyr) increased in neurons of AD patients and AD neurons that exhibited high pAPP-Tyr also had higher Fyn TK activity. Fyn TK inhibition abolished the pAPP-Tyr and reduced Aß42 secretion in AD neurons. In addition, the multidomain adaptor protein Fe65 controlled the Fyn-mediated pAPP-Tyr, warranting the possibility of targeting the Fe65-APP-Fyn pathway to develop innovative strategies in AD. Altogether, these results strongly emphasize the relevance of focusing on pAPP Tyr682 either for diagnostic purposes, as an early biomarker of the disease, or for pharmacological targeting, using Fyn TKI.

The Medial Septum Is Insulin Resistant in the AD Presymptomatic Phase: Rescue by Nerve Growth Factor-Driven IRS1 Activation.

Basal forebrain cholinergic neurons (BFCN) are key modulators of learning and memory and are high energy-demanding neurons. Impaired neuronal metabolism and reduced insulin signaling, known as insulin resistance, has been reported in the early phase of Alzheimer's disease (AD), which has been suggested to be "Type 3 Diabetes." We hypothesized that BFCN may develop insulin resistance and their consequent failure represents one of the earliest event in AD. We found that a condition reminiscent of insulin resistance occurs in the medial septum of 3 months old 3×Tg-AD mice, reported to develop typical AD histopathology and cognitive deficits in adulthood. Further, we obtained insulin resistant BFCN by culturing them with high insulin concentrations. By means of these paradigms, we observed that nerve growth factor (NGF) reduces insulin resistance in vitro and in vivo. NGF activates the insulin receptor substrate 1 (IRS1) and rescues c-Fos expression and glucose metabolism. This effect involves binding of activated IRS1 to the NGF receptor TrkA, and is lost in presence of the specific IRS inhibitor NT157. Overall, our findings indicate that, in a well-established animal model of AD, the medial septum develops insulin resistance several months before it is detectable in the neocortex and hippocampus. Remarkably, NGF counteracts molecular alterations downstream of insulin-resistant receptor and its nasal administration restores insulin signaling in 3×Tg-AD mice by TrkA/IRS1 activation. The cross-talk between NGF and insulin pathways downstream the insulin receptor suggests novel potential therapeutic targets to slow cognitive decline in AD and diabetes-related brain insulin resistance.

Correction to: The Medial Septum Is Insulin Resistant in the AD Presymptomatic Phase: Rescue by Nerve Growth Factor-Driven IRS1 Activation.

The authors, due to the change in one of the University name at the affiliation, hereby correct the proof.

Free d-aspartate triggers NMDA receptor-dependent cell death in primary cortical neurons and perturbs JNK activation, Tau phosphorylation, and protein SUMOylation in the cerebral cortex of mice lacking d-aspartate oxidase activity.

In mammals, free d-aspartate (D-Asp) is abundant in the embryonic brain, while levels remain very low during adulthood as a result of the postnatal expression and activity of the catabolizing enzyme d-aspartate oxidase (DDO). Previous studies have shown that long-lasting exposure to nonphysiological, higher D-Asp concentrations in Ddo knockout (Ddo-/-) mice elicits a precocious decay of synaptic plasticity and cognitive functions, along with a dramatic age-dependent expression of active caspase 3, associated with increased cell death in different brain regions, including hippocampus, prefrontal cortex, and substantia nigra pars compacta. Here, we investigate the yet unclear molecular and cellular events associated with the exposure of abnormally high D-Asp concentrations in cortical primary neurons and in the brain of Ddo-/- mice. For the first time, our in vitro findings document that D-Asp induces in a time-, dose-, and NMDA receptor-dependent manner alterations in JNK and Tau phosphorylation levels, associated with pronounced cell death in primary cortical neurons. Moreover, observations obtained in Ddo-/- animals confirmed that high in vivo levels of D-Asp altered cortical JNK signaling, Tau phosphorylation and enhanced protein SUMOylation, indicating a robust indirect role of DDO activity in regulating these biochemical NMDA receptor-related processes. Finally, no gross modifications in D-Asp concentrations and DDO mRNA expression were detected in the cortex of patients with Alzheimer's disease when compared to age-matched healthy controls.

PC4 coactivates MyoD by relieving the histone deacetylase 4-mediated inhibition of myocyte enhancer factor 2C.

Histone deacetylase 4 (HDAC4) negatively regulates skeletal myogenesis by associating with the myocyte enhancer factor 2 (MEF2) transcription factors. Our data indicate that the gene PC4 (interferon-related developmental regulator 1 [IFRD1], Tis7), which we have previously shown to be required for myoblast differentiation, is both induced by MyoD and potentiates the transcriptional activity of MyoD, thus revealing a positive regulatory loop between these molecules. Enhancement by PC4 of MyoD-dependent activation of muscle gene promoters occurs selectively through MEF2 binding sites. Furthermore, PC4 localizes in the nucleus of differentiating myoblasts, associates with MEF2C, and is able to counteract the HDAC4-mediated inhibition of MEF2C. This latter action can be explained by the observed ability of PC4 to dose dependently displace HDAC4 from MEF2C. Consistently, we have observed that (i) the region of PC4 that binds MEF2C is sufficient to counteract the inhibition by HDAC4; (ii) PC4, although able to bind HDAC4, does not inhibit the enzymatic activity of HDAC4; and (iii) PC4 overcomes the inhibition mediated by the amino-terminal domain of HDAC4, which associates with MEF2C but not with PC4. Together, our findings strongly suggest that PC4 acts as a coactivator of MyoD and MEF2C by removing the inhibitory effect of HDAC4, thus exerting a pivotal function during myogenesis.

Maternal exposure to low levels of corticosterone during lactation protects adult rat progeny against TNBS-induced colitis: A study on GR-mediated anti-inflammatory effect and prokineticin system.

The early phase of life represents a critical period for the development of an organism. Interestingly, early life experiences are able to influence the development of the gastrointestinal tract and the reactivity to colonic inflammatory stress. We recently demonstrated that adult male rats exposed to low doses of corticosterone during lactation (CORT-nursed rats) are protected against experimental colitis induced by the intracolonic infusion of 2,4,6-trinitrobenzenesulfonic acid (TNBS). Based on these interesting results, we wanted to better investigate which cellular actors could be involved in the protection of CORT-nursed rats from TNBS-induced experimental colitis. Therefore, in the present work, we focused our attention on different factors implicated in GR-mediated anti-inflammatory effect. To address this issue, colonic tissues, collected from control and CORT-nursed healthy animals and from control and CORT-nursed colitic rats, were processed and the following inflammatory factors were evaluated: the expression of (i) glucocorticoid receptors (GR), (ii) glucocorticoid-induced leucine zipper (GILZ), (iii) phospho-p65NF-κB, (iv) the pro-inflammatory cytokines IL-1ß and TNF-α, (v) the prokineticins PK2 and PK2L and (vi) their receptors PKR1 and PKR2. We found that adult CORT-nursed rats, in comparison to controls, showed increased expression of colonic GR and reduced expression of pro-inflammatory molecules (IL-1ß, TNF-α, PK2 and PK2L) in response to inflammatory colitis. The observed changes were associated with an increase in GILZ colonic expression and with a reduction in phospo-p65NF-κB colonic expression.

Association of TrkA and APP Is Promoted by NGF and Reduced by Cell Death-Promoting Agents.

The amyloid precursor protein (APP) interacts with the tropomyosin receptor kinase A (TrkA) in normal rat, mouse, and human brain tissue but not in Alzheimer's disease (AD) brain tissue. However, it has not been reported whether the two proteins interact directly, and if so, which domains are involved. Clarifying these points will increase our understanding of the role and regulation of the TrkA/APP interaction in normal brain functioning as well as in AD. Here we addressed these questions using bimolecular fluorescence complementation (BiFC) and the proximity ligation assay (PLA). We demonstrated that exogenously expressed APP and TrkA associate through their juxtamembrane/transmembrane domains, to form a complex that localizes mainly to the plasma membrane, endoplasmic reticulum (ER) and Golgi. Formation of the complex was inhibited by p75NTR, ShcC and Mint-2. Importantly, we demonstrated that the association between endogenous APP and TrkA in primary septal neurons were modified by NGF, or by drugs that either inhibit ER-to-Golgi transport or perturb microtubules and microfilaments. Interestingly, several agents that induce cell death [amyloid ß (Aß)-peptide, staurosporine and rapamycin], albeit via different mechanisms, all caused dissociation of APP/TrkA complexes and increased production of C-terminal fragment (ß-CTF) APP fragment. These findings open new perspectives for investigating the interplay between these proteins during neurodegeneration and AD.

NGF controls APP cleavage by downregulating APP phosphorylation at Thr668: relevance for Alzheimer's disease.

NGF has been implicated in forebrain neuroprotection from amyloidogenesis and Alzheimer's disease (AD). However, the underlying molecular mechanisms are still poorly understood. Here, we investigated the role of NGF signalling in the metabolism of amyloid precursor protein (APP) in forebrain neurons using primary cultures of septal neurons and acute septo-hippocampal brain slices. In this study, we show that NGF controls the basal level of APP phosphorylation at Thr668 (T668) by downregulating the activity of the Ser/Thr kinase JNK(p54) through the Tyr kinase signalling adaptor SH2-containing sequence C (ShcC). We also found that the specific NGF receptor, Tyr kinase A (TrkA), which is known to bind to APP, fails to interact with the fraction of APP molecules phosphorylated at T668 (APP(pT668) ). Accordingly, the amount of TrkA bound to APP is significantly reduced in the hippocampus of ShcC KO mice and of patients with AD in which elevated APP(pT668) levels are detected. NGF promotes TrkA binding to APP and APP trafficking to the Golgi, where APP-BACE interaction is hindered, finally resulting in reduced generation of sAPPß, CTFß and amyloid-beta (1-42). These results demonstrate that NGF signalling directly controls basal APP phosphorylation, subcellular localization and BACE cleavage, and pave the way for novel approaches specifically targeting ShcC signalling and/or the APP-TrkA interaction in AD therapy.

Design and cloning of short hairpin RNAs (shRNAs) into a lentiviral silencing vector to study the function of selected proteins in neuronal apoptosis.

Double-stranded RNA -mediated interference (RNAi ) is a new simple and fast research tool for shutting down genes and characterizes function of their respective proteins. Many strategies for design and delivery of siRNA to target cells are available. Here, we describe the use of lentiviral short hairpin RNA (shRNA) RNA silencing to identify the involvement of d-serine racemase (SR )- an enzyme that syntheses d-serine to modulate glutamate- N-methyl-D-aspartate receptor- in regulating rat cerebellar granule neurons (CGN ) apoptosis. Apoptosis is induced by serum and KCl withdrawal and is detected with fluorometric caspase 3 assay.

Proteasome inhibitors prevent cytochrome c release during apoptosis but not in excitotoxic death of cerebellar granule neurons.

In order to find out whether and how proteasomes participate in the processes leading cerebellar granule cells to death either in necrosis, due to glutamate neurotoxicity, or in apoptosis, due to K(+) shift, we measured the three proteasome activities by using specific fluorescent probes and investigated the effect of several proteasome inhibitors, including MG132, on the cytochrome c release taking place in the early phase of both apoptosis and necrosis. We show that differently from apoptosis, the early phase of necrosis does not require proteasome activation. Inhibition of proteasome activity can prevent cytochrome c release in cerebellar granule cells undergoing apoptosis, thus improving cell survival, but not necrosis. These findings show that proteasomes play an important role in the early phase of apoptosis but not that of necrosis, and that these two types of cell death differ from each other in their mechanism of cytochrome c release.

Serine racemase: a key player in apoptosis and necrosis.

A fine balance between cell survival and cell death is required to sculpt the nervous system during development. However, an excess of cell death can occur following trauma, exposure to neurotoxins or alcohol, and some developmental and neurodegenerative diseases, such as Alzheimer's disease (AD). N-Methyl-D-aspartate receptors (NMDARs) support synaptic plasticity and survival of many neuronal populations whereas inappropriate activation may promote various forms of cell death, apoptosis, and necrosis representing the two extremes of a continuum of cell death processes both "in vitro" and "in vivo." Hence, by identifying the switches controlling pro-survival vs. apoptosis and apoptosis vs. pro-excitotoxic outcome of NMDAR stimulation, NMDAR modulators could be developed that selectively block the cell death enhancing pro-survival signaling or synaptic plasticity mediated by NMDAR. Among these modulators, a role is emerging for the enzyme serine racemase (SR) that synthesizes D-serine, a key co-agonist with glutamate at NMDAR. This review summarizes the experimental evidence from "in vitro" neuronal cultures-with special emphasis on cerebellar granule neurons (CGNs)-and "in vivo" models of neurodegeneration, where the dual role of the SR/D-serine pathway as a master regulator of apoptosis and the apoptosis-necrosis shift will be discussed.

Brain cholinergic markers and Tau phosphorylation are altered in experimental type 1 diabetes: normalization by electroacupuncture.

Diabetes often correlates with tau phosphorylation and the development of Alzheimer's disease. Both are associated with brain cholinergic dysfunction that could benefit from nerve growth factor (NGF)-based therapies. Electroacupuncture (EA) improves brain NGF availability and action. Here we assessed the variations of NGF and tau phosphorylation in the cortex and hippocampus, as well as the expression of choline acetyltransferase in the basal forebrain following diabetes induction and EA in adult rats. We found that EA counteracts diabetes-associated tau hyperphosphorylation and decreases in NGF and choline acetyltransferase, suggesting a possible beneficial effect of EA on brain cholinergic system in diabetes.

Over-expression of N-type calcium channels in cortical neurons from a mouse model of Amyotrophic Lateral Sclerosis.

Voltage-gated Ca(2+) channels (VGCCs) mediate calcium entry into neuronal cells in response to membrane depolarisation and play an essential role in a variety of physiological processes. In Amyotrophic Lateral Sclerosis (ALS), a fatal neurodegenerative disease caused by motor neuron degeneration in the brain and spinal cord, intracellular calcium dysregulation has been shown, while no studies have been carried out on VGCCs. Here we show that the subtype N-type Ca(2+) channels are over expressed in G93A cultured cortical neurons and in motor cortex of G93A mice compared to Controls. In fact, by western blotting, immunocytochemical and electrophysiological experiments, we observe higher membrane expression of N-type Ca(2+) channels in G93A neurons compared to Controls. G93A cortical neurons filled with calcium-sensitive dye Fura-2, show a net calcium entry during membrane depolarization that is significantly higher compared to Control. Analysis of neuronal vitality following the exposure of neurons to a high K(+) concentration (25 mM, 5h), shows a significant reduction of G93A cellular survival compared to Controls. N-type channels are involved in the G93A higher mortality because ω-conotoxin GVIA (1 µM), which selectively blocks these channels, is able to abolish the higher G93A mortality when added to the external medium. These data provide robust evidence for an excess of N-type Ca(2+) expression in G93A cortical neurons which induces a higher mortality following membrane depolarization. These results may be central to the understanding of pathogenic pathways in ALS and provide novel molecular targets for the design of rational therapies for the ALS disorder.