modulation-tau-subcellular-localization-as-tool-to-investigate

Modulation of Tau Subcellular Localization as a Tool to Investigate the Expression of Disease-related Genes

Tau is a microtubule binding protein expressed in neurons and its main known function is related to the maintenance of cytoskeletal stability. However, ...

Scuola Normale Superiore

Recent evidence points to homeotic proteins as actors in the crosstalk between development and DNA replication. The present work demonstrates that HOXC13, previously identified as a new member of human DNA replicative complexes, is a stable component of early replicating chromatin in living cells: it displays a slow nuclear dynamics due to its anchoring to the DNA minor groove via the arginine-5 residue of the homeodomain. HOXC13 binds in vivo to the lamin B2 origin in a cell-cycle-dependent manner consistent with origin function; the interaction maps with nucleotide precision within the replicative complex. HOXC13 displays in vitro affinity for other replicative complex proteins; it interacts also in vivo with the same proteins in a cell-cycle-dependent fashion. Chromatin-structure modifying treatments, disturbing origin function, reduce also HOXC13-origin interaction. The described interactions are not restricted to a single origin nor to a single homeotic protein (also HOXC10 binds the lamin B2 origin in vivo). Thus, HOX complexes probably contribute in a general, structure-dependent manner, to origin identification and assembly of replicative complexes thereon, in presence of specific chromatin configurations.

Homeotic proteins participate in the function of human-DNA replication origins.

We present a toolbox for the study of molecular interactions occurring between NGF and its receptors. By means of a suitable insertional mutagenesis method we show the insertion of an 8 amino acid tag (A4) into the sequence of NGF and of 12 amino acid tags (A1 and S6) into the sequence of TrkA and P75NTR NGF-receptors. These tags are shortened versions of the acyl and peptidyl carrier proteins; they are here covalently conjugated to the biotin-substituted arm of a coenzyme A (coA) substrate by phosphopantetheinyl transferase enzymes (PPTases). We demonstrate site-specific biotinylation of the purified recombinant tagged neurotrophin, in both the immature proNGF and mature NGF forms. The resulting tagged NGF is fully functional: it can signal and promote PC12 cells differentiation similarly to recombinant wild-type NGF. Furthermore, we show that the insertion of A1 and S6 tags into human TrkA and P75NTR sequences leads to the site-specific biotinylation of these receptors at the cell surface of living cells. Crucially, the two tags are labeled selectively by two different PPTases: this is exploited to reach orthogonal fluorolabeling of the two receptors co-expressed at low density in living cells. We describe the protocols to obtain the enzymatic, site-specific biotinylation of neurotrophins and their receptors as an alternative to their chemical, nonspecific biotinylation. The present strategy has three main advantages: i) it yields precise control of stoichiometry and site of biotin conjugation; ii) the tags used can be functionalized with virtually any small probe that can be carried by coA substrates, besides (and in addition to) biotin; iii) above all it makes possible to image and track interacting molecules at the single-molecule level in living systems. 

Site-specific labeling of neurotrophins and their receptors via short and versatile peptide tags.

The classical view of nerve growth factor (NGF) action in the nervous system is linked to its retrograde axonal transport. However, almost nothing is known on the trafficking properties of its unprocessed precursor proNGF, characterized by different and generally opposite biological functions with respect to its mature counterpart. Here we developed a strategy to fluorolabel both purified precursor and mature neurotrophins (NTs) with a controlled stoichiometry and insertion site. Using a single particle tracking approach, we characterized the axonal transport of proNGF versus mature NGF in living dorsal root ganglion neurons grown in compartmentalized microfluidic devices. We demonstrate that proNGF is retrogradely transported as NGF, but with a lower flux and a different distribution of numbers of neurotrophins per vesicle. Moreover, exploiting a dual-color labelling technique, we analysed the transport of both NT forms when simultaneously administered to the axon tips. 

Precursor and mature NGF live tracking: one versus many at a time in the axons.

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

The chemokine CXCL12 mediates the anti-amyloidogenic action of painless human nerve growth factor.

The ability to selectively interfere with post-translationally modified proteins would have many biological and therapeutic applications. However, post-translational modifications cannot be selectively targeted by nucleic-acid-based interference approaches. Here we describe post-translational intracellular silencing antibody technology (PISA), a method for selecting intrabodies against post-translationally modified proteins. We demonstrate our method by generating intrabodies against native acetylated proteins and showing functional interference in living cells.

Post-translational selective intracellular silencing of acetylated proteins with de novo selected intrabodies.

Neurotrophins are growth factors of fundamental importance for the development, survival and maintenance of different neuronal and non-neuronal populations. Over the years, the use of labeled neurotrophins has helped in the study of their biological functions, leading to a better understanding of the processes that regulate their transport, traffic, and signaling. However, the diverse and heterogeneous neurotrophin labeling strategies adopted so far have often led to poorly reproducible protocols and sometimes conflicting conclusions. Here we present a robust, reliable, and fast method to obtain homogeneous preparations of fluorescent proNGF and NGF with 1:1 labeling stoichiometry. This strategy is well suited for several applications, ranging from advanced imaging techniques such as , to analyses that require large amounts of neurotrophins such as  monitoring of protein biodistribution. As a proof of the quality of the labeled NGF and proNGF preparations, we provide a quantitative analysis of their colocalization with proteins involved in the  function and sorting. This new analysis allowed demonstrating that proNGF localizes at a sub-population of endosomes not completely overlapped to the one hosting NGF.

An Optimized Procedure for the Site-Directed Labeling of NGF and proNGF for Imaging Purposes.

The morphogen FGF8 plays a pivotal role in neocortical area patterning through its inhibitory effect on COUP-TFI/Nr2f1 anterior expression, but its mechanism of action is poorly understood. We established an in vitro model of mouse embryonic stem cell corticogenesis in which COUP-TFI protein expression is inhibited by the activation of FGF8 in a time window corresponding to cortical area patterning. Interestingly, overexpression of the COUP-TFI 3'UTR reduces the inhibitory effect of FGF8 on COUP-TFI translation. FGF8 induces the expression of few miRNAs targeting COUP-TFI 3'UTR in silico. We found that the functional inhibition of miR-21 can effectively counteract the inhibitory effect of FGF8 in vitro and regulate COUP-TFI protein levels in vivo. Accordingly, miR-21 expression is complementary to COUP-TFI expression during corticogenesis. These data support a translational control of COUP-TFI gradient expression by FGF8 via miR-21 and contribute to our understanding of how regionalized expression is established during neocortical area mapping.

The microRNA miR-21 Is a Mediator of FGF8 Action on Cortical COUP-TFI Translation.

The neurotrophin Nerve growth factor (NGF) plays a critical role in the mature and developing nervous system. A point mutation (R100W) in the NGFB gene was found in patients with Hereditary Sensory and Autonomic Neuropathy type V (HSAN V), which leads to pain insensitivity. In a previous work it has been shown that the mutation provokes a reduced secretion of mature NGF. In this study we generated and analyzed homozygous NGF mice to understand whether the reduced NGF bioavailability can contribute to the clinical phenotype of the homozygous condition. We found that the majority of NGF mice were born normal but failed to reach the first month of age. This early lethality was rescued by daily treatment with wild type NGF. In addition, we found that the density of cholinergic neurons of homozygous mice was unaffected in the medial septum and in the nucleus basalis of Meynert, whereas, suprisingly, it was increased specifically in the striatum. Due to the known action of the striatal cholinergic tone in modulating pain, our findings support the hypothesis that a central mechanism, linked to the NGF-dependent increase of the striatal cholinergic tone, can contribute to the pain insensitivity observed in HSAN V patients.

Cholinergic striatal neurons are increased in HSAN V homozygous mice despite reduced NGF bioavailability.

Formation of Tau aggregates is a common pathological feature of tauopathies and their accumulation directly correlates with cytotoxicity and neuronal degeneration. Great efforts have been made to understand Tau aggregation and to find therapeutics halting or reversing the process, however, progress has been slowed due to the lack of a suitable method for monitoring Tau aggregation. We developed a cell-based assay allowing to detect and quantify Tau aggregation in living cells. The system is based on the FRET biosensor CST able to monitor the molecular dynamic of Tau aggregation in different cellular conditions. We probed candidate compounds that could block Tau hyperphosphorylation. In particular, to foster the drug discovery process, we tested kinase inhibitors approved for the treatment of other diseases. We identified the ERK inhibitor PD-901 as a promising therapeutic molecule since it reduces and prevents Tau aggregation. This evidence establishes the CST cell-based aggregation assay as a reliable tool for drug discovery and suggests that PD-901 might be a promising compound to be tested for further preclinical studies on AD.

Identification of an ERK Inhibitor as a Therapeutic Drug Against Tau Aggregation in a New Cell-Based Assay.

The p75 neurotrophin (NT) receptor (p75) plays a crucial role in balancing survival-versus-death decisions in the nervous system. Yet, despite 2 decades of structural and biochemical studies, a comprehensive, accepted model for p75 activation by NT ligands is still missing. Here, we present a single-molecule study of membrane p75 in living cells, demonstrating that the vast majority of receptors are monomers before and after NT activation. Interestingly, the stoichiometry and diffusion properties of the wild-type (wt) p75 are almost identical to those of a receptor mutant lacking residues previously believed to induce oligomerization. The wt p75 and mutated (mut) p75 differ in their partitioning in cholesterol-rich membrane regions upon nerve growth factor (NGF) stimulation: We argue that this is the origin of the ability of wt p75 , but not of mut p75, to mediate immature NT (proNT)-induced apoptosis. Both p75 forms support proNT-induced growth cone retraction: We show that receptor surface accumulation is the driving force for cone collapse. Overall, our data unveil the multifaceted activity of the p75 monomer and let us provide a coherent interpretative frame of existing conflicting data in the literature.

Article	Year
Homeotic proteins participate in the function of human-DNA replication origins. Nucleic acids research\| PubMed ID: 20693533	2010
Site-specific labeling of neurotrophins and their receptors via short and versatile peptide tags. PloS one\| PubMed ID: 25426999	2014
Precursor and mature NGF live tracking: one versus many at a time in the axons. Scientific reports\| PubMed ID: 26829890	2016
The chemokine CXCL12 mediates the anti-amyloidogenic action of painless human nerve growth factor. Brain : a journal of neurology\| PubMed ID: 28031222	2017
Post-translational selective intracellular silencing of acetylated proteins with de novo selected intrabodies. Nature methods\| PubMed ID: 28092690	2017
An Optimized Procedure for the Site-Directed Labeling of NGF and proNGF for Imaging Purposes. Frontiers in molecular biosciences\| PubMed ID: 28210618	2017
The microRNA miR-21 Is a Mediator of FGF8 Action on Cortical COUP-TFI Translation. Stem cell reports\| PubMed ID: 30174317	2018
Cholinergic striatal neurons are increased in HSAN V homozygous mice despite reduced NGF bioavailability. Biochemical and biophysical research communications\| PubMed ID: 30612733	2019
Identification of an ERK Inhibitor as a Therapeutic Drug Against Tau Aggregation in a New Cell-Based Assay. Frontiers in cellular neuroscience\| PubMed ID: 31496937	2019
Fast-diffusing p75 monomers support apoptosis and growth cone collapse by neurotrophin ligands. Proceedings of the National Academy of Sciences of the United States of America\| PubMed ID: 31515449	2019

Mariantonietta Calvello

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