The development of new therapeutic avenues that target the early stages of Alzheimer's disease (AD) is urgently necessary. A disintegrin and metalloproteinase domain 10 (ADAM10) is a sheddase that is involved in dendritic spine shaping and limits the generation of amyloid-β. ADAM10 endocytosis increases in the hippocampus of AD patients, resulting in the decreased postsynaptic localization of the enzyme. To restore this altered pathway, we developed a cell-permeable peptide (PEP3) with a strong safety profile that is able to interfere with ADAM10 endocytosis, upregulating the postsynaptic localization and activity of ADAM10. After extensive validation, experiments in a relevant animal model clarified the optimal timing of the treatment window. PEP3 administration was effective for the rescue of cognitive defects in APP/PS1 mice only if administered at an early disease stage. Increased ADAM10 activity promoted synaptic plasticity, as revealed by changes in the molecular compositions of synapses and the spine morphology. Even though further studies are required to evaluate efficacy and safety issues of long-term administration of PEP3, these results provide preclinical evidence to support the therapeutic potential of PEP3 in AD.

Very few cases of gastric paragangliomas have been reported in the literature to date. We report a rare case of parietal gastric paraganglioma fortuitously detected during intraoperative exploration. A 82-years-old woman presented to our emergency room for abdominal pain. On physical examination abdomen was painful on palpation and Blumberg's sign was present. The laboratory exams showed a neutrophilia in absence of leukocytosis. Acute appendicitis was suspected and a laparoscopy was performed. At exploration, the vermiform appendix was normal while a lumpy, hard-fibrous and white-pinkish extraluminal lesion of the anterior wall of the gastric body near the greater curvature of about 2 cm in diameter was present. Laparoscopic resection of the gastric lesion was performed. The patient was discharged in good condition in the fourth postoperative day. Pathologic examination revealed a gangliocitic paraganglioma. The patient is alive and well without evidence of relapse 6 months after surgery. Gastric paraganglioma is a very rare tumor and its diagnosis is very difficult. Surgical excision is the treatment of choice which can be performed successfully with laparoscopy.

Amyloidβ oligomers (AβOs) are crucial neurotoxic species in Alzheimer’s Disease (AD) but they are still difficult targets for therapy. We demostrated that recombinant antibody fragments can be exploited as intracellular antibodies to block or modulate the toxicity of endogenous AβOs in living cells. In this way, we established a new experimental paradigm of subcellular-localized conformational-selective interference (CSI) (Meli et al., Nature Comm 2014). The intrabody-based CSI approach, previously established in mammalian fAD cell lines, was here developed through the use of new lentiviral systems, inducible or not, exploited in different murine and human primary cell systems. In detail, we investigated: i. primary neuronal stem cells (NSC) derived from neurogenic niches of the adult brain of AD mouse model Tg2576; ii. primary human fibroblasts from different AD patients (some of them carrying the V717I fAD mutation); iii. human iPS cells derived from primary fibroblasts. NSC were mantained in vitro as neurosphere cultures and differentiated in neurons or astrocytes. We demostrated that subcellular AβOs are responsible for alterations on growth and differentiation of NSC, and for dysfunctions of mitochondria and of homeostatic mechanisms in primary cells. These alterations and dysfunctions are functionally rescued by our CSI approach, in which the anti-AβOs intrabodies were targeted to the Endoplasmic Reticulum and the early secretory pathway. The subcellular localization of the intrabody-based CSI for AβOs in the Endoplasmic Reticulum and the early secretory pathway, strongly suggests the role of these subcellular compartments in determining the toxic activity of endogenously produced AβOs. In mammalian fAD cell lines we found an AD-relevant functional link between the ER and mitochondria, which seems to be confirmed in human primary fibroblasts. Thus, the intrabody-based CSI for AβOs is an useful approach to study AβO actions inside living cells and it gives a new perspective for in vivo immunotherapy. Supported by Alzheimer's Association NIRG-12-237751, Human Brain Project Neuroantibodies #604102.

Hypothalamic orexin/hypocretin neurons send long axonal projections through the dorsal spinal cord in lamina I-II of the dorsal horn (DH) at the interface with the peripheral nervous system (PNS). We show that in the DH OXA fibers colocalize with substance P (SP) positive afferents of dorsal root ganglia (DRG) neurons known to mediate sensory processing. Further, OR1 is expressed in p75(NTR) and SP positive DRG neurons, suggesting a potential signaling pathway between orexin and DRG neurons. Interestingly, DRG sensory neurons have a distinctive bifurcating axon where one branch innervates the periphery and the other one the spinal cord (pseudo-unipolar neurons), allowing for potential functional coupling of distinct targets. We observe that OR1 is transported selectively from DRG toward the spinal cord, while OXA is accumulated retrogradely toward the DRG. We hence report a rare situation of asymmetrical neuropeptide receptor distribution between axons projected by a single neuron. These molecular and cellular data are consistent with the role of OXA/OR1 in sensory processing, including DRG neuronal modulation, and support the potential existence of an OX/HCRT circuit between CNS and PNS.

Aβ oligomers (AβOs) are crucially involved in Alzheimer’s Disease (AD). However, the lack of selective approaches for targeting these polymorphic Aβ assemblies represents a major hurdle in understanding their biosynthesis, traffic and actions in living cells. Here, we established a subcellularly localized conformational-selective interference (CSI) approach, based on the expression of a recombinant antibody fragment against AβOs in the endoplasmic reticulum (ER). By CSI, we can control extra- and intracellular pools of AβOs produced in an AD-relevant cell model, without interfering with the maturation and processing of the Aβ precursor protein. The anti-AβOs intrabody selectively intercepts critical AβO conformers in the ER, modulating their assembly and controlling their actions in pathways of cellular homeostasis and synaptic signalling. Our results demonstrate that intracellular Aβ undergoes pathological oligomerization through critical conformations formed inside the ER. This establishes intracellular AβOs as key targets for AD treatment and presents CSI as a potential targeting strategy.

Amyloidß oligomers (AßOs) are considered crucial players in the early pathogenesis of Alzheimer's Disease (AD) but their intracellular formation and actions are still poorly understood. The use of intracellular antibodies (intrabodies) allows selectively interfering in living cells with post-translationally modified, or processed, proteins, such as the Amyloid Precursor Protein (APP) and its products Aß/AßOs, in a way that cannot be performed with the RNAi-based methods. Here, conformation-sensitive anti-AßOs antibody fragments (Meli et al., J Mol Biol 2009) are exploited as intrabodies. We expressed recombinant anti-AßOs intrabodies targeted to distinct subcellular compartments of different types of cultured cells. The functional effects of intrabody expressed in familial AD (fAD) CHO cell models (Meli et al., Alz&Dementia 2011) were investigated in terms of mitochondrial physiology and intracellular signalling pathways. Furthermore we targeted intrabodies in primary cells derived different from the AD mouse model Tg2576: mouse embryonic fibroblasts (MEF); mouse adult fibroblasts (MAF); neuronal stem cells (NSC) from embrionic spinal cord or adult brain subventricular zone (SVZ), mantained in vitro as neurosphere cultures. Recently we demonstrated some AD-related mitochondrial dysfunctions and bioenergetic deficit in CHO cell models of Aß oligomerization (Krako et al., J Alz Disease 2013). Here, we demostrate that the anti-AßOs intrabodies targeted to the Endoplasmic Reticulum and to early secretory compartments, through their modulatory effects on the levels and assembly of AßOs, trigger a strong rescue of mitochondrial dysfunctions and bioenergetic deficits in fAD CHO cell models. In these cells, we also demonstrated that the AßOs targeted to the secretory pathway, mitochondria and to an ER-associated degradation pathway, differentially modulate the biochemical patterns of intracellular and extracellular Aß/AßOs. Finally, we are investigating the actions of anti-AßOs intrabodies in MAF, MEF and NSC. Different pools of subcellular AßOs can be selectively targeted by intrabodies. The anti-AßOs intrabodies are unique tools to study the intracellular actions of AßOs; in this way we established an AD-relevant functional link ER-mitochondria by targeting AßOs in the ER. In conclusion, subcellular pools of AßOs are relevant targets for new therapeutics and anti-AßOs intrabodies can be exploited as new diagnostics and therapeutics.

The 7WD4 and 7PA2 cell lines, widely used as cellular models for Alzheimer's disease (AD), have been used to investigate the effects of amyloid-β protein precursor overexpression and amyloid-β (Aβ) oligomer accumulation on mitochondrial function. Under standard culture conditions, both cell lines, compared to Chinese hamster ovary (CHO) control cells, displayed an ~5% decrease of O2 respiration as sustained by endogenous substrates. Functional impairment of the respiratory chain was found distributed among the protein complexes, though more evident at the level of complex I and complex IV. Measurements of ATP showed that its synthesis by oxidative phosphorylation is decreased in 7WD4 and 7PA2 cells by ~25%, this loss being partly compensated by glycolysis (Warburg effect). Compensation proved to be more efficient in 7WD4 than in 7PA2 cells, the latter cell line displaying the highest reactive oxygen species production. The strongest deficit was observed in mitochondrial membrane potential that is almost 40% and 60% lower in 7WD4 and 7PA2 cells, respectively, in comparison to CHO controls. All functional parameters point to a severe bioenergetic impairment of the AD cells, with the extent of mitochondrial dysfunction being correlated to the accumulation of Aβ peptides and oligomers.

Nerve growth factor (NGF) was discovered because of its neurotrophic actions on sympathetic and sensory neurons in the developing chicken embryo. NGF was subsequently found to influence and regulate the function of many neuronal and non neuronal cells in adult organisms. Little is known, however, about the possible actions of NGF during early embryonic stages. However, mRNAs encoding for NGF and its receptors TrkA and p75NTR are expressed at very early stages of avian embryo development, before the nervous system is formed. The question, therefore, arises as to what might be the functions of NGF in early chicken embryo development, before its well-established actions on the developing sympathetic and sensory neurons. To investigate possible roles of NGF in the earliest stages of development, stage HH 11–12 chicken embryos were injected with an anti-NGF antibody (mAb αD11) that binds mature NGF with high affinity. Treatment with anti-NGF, but not with a control antibody, led to a dose-dependent inversion of the direction of axial rotation. This effect of altered rotation after anti NGF injection was associated with an increased cell death in somites. Concurrently, a microarray mRNA expression analysis revealed that NGF neutralization affects the expression of genes linked to the regulation of development or cell proliferation. These results reveal a role for NGF in early chicken embryo development and, in particular, in the regulation of somite survival and axial rotation, a crucial developmental process linked to left–right asymmetry specification.

Increasing evidence supports the role of intracellular Aß oligomerization as an early event in the pathogenesis of Alzheimer's Disease in humans and in transgenic mice, but little is known about the intracellular processing and trafficking events of the different forms of AßOs. Here, we propose the use of intracellular antibodies (intrabodies) to dissect the cellular pathways leading to AßOs formation and actions, exploiting new conformational anti-AßOs antibody fragments (Meli et al., J. Mol. Biol. 2009), which are suitable for intracellular expression. The use of specific conformational intrabodies allows interfering in cells with post-translationally modified, or processed, proteins (as APP and its products Aß/AßOs) in a way that cannot be performed with the RNAi-based methods. In this study, we propose an intrabody-mediated targeting of AßOs in cultured cells. The intracellular expression of the anti-AßOs intrabodies was performed in a well established cell model for AßOs production and secretion, referred to as 7PA2 cells (Walsh et al., Nature, 2002), generating cells stably transfected with intrabodies in different formats. The in vivo binding and the modulation of AßOs generation were studied by analysis of cellular media and by experiments of sub cellular fractionation and intrabody-AßOs pull-down. The anti-AßOs intrabodies targeted to specific intracellular compartments act as specific interference tools. In particular, we performed the specific and selective sub cellular targeting of AßOs in the Endoplasmic Reticulum (ER), through the expression of an intrabody with an ER retention signal (KDEL). The use of derivative KDEL form of an anti-AßOs intrabody shows: i) the efficacy in AßOs-binding, trapping and retargeting in specific ER compartments; ii) the ability to selectively reduce the secretion of small AßOs; iii) no interference in the post-translational maturation of the Aß precursor protein (APP). The specific sub cellular localization of anti-AßOs intrabodies and their efficacy in vivo in AßOs-binding, trapping and in the reduction of AßOs secreted shows that Aß principally do not oligomerize post-secretion, but intracellularly. Thus, intracellular AßOs are a relevant target for new therapeutics. We confirmed the anti-AßOs intrabodies as unique tool to study the intracellular Aß oligomerization and also to be exploited as new diagnostics and therapeutics.

The immunochemical occurrence and localization of the Glial cell line-derived neurotrophic factor (GDNF) family ligands neurturin (NTN), persephin (PSP), and artemin (ART) is described in the human postmortem hippocampus and fascia dentata from subjects aged 21 weeks of gestation to 88 years. The detectability of NTN, PSP, and ART is shown in the rat by Western blot and immunohistochemistry up to 70 h postmortem. In the human tissue, labeled neuronal perikarya were detectable for each trophin at all examined ages, with prevalent localization in the pyramidal layer of the Ammon's horn and hilus and granular layer of the fascia dentata. In the adult subjects, punctate elements were also present. Comparison of the pattern of immunoreactive structures among young and adult subjects suggests that intracellular distribution and/or trafficking of the GDNF family ligands may undergo age-related changes. Labeled glial elements were also identifiable. Western blot analysis indicates that the availability of the dimeric and monomeric forms of the trophins may vary with age and postmortem delay. The results obtained suggest the involvement of NTN, PSP, and ART in processes subserving both the organization of this cortical region during development and the functional activity and maintenance of the mature human hippocampal neurons.

The immunohistochemical occurrence and localization of the receptor components of the glial cell line-derived neurotrophic factor (GDNF) family ligands, the Ret receptor tyrosine kinase and GDNF family receptor (GFR) alpha-1 to -3, is described in the human post-mortem hippocampal formation at pre- and full-term newborn, and adult age. Two different antibodies for each of the four-receptor molecules were used. Western blot analysis indicates that the availability of GFRalpha receptor proteins may vary with age and post-mortem delay. The immunohistochemical detectability of GFRalpha-1, GFRalpha-2, GFRalpha-3 and Ret receptor molecules is shown in the rat up to 72 h post-mortem. In the human specimens, labelled neuronal perikarya were detectable for each receptor protein at all examined ages, with prevalent localization in the pyramidal layer of the Ammon's horn and hilus and granular layer of the fascia dentata. In the adult subjects, abundant punctate-like structures were also present. Labelled glial elements were identifiable. Comparison of the pattern of immunoreactive elements among young and adult subjects suggests that the intracellular distribution of the GDNF family ligands may vary between pre- and perinatal life and adult age. The results obtained suggest the involvement of the Ret and GFRalpha receptors signalling in processes subserving both the organization of this cortical region during development and the functional activity and maintenance of the mature hippocampal neurons.

The immunohistochemical occurrence of the neurotrophin (NT) proteins nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), neurotrophin-4 (NT-4), and neurotrophin-3 (NT-3) is shown in the pre-term newborn, infant, and adult human post-mortem cerebellum. The NT-like immunoreactive structures were unevenly distributed and showed regional differences among cerebellar lobules and folia. NGF-, NT-4-, and NT-3-positive neuronal perikarya were observed in all specimens examined. At variance with the other neurotrophins, the BDNF antiserum labelled neuronal cell bodies only in newborn life and infancy, as well as extensive nerve fibre systems, whose density increased with age. The NT-antibodies, tested by Western blot on human cerebellum homogenates, revealed immunoreactive bands corresponding to proteins of heterogenous molecular weight. The results obtained provide a first demonstration of the tissue localization of the NTs in the human cerebellum from perinatal to adult age, thus suggesting their involvement in the development, differentiation and maintenance of the cerebellar connectivity. Codistribution of the four NTs or sets of them was observed in cortical and deep nuclei neurons. Multiple trophic roles for NTs, encompassing the classic target-derived and local mechanisms of support, are envisaged as significant in development, differentiation, and maintenance of the human cerebellar connectivity.

The immunohistochemical occurrence of the high affinity neurotrophin (NT) receptors trkA, trkB, and trkC is shown in the pre-term newborn, infant, and adult human post-mortem cerebellum. Immunoreactive neuronal perikarya and processes were observed in all specimens examined, where they appeared unevenly distributed in the cerebellar cortical layers and deep nuclei, and showed regional differences among cerebellar lobules and folia. The trk receptor-antibodies, tested by Western blot on human cerebellum homogenates, revealed multiple immunoreactive bands for trkA and single bands for trkB and trkC. The results obtained show the tissue localization of the trk receptor-like immunoreactivity in the human cerebellum from prenatal to adult age. The analysis for codistribution of the receptors with the relevant ligand and among the receptors in discrete cortical and deep nuclei tissue fields shows a wide variety of conditions, from a good similarity in terms of type and density of labeled structures, to a lack of correspondence, and suggests the possibility of colocalization of trk receptors with the relevant neurotrophin and among them in the cerebellar cortex. These results sustain the concept that the neurotrophin trophic system participates in the development, differentiation, and maintenance of the human cerebellar connectivity and support the possibility of a multifactorial trophic support for the neurotrophins through target-derived and local mechanisms.